CN100560708C - Associativity zymotechnique continuously and in batches - Google Patents

Abstract

Employing continuously ferments that the stage adds and/or initial fermentation contains the wort of fermentable carbohydrate, produces drinkable alcohol.The exemplary stage of continuously fermenting comprises utilizes super flocculating yeast bacterium and strict oxygen control, and adopts the air lift type biological reactor.The ejecta to the small part fermentation of processing continuously can be delivered in batches the process segment after this successive stage to finish fermentation.

Description

Associativity zymotechnique continuously and in batches

Invention field

The present invention relates to the production technique of potable alcohol product, particularly beer, specifically is to adopt hybrid technique continuous and that the batch fermentation segmental machining is formed.

Background of invention

The lot of documents that deliver in recent years in the wine fermentation field has illustrated the very big interest of wine brewing industry to the immobilization production technique.(Enari 1995 several pieces of summaries; Iserentant 1995; Masschlein 1997; Mensour etc., 1997; Stewart 1996; Virkajarvi ﹠amp; Linko 1999) in emphasis narrated immobilized cell and produced the beer revolutionary character effect possible the wine-making industry overall state.Except the research group that mentions in the 3.1-3.5 joint, many other mechanisms have also participated in the research and development of the immobilized cell that is used to make wine.U.S. Miller brewages company (Duncombe etc., 1996; Tata etc., 1999) the fluidized bed bio retort that Meura Delta test unit and Schott engineering corporation are provided has been carried out some preliminary assessments.Coors brewages company and has also carried out preliminary experiment with Meura Delta system.

Slovakia technology university and Heineken joint study in the air lift type system, adopt the calcium citrate malate gel to produce beer (Domeny, 1996).Several years ago, this research group of Slovakia technology university has delivered several pieces about article (Smogrovicova etc., 1997 that they did to study; Smogrovicova ﹠amp; Domeny1999).Guinness had before studied and has adopted different absorption carriers to carry out cell fixation, fermentation (Donnelly 1998) in the fluidized bed bio retort subsequently.Donnelly in 1999 and colleague have delivered one piece of article and have described glycometabolic kinetics in its fluidized bed bio retort (Donnelly etc. 1999).Their experiment setting comprises adopts the immobilization carrier of Siran Bio-Glas as top fermented yeast bacterium.Guinness became the part of Immocon financial group described in the 2.2.5 joint afterwards.

The pilot plant (Dziondziak, 1995) that Germany Holsten Brauerei AG and Lurgi AG had developed and managed the continuous production alcohol-free beer jointly.In a step circulation bed fermentor tank (130 liters), adopt the Protanal TXF 200 pearl to ferment, used screen cloth foundation (at the bottom of 7 layers of screen cloth) to slough the ethanol in the beer simultaneously.This technology took time 8.5 hours altogether.

The Sapporo that is positioned at Japan brewages a group of company wine brewing research laboratory, has studied the Primary Fermentation of using fixed cells produce beer in the fluidized-bed reaction jar.Their research relates to adopts polyvinyl alcohol gel pearl (Shindo﹠amp; Kamimur 1990), calcium alginate gel pearl 1994a such as () Shindo, double-deck gelled fibre 1994b such as () Shindo and chitosan (chitosan) gel beads (Shindo etc. 1994) be as fixing matrix.The wort continuously feeding of having adopted glucoamylase to handle in one research of back is wherein in the biological reactor of one liter of working volume The volume of II type pearl (chitosan pearl) accounts for 25%.This enzyme is handled and is able to form acetic ester in this immobilized cell system, and this is similar to conventional batch fermentation, so this step more approaches to match with product.

Sapporo research group now will concentrate on to develop the chitosan pearl fluidized-bed fermentor tank that is used for repeated batch process production.This system has turned round big problem did not take place in 75 days, and the beer quality of production is similar to market product.Non-flocculence yeast strain shows that much higher (Maeba etc. 2000 than flocculence bacterial strain efficient; Umemoto etc., 1998).

Two kinds of other di-acetyl method of reducing were proposed in the EBC meeting that Maastricht holds in 1997.France scholar (Dulieu etc., 1997) proposition adopts the acetolactate decarboxylase of packing to transform acetylactis fast becomes 3-hydroxyl-2-butanone.Meura Delta emphasis has been reported with aluminosilicate zeolite as the PRELIMINARY RESULTS of catalyzer with the direct cold 3-of the being converted into hydroxyl-2-butanone (Andries etc., 1997) of acetylactis.Accept if these treatment process proofs effectively also are the human consumer, the low-cost alternative method to this mature system that Cultor and Alfa Laval propose just may become a reality.

Immobilized cell is produced other the non-study on the industrialization in the beer field, comprises that the line style alginic acid gel particle of Singapore " standard and study on the industrialization institute " is used for the research of packed bed retort, finds than alginic acid pearl better (Que1993).The Mafra of Portugal Universidade do Minho has reported that with reaching to work together the super flocculating yeast bacterial strain of employing carries out the continuous maturation (Mafra etc. of beer, 1997), this research group has also delivered about the flocculating yeast bacterium of adopting them in the air lift type biological reactor and has produced alcoholic acid paper (Vicente etc., 1999; Domingues etc. 2000).

The scholars of each academic institution have also studied in recent years with immobilized cell and have produced beer (Argiriou etc., 1996; Bardi etc., 1996; Cashin1996; Moll ﹠amp; Duteurtre1996; Nedovic etc., 1996; Nedovic etc., 1996b; Norton etc., 1995; Scott etc., 1995; Wackerbauer etc., 1996a; Wackerbauer etc., 1996b).China (Chao etc., 1990; Yuan1987; Zhang etc., 1988) Russia (Kolpachki etc., 1980; Sinitsyn etc., 1986) and research group (Chladek etc., 1989 of CZECHOSLOVAKIA; Curin etc., 1987; Polednikova etc., 1981) also study immobilized cell technology and delivered the result the eighties.

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Summary of the invention

The present invention relates to the production technique of potable alcohol product, comprise be used to drop into and/or at least initial fermentation contain the wort of fermentable sugars and continuously ferment the stage.Specifically, the invention provides a kind of selection process, wherein use the air lift type biological respinse, adopt flocculence (particularly high flocculence or super flocculence) yeast strain and strict oxygen control to continuously ferment.The present invention's one special preferred form is that the ejecta to the small part fermentation that will continuously ferment is transported to the batch fermentation stage to finish fermentation (can include but not limited to that in claims of the present invention fermenting process makes carbohydrate fermentation become alcohol fully).

The present invention relates to beer production (specifically comprising particularly North America type beer of pale beer, storage old (lager) beer).About this point referring to for example: " the Essentials of Beer Style " of F.Eckhart.

Technology according to claim 1, the stage of wherein continuously fermenting carries out in the air lift type biological reactor.Be used for continuously fermenting the stage of the various practices of the present invention, preferably adopt immobilized cell (opposite with pure free cell), this can select for use one of carrier immobilized yeast or flocculence yeast to carry out.Although (forging) as previously mentioned preferably replaces carrier immobilized cell with the flocculence yeast, it is particularly preferred that super flocculating yeast bacterium is used for this purpose.

About being described in more detail of preferred embodiment relevant and advantage with continuous processing, be provided among the detailed content of the present invention, comprise and adopt artificial (promptly in check) mixed gas and adopt nitrogen, carbon dioxide gas, oxygen and air.In addition, this paper also provides about the more detailed description of body temperature (hold) stage process in batches.Please note in certain embodiments of the present invention, the concern of heat preservation method is in batches surpassed fermentable saccharide " fully " is transformed into alcohol (this in fact can finish in the stage of continuously fermenting of this technology in a word).In this class embodiment, be that taste is complementary (or correction) to the main concern of holding stage in batches, particularly about di-acetyl and acetaldehyde.The preferred embodiments of the invention provide and drop into and/or continuously ferment after the stage by distributing pipelines (no matter being the fixed pipeline or the conduit of alternative connection and disengagement) in a plurality of distribution that are incubated in the basin in batches to the wort of small part fermentation.In a series of distribution processs, fill one jar, fill next jar again ....In a particularly preferred embodiment, the through-put of this successive reaction jar and the capacity that in batches is incubated are complementary with regard to the size of the insulating container of retort/in batches and quantity, and product flow and capacity/time ratio mate like this.It is desirable to, insulation time of jar discharging the finished product is just cleaning in batches, connects and the stage ejecta refitting of continuously fermenting when filling out insulation in batches jar again.

Another aspect of the present invention content is that some embodiment is about the concrete processing of oxygen level in wort/beer.What this processing was applied to this technology is incubated two stages continuously and in batches.The stage oxygen concn that continuously ferments has multiple influence.But be significantly make its reduce to minimum and optimize and make higher alcohols be transformed into the ester of features good taste may be comparatively desirable.About this point, notice if, can keep higher alcohols concentration not to be subjected to the influence of holding stage processing in batches basically with the desirable strict balance that oxygen is handled the taste of fusel ester of controlling.This selects can be with carbonic acid gas pre-washing wort before continuously fermenting.

In the one embodiment of the present invention, the continuously ferment main purpose in stage of this technology provides feeding method at the downstream batch fermentation that takes place in the insulation processing thereafter in batches.

For more sure, all include the content of above preference file the part of this paper in as this specification sheets, duplicate fully in this article as these files.

Detailed Description Of The Invention

The two portions (zymotechnique) that below are aspects content of the present invention describe in detail.

Described explanation comprises: chart, formula, accompanying drawing etc.There is a particular number curve " figure " word back.Every accompanying drawing has explanation, in " figure " word back one particular number is arranged.The article of explanation may not be standard unit's yardsticks during these were drawn.

Accompanying drawing is as follows:

Fig. 1. the continuously ferment schema of system process of pilot-scale, each parts of equipment see Table 5.1 brief summaries.

Fig. 2 .50 rises the mode chart of pilot-scale air lift type draft tube (GLDT) formula biological reactor.

Fig. 3. be Fig. 2 biological reactor sectional view, comprise the position of inner draft tube and inner separator.

Fig. 4. be Fig. 2 biological reactor top board detail drawing.

Fig. 5. be the tank body figure of Fig. 2 biological reactor.

Fig. 6. be Fig. 2 biological reactor tapered bottom detail drawing.

Fig. 7. be the detail drawing of Fig. 2 biological reactor gas pipe line shower nozzle, bored 160 holes (0.16 centimetre of diameter) altogether in this duct type shower nozzle of 1.27 cm diameters, longitudinal interval is 0.8 centimetre between the center, hole, 0.6 centimetre of lateral separation.

Fig. 8. adopt motionless mixer (the Labatt number of patent application 2.133.789) graphic extension of pearl method fermentative production continuously.

Fig. 9 .Schott Engineeing provides

The photo of granulated glass sphere.

Figure 10 .World Minerals provides

Diatomite pearl photo.

Figure 11 .Labatt brewages the κ-carrageenan gel beads photo of company limited (" Labatt ") Laboratory Production.

Figure 12. represent non-flocculating yeast bacterium, the yeast that forms chain and the photo of flocculating yeast bacterium respectively.These photos are taken with focusing on photomicroscope, amplify 100 times.

Figure 13. the Photomicrograph of medium flocculence yeast strain LCC3021, amplify 100 times.

Figure 14. the Photomicrograph of super flocculating yeast bacterial strain LCC290, amplify 100 times.

Figure 15. the graphic extension of preparation κ-carrageenan gel beads motionless mixer processing.Liquid flow makes liquid mix when being pumped to pipeline through this mixing tank (rather than agitator moves in liquid) in motionless mixer.

Figure 16. just ferment another mode chart of air lift type draft tube biological respinse can system of usefulness of beer.

Figure 17. be the photo of Figure 15 air lift type draft tube biological reactor.

Figure 18. be the figure of 13 liters of (containing 8 liters of working capacitys) air lift type pipe reaction can container of Figure 16.

Figure 19. biological reactor container top board detail drawing, wherein 1, the liquid assimilating mouth of oxygen sensor; 2, the thermowell of temperature sensor is connected in radiator valve; 3, temp probe; 4, the liquid return port of oxygen sensor; 5, inoculation mouth; 6, the film thief hatch that has the stainless steel lid.

Figure 20. the general picture figure of oxygen sensor liquid assimilating mouth has filtration (filler) element that is immersed in the biological reactor liquid.

Figure 21. adopt the detailed device and the frontview (seeing Table the detailed description of 5 pairs of devices) of the first fermentative production of continuous beer of air lift type biological respinse can system.

Figure 22. the graphic extension of carrageenan gelling mechanism (picking up from Rees, 1972).

Figure 23. immobilized yeast utilizes the graphic extension of wort composition in just fermenting.

Figure 24. contain immobilization and store the old saccharomycetic κ-photo of carrageenan gel beads when fermenting zero point.

Figure 25. the pod-like thing photo that the old yeast batch fermentation of storage forms two days later in κ-carrageenan gel beads shows the bud trace on the single yeast cell.

Figure 26. κ-carrageenan gel beads outer rim photo shows the old yeast Weiberg process of storage that continuously ferments after two months.

Figure 27. the photo of old yeast cell is store in the κ-carrageenan gel beads external region of continuously fermenting after two months.

Figure 28. the photo of old yeast cell is store in the κ-carrageenan gel beads center of continuously fermenting after two months.

Figure 29. the photo of whole κ-carrageenan gel beads after continuously fermenting six months, many pearls of breaking are hollow.

Detailed Description Of The Invention one first part

Yeast strain and inoculum preparation

The polyploid yeast (being also referred to as Sucus Vitis viniferae sugar yeast and/or Ka Ersibai sugar yeast) of yeast saccharomyces cerevisiae pedigree is adopted in the fermentation that this paper carried out.Brewage the boundary and usually this yeast is called the submerged fermentation bacterium of production storage type beer.This feature is attributable to store old yeast can sedimentation from liquid nutrient medium when fermentation is finished.And pale beer Ale yeast does not resemble the old yeast of storage, can be raised to the top of fermentor tank thereby be called the top fermentation bacterial strain.Yeast sinks to the bottom or the ability that floats depends on that not necessarily yeast is old type of storage or ale type, and tool bacterial strain specificity.Store old yeast usually in temperature nonfermented more than 34 ℃.And the unfermentable melibiose of ale yeast.Scientists will utilize these feature differentiation to store old bacterial strain and ale yeast (McCabe 1999).

Medium flocculence yeast strain is the Wine brewing yeast strain 3021 of Labatt culture collection institute (LCC), is used for the fermentation of free cell self aggregation and κ-carrageenan immobilization fermentation.Fixedly bacterial strain (immobilizant) of super flocculence yeast conduct is used in this test, and pure yeast culture is frozen in-80 ℃ of cryogenic refrigerators of Labatt Technology Development Department.When needs, the yeast culture of getting several rings with the aseptic inoculation ring carries out 21 ℃ of aerobic pre-cultivations on the PYG agar plate.This agar plate restrains peptones with 3.5,3 gram yeast extracts, 2 gram KH ₂PO ₄, 1 the gram MgSO ₄7H ₂O, 1.0 gram (NH ₄) ₂SO ₄, 20 the gram glucose, 20 the gram agar be dissolved in the distilled water to 1 liter of volume.

Then with isolating yeast colony lift in the test tube of the wort that contains 10ml pasteur's method sterilization, in 21 ℃ of mixed culture 24 hours.This culture progressively is expanded to 5 liters by (10ml join 190ml, 200ml join 800ml, 1 liter join in 4 liters) in the wort that last culture is added proper volume.Then yeast culture is moved in the tophan pot,, from gained yeast moist precipitate thing (30%w/v), take out the yeast cenobium that all its secondary fermentation need in centrifugal 10 minutes of 4 ℃ of 10000rpm.

Fermention medium

The technical grade that adopts Labatt London ferment to make factory's production is store the nutritional medium of old wort as all fermentation usefulness.The wort proportion of this paper is expressed as Plato degree (° P), the relation between formula 4.1 explanation proportions and ° P.

°P＝135.997·SG ³-630.272·SG ²+1111.14·SG-616.868 (4.1)

The used wort of the present invention is 17.5 ° of P, is equivalent to proportion 1.072.

Table 4.1 provides the typical carbohydrate of this wort to form general picture, saves described high performance liquid chromatography (HPLC) method with 4.7.2 and measures.About 73% carbohydrate is fermentable in this wort, and the S. cervisiae of this research usefulness is difficult to absorb 27% long-chain sugar.

The typical carbohydrate of the used wort of table 4.1 fermentation test is formed.This wort is produced by Labatt London brewery, and the proportion that records is 17.5 ° of P

The variation coefficient of the materials that great majority are analyzed is between 10%～20%, and this variation major part is to brewage raw materials used difference and cause owing to used industrial manufacture process and each time.

The immobilization type

---embedding, absorption and self aggregation---tested in this Ph D dissertation research three types.For the carrier of industrial source, give information earlier by the supplier, the chamber is analyzed additional by experiment then.Photo and particle size distribution data (in the time can obtaining) are provided herein to the carrier of being studied.

Tested two kinds of adsorbing bases in the through-flow tubular type biological reactor of the gas lift of prerun scale, this paper provides the photo of these two kinds of carriers.Schott Engineering provides agglomerating sintered glass bead carrier

Select the particle of 1～2 millimeter of diameter, have for the immobilized open aperture of yeast, pore volume accounts for 55～60%, and pore size distribution is equivalent to the yeast cell size between 60～300 microns.It is reported this kind carrier organism and chemically stable, easy to clean, reusable, but vapor sterilization, do not compress, taste neutrality, therefore ratify to be used for food mfg.

The ball type carrier that the World Minerals of California provides a kind of diatomite to form, this carrier advantage are heat and chemically stable, physical strength height and tool rigidity.Diatomite is to brewage the basic material that industry is usually used in filtered beer.

The R-632 carrier is that specialized designs is used for full cell fixation.

Supplier's specification sheets is as follows:

The granular size scope	0.595 millimeter～1.41 millimeters (14/30 eye mesh screen is held back)
		Mean pore size	7.0 micron
The cumulative volume in hole	1.19 centimetre 3/ gram
		Bed density under the compact state	0.334 kilogram/rice 3

κ-carrageenan gel beads is to be brewageed a kind of embedding carrier of company limited's Laboratory Production by Labatt.Its production technique is described in 5.2 joints, produces to the results are shown in the 6.2.1 joint.

The simplest immobilization pattern is a self aggregation, can produce by the yeast strain of selecting to flocculate.The old yeast LCC3021 of the storage of industrial usefulness is natural to have flocculation ability, is considered to a kind of medium flocculation bacterial strain.Along with the little flora of 0.5-1.0 millimeter yeast that carries out that ferments will form in the liquid medium within.The LCC290 yeast is that LCC3021 stores an old saccharomycetic variant, will form much bigger throw out (the 1.0-5.0 millimeter depends on degree of mixing), thereby classifies as super flocculating yeast bacterium, and this paper provides the photo of various yeast throw outs.

The method of sampling

Along with the carrying out of fermentation, repeatedly fermentation broth sample is taked in the compartment of terrain.Bought Scandi-in order to carry out this task

Aseptic sampling valve, these valves are Stainless Steel Products, are equipped with to store the cell (is the boundary with apical pore and bottom outlet) that ethanol keeps gnotobasis.Remove the retaining cap of outlet at bottom pipe before the sampling, from the chamber, emit ethanol.Fresh ethanol (by volume the accounting for 75%) cell of flowing through covers the valve apical pore then.The pulling valve lever is collected about 50ml liquid sample in the sterile chamber, and second part of fermented sample of collection contains super flocculating yeast bacterium, therefore will carry out the suitable flocculation of taking off before cell counting.In case finish sampling, Peracetic Acid is used in the hot water drip washing of valve cell then, washes with ethanol at last.Cover outlet at bottom pipe retaining cap, cell is filled ethanol and is ready to sampling next time.

The microbiology monitoring

Free yeast cell counting and methylene blue method viability are checked

Collect the liquid sample that fermented liquid contains free suspension yeast cell with the above-mentioned method of sampling earlier.Adopt opticmicroscope and 10 ^-4The hematimeter of the Hauser scientific company of ml volume carries out cell counting.Use the distilled water diluting liquid sample, reach that yeast adds up to 150～200 cells in the counting visual field.Heggart etc. (1999) described the factor of influential yeast viability and vigor.In order to assess saccharomycetic vitality degree in the sample, adopt the said methylene blue staining technology of ASBC (ASBC Technical Committee and editorial board 1992).Viable cell can oxidized methylene blue make it colourless, and dead cell will be dyed blueness on the other hand.The methylenum coeruleum liquid that preparation is used for the viability assessment has adopted following reagent:

Solution A: 0.1 gram methylenum coeruleum is dissolved in 500 ml distilled waters

Solution B: 13.6 gram KH ₂PO ₄Be dissolved in 500 ml distilled waters

Solution C: 2.4 gram Na ₂HPO ₄12H ₂O is dissolved in 100 ml distilled waters

With 500 ml soln A, 498.75 ml soln B and 1.25 ml soln C are mixed with Fink-Kuhles methylenum coeruleum damping fluid, and final blending liquid pH is 4.6.The cell suspension and the methylenum coeruleum of thorough mixing dilution in a test tube.This mixture is left standstill several minutes (guaranteeing that cell contacts with dyestuff), get a mixed solution and place (prescribed volume) between glass for haemocytometer,cover and the cover glass.That live in the count visual field and dead cell count, then with viable count divided by total cellular score, measure viable cell per-cent.

4.5.2 immobilized yeast cell counting---self aggregation

When employing was tended to form the yeast cell of flocculation piece, difficulty was the cell per-cent in the accurate assessment liquid sample, because cell can sedimentation in sampling jug.In order to obtain representative sample, adopted to take off flocculation agent.In these experiments, adopt the sulphuric acid soln of 0.5% volume to destroy the stable of flocculating yeast cell, thereby obtain representational yeast cell counting.Adopt 4.5.1 to save described same counting and viability mensuration program, replace distilled water to make thinner with sulfuric acid.

Fixed yeast cell counting---gel beads

Before the yeast cell of counting gel embedding, must use

Device (Brinkmann Instruments) destroys gel matrix.Make the pearl sample by a sterilization screen cloth (500 microns of screen sizes) earlier, use aseptic water washing then.Get the cell pearl and 19 ml distilled waters of 1 milliliter of gel embedding, add in the 50 ml sample containers, use

Physical property is destroyed gel yeast is discharged in the solution.On gel destroyed sample, carry out 5.5.1 and save described counting and viability mensuration.

Pollution monitoring

All fermenting processs that this paper carried out are the periodic monitoring pollution condition all.Monitoring facilities comprise verify at least weekly one time in 50 liters of jars that continuously ferment liquid and the wort in the accumulator tanks.The liquid sample of aseptic collection is coated culture plate (containing Universal Beer agar (UBA Difco Laboratories) and 10mg/L cycloheximide).With specimen under anaerobism and aerobic conditions 28 ℃ cultivated 10 days.The flat board of selecting put into contain

In the jar of bag (Oxoid), it removes a jar interior residual oxygen, forms required anaerobic growth environment.Verify whether really anaerobism of this environment with a bar (as there being oxygen meeting pulverize redness).As existing polluted bacteria can detect in the liquid sample with this method.

Detection wild or non-yeast saccharomyces cerevisiae needs a kind of isolation medium, and it is unfavorable for the growth of bacterium and/or S. cervisiae.0.4 grams per liter CuSO will have been added ₄Microzyme culture medium (YM.Difco Laboratories) preparation pour plate be used for optionally allowing potential wild yeast bacteria growing (cultivating 7 days for 25 ℃).The liquid sample that is seeded on PYN agar (PeptoneYeast-Extract Nutrient.Difco) flat board was cultivated 7 days for 37 ℃, carried out the inspection of non-storage ageing brewer yeast bacterium.The old yeast growth of storage is suppressed under the temperature more than 34 ℃, therefore should have the ale yeast to pollute if there is any growth to show on the flat board.

Analytical procedure

By the standard industry schedule of operation all relevant devices are suitably calibrated.

Ethanol

With the alcohol concn in the described vapor-phase chromatography of Technical Committee of ASBC and editorial board (1992) analysis beer and the fermented sample.With the liquid sample of the degassing with get this mixture 0.2 microlitre injection Perkin Elmer8500 gas-chromatography (GC) instrument after 5%v/v Virahol internal standard product mix.Following table further provides the definite setting of GC in detail.

Flame ionic detector (FID)

The automatic sample thief of Dynatech

Chromosorb 102, and 80-100 order carrier is filled

Helium carrier gas flow velocity 20 ml/min

175 ℃ of injector temperature, 250 ℃ of detector temperatures, 185 ℃ of isothermals of column temperature.

Carbohydrate

With high performance liquid chromatography (Spectra-Physics SP8100XR HPLC) systems measurement glucose, fructose, wort, trisaccharide maltose, maltotetrose, polysaccharide and glycerol concentration.Cationic exchange coloum (Bio-Rad Aminex HPX-87K) as moving phase, separates these carbohydrates with dipotassium hydrogen phosphate when from this system wash-out.Produce the amount that suitable compound peaks is determined compound with RI-detector then.HPLC operation: counterpressure 800psi, 85 ℃ of column temperature, 40 ℃ of detector temperatures.With the sample degassing and be diluted to proper level.Get 10 microlitres and inject this system, flow velocity 0.6 ml/min.

Brewing industry is assessed the total glucides level of liquid usually with other method.The Plato kilsyth basalt that the proportion of liquid is measured with Anton PaarDMA-58 density instrument shows.Carry out electronic oscillation with moving into special glass U-shaped test tube with the sample that outgases after filtration, measure the oscillation frequency of this liquid, then with liquid specific gravity (gram/100 gram or ° P) associated.Should notice that this kind mensuration is the approximation of sample carbohydrate total concn (or proportion), because be that 20 ℃ aqueous sucrose solution is calibrated, its proportion is identical with described wort.

The ortho position diketone

Detect di-acetyl (2, the 3-dimethyl diketone) total concn and 2,3-diacetylmethane total concn with Perking Elmer 8310 gas chromatographs that have been equipped with electron capture detector.Employing contains the argon carrier gas (flow velocity 1.0 ml/min) of 5% methane as carrier gas, makes sample pass through J ﹠amp; W DB-Wax post.Injector temperature remains on 105 ℃, and detector temperature is set to 120 ℃ simultaneously.Hewlett Packard 7694E head space self-actuated sampler has been accelerated analysis.Estimate selected sample composition peak area and with itself and 2,3-hexanedione internal standard product calibration value cross-reference calculates content.In order to assess the total concn of these compounds, these samples need be equilibrated at 65 ℃ and kept 30 minutes earlier.Sample preparation before this analysis is able to acetylactis and hydroxybutyric acid are transformed into its each oneself diketone, di-acetyl and 2,3-dimethyl diketone.

Ester and higher alcohols

Adopt some the most important aromatic compounds in the Headspace Gas Chromatography beer.The positive alcohol of the quantitative usefulness of acetaldehyde, ethyl acetate, isopropylcarbinol, 1-propyl alcohol, Isoamyl Acetate FCC, primary isoamyl alcohol, ethyl hexanoate and ethyl octylate is as the internal standard product.Used Hewlett Packard 5890 gas chromatographs are equipped with flame ionization detector, HP7994 head space automatic sampler and J﹠amp; W DB-Wax capillary column.Injector temperature is made as 200 ℃, and detector is 220 ℃.Furnace temperature is as follows: 40 ℃ 5 minutes, be increased to 200 ℃ with 10 ℃ of/minute speed from 40 ℃, be increased to 220 ℃ with 50 ℃ of/minute speed from 200 ℃.Kept 5 minutes in 220 ℃ at last.Replenish gas with 30 ml/min (28psig) helium, hydrogen stream 50 ml/min (25psig), airflow 300 ml/min (35psig) are replenished the helium carrier gas stream of 6.0 ml/min.The whole GC circulation of 1 ml sample endless tube 40 minutes.

Other analytes

On the basis of needs, other several analyte determinations have been carried out to experiencing fermented liq aging and packing, Labatt Quality Control portion has carried out the analysis of the finished product by various final beer standards, and these are measured based on the described method of Technical Committee of ASBC and editorial board (1992).Table 4.2 provides to be analyzed table look-up and measures relevant brief description with these.

Table 4.2 Quality Control Analysis and explanation

Yeast sedimentation method-LCC290

Preparation yeast specimen

As described in 4.1 joints, in wort, allow super flocculating yeast bacterium (LCC290) grow.In 4 ℃ with the centrifugal inoculum of 10000rpm 15 minutes, obtain the yeast spheroid and be used for next step inoculation.As described in 4.2 joints, in 100 ℃ of sterilization worts 60 minutes, be transferred to 62 liters of sterilizations and shake in the bottle 1 liter of wort is aseptic then, every bottle graft kind 4 grams are through the centrifugal yeast.Bottle is put into 21 ℃ of room temperatures of shaking table make fermentation with 135rpm.In the following timed interval: 24, respectively got one bottle in 40,48,64,71 and 192 hours and come out, get the liquid small sample and make carbohydrate analysis and yeast concentration and viability mensuration (concrete grammar is described by the 4th chapter).Remaining liquid/yeast mixt saves described yeast sedimentation method by 4.7.2 to carry out.

The yeast sedimentation method

Adopt following method to measure the flocculation rate of the super flocculating yeast bacterial strain of LCC290.Make every duplicate samples fermentation save the described time until 4.7.1.Take out suitable sample bottle in the described time, mix this sample and guarantee all particle suspensions, immediately the bottle content is moved in the 1000ml measuring graduates, along with the throw out sedimentation, distance between per 30 seconds measuring space fluid surfaces and the throw out-liquid surface, settling velocity is calculated with following formula.

The Kynch method (1952) of employing standard, the subsidence curve that obtains from each sweathouse interval draws the curve of settling velocity pair cell concentration.

Circulation and mixing velocity measuring method

In order to measure mixing time and the cycle rate in the through-flow tubular type biological reactor of three-phase gas lift, adopted an acid solution injected system that is connected with data acquisition system.This biological reactor is injected in the pulse of strong acid liquid, can then itself and 3.2.2 be saved the formula associated that proposes by monitoring pH over time, calculate cycle rate and mixing time.This data acquisition system comprises:

The Ingold pH probe that links to each other with Ingold microprocessor pH transmitter (2300 type) (Cole-Parmer, cat#P-05990-90).

Data translation DT2805 card

The 386DX computer

With Quick Basic data acqrisition program (J.1994 Hudson C and Beltrano write, and N.Mensour1998 revises).

That gets that 10 milliliters of 10N hydrochloric acid inject the through-flow tubular type biological reactors of gas lift (seeing Fig. 5 .5) just is positioned at annular space under the pH probe.Be equivalent to following 26 centimetres of top board herein, use through the standard buffer solution (green damping fluid of Beckman pH7.0 and the red damping fluid of pH4.0) of calibrating before all mixed experiments the pH probe is done 2 calibrations.The 4-20 milliampere electric current that pH meter is produced is connected in the screw terminal plate, and electric current is converted into voltage, obtains card by the data of computer and measures.Log-on data is obtained program when injecting acid solution, collects 5-minute data with the sampling frequency of 50Hz.The array size of this program is set at data and obtains one group on card and obtain 3750 points (collect always count 15000 points).

The data of collecting are conveyed into stronger computer (Pentium II microprocessor) from laboratory computer to be done further to analyze.Extensively adopt TableCurve 2D (Jandel Scientific Software, Labtrouics Canada) does data analysis, because of its can handle big data set with and the many data splitting processing capacities of inherent (data filter is level and smooth, fitting of a curve etc.).With the Savitzky-Golay algorithm, a kind of to cross over the time domain filtration smoothing method that moving window least squares quartic polynomial fits to the basis, be applied to raw data to eliminate noise.Adjust then through the data of smoothing but not the variation of true pH measured value reflection pH.With decay sinusoidal function and the data fitting through adjusting, the calculation of parameter from match goes out mixing time and cycle rate then.

In 50 liters of air lift type biological reactors, carry out these combined experimentses during real attenuation with one of three kinds of fixation supports (super flocculating yeast LCC290, medium flocculating yeast LCC3021 or κ-carrageenan gel beads).Liquid phase is the fermentation beer of 2.5 ° of P of proportion, and gas phase comprises carbon dioxide jet gas.The control leavening temperature is at 15 ℃.Spraying the gas meter face velocity changes between 2.0～6.0 mm/second.Make this system balancing 10 minutes with certain given gas flow rate.Begin the acid solution injection test then, triplicate is selected next gas flow rate, and the mixture pH value in the retort is adjusted to original level again.

The design of tubular type biological reactor system that the experimental scale gas lift is through-flow

Gas lift is through-flow tubular type biological reactor fermentation system

Through-flow tubular type liquid bed (DTFB) system uses in three-phase system and has shown its value.Designed the through-flow tubular type biological reactor of gas lift of two identical experimental scale, be installed in the wine brewing laboratory that Labatt brewages company to carry out cut-and-try work described herein.In addition, several existing containers have been reequiped for storing wort and collecting beer.Fig. 1 flowchart text the experimental scale used whole technology of continuously fermenting.Table 5.1 has been listed being described in more detail of Fig. 1 equipment.The wort that the London distillery provides is imported in the wort basin (WT1 and WT2) of 1600 liters of working volumes by 5.08 centimetres of stainless steel pipes.The continuous supply of jar (R1 and R2) nutritional medium but the two can system warranty test scales of employing are continuously fermented.Each jar is equipped with the carbon dioxide jet system with control oxygen and homogeneity, and a glycol-cooled chuck system controlled temperature.Nutritional medium central supply chamber is set, by valve manifold system (V7, V8 and V9) to 3 independently fermentor tank chargings.Masterflex peristaltic pump (P1 and P2) is used for making described wort to flow into experimental scale biological reactor (R1 and R2).

The explanation of each equipment shown in table 5.1 Fig. 5 .1

Carbon dioxide gas and air are sprayed into this air lift type system (Fig. 7) by stainless steel gas pipeline shower nozzle.Inject the flow velocity of this system with spinner-type flowmeter (RM3, RM4, RM5 and RM6) monitoring.Sterilization filter (0.2 micron mesh) is housed to guarantee pollutent not to be imported biological reactor in the gas pipeline.Product flows out retort (Fig. 4) by overflow system.Only use the fresh feed pump controlled liq residence time.Two retort (R1 and R2) are connected in waste beer jar (WBT ₁) to collect overflowing liquid.Adopt 50 liters of specific holding tanks to collect product and processing on demand.

5.1.1 the more detailed graphic extension and the definite size of 50 liters of experimental scale production retort are provided in the joint.Wort is handled and storage procedures provides in the 5.1.2 joint, and the cleaning of the system of continuously fermenting and sterilising method see that the 5.1.3 joint is described.5.1.4 joint comprises fermentation scheme described herein.

Retort design and explanation

50 liters of working volume biological reactors that are designed for this work are all used the manufacturing of 304L stainless steel, and being positioned at the retort body has 4 Plexiglas viewing windows can observe particle and liquid movement.Selected structure material should be able to tolerate chemostefilant (corrodibility and acidity) and tolerance vapor sterilization.Another importance of design be with use the threaded connector accessory less during fermention medium directly contacts as far as possible, and with welding.Adopt sterilizable Tri Clover accessory when needing.The retort of design enlarges zone on the liquid level, thereby at utmost helps gas delivery and promote liquid-solid mass transfer (Chisti and Moo-Yong 1993).The retort bottom of design is that 90 degree bevel angles can at utmost reduce the situation that solid is not collected the bottom.Fig. 2 is mounted in the graphic extension of 50 liters of experimental scale systems in the Labatt wine brewing laboratory.This icon is understood jet entry position, liquid inlet, glycol-cooled chuck, product outlet, temperature sensing and Controlling System, and the position of two sterilizable thief holes.Fig. 3 is the explanation of same GLDT biological reactor, is of a size of centimetre.Fig. 4-6 is the detailed zone chart of 50 liters of through-flow tubular type biological reactors of gas lift, and Fig. 7 is the explanation of the used air jet system of these experiments.

Fig. 3 has illustrated inner draft tube and particle separator (baffle plate).Selecting draft tube diameter and retort diameter ratio according to documents and materials (Chisti 1991) is 2/3.The size of particle separator is divided gas flow and solid-liquid mixtures better, diameter by increasing this baffle plate (20.32 centimetres and the draft tube diameter is 10.16 centimetres) may obtain bubble and produce bigger difference between speed and the liquid-solid fluid lowering speed, will reduce gas entrainment and advance the annular space of draft tube system, and cause better solid-liquid mass transfer.

Design a tubulose shower nozzle (Fig. 7) carbon dioxide mix gas is injected through-flow area under control.160 holes in the shower nozzle of 1.27 cm diameters, have been bored altogether, 0.16 centimetre in aperture, 0.8 centimetre of longitudinal interval in the heart in these holes, 0.6 centimetre of lateral separation (20 holes of the 8 every row of row).Because jet main effect is to mix, select 0.16 centimetre of gas orifice diameter.

Wort is handled and storage procedures

Traditional zymotic is not when adding yeast, and wort is not preserved for a long time.The wort of oxygenation is the good growth medium of many microorganisms (comprising yeast).Because the fermentation of air lift type system requires a large amount of worts continuously, must carry and store method by the exploitation wort.By researchist's view, the cold wort of oxygenation such as suitable input are not preserved in the jar and can store for two weeks when not being polluted.In case also think to import to preserve and to guarantee the appropriately temperature of control wort in the jar.The jar of brewageing in the laboratory originally was designed for fermentation but not the preservation wort.Test these jars and kept the not performance of fluxion temperature.Require to keep 4 ℃ of constant temperature if Fig. 2 clearly demonstrates, just not mixing can not be with these jar.These jars of in the time of 4 ℃ wort being packed into earlier, the temperature in several points of whole jar are measured jar is with better understanding true temperature.When liquid is stayed in the jar when not mixing in 24 hours, near the liquid temperature rise of top layer to about 20 ℃.Jar middle part liquid temperature also slightly rises (rising about 3 degree), and jar cone angle part is still near initial 4 ℃.

Graphic representation 5.8 shows that jet mixing is to a conical wort preservation jar Effect on Temperature Distribution.In the time of 4 ℃ with the wort tank filling in, fill and measured temperature in back 24 hours.With CO ₂Gas sprays in the jar with 0.113 cubic centimetre/hour flow velocity.Room temperature is 19.8 ℃.Thermometric position: jar tank skin middle part, top, center (3) (4), (1) tank skin top (2) jar top, cone center, cone wall top, center (5) (6) (7), middle part cone base.

By injecting the CO of 0.133 cubic centimetre/hour of flow velocity ₂The gas gentle agitation may will be preserved jar interior wort temperature maintenance at 4 ℃.Because these find that but loading onto 2.54 centimetres of decontamination duct in the awl bottom of two worts preservation jars is used to stir wort.

The wort of not ventilation is transported to the surge tank by 5.08 centimetres of stainless steel pipes from Labatt London factory.From then on the surge tank wort is transported into wort through Pasteur's instantaneous sterilization device and is preserved one of jar (WT1 or WT2), is stored in 2 ℃ of two week therein.Be provided with the pasteurization step carefully to guarantee in whole preservation period, to eliminate the bad microorganism in the wort.Owing to adopt the not wort of ventilation, the infringement (produce outmoded aldehyde) of oxygen to hot wort can be reduced as far as possible.In addition, can strictly realize the oxygen of the jar that continuously ferments is controlled with jet introducing air.

Wort is kept in the jar, once measures the dissolved oxygen in the wort.Graphic representation 5.9 explanations by three kinds of transfer method wort oxytys over time.First example is transferred to the preservation jar with wort and begins to spray the appropriate temperature control of carbon dioxide gas (0.085 cubic metre/hour) assurance.The wort oxyty increased to about 1.3 mg/litre in first day, dropped to the 5th day then and was about 0.1 mg/litre.The test second time, the carbonic acid gas with 0.85 cubic metre/hour before filling cleaned wort preservation jar totally 3 hours, and original oxygen level significantly reduces, and wort just reaches desired oxygen level (＜0.1 mg/litre) after two days.

Fig. 5 .9 shows when London factory transports the control to dissolved oxygen the wort.The third method only is to have made up the first two kind method, estimates three kinds of can schemes altogether.The wort temperature remains on 4 ℃.

In the end in the test, clean this jar as mentioned above in advance, spray into (0.085 cubic metre/hour) and jet in the phase is stayed in storage by continuous CO 2 in the grave process.Whole storage stays phase dissolved oxygen content to keep minimum level (＜0.1 mg/litre).Therefore, adopt this method and further collect the scheme of wort as all.

5.1.2 cleaning and sterilization scheme

Make the pre-drip washing of wort hold tank experience hot water (85 ℃), the drip washing (60 ℃, 40% caustic alkali) of corrodibility cleaning, the wash phase of hot water (85 ℃) back drip washing makes the contacted fluoroacetic acid liquid of tank skin (2%w/v) realize these basin sterilizations then.The wort transport pipe also experiences same cleaning and disinfection method.

Then 50 liters of biological reactors are carried out different cleanings and sterilization process,, fill 40 ℃ of warm water then to the top with 60 ℃ of these systems of hot water drip washing.(Diverseylever Canada) forms 2%w/v solution to add a kind of industrial cleaners DiversolCX/A in water.Make air spray into this retort bottom with surface gas flow velocity 5 mm/second to guarantee suitably dissolving and in retort, suitably to contact.Contact after 1 hour, the emptying retort, with fresh tap water flushing, this cleaning procedure is secondary repeatedly.Fill with-the emptying secondary with tap water cold water at last.

The waste beer jar cleans with 2%w/v Diversol CX/A liquid, and is different with the air lift type biological reactor, without air-jet method, because WBT is not furnished with nozzle.This jar side rolling carry out mechanical stirring, repeat secondary, water is filled with-the emptying secondary then.

Before the vapor sterilization, 50 liters of biological reactors are connected with the waste beer jar, and disconnect the wort feed pipe, the gas atomization pipeline also disconnects.Open valve V10, V11, V12, V13, V14, V15, V16, V17 and V18, remove filter F5, F6 and F7, with these gas pipelines and filter respectively at 121 ℃ of autoclavings 15 minutes.Valve V12 is connected steam supply with V16.Slowly open this steam valve and reduce infringement as far as possible, pay close attention to the retort internal temperature equipment.In case internal temperature reaches 100 ℃, carry out sterilization in a hour.The valve-off V10 of elder generation, V11, V14, V15 and V18 close steam supply then and connect sterilized filter F7.Immediately will be sterilization filter F5 and F6 be connected in steam line, the carbon dioxide gas surface velocity is 3 mm/second during beginning, this air-flow guaranteed that not only retort can not subside when cooling, and replaced the air that exists in 50 liters of air lift type biological reactors.

After temperature reaches 20 ℃ in jar, the wort feed-pipe is communicated with biological reactor.Valve V2, V5, V10 and V14 still close, and valve V6, V7, V8, V9, V11 and V15 open.Which connection steam air feed of V3 or V6 depends on the wort supply that is utilized.Air-flow continues 1 hour, and after this valve-off V9, V11 and V15 supply steam simultaneously.In case pipeline reaches room temperature (20 ℃) valve-off V3 and V6 cuts off steam supply.This moment, the whole system of continuously fermenting comprised the wort supply line, and 50 liters of biological reactors and waste beer jar are sterilized, and prepared to ferment.

5.1.3 fermentation scheme

Adopt 50 liters of through-flow tubular type biological reactors of experimental scale gas lift, distiller's wort is just fermented continuously is beer.Ethylene glycol temperature control chuck is controlled at 15 ℃ with the fluid temperature in the whole fermentation test.Each retort is equipped with a temp probe, a temperature thermopair and regulates the ethylene glycol electromagnetic valve that ethylene glycol is fed to retort.This airlift fermentor also has preliminary gas mixture (carbonic acid gas or nitrogen), and the air of supply oxygen.By regulating corresponding spinner-type flowmeter/needle-valve combination, the mixed gas that selection needs allows gas mixture pass through sterilization filter (Millipore. then

-FG50,0.2 micron filtering unit) enters the draft tube of biological reactor, the air of surface velocity 0.39 mm/second (0.4scfh) is injected retort carry out all fermentations, regulate preliminary gas mixture flow velocity simultaneously to be fit to specific immobilization type.

Before continuously fermenting, when beginning, 50 liters of air lift type biological reactors start according to traditional batch-type.After cleaning as described in the 5.1.2 and sterilization, fill with 50 liters for this air lift type biological reactor and preserve the worts that jar (WT1 or WT2) transports from wort.Pass through Scandi-then The sterilization thief hole injects 200 gram yeast (4 grams per liter).With regard to κ-carrageenan gel beads, 20 liters of pearls are injected retort, produce the initial concentration of the medium flocculating yeast bacterium of LCC3021 of every liter 4 gram.Sample from biological reactor every day, and monitor di-acetyl and tintometric variation closely.In case proportion reaches its minimum value, di-acetyl concentration falls to below 30 micrograms per litre, thinks that this system can be provided with to enter to continuously ferment.Fermention medium (wort) is by retort bottom continuously feeding, and " life " beer overflows by the retort top funnel simultaneously.Because this retort working volume is a fixed, selects new sweet wort to enter the flow velocity of retort, with the controlled liq average retention time.Every day is by sterilization sampling valve (Scandi-

) export and get the liquid sample from retort, be used for chemistry and microbiologic analysis (the described method of the 4th chapter).

In the time of selecting, the fermenting organism retort is collected the product that continuously ferments in a large number (the 40 liters of sterilizing stainless steel jars) post-treatment that ferments at the beginning of 50 liters, to produce the final beer sold, estimates and makes comparisons with the contrast beer of suitability for industrialized production.Disconnect being connected of selected 50 liters of biological reactors and waste beer jar, be connected with the beer holding tank immediately.Biological reactor is connected with the waste beer jar in case collect required liquid, insulation after " life " beer of collecting is fermented is to drop to its di-acetyl level below 30 micrograms per litre again.The yeast sedimentation that liquid is stayed, and make liquid (every milliliter contains 1～500 ten thousand cell) cold be housed in 2 ℃ 7 days and wear out.Aging time after-filtration liquid, be diluted to alcohol and account for volume 5%, carbonating gas, can 341ml Beer Bottle.The wine liquid of all cans is done the pasteurization sterilization with the Labatt shop equipment.

5.2 continuous gel beads production technique

The target of this joint cut-and-try work is to estimate continuous pearl method production technique, in order to provide immobilization LCC3021 yeast cell to the through-flow tubular type biological reactor of 5.1 described 50 liters of continuous gas lifts and to produce and inoculated saccharomycetic gel beads.

This production technique (Fig. 8) at first requires to form emulsion with motionless mixer between non-water continuous phase (vegetables oil) and water-dispersion phase (and yeast cell blended κ-carrageenan gelating soln).Next step is to cool off fast with the induced polymer gelling, the gel beads that forms is joined promote its hardening in the potassium chloride liquid and make gel beads and separation of oil.

In the water-bath of 37 ℃ of controlled temperature, carry out the formation of κ-carrageenan gel beads emulsion, to prevent before carrageenan glue maturation gelling taking place.The polymkeric substance of sterilization is maintained in 37 ℃ of water-baths.Before the immobilization yeast inoculum is maintained 20 ℃.(Cole Parmer company USA) pumps into 24 members of gel and yeast slurry by 6.4 mm dia motionless mixers so that cell is dispersed in the gel with the Masterflex peristaltic pump.The aseptic oil pump (Masterflex peristaltic pump) of room temperature storage is gone into hot water bath also reach 37 ℃.

Make by another group motionless mixer then and inoculated saccharomycetic polymkeric substance (water) and mix the required emulsion of generation with oil (external phase).The emulsion that produces is cooled to 5 ℃ rapidly in ice-water bath, makes polymkeric substance drop gelling Cheng Zhu.Again pearl is put into 22 aseptic grams per liter Klorvess Liquids, help its hardening and and separation of oil.Used oil recirculation is returned processing and water (pearl and Klorvess Liquid) is moved into separating tank and carries out magnitude classification, in the 50 liters of biological reactors of packing into then.

5.2.1 motionless mixer-Kenics type

The heart of the pearl method production technique that this is novel be the Kenic motionless mixer (Cole Parmer InstrumentCompany, Niles, Illinois, USA), they are formed by being positioned at one group of stationary member that the pipeline internal diameter is equivalent to the motionless mixer diameter.These members have formed cross aisle, make by the liquid diverting flow of motionless mixer and vertically reorganization.The streamline of these meticulous generations be subjected to this mixing system urgent transverse breakage and become the emulsion that homogeneity improves.Table 5.2 has been listed three types of motionless mixers that are used for this research.

Table 5.2Kenics motionless mixer explanation (Cole Parmer provides)

5.2.2 the material of production gel beads

Two kinds of basic materials of production gel beads are oil and polymkeric substance.(model X-0909, lot number 330360 Copenhagen Pectin Denmark) are the polysaccharide polymer of a kind of extraction from red algae, for CopenhagenPectin A/S gives to κ-carrageenan.This polymkeric substance has hot glue and coagulates characteristic, and gelation temperature depends on the two concentration of κ-carrageenan ([Car]) and Repone K (KCl).This polymkeric substance is dissolved in 80 ℃ of distilled water that contain 2 grams per liter KCl concentration 30 grams per liters.The gelating soln gelation temperature that produces is 28 ℃.121 ℃ of these gels of autoclaving 1 hour place 40 ℃ of water-baths can hardening then.Commercial grade Semen Maydis oil (Pasquale Bros.Inc.Canada) also in 121 ℃ of sterilizations 1 hour, is deposited room temperature (20 ℃) until use, is ready to the yeast slurry as described in 4.1.

5.2.3 gel beads diameter measurement

In containing 100 milliliters the flask of 22 grams per liter KCl solution, collect the pearl samples, pearl is immersed in this solution 2 hours makes it hardening, by separating fuel-displaced from aqueous phase with KCl liquid continuous washing in 5 ℃ of heat exchanger outlet places.Sample retention prevents microbial contamination at 4 ℃ before analyzing.The pearl measuring diameter adopts imaging analysis software Optimas (4.02 editions, BioScan company, the U.S.) to be connected with videocorder (PentaxMacro 50mm), and the pearl sample is moved in the plate that contains thin moisture film (be used for separating gel pearl), places under the shooting hole.Measure 300-400 pearl altogether with this system.The capacity of Optimas software between 100 microns to several millimeters, 30 microns of the highest absolute error.Further analyze the data that Optimas obtains with Microsoft Excel.By sample average diameter (D _B) and the variation coefficient (COV), the sample size of signature analysis gained distributes.

5.2.4 gel beads system evaluation-experimental plan

Totally 3 kinds of motionless mixer diameter (D have been compared _S=6.4mm, 9.5mm and 12.7mm), can be according to the average pearl footpath of sample and such production of size distribution variation coefficient mensuration with the pearl of assessing the sort of type.Motionless mixer scantling numeral (Ns) changes between 12-120, and the polymer volume mark (ε that measures _c) between gel/oil solution volume ratio 8.3%～50%.ε _cBe higher than at 50% o'clock, disperse phase (gel) and external phase (oil) reversing, promptly oil droplet is included in the polymeric matrix, but not gel drops wraps in the oil matrix.The surface liquid flow rate regulation of oil/gel emulsion by the emulsion district to 3.6cm/ second and 17.8cm/ between second, calculated surface liquid speed (V by the emulsion motionless mixer with following formula _SL):

V _SL=(Q _Oil+ Q _{The angle})/S

Wherein S is the cross-sectional area that the pipe of motionless mixer is housed, Q _OilBe the volumetric flow rate of oil phase, Q _{The angle}It is the volumetric flow rate of carrageenan glue.

Result and discussion: the fermentation of 50 liters of air lift type biological reactors and hybrid dynamics

Adopting immobilized cell to produce alcoholic acid research delivers.Scholars attempted immobilized cell technology and continuous processing are combined optimization alcohol production technology (Kuu, 1982 in past 20 years; Gil, 1991; Maiorella, 1983), many people achieve success, and adopt the industrialization of continuous immobilized cell system's production ethanol.Yet Brewing industry will be implemented this continuous processing and carry out fermentative production beer just, and is so not simple.Beer not only contains ethanol, also contains countless aromatic compounds, has increased the complicacy and the degree of depth of the finished product, and following chapters and sections are described the author and produce the well balanced result that beer obtained in experimental scale GLDT fermentor tank.

6.1 the batch fermentation of experimental scale GLDT system

Batch fermentation adopts free suspension yeast cell, carries out in 50 liters of through-flow tubular type biological reactors of experimental scale gas lift.These tests provide chance to assess the feasibility that this system's fermenting wort of employing is produced beer.In addition, this test is for further relatively having set up a benchmark with the liquid phase of continuously fermenting.The old yeast strain of storage (LCC3021) with Labatt Cultrue Collection in 50 liters of biological reactors has carried out two batch fermentations.Monitored the yeast speed of growth in the fermentation whole process, and nutrient consumption and product release.

Fig. 6 .1 and 6.2 provides the yeast concentration and the viability variation diagram of batch fermentation 1 and 2 respectively.In the typical rate document of both of these case yeast growth report is arranged all.The vigor that methylenum coeruleum is measured is high all the time, and the viability value remains on about 90%.Batches 1 and 2 concentration of saccharide is seen graphic representation 6.3 and 6.4.Yeast is picked-up simple sugar glucose and fructose earlier, consumes maltose and trisaccharide maltose then, and maltotetrose and bigger polysaccharide level remain unchanged in the whole fermentation process.Ethanol is one of most important by product of metabolism of yeasts.The every metabolism 100 gram glucose of the aerobic fermentation of optimizing will produce about 48 gram ethanol and 47 gram carbonic acid gas.Also produce glycerine (per 100 gram glucose produce 3.3 grams) in a small amount, because this by product can be kept the redox equilibrium of fermented yeast bacterium, and the osmotic pressure balance of sustenticular cell, particularly ooze in the substratum at height.The variation of ethanol and glycerol concentration when graphic representation 6.5 and 6.6 has illustrated fermentation, alcohol concn raises very slowly during the fermentation beginning, because be in its grow aerobically during the phase when yeast cell, has oxygen in the fermention medium.In case exhaust oxygen, the ethanol level is exponential rising and exhausts up to metabolizable sugar, and after this its concentration level descends.The ortho position diketone also is the very important by product of metabolism of yeasts.Graphic representation 6.7 and 6.8 provides batches 1 and 2 total di-acetyl and diacetylmethane concentration.These compounds were increased to about 40 hours, corresponding to the peak value of yeast concn, be carry out the yeast growth phase amino acid synthetic due to.Fermentation later stage yeast is transformed into the lower active glycol of corresponding fragrance with di-acetyl, diacetylmethane and their precursor α-acetylactis and α-ketone butyric acid.The result who provides from this section knows that two batch fermentations carry out normally the through-flow tubular type biological reactor of gas lift.Expecting way is followed in Yeast proliferation and carbohydrate picked-up, and by product ethanol, di-acetyl and diacetylmethane are too.Each batch data comparison shows that two batch fermentation modes are very similar.Graphic representation 6.9 has compared batches 1 and 2 alcohol concn, and curve overlaps each other by identical form in many data points, shows that level repeats.Though base consumption and the order that produces product subsequently do not change in the gas lift system, fermenting speed changes.Two batches all reach peak value at about 80-85 hour alcohol concn, and representative just fermentation is finished.Be reduced to below the 30 μ g/L after 20 hours di-acetyls.The first fermentation of these results suggest heavy wort juice can be finished in about 100 hours, compared, and the old batch fermentation of traditional storage is wanted 120-168 hour.Gas lift is through-flow, and mixing that the tubular type biological reactor provides has contribution to reducing fermentation time, because improved the mass transfer of this system.Utilize these data to carry out further fermentation test surely, believe that gas lift draft tube biological reactor can significantly not change saccharomycetic fermentating metabolism.This system can reduce at least 20 hours batch fermentation time with the free saccharomycetes to make fermentation that suspends.

The cell total concn of LCC3021 yeast batch fermentation 1 and viability are with the variation of fermentation time in the graphic representation 6.1 experimental scale GLDT biological reactors

The cell total concn of LCC3021 yeast batch fermentation 2 and viability are with the variation of fermentation time in the graphic representation 6.2 experimental scale GLDT biological reactors

The carbohydrate concentration of LCC3021 yeast batch fermentation 1 is with the variation of fermentation time in the graphic representation 6.3 experimental scale GLDT biological reactors

The carbohydrate concentration of LCC3021 yeast batch fermentation 2 is with the variation of fermentation time in the graphic representation 6.4 experimental scale GLDT biological reactors

The ethanol of LCC3021 yeast batch fermentation 1 and glycerol concentration are with the variation of fermentation time in the graphic representation 6.5 experimental scale GLDT biological reactors

The ethanol of LCC3021 yeast batch fermentation 2 and glycerol concentration are with the variation of fermentation time in the graphic representation 6.6 experimental scale GLDT biological reactors

Two two acyls of LCC3021 yeast batch fermentation 1 and diacetylmethane concentration are with the variation of fermentation time in the graphic representation 6.7 experimental scale GLDT biological reactors

Two two acyls of LCC3021 yeast batch fermentation 2 and diacetylmethane concentration are with the variation of fermentation time in the graphic representation 6.8 experimental scale GLDT biological reactors

The comparison that the alcohol concn of the 1st batch in LCC3021 yeast and the 2nd batch changes with fermentation time in the graphic representation 6.9 experimental scale GLDT biological reactors.

6.2 fixation support

This research project has been studied several carriers and has been identified the carrier that application prospect is arranged most for next step development, has tested three kinds of multi-form immobilizations in 50 liters of through-flow tubular type biological reactors of gas lift.Estimated the commercially available absorption carrier of two kinds of sizes between 1～2mm. Be a kind of granulated glass sphere carrier (Fig. 9) that Schott Engineering provides,

It is a kind of diatomite pearl (Figure 10) that World Minerals provides.Because easily handling and can buy, they test.Absorption carrier provides the chance of easier aseptic technique, because retort can be loaded onto carrier earlier, and in-situ sterilization, direct inoculation yeast in retort at last again.Very attractive from this selection of industrialization position, because this kind carrier does not need special storage, factory needn't obviously change its inoculation method.

Result with these two kinds of carriers initial fermentation in 50 liters of through-flow tubular type biological reactors of gas lift is bad.The problem that occurs mainly is

With Particulate density is compared too high with liquid nutrient medium.When carrier and fluid density than keeping near 1 the time, this three-phase gas lift draft tube system works is good.Just

This ratio is 1.34, and Ratio is 1.31.The result that high like this density variation is arranged between the solid-liquid phase is the 50 liters of desired minimum gas fluidized speed of the through-flow tubular type biological reactor of gas lift of operation that obviously raise.For 4 liters Carrier (8%v/v solid loading) needs the gas flow rate of 21.5 mm/second (according to the vapor pipe diameter) to realize circulation.This higher gas flow rate is not a major issue when test in the aqueous solution, yet in case when liquid nutrient medium is wort, in gas lift draft tube (LGDT) system destructive failure can take place.This gas flow rate excess foaming in the jar that can induce reaction makes liquid level reduce to below the draft tube at last, has in fact stopped liquid and solid circulation.Similarly failure is being used

Replace

Also can run into during as immobilization material.Because these results abandon using these adsorptive supports in next step gas lift fermentation test.

Adopt the embedding carrier of similar κ-carrageenan to make this system can per hour need 0.17 standard cubic meter gas (superficial gas velocity of 5.8 mm/second) flow velocity with solid loading 40% (v/v) and in its fluidisation and circulation subsequently.Yeast cell pearl with the carrageenan embedding is operated this system, has obtained good result, is because this support density lower (about 1110 kilograms/cubic metre) and fluidisation are easy.Similarly, self aggregation yeast LCC3021 (medium flocculence) and the required solid loading of LCC290 (super flocculence), the gas fluidized speed of available desired about 3 mm/second that guarantee suitably to circulate and reaching, 6.2.1 understands κ-carrageenan gel carrier in more detail.6.2.2 described self aggregation yeast-LCC3021 and LCC290 throw out, estimated them and be used as the effect of the first continuously fermentation immobilization matrix of 50 liters of GLDT fermentor tanks.

6.2.1 κ-carrageenan gel beads

Embedding immobilization method requires yeast cell is wrapped in the matrix, moves in the fermentor tank then.Because this moment, the retort in-situ inoculating was infeasible, must be before beginning to ferment these gel beads of production.It is unclear that long storage what influence is the gel beads of inoculating had.In order to reduce any possible bad storage effect as far as possible, a large amount of gel beads are produced in decision in the short period of time (8 hours).The pearl technology of motionless mixer is seen that 5.2 joints are described and is used for this purpose.The ideal pearl should have the particle diameter (D of 0.8-1.4 millimeter _B), it is minimum that the variation coefficient of size distribution keeps.Must adjust the Several Parameters production desired number of pearl preparation processing and the pearl of homogeneous.Following chapters and sections brief summary the parameter of pearl technology select, 6.2.1.2 has described the pearl of the test usefulness of continuously fermenting.

6.2.1.1 pearl production technique: Variables Selection

Cooperate to carry out the feature of pearl manufacturing process with other scholars, emphasized following processing parameter, the diameter of motionless mixer (Ds), motionless mixer number of components (Ns), surface liquid flow velocity (V _SL) and polymer volume mark (ε _c).Graphic representation 6.12-6.21 brief summary the result that obtains of experiment.The graphic representation 6.12 explanations motionless mixer technology typical particle size distribution that immobilized yeast obtains in the carrageenan gel.Adopt following parameter in this example: 12.7 millimeters of motionless mixer diameters, 60 motionless mixer members, surface liquid flow velocity 10.5 cels, polymer volume mark 0.25.The average pearl of measuring directly is 701 microns, the variation coefficient 45%.It seems that graphic representation 6.13 shows the accumulation size distribution, be consistent with the normal state cumulative distribution of calculating with sample average and standard deviation.Checked normality with de KolmorogofSmirnov method (Scheaffer et Mc Clave, 1990), the ultimate range (K-S statistic D) that calculates between experimental data and the fitting data is 0.0274.Its 95% fiducial limit level of D value corresponding to the data correction of normal distribution must be lower than 0.895.The D value that our experimental calculation goes out to revise is 0.174, and below 0.895 limit value, can draw a conclusion: our data conform to normal distribution.Make the size distribution that be data show of processing collection from current pearl and have only a peak.Yet Poncelet etc. (1992) show several attached peaks and/or a secondary peak, corresponding to the alginic acid pearl of the diameter that disperses to produce in stirred pot less than 200 microns.What produce in possible our technology loses in the pearl washing step than globule, thereby does not appear in our particle size distribution data.

Graphic representation 6.14 and 6.15 has illustrated the influence to the variation coefficient of average pearl footpath and size distribution of surface liquid flow velocity and motionless mixer diameter respectively.The average pearl of all three kinds of motionless mixers footpath is reduced with the increase of surface liquid flow velocity, and 12.7 millimeters motionless mixers influences are the most obvious.With all not producing the pearl of mean diameter greater than 700 microns when the flow rate of liquid of all tests than the motionless mixer (6.4 and 9.5 millimeters) of minor diameter, and 12.7 mm dia motionless mixers produce the pearl greater than 700 microns when flow rate of liquid is lower than 11 cels.The pearl variation coefficient that all three kinds of fixed diameter mixing tanks produce is 38%～58%, shows that also the variation coefficient reduced when flow velocity increased under these three kinds of situations.The variation coefficient becomes with the motionless mixer diameter, produces minimum numerical value with the motionless mixer of minimum diameter.

When 3.5 cel surface velocities, the polymer volume mark be it seems can the average pearl of influence footpath, and flow velocity 7 cels ε when above _cExperimental value changes between 0.083～0.5, does not produce difference (graphic representation 6.16) relatively.It seems that the polymer volume mark does not influence or seldom influence the variation coefficient (graphic representation 6.17) when improving the surface liquid flow velocity.Graphic representation 6.18 and the 6.19 surperficial flow rate of liquid of explanation and motionless mixer scantling numeral are to the influence in average pearl footpath.Along with flow rate of liquid increases, the average pearl footpath that all different fixed number mixing tank members produce all reduces (graphic representation 6.18).The average pearl footpath of 24-120 member generation under given flow rate of liquid is similar, and the pearl footpath of 12 member generations is greater than the structure of other 5 kinds of tests.Fig. 6 .19 shows that also the average pearl of 24 above motionless mixer members directly reaches minimum.

The surperficial flow rate of liquid of graphic representation 6.20 explanations is to the influence of the variation coefficient of the motionless mixer member generation of different numbers.It seems that flow rate of liquid does not influence the variation coefficient of the structure of all tests.The motionless mixer number of components is to the influence of the variation coefficient more remarkable (Fig. 6 .21), the increase of motionless mixer member reduces the variation coefficient, reaching minimum value 45% during 60 members, is consistent in the surface liquid flow rates of these results between the 3.6-17.8 cel.

Proposed following hypothesis, motionless mixer diameter (Ds) increases can produce uneven shearing force in agitator, shows as the mensuration of the variation coefficient to cause the increase of particle size dispersion degree.Ds increases and will reduce the shearing intensity of force simultaneously, thereby improves average pearl footpath.These two kinds of effects see when adopting minimum diameter motionless mixer production gel beads, and the minimum average B configuration pearl directly is 400～500 microns, the minimum size distribution variation coefficient about 40%.

It is proportional to generate emulsion institute's energy requirement and polymkeric substance and oil phase interfacial area.Pearl is more little, and the energy consumption that forms pearl is big more.Berkman and Calabrese (1988) prove that average surface flow velocity (Vs) increase can make the power consumption of the liquid of per unit mass increase, and directly reduces so help pearl.Average surface flow rate of liquid (measuring between the 3.6-17.8 cel) increases directly reduces average pearl.The flow velocity increase causes the pressure difference between the motionless mixer entrance and exit, and this pressure difference and the power consumption of per unit mass liquid are proportional.Flow velocity increases thereby causes system's power consumption to increase, and helps reducing the pearl footpath.When increasing, the surface liquid flow velocity observes (generation) pearl footpath (D _B) reduce.When A1Taweel and Walker (1983) prove the high flow rate that is equivalent to remarkable turbulent flow level, assemble between formation of pearl and pearl and set up running balance.For constant motionless mixer diameter (Ds) and number of components (Ns), surface velocity is to almost not effect of the variation coefficient.Therefore flow velocity is one can handle and select average pearl footpath, but does not significantly change the parameter of size distribution.

In this research range, carrageenan gel volume mark (ε _c) the average pearl footpath or the variation coefficient are had no impact, except average pearl footpath when minimum speed 3.6 cels of research with ε _cReduction and reduce.Audet and Lacroix (1989) studied this parameter that produces the carrageenan gel beads at two-phase dispersion system (stirred pot rather than motionless mixer technology in batches continuously), and their conclusion is ε _cDo not influence the average pearl footpath of the polymers soln of carrageenan concentration 3% (w/v).Audet and Lacroix (1989) have studied κ-carrageenan gel strength to the special influence that pearl directly distributes, and show that this parameter influences pearl strongly and directly distributes.The gel strength increase causes average pearl footpath (D _B) and the variation coefficient (COV) increase.This kind influence during owing to high density gel viscosity improve and cause the emulsion shearing force is reduced and forms bigger pearl.Though this paper does not study the influence of gel strength to the pearl footpath, if need can be used as another means in control pearl footpath.

Mixing tank scantling numeral (Ns) increases, and the average retention time of liquid increases in the motionless mixer, causes more uniform mixing and forms the littler more intensive pearl of distribution.In an experiment, about 60-72 member has reached distribution equilibrium (measuring with the variation coefficient).Middleman (1974) proves that (10 members of 0.6～1.0cP) emulsion are enough to obtain this balance to low viscosity.Carrageenan solution (3%w/v) average viscosity of this experiment usefulness is 200cP, and oil viscosity is 25cP.Therefore this higher viscosity needs liquid to stop the long period to reach roughly homogeneous in mixing tank.

The typical pearl of the gel beads that the processing of graphic representation 6.12 usefulness motionless mixers produces directly distributes, and the machined parameters in this example is: Ds=12.7mm, N _S=60, V _SL=10.5cm/s, ε _c=0.25.

The typical case of the gel beads that the processing of graphic representation 6.13 usefulness motionless mixers produces accumulates pearl and directly distributes, and the machined parameters in this example is: Ds=12.7mm, N _S=60, V _SL=10.5cm/s, ε _c=0.25.

Graphic representation 6.14 surface liquid flow velocitys (cel) are to the graphic representation in average pearl footpath (micron).Estimated three kinds of different motionless mixer diameters (Ds12.7mm, 9.5mm and 6.4mm), it is constant that motionless mixer scantling numeral (Ns) keeps, and is 48, fraction of polymer ε _cBe set at 0.25.

Graphic representation 6.15 surface liquid flow velocitys (cel) are to the graphic representation of the pearl footpath variation coefficient (%).Estimated three kinds of different motionless mixer diameters (Ds12.7mm, 9.5mm and 6.4mm), it is constant that motionless mixer scantling numeral (Ns) keeps, and is 48, fraction of polymer ε _cBe set at 0.25.

Graphic representation 6.16 surface liquid flow velocitys (cel) are to the graphic representation in average pearl footpath (micron).4 kinds of fraction of polymer (ε have been estimated _c0.5,0.25,0.125 and 0.083).It is constant that motionless mixer scantling numeral (Ns) keeps, and is 48, and motionless mixer diameter (Ds) is set at 12.7 millimeters.

Graphic representation 6.17 surface liquid flow velocitys (cel) are to the graphic representation of the pearl footpath variation coefficient (%).4 kinds of fraction of polymer (ε have been estimated _c0.5,0.25,0.125 and 0.083).It is constant that motionless mixer scantling numeral (Ns) keeps, and is 48, and motionless mixer diameter (Ds) is set at 12.7 millimeters.

Graphic representation 6.18 surface liquid flow velocitys (cel) are to the graphic representation in average pearl footpath (micron).Motionless mixer scantling numeral (Ns) is changed to 12,24,48,60, and 72 and 120.Fraction of polymer (ε _c) keep constant, be 0.25, motionless mixer diameter (Ds) is 12.7 millimeters.

Graphic representation 6.19 surface liquid flow velocitys (cel) are to the graphic representation of pearl (micron) variation coefficient.Motionless mixer scantling numeral (Ns) is changed to 12,24,48,60, and 72 and 120.Fraction of polymer (ε _c) keep constant, be 0.25, motionless mixer diameter (Ds) is 12.7 millimeters.

Graphic representation 6.20 average pearl footpaths (micron) are to the figure of motionless mixer scantling numeral (Ns).Surface liquid flow velocity (cel) is changed to 3.6,7.0,10.6,13.2 and 17.8.Fraction of polymer (ε _c) keep constant, be 0.25, motionless mixer diameter (Ds) is 12.7 millimeters.

The graphic representation 6.21 pearls footpath variation coefficient (%) are to the figure of motionless mixer scantling numeral (Ns).Surface liquid flow velocity (cel) is changed to 3.6,7.0,10.6,13.2 and 17.8.Fraction of polymer (ε _c) keep constant, be 0.25, motionless mixer diameter (Ds) is 12.7 millimeters.

6.2.1.2 pearl production technique: κ-carrageenan characteristic

From the described data of last joint, might select the gel beads processing parameter of producing pearl and use for fermentation test with required feature.For reduce the variation coefficient of certain particular fixed mixing tank diameter as far as possible, selected 60 hybrid components to produce oil-gel dispersion.Select the on average about 1 millimeter pearl of diameter to reduce outside mass transfer and to promote separating of pearl and fermented liquid with mechanical means as far as possible.Selected the maximum flexibility mixing tank (12.7 millimeters) and the minimum speed (3.6 cel) of being tested for realizing this purpose.Because fraction of polymer is to almost not effect of the variation coefficient, so adopted gel: oil is 50/50 ratio (ε _c=0.5) to reach maximum pearl productivity.

Save described technology: Ds=12.7mm, N in this laboratory with 5.2 _S=60, V _SL=3.9cm/s, and ε _c=0.5 saccharomycetic gel beads of LCC3021 of having produced several embeddings.The pearl (Figure 11) that makes generation is removed greater than 2.0 millimeters with less than 0.5 millimeter pearl by a series of screen clothes.Fig. 6 .23 provides the particle size dispersion of the pearl that produces.Fig. 6 .24 illustrates the accumulation size distribution of these pearls.This is a used exemplary distribution in whole 50 liters of airlift fermentations test.

The bead size distribution that 6.23 50 liters of through-flow tubular type biological reactors of gas lift of graphic representation continuously ferment and test used κ-carrageenan gel beads.

The accumulation bead size distribution that 6.24 50 liters of through-flow tubular type biological reactors of gas lift of graphic representation continuously ferment and test used κ-carrageenan gel beads.

6.2.1.3 the technical scale restriction of pearl production technique

Develop and per hour produce the technology of 10 liters of pearls at every motionless mixer of pilot plant level implementation.May think that this technology also will further be developed and/or be optimized before industry size is produced pearl aspect several.The volumetric productivity that must increase this system is to provide extensive biological reactor charging needed a large amount of immobilized cells.For example the through-flow tubular type biological reactor of 2000hL gas lift needs about 800hL pearl.Must increase the flow velocity of gel and oil for reaching such volume.The data prompting increases with the flow velocity of the motionless mixer generation of diameter 6.4-12.7 millimeter, can form little of the obsolete pearl of this fermentation system.Therefore must increase the diameter of motionless mixer, thereby improve average pearl footpath.Yet adopt the bigger motionless mixer of diameter also can increase the dispersity of bead size, the size that produces big per-cent surpasses the pearl of required scope.Another kind method is to adopt the motionless mixer system of the median size (12.7 millimeters) of parallel placement, believes that with 10 motionless mixer systems productivity can reach 100 liters/hour.For the industrialization production of 2000hL, this technology implemented continuously 800 hours or about 34 days to produce volume required pearl.Can start several cover systems to reduce the production time but this has produced other complicacy again.If can keep the yeast viability simultaneously by long time stored pearl, the production time may not be any problem just.It is believed that and to develop dry pearl or pearl is stored in method in the vacuum-tight container.The article that Poncelet and colleague (1993) deliver proposes to consider that this kind of used motionless mixer can produce deviation, their system of suggestion adopts another type motionless mixer, the Kenics type of using with this institute is opposite, can adopt larger-diameter agitator and can not damage bead size and distribute (keeping the minimum variation coefficient).

To another worry of present tentative technology, be included in 40 ℃ of these systems of operation and adopt vegetables oil and Klorvess Liquid is produced pearl.Because higher production temperature requires to have heating and cooling system in technology.Thereby require further research is done in the potential thermal shocking that yeast cell must expose, negative impact is assessed to potential.Yeast viability very high (more than 90%) in the immobilized cell pearl that is produced in this research, but whether this technology may cause not research of other effect to yeast flora.Because produce required emulsion with oil, and then form pearl, can suppress foam formation because oil plays tensio-active agent, the bead surface oil residues is a problem.Though residual helpful at fermentation stage oil, bringing into then is deleterious in the last beer, because foaming is that the finished product are needed.Separate solid phase pearl and oil with a large amount of potassium chloride liquids, also need to study pearl packed into and from the pearl slurry, remove the method for saline solution before the biological reactor, remove this solution otherwise after pearl adds retort, also must from retort, wash.

Because the pearl that produces immobilized cell outside biological reactor must be adopted Aseptic technique and keep aseptic up to pearl is added biological reactor in pearl preparation processing whole process.Each reloading point between each jar provides opportunities for contamination, must monitor, because the contaminated bacteria that exists may be fixed in the pearl together.The result that make great efforts in this laboratory is the aseptic pearl that can produce homogeneous.Yet the environment of plant is not suitable for the laboratory, thereby requires control more strictly.

6.2.2 flocculence yeast cell

One of modal natural immobilization form is that microorganism self is gathered into the cell floss.Calleja and Johnson proposition cell contact with each other the formation aggregate three kinds of possible causes, has different binding characteristics.First relates to different sexes cell releaser pheromone (and the A factor) attracts each other, and this kind combination is temporary, relates to the that is fixed in the complementary cell walls and the protein-protein bound between the A lectin.Cell also may assemble in the process of sprouting because of not separating with its mother cell, and this may be that special yeast strain inherent maybe may be due to nutritional deficiency or some transgenations.This phenomenon is called chain formation but not flocculation, and these intercellular combinations can irreversibly destroy (Stratford, 1996) because of mechanical shear stress.The third is more common is called flocculation.Stewart and Russell (1981) definition flocculation is for " a kind of reversible phenomenon, yeast cell adhesion become flora, can rapid subsidence in the substratum that their suspend or rise to media surface." widely evidence show that flocculation is to be subjected to genetically controlledly, the mechanism of flocculation depends on selected molecule and plays a part to adjoin bridge between the cell walls.More specifically say, it is believed that the specific agglutination element when calcium ion exists, be incorporated into the mannosans (Calleja and Johnson, 1977) that adjoins cell.Found that this protein/sugar suppresses in conjunction with the reversibility that is subjected to sequestrant or specificity sugar.

Figure 12 has described three kinds of possible yeast cell structures, is called non-flocculating yeast bacterium, chain formation yeast and flocculating yeast bacterium.Though the cell aggregation of chain formation yeast does not think that it is a kind of flocculation, because originally cell is not one just.Flocculation means individual cells and forms group because environment favourable (calcium ion and low-level inhibition sugar) is got together.With regard to the flocculation cell, the specific size of throw out may depend on the hereditary instinct and the cell institute fluid in contact dynamics environment (shear environment) of cell.Figure 13 and 14 has stressed that two kinds of Labatt with different flocculating degrees store old yeast strain, and Figure 13 is medium flocculence yeast strain LCC3021.Glucose when having calcium ion in this bacterial strain liquid medium within just forms 0.5～1.0 millimeter aggregate when in a single day exhausting.Figure 14 is the photo of super flocculating yeast bacterial strain LCC290, and the throw out of formation is gathered into the group of 5 millimeters of diameters greater than 1 millimeter under the low-shearing power environment.Under the mild stirring situation, LCC290 throw out diameter is between 1～2 millimeter.Several measuring methods (Speer and Ritcey, 1995 of assessment yeast flocculence have been proposed; Akiyama-Jibiki etc., 1997; Teixera etc., 1991, Stewart and Russell2000).Proposing yeast flocculation measuring method in " Brewer ' s Yeast " (Stewart and Russell, 2000) can be divided three classes: settling process, the throw out in static fermentation method and the direct viewing growth medium forms.

Nineteen thirty-seven Burns has at first described settling process, and nineteen fifty-three Helm and colleague improve, and becomes the part (1992) of the present standard method of analysis that Technical Committee of ASBC and editorial board admit.This technology is called ex vivo technique, because saccharomycetic settling characteristics is to assess with the calcium sulfate damping fluid rather than with real fermention medium.Static fermentation method (being also referred to as the Gilliland method) relates in the wort of hope the culturing yeast bacterium and measures its flocculation feature in vivo.The absorbancy that these two kinds of methods all will be measured settled yeast sample and take off the yeast sample of flocculation adopts the ultraviolet spectrophotometer.Stewart and Russell (2000) propose a kind of yeast flocculation measuring method: the flocculation level that the yeast sample of growing in 20 milliliters of threaded cap vials of visual observation is taken place.For flocculating degree is described, they adopt the subjective scoring method, for example: 5 extremely strong flocculations, the semi-finals flocculation, 3 medium flocculations, 2 weak flocculations, 1-does not have flocculation with 0-slightly.Super flocculating yeast bacterial strain LCC290 classifies as 4-and flocculates by force, and the LCC3021 bacterial strain classifies as 3 medium flocculations.

Flocculation is a kind of key character in Brewing industry, tends to sedimentation or floats on the surface because yeast is natural, can be used as the isolating method of this yeast and fermented liquid usually.Yet the yeast strain of having finished flocculation before fermentation is unfavorable, because nutrient solution does not also produce the alcohol of desired quantity and have sugar residual.Continuously fermenting, specifically the flocculating yeast bacterium plays the immobilization matrix effect in the through-flow tubular type of gas lift is continuously fermented.Its sedimentation tendency can make it keep suspending by injecting jet compensation.Adopt this system to eliminate to fail the worry of abundant fermentation broth, keep contacting closely with fermented liquid because solid particulate constantly circulates.

6.2.2.1 examined and determine settling property and the leavening property of super flocculating yeast bacterium LCC290 in the joint.Interesting is to identify the flocculation that this special yeast strain is taken place.In addition, measured this saccharomycetic settling velocity, the valuable information that can be used for yeast slurry tank design in the future is provided.

6.2.2.1 the calibrating of super flocculating yeast bacterium LCC290

Before continuously fermenting in the through-flow tubular type biological reactor of gas lift with super flocculating yeast bacterium LCC290, decision is examined and determine this yeast with the laboratory scale shake flask fermentation.Graphic representation 6.28 shows yeast flora over time.As expecting, bacteria concentration sharply rose in preceding 48 hours, and stopping then increasing, and level is slightly fallen during fermentation ends.Enough nutriment and oxygen are arranged for the yeast growth in the preceding 48 hours worts, yet along with yeast constantly consumes carbohydrate and anoxic, it can not be bred but enter the anaerobically fermenting phase.In case the carbohydrate supply exhausts that the small portion yeast begins death.This phenomenon sees graphic representation 6.29, and cell viability drops to a little more than 90% from about 97% among the figure.

Graphic representation 6.30 shows the consumption of carbohydrate between yeast phase.Yeast consumes simple sugar glucose and fructose earlier.Absorb maltose and trisaccharide maltose then in succession.Yet yeast saccharomyces cerevisiae is energy metabolism maltotetrose or long-chain polysaccharide (poly 1 and 2) more not.Along with total sugar concentration reduces (seeing shown in graphic representation 6.31 specific gravity curves) proportional rising of alcohol concn, to ferment about 37 hours the time, ethanol and concentration of saccharide equate.As if from growth and sugar metabolism, the performance of super flocculating yeast bacterium LCC290 is as industrial yeast bacterial strain LCC3021.When liquid specific gravity reached about 2.7 ° of P, it seems that fermentation reach end.The preceding flocculating yeast bacterial strain of common fermentation ends forms bulk (throw out) and settles from solution.This phenomenon is called " hung " fermentation in Brewing industry.In our batch test, we can finish fermentation, because can shake bottle the yeast suspension are contacted closely with nutrition supply.Another key character of being studied is the ability of this yeast flocculation.Specifically, we are interested to be to determine the settled speed of this yeast, and obtain this special yeast strain of a kind of indication when begin the flocculation, these two kinds of features to continuously ferment the test all important because play a role in their healthy yeast floras in keeping the gas lift fermentor tank.Graphic representation 6.32 shows yeast subsidence curve in the fermenting process.Sedimentation appears in fermented sample hardly that tested 24 hours, the inhibition of some sugar that flocculation is existed, and glucose is a kind of known inhibitor, in case therefore the depleted flocculation of this inhibitor just begins.In 40 hours the sample of batch fermentation when adopting the described method test of 4.7 joints cell begin flocculation and from the solution sedimentation.The time settlement of all tests is all very fast, except sedimentation did not take place in 24 hours.In the 40 hours the slowest settling tests that carry out, this yeast has been spent sedimentation fully from testing apparatus in 90 seconds.Sedimentation need be less than 50 seconds in the time of 71 hours.

Scholars propose the function that settling velocity is a solids concn (Coe and Clevenger, 1916).Solid settling velocity during certain given barm cell concentration of graphic representation 6.33 expression.The method (1952) that adopts Kynch to propose, the settling data of collecting at interval according to each fermentation time has produced the data point of this curve.These results obtain roughly the same curve, have confirmed the observed same phenomenon of Coe and Clevenger (1916).

The result that this settling test is collected shows, super flocculating yeast bacterial strain LCC290 will flocculate at 6 ° of P of liquid specific gravity with when lower.This numerical value can be used as a guide that continuously ferments, and shows to keep the cell flocculation if desired, should keep this quasi-stable state liquid specific gravity.Retort is higher than 6 ° of P during operation will have the flocculation cell to lose stable risk, may cause the fixed yeast flora to be washed off.The settling characteristics of super flocculating yeast bacterium shows, if leave standstill this yeast flora that do not flow with rapid sedimentation.With the through-flow tubular type biological reactor of three-phase gas lift the time, these cells are remained in the circulation, yet do not having under the situation of gas supply system, this cell mass is rapid sedimentation, adopts the complementary jet resuspension solid that comes possibly.For the fermentation post-treatment, this kind rapid subsidence feature is useful, can adopt solid separating device such as gravitational settler to remove solid in a large number.Brewageing industry, this will alleviate the solid loading of centrifugation apparatus, thereby be able to use the longer time before centrifugal barrel is scrapped.Estimate that the beer loss can be less centrifugal, make beer fragrance lose (though minimum) and also can minimize, because flat lower by the yeast bio water gaging of whizzer.

Graphic representation 6.28 stirs the figure of the LCC290 yeast cell total concn of batch fermentation to fermentation time, and leavening temperature remains on 15 ℃.

The yeast bacteria living power that graphic representation 6.29 methylenum coeruleum are measured is to the figure of fermentation time.

The concentration of saccharide of graphic representation 6.30LCC290 yeast batch fermentation is to the figure of fermentation time.

The ethanol of graphic representation 6.31LCC290 yeast batch fermentation and glycerol concentration and liquid specific gravity are to the figure of fermentation time.

The interfacial level of graphic representation 6.32 yeast suspensions is to the figure of settling time.The numerical value of this figure is collected in several fermentation times at interval.

The figure of the settling velocity pair cell concentration of graphic representation 6.33 yeast suspensions.The numerical value of this figure is collected in several fermentation times at interval.

6.3 the gas lift technology evaluation of continuous beer fermentation

This paper first and most important purpose are to estimate the feasibility that adopts κ-carrageenan gel beads embedding Ka Ersibai sugar yeast cell (seeing that the 6.2.1 joint is described) to operate 50 liters of experimental scale air lift type biological reactors in a continuous manner.In addition, we need investigate with this system whether to produce the North America type lager beer with fragrance that people take like a shot.We also want to determine the minimum residence time that heavy wort juice (17.5 ° of P) fully fermenting in this system of continuously fermenting is required and set up the operating restraint of oxygen.

The minimum residence time that sugar in all worts all exhausts is 24 hours.Compare with it, the classical batch fermentation time is 5-7 days, although add oxygen (0-20%v/v) in jet, the oxyty that the original position Ingold oxygen probe in the biological reactor records is still near zero.This shows that the oxygen in the wort is consumed fast or discharges simply by yeast cell in waste gas.Free cell level in the beer overflowing liquid is every milliliter of unpasteurized beer 10 ⁸Individual cell.Ortho position diketone, di-acetyl and 2, the level of 3-diacetylmethane and acetaldehyde level are with the oxygen ratio in jet descend (graphic representation 6.34 and 6.35).The influence (Fig. 6 .36) that as if ester that records (ethyl acetate and Isoamyl Acetate FCC) and higher alcohols (propyl alcohol, isopropylcarbinol, primary isoamyl alcohol) not changed by oxygen supply

Graphic representation 6.37 has compared various fragrant active compounds in the beer of beer that two batches of producing with continuous immobilized cell system finish test and contrast suitability for industrialized production (free cell batch fermentation).Oxygen supply level between beer and the contrast is all observed some difference of ester (ethyl acetate and Isoamyl Acetate FCC) and higher alcohols (acetone) no matter continuously ferment.Judge quite near contrast beer (suitability for industrialized production) by one group of trained teacher of sampling wine with the taste of the beer of 2% oxygen production, yet be judged as that sign with fragrance oxidation and " cardboard flavor " and " wine " are distinguished the flavor of and unacceptable with the beer of 20% oxygen production.

In 6 weeks, the residence time of experimental scale biological reactor running is 24 hours, notices that " life " beer has acceptable taste and shows do not have significant deficiency (sulfury) when quasi-stability.The oxygen amount that provides during this when test is jet is a key factor, has obtained optimum taste with the beer of the jet production that contains oxygen 2～5%.This critical reference mark need further concentrate on attention the more accurate oxygen determination techniques to analyte before and after a series of fermentations.

In traditional fermentation just in batches, wort is to add oxygen before being conveyed into fermentor tank.Behind the inoculation medium because by yeast cells consume, oxyty descend rapidly (just 24 hours yeast propagation of fermentation).All the other times of fermentation are almost carrying out under the anaerobic condition.Adopt the first fermentation of continuous homogeneous system that this oxygen concn time to time change can not take place.Therefore, with continuously and the beer of batch fermentation production may be difficult to obtain that taste is consistent completely.Although these difference is arranged, the biological respinse jar structure of testing in this entry evaluation has been produced the beer with acceptable taste and analytic curve figure.By adopting air lift type biological reactor and less pearl (about 1 mm size), can improve the volumetric productivity of biological reactor and reduce fermentation time several days just.The biomass level that is released in the gained beer shows that the yeast level of growth is equivalent to the level of free cell batch fermentation under simulated condition in the immobilized cell biological reactor.These are observed the formation that confirms fragrant substance and depend on the yeast level of growth.Attempt to fail to produce the reason that to accept beer before this is soluble with the immobilized cell system of limiting growth.It may be a kind of strong method of meticulous adjusting beer taste performance in continuous immobilized cell fermentation that in check mixed gas is provided.Other scholar also observes high-caliber di-acetyl (Virkajarvi ﹠amp in the gained beer; Pohjala, 1999; Kronlof etc., 2000).The di-acetyl target level that old type beer is store in the North America is 30 micrograms per litre, and the continuously ferment level of liquid of this of Pai Chuing is the 400-800 micrograms per litre by comparison.Adopt traditional aging technology (2 ℃ cold aging 14 days) di-acetyl can be reduced to desired level, but damaged the productivity of whole technology.Adopt the quick secondary fermentation technology of the 2nd chapter report to help to reduce di-acetyl and not obvious reduction productivity (processing in 2 hours).Yet increasing cost may be that people are loth, and all brewers may not want to allow its beer experience high temperature (80-90 ℃).

Graphic representation 6.34 ortho position diketone concentration and jet in the relation of oxygen per-cent.In 50 liters of air lift type systems that 40% (v/v) carrageenan gel beads is housed, ferment.It is constant that total efflux velocity keeps, and is 6.4scfh, 24 hours residence times.

The relation of graphic representation 6.35 acetaldehyde concentrations and jet middle oxygen per-cent.In 50 liters of air lift type systems that 40% (v/v) carrageenan gel beads is housed, ferment.It is constant that total efflux velocity keeps, and is 6.4scfh, 24 hours residence times.

Graphic representation 6.36 esters and fusel concentration and jet in the relation of oxygen per-cent.In 50 liters of air lift type systems that 40% (v/v) carrageenan gel beads is housed, ferment.It is constant that total efflux velocity keeps, and is 6.4scfh, 24 hours residence times.

Fig. 6 .37 the finished product beer of the jet production that contains 2% oxygen and 20% oxygen and the comparison of Industrial products beer and taste threshold.The production of continuously fermenting is to ferment in 50 liters of air lift type systems that 40% (v/v) carrageenan gel beads is housed.It is constant that total efflux velocity keeps, and is 6.4sefh, 24 hours residence times.

6.4 the mixing and the cycle rate of tubular type biological reactor fermentation that the three-phase gas lift is through-flow

Adopt the described acid solution injection method of 4.8 joints to carry out combined experiments.The purpose of this phase experiment has two.Whether we at first will assess this gas lift system with multiple curing carrier by the mixing time of calculating liquid circulation velocity and draw and can finely mix under different surperficial fluidization gas velocity.These tests and the test of bibliographical information difference are that all experiments are carrying out with living yeast on the real fermention medium rather than carrying out on model solution (Gu water--gas system).These combined experimentses also are fit to the gauging surface flow rate of liquid, can be used for further amplifying this pilot system.

6.4.1 the calibration of probe

Adopt the pH probe of using in the described method calibration of the 4.8 joints bulk testing.The 4-20 milliampere signal that pH probe is sent changes the 1-5 vor signal into by 250 ohmic resistances, and it can be data and adopts and sell card (DAC) institute record.The pH probe is immersed in following a series of damping fluid successively: in the damping fluid of pH4.6, pH4.0, pH5.0, the last pH7.0 of pH6.0 (Beckman standard verification product, Cole Parmer).Graphic representation 6.38 is collected the data of Ingold pH probe to 5 kinds of standard pH solution reactions for this acquisition system.Graphic representation 6.39 is pH probe working curves, draws with true pH value and the signal voltage of measuring.The best-fit working curve of this system (relation conefficient is 0.9996) is determined by following formula:

PH=3.68 * voltage-3.53 (6.1)

With blended data conversion the true pH value in reflection gas lift biological reactor to record of formula 6.1 with collection.

Also measured the reaction times that the pH probe changes pH.Fig. 6 .40-6.43 shows that the pH probe changes the i.e. type reaction of 0.6,1.2,2.3 and 3.4 conversion steps to different pH of 4 steps.Adopt Table Curve 2D (JandelScientific) data to draw and analysis software, raw data is fitted to an impulse function, the relation conefficient of gained matched curve is greater than 0.994.Calculate 98% the reaction times of this probe from these opisometers, then to pH conversion step draw (graphic representation 6.44) to this conversion step.Reduce with the pH conversion step in the scope internal reaction time of being measured.To the pH0.6 conversion step of minimum, the pH probe reaction times is about 6.4 seconds.PH changes 3.4 o'clock time of response and is reduced to about 4.2 seconds.

This importance of probe is especially when this system is used to estimate mixing time and cycle rate.Should have the low reaction time for the probe of measuring the variation of reflection medium pH.If accurately be used for this mensuration, the pH probe reaction times must be lower than the cycle rate in the retort specifically.Yet do not need to intend the moment reactivity,, thereby can offset because the hysteresis a little of reaction will be reflected in the continuous cycle rate measured value simply.

Graphic representation 6.38 data collecting systems are to raw data that reaction obtained and the time relation of Ingold pH probe to each buffered soln.Frequency acquisition is 50Hz 15000 points altogether.

Graphic representation 6.39pH probe working curve shows the relation of the voltage of pH and mensuration (V).Best-fit song or working curve have following formula: pH=3.68 * voltage-3.53, relation conefficient 0.9996 (N=2500)

Graphic representation 6.40pH probe is to the representative data of the time of response of pH one step change about 0.6.React with the match of Table Curve software.The relation conefficient of this best-fit impulse function is 0.995.

Graphic representation 6.41pH probe is to the representative data of the time of response of pH one step change about 1.2.React with the match of Table Curve software.The relation conefficient of this best-fit impulse function is 0.999.

Graphic representation 6.42pH probe is to the representative data of the time of response of pH one step change about 2.3.React with the match of Table Curve software.The relation conefficient of this best-fit impulse function is 0.999.

Graphic representation 6.43pH probe is to the representative data of the time of response of pH one step change about 3.4.React with the match of Table Curve software.The relation conefficient of this best-fit impulse function is 0.999.

The time of response (N=3) that graphic representation 6.44Ingold pH probe changed a step.Be immersed in immediately in low 4.0 the low pH damping fluid being immersed in probe in the higher pH damping fluid.Calculating this probe with the best-fit line of each response curve reaches this step and changed for 98% required time.

6.4.2 mixing time and speed of circulation

Mixing time and speed of circulation experiment are carried out three types of immobilized cell fermentations.In 50 liters of through-flow tubular type biological reactors of experimental scale,, in the fermentation culture that contains κ-carrageenan gel beads, super flocculation LCC290 yeast or medium flocculation LCC3021 yeast, 2.7 ° of P of proportion, carry out this experiment then not contain the solid model aqueous solution.Graphic representation 6.45 and 6.46 is to inject the sampling explanation of acid solution (the described method of 4.8 joints) back with the raw data of this pH detection system collection in pulse.Graphic representation 6.45 explanation pH probes inject the reaction that does not contain the solid aqueous solution to acid solution, and graphic representation 6.46 is the reactions of acid solution being injected the fermentation culture that contains high flocculence yeast LCC290.With the sinusoidal curve mutually match of signal with decay.Calculate relation conefficient with respective formula, be respectively 0.96 and 0.90.Fitting parameter among the figure " b " and " c " are corresponding to " a " and " " value described in the decay sine formula (3.1).In formula 3.2 and 3.3, utilize these numerical evaluation to go out the speed of circulation and the ijwg time of this system.Appendix B includes the fitting of a curve of remaining ijwg data and all experiments.

Graphic representation 6.47 and 6.48 is that acid solution is injected mixing time and speed of circulation figure when not containing the solid aqueous solution, graphic representation 6.49 and 6.50 is to carry out the corresponding of combined experiments with high flocculating yeast bacterium LCC290 to illustrate, and graphic representation 6.51 and 6.52 provides the result of κ-carrageenan bulk testing.At last, graphic representation 6.53 and 6.54 has shown mixing time and the cycling time of medium flocculating yeast bacterium LCC3021.No matter the solid type of test, along with the corresponding increase of surface gas flow velocity, mixing time and speed of circulation reduce.For all four kinds of test macros, the relation between speed of circulation and the surface liquid flow velocity is followed the formula 3.11 of Kennard and Janekah (1991) proposition.Water/no solid system and κ-carrageenan gel beads system mixing time is followed formula 3.11.Adopt the flocculating yeast bacterium not demonstrate strong correlation with Kennard and Janekah theoretical model as these two kinds of systems of immobilization matrix.The initial mixing time of LCC290 and LCC3021 system (surpassing 4 mm/second up to the surface gas flow velocity) is lower than this model predication value.Yet the speed that mixing time reduces slows down when the surface gas flow velocity is higher than 4 mm/second, and the value of this model is lower.Table 6.1 provides cycling time of drawing and the mixing time calculation formula with respect to the surface gas flow velocity.

The relation of graphic representation 6.55 all 4 kinds of system's mixing times of explanation and surface gas flow velocity.Water/no solid system shows that a pH pulse value mixes 98% required time the highest (during Vsg 3 mm/second about 220 seconds), and the LCC290 system can make the effect of the pulse of a hypo acid reduce to minimum (during Vsg 3 mm/second about 110 seconds) in this system.The numerical value of LCC3021 and κ-carrageenan system is between water/no solid system and LCC290 system.Produce eddy current and promote coaxial mixing by exciting, the solid in the gas lift biological reactor helps the dispersion of liquid phase flow composition.Though super flocculating yeast bacterium LCC290 solid loading (16%w/v) is identical with medium flocculating yeast bacterium LCC3021, when the surface gas flow velocity of all tests, its mixing time is very fast.The cycling time of graphic representation 6.56 all 4 kinds of test macros of explanation and the relation of surface gas flow velocity.When the surface gas flow velocity was 2 mm/second, cycling time, water/no solid system had the fastest speed of circulation between 28～35 seconds, and the LCC290 system shows the slowest speed of circulation.When higher gas flow rate, the difference between 4 kinds of systems was reduced to about 3 seconds.Yet for the flow velocity of all tests, the LCC290 system shows slightly slow speed of circulation, and water/no solid system speed of circulation is the fastest.

Relation between graphic representation 6.57 surperficial gas flow rates of explanation and the surface liquid flow velocity.Surface liquid flow velocity when the formula 3.7 that proposes with Livingston and Zhang (1993) calculates given speed of circulation and solid type.Corresponding increase surface liquid flow velocity increase along with the surface gas flow velocity.LCC3021 and water/no solid system has similar superpotential, and LCC290 and κ-carrageenan system shows certain similarity.The simulation formula that Kennard and Jenekah propose is suitable for the curve (Fig. 6 .57) of surface liquid flow velocity to surperficial gas flow rate.Fig. 6 .58 is the surface liquid flow velocity that draws with the formula 3.10 experiments with computing figure to the surface liquid flow velocity that calculates in theory.All 4 kinds of systems all meet slope be 1 and starting point be the formula shown in the linear functional relation of y=0.Table 6.1 has been listed the dependency that system that this research work tests draws.

The response data of pH probe in 6.45 50 liters of through-flow tubular type biological reactors of gas lift of graphic representation.The injection of acid solution pulsed is not contained the solid aqueous solution.The surface gas flow velocity is made as 1.94 mm/second.Raw data matches with decay property sinusoidal curve.Relation conefficient is 0.96.

The response data of pH probe in 6.46 50 liters of through-flow tubular type biological reactors of gas lift of graphic representation.The acid solution pulse feature is injected the fermention medium that contains high flocculating yeast bacterium LCC290.Surface gas institute rate of flow of fluid is made as 1.94 mm/second.Raw data matches with decay property sinusoidal curve.Relation conefficient is 0.90.

The relation of mixing time and surface gas flow velocity in 6.47 50 liters of through-flow tubular type biological reactors of gas lift of graphic representation.The injection of acid solution pulse feature is not contained the solid aqueous solution.Mixing time equals to make pH should go on foot change cancellation 98% required time.(N＝3)

The relation of cycling time and surface gas flow velocity in 6.48 50 liters of through-flow tubular type biological reactors of gas lift of graphic representation.The injection of acid solution pulse feature is not contained the solid aqueous solution.(N＝3)

The relation of mixing time and surface gas flow velocity in 6.49 50 liters of through-flow tubular type biological reactors of gas lift of graphic representation.The acid solution pulse feature is injected the fermention medium contain high flocculating yeast bacterium LCC290 (throw out size＞1.0 millimeter).Mixing time equals to make pH should go on foot change cancellation 98% invalid required time.(N＝3)。

The relation of cycling time and surface gas flow velocity in 6.50 50 liters of through-flow tubular type biological reactors of gas lift of graphic representation.The acid solution pulse feature is injected the fermention medium contain high flocculating yeast bacterium LCC290 (throw out size＞1.0 millimeter).(N＝3)

The relation of mixing time and surface gas flow velocity in 6.51 50 liters of through-flow tubular type biological reactors of gas lift of graphic representation.The acid solution pulse feature is injected the fermention medium that contains useful κ-carrageenan gel beads (pearl footpath 1-2 millimeter) fixed yeast (LCC3021).Mixing time equals to make pH to change cancellation 98% required time step by step.(N＝3)

The relation of cycling time and surface gas flow velocity in 6.52 50 liters of through-flow tubular type biological reactors of gas lift of graphic representation.The acid solution pulse feature is injected the fermention medium that contains useful κ-carrageenan gel beads (pearl footpath 1-2 millimeter) fixed yeast (LCC3021).

The relation of mixing time and surface gas flow velocity in 6.53 50 liters of through-flow tubular type biological reactors of gas lift of graphic representation.The acid solution pulse feature is injected the fermention medium contain medium flocculating yeast bacterium LCC3021 (throw out size＜0.5 millimeter).Mixing time equals to make this step of pH to change cancellation 98% required time.(N＝3)

The relation of cycling time and surface gas flow velocity in 6.54 50 liters of through-flow tubular type biological reactors of gas lift of graphic representation.The acid solution pulse feature is injected the fermention medium contain medium flocculating yeast bacterium LCC3021 (throw out size＜0.5 millimeter).(N＝3)

The relation of mixing time and surface gas flow velocity in 6.55 50 liters of through-flow tubular type biological reactors of gas lift of graphic representation.The injection of acid solution pulse feature is contained different solid fermention mediums.Mixing time equals to make this step of pH to change cancellation 98% required time (N=3).

The relation of cycling time and surface gas flow velocity in 6.56 50 liters of through-flow tubular type biological reactors of gas lift of graphic representation.The injection of acid solution pulse feature is contained different solid fermention mediums.(N＝3)。

The surface liquid flow velocity (mm/second) of graphic representation 6.57 4 kinds of test macro-LCC3021 yeast, LCC290 yeast, carrageenan gel beads and water/no solid systems and the relation of surface gas flow velocity (mm/second).In 50 liters of through-flow tubular type biological reactors of experimental scale gas lift, carry out this test.

The theoretical surface flow rate of liquid (mm/second) of 6.58 4 kinds of test macros of graphic representation and the surface liquid flow velocity (mm/second) of test determination.Following relation formula theory of computation value with Kennard and Janekah (1991) proposition: V _SLα V _SG ^M, straight slope is 1, the Y-axis intercept is 0.

The dependency brief summary of mixing time, speed of circulation and surface liquid flow velocity that 4 kinds of test macros of table 6.1 calculate

For the surface liquid dependency, index is 0.41 in Kennard and Janekah (1991) the proposition distilled water, when this solution contains carboxymethyl cellulose and ethanol, is 0.64.LCC290 and this index of LCC3021 system are respectively 0.419 and 0.427, and κ-carrageenan system and water/no solid system index is 0.283.

Basic assumption to the through-flow tubular type technology of gas lift is that this system can transmit enough whipping forces, and the liquid component in the retort is mixed fully.When 50 liters of experimental scale systems continuously fermented, fresh nutritional medium was injected into 50 liters of cumulative volumes with 36 ml/min flow velocitys at the bottom of retort.This is representing the about 1000 times of dilutions of charging composition.Calculate the composite character of LCC290 Yeast system, about 3 circulations liquid component that has been mixing in the retort, and water/no solid system needs to circulate for 10 times.In addition, the residence time (24 hours) is about higher 1000 times than mixing time (180 seconds).Short mix combines with the nutrition dilution in this system, and mixing time and residence time differ greatly and point out it strongly is a kind of well-mixed system.The initial prerequisite of employing gas lift biological reactor provides a kind of ideal hybird environment of beer fermentation.The mixing result of study of carrying out on all three kinds of fermentation carriers is supported this prerequisite.

6.5 estimate several process for fixation of successive fermentation usefulness in the gas lift system

Use three kinds of fixation support-κ-carrageenan gel beads, super flocculation LCC290 yeast and medium flocculation LCC3021 yeast in 50 liters of through-flow tubular type biological reactors of experimental scale gas lift, to continuously ferment.The initial yeast inoculum (4 grams per liter) that drops into par of all fermentations, the industrial old wort of storage that produces are as nutritional medium, and fermentation begins to be able to fast restore wort carbohydrate and promotes the yeast in the fermentor tank to breed with batch mode.This batch mode is formed the yeast throw out during with the flocculating yeast bacterium, and it can remain on continuously feeding Once you begin in the fermentor tank.When the di-acetyl level in the fermented liquid drops to below 30 micrograms per litre, wort begins continuous charging.The analysis of the fermented product that following chapters and sections more detailed description obtains these three kinds of immobilization matrix.

6.5.1 κ-carrageen condenses sugared gel beads: embedding

Adopt κ-carrageenan gel beads to help the continuously first fermentation beer of gas lift technology (6.3 joint) is assessed as immobilization matrix.Still whether for a long time this system of needs assessment (more than February) running and do not have great operational difficulty comprises that fermentation is unstable and pollutes.Graphic representation 6.59 shows the operating parameters of this fermentation test, carbonic acid gas surface gas flow velocity is made as 5.5 mm/second, and the air that imports in fermentor tank jet is 0.9 mm/second, oxygenate rate in always jet is 3%, by chance consistent with the result in 6.3 joints, show when this system imports the oxygen of 2-5%, to produce good beer.Leavening temperature is controlled at 15 ℃, visible some fluctuation in the data, and they relate to the performance of used control loop.

Graphic representation 6.60 explanation free yeast mycetocyte groups development and saccharomycetic viability in time.Viability is kept quite highly between 2 months yeast phases, and transience reduces during with about 200 hours.This is corresponding to the moment before the beginning continuously feeding wort.Descend in viability during common fermentation ends in the batch fermentation, because cell has lost nutriment.Once you begin continuity wort charging viability is returned and is risen to more than 90%.Fermentation just 400 hours, the free cell yeast flora was low, and in after this 300 hours, it increases about 10 times, is raised to 1,500,000,000 cells/ml from 100,000,000 cells/ml.In case arrive this peak concentration, free cell yeast group keeps this kind quasi-stable state value in remaining fermentation time.The unexpected increase of free yeast concentration may be related to the fixed yeast cell group.During the fermentation beginning, the yeast that is embedded in the gel will all be occupied up to available space in the gel growth inside.In case gel beads is full of by yeast, the flora of expansion will enter liquid nutrient medium.As if our result is explanation, and at initial 400 hours, immobilized yeast was grown in gel, about 700 hours, yeast has not had the more location to grow in pearl, therefore begins to discharge a large amount of cells in substratum.The instability of yeast flora is reflected in ethanol and the specific gravity curve (graphic representation 6.61).Fermented initial 200 hours, and estimated that ethanol rose in time, proportion is then followed traditional kinetics in batches and is descended.Ethanol rises to 45 grams per liter platforms between 200～600 hours, and proportion is maintained at about 6 ° of P.This result is not desired, and the proportion of liquid is about 2.5～2.7 ° of P because ferment finally.When the free yeast flora reached its maximum value in about about 600 hours, the ethanol level rose to about 70 grams per liters, and wort proportion is reduced to about 2.2 ° of P.The more time dependent characteristic curve of close observation carbohydrate (Fig. 6 .62) shows that the maltose concentration instability was up to about 600 hours.Other carbohydrate reduces as expectedly.

Other two important analytes-di-acetyls and 2 in the process of continuously fermenting in 2 months have been monitored, 3-diacetylmethane (graphic representation 6.63).Low spot in the time of about 180 hours finished corresponding to the initial phase of batch-wise, continuously feeding Once you begin, and di-acetyl and 2,3-diacetylmethane are increased to 500 micrograms per litre and 400 micrograms per litre respectively rapidly.This initial rising is as expected, because fresh nutrition supply has stimulated yeast propagation, thereby has improved the metabolite level of discharging, and produces di-acetyl and 2, the 3-diacetylmethane.Between whole yeast phase 2, it is above and di-acetyl concentration drops to 275 micrograms per litre in the mid-term of continuously fermenting from 500 micrograms per litre that 3-diacetylmethane horizontal dimension is held in 400 micrograms per litre.This point also with the feasibility assessment of 6.3 joint reports in viewed di-acetyl level fall to 2, match under the 3-diacetylmethane level.

The operating parameters of Fig. 6 .59 carrageenan immobilization fermentation and the relation of fermentation time

The total yeast concentration of Fig. 6 .60 carrageenan immobilized yeast and the relation of viability and fermentation time

The alcohol concn that Fig. 6 .61 carrageenan immobilized yeast continuously ferments and the relation of proportion and fermentation time

The carbohydrate changing conditions that Fig. 6 .62 carrageenan immobilized yeast continuously ferments and the relation of fermentation time

Ortho position diketone concentration and fermentation time that Fig. 6 .63 carrageen fixed yeast continuously ferments

6.5.2 the application of super flocculating yeast bacterial strain: self flocculates

Continuously fermented 3 months with 50 liters of experimental scale air lift type biological reactors that the super flocculating yeast bacterium of LCC290 is housed.CO ₂Jetly be made as about 2.5 mm/second and import air to promote some yeast growth (this be equivalent to import in 1.51 liters of/minute total gases contain 3% oxygen) with about 0.4 mm/second.The whole on-stream period of the leavening temperature of retort remains on about 15 ℃.A power failure forces us that fermentation jar temperature is reduced to 4 ℃ of 3 days (ferment about 1700 hours time) (graphic representations 6.64).The fermentor tank cooling is to keep its viability for the metabolism of yeasts that slows down at turnoff time.This unscheduled event provides a chance to estimate the ability of recovering the common incident of this system possibility under industrial situations.The retort temperature transfers to 15 ℃ again and fermented 30 days again in case restore electricity.

In case finished the initial in batches phase (about 180 hours), supply with wort with 2.16 liters of/hour flow velocitys continuously to this system, thereby 24 hour residence time was provided on the basis of 50 liters of retort working capacitys.Initial in batches after date, cell concn rise, and ferment and reach 3,000,000,000 cells/ml (graphic representation 6.65) about 750 hours the time, are reduced to about 1,000,000,000 cells/ml and keep this level up to fermentation ends yeast cell group about 1000 hours then.The yeast viability is (Fig. 6 .65) more than 90% at whole yeast phase.

Very fast alcohol concn and the fermented liquid ratio quasi-stable state (graphic representation 6.66) that weighs after continuously feeding begins.All the other time alcohol concn that ferment rise to about 70 grams per liters, and liquid specific gravity is about 2.3 ° of P.The carbohydrate change curve of graphic representation 6.67 confirms to continuously ferment to enter quasi-stable state about 270 hours.About the 1400 hours polysaccharide concentrations that ferment are reduced to about 33 grams per liters from 42 grams per liters approximately, and this result is because the difference between each batch of wort.Can not consume these polysaccharide because store old yeast, the unusual performance to first fermentor tank of this wort nutrition does not make significant difference.The difference of this nonfermented sugar composition can be detected by housebroken teacher of the sampling wine member, and they can inform that this product has " light refreshing " wine body.

Graphic representation 6.68 provides di-acetyl and 2,3-diacetylmethane concentration over time, as continuously fermenting with κ-carrageenan, continuously feeding wort Once you begin, di-acetyl and 2 is just 3-diacetylmethane level raises.Di-acetyl reaches about 375 micrograms per litre, and 2,3-diacetylmethane concentration is about 600 micrograms per litre.Whole yeast phase is kept this concentration level and is cut off the power supply up to about 1700 hours, because fermented liquid leaves standstill in batches and do not have further metabolism of yeasts (nutrition for want of) in 3 days, ortho position diketone level descends.In case begin the charging wort again, di-acetyl and 2,3-diacetylmethane recover its quasi-stable state value.

The operating parameters of graphic representation 6.64LCC290 fermentation and the relation of fermentation time.

The relation of the saccharomycetic total barm cell concentration of graphic representation 6.65LCC290 and viability and fermentation time.

The alcohol concn that graphic representation 6.66LCC290 yeast continuously ferments and the relation of proportion and fermentation time.

The concentration of saccharide that graphic representation 6.67LCC290 yeast continuously ferments and the relation of fermentation time.

Ortho position diketone concentration and fermentation time that Fig. 6 .68LCC290 yeast continuously ferments.

6.5.3 the application of medium flocculating yeast bacterium: self flocculates

In 50 liters of experimental scale air lift type biological reactors, adopt medium flocculating yeast bacterial strain LCC3021 to carry out fermentation several times as immobilization matrix.Two kinds of immobilization patterns are such as the aforementioned, and initial yeast concentration is set at 4 grams per liters.This yeast is dropped in the old wort of industrial storage (see 4.2 joint described), carry out batch fermentation and exhaust with the di-acetyl level up to all fermentable saccharides and reduce to below 30 micrograms per litre.Leavening temperature is controlled at 15 ℃, and efflux velocity is controlled at and LCC290 fermentation par (surface C O ₂Gas velocity～2.5 mm/second, air～0.4 mm/second, total jet middle oxygen accounts for about 3%) (Fig. 6 .69).

This first batch fermentation makes yeast cell that flocculation take place and is easy to keep flocculation in the gas lift system.In the initial in batches end of term, the wort input speed is made as 2.16 liters/hour, is equivalent to about 24 hours of the residence time in 50 liters of retort working capacitys.Yeast flora (graphic representation 6.70) increases to about 1,000,000,000 cells/ml and keeps this level in whole 1000 hours (continuously fermenting 500-1500 hour that begins).About the 1500 hours yeast floras that ferment double suddenly, and horizontal stable is in 2,000,000,000 cells/ml, and this variation of yeast flora is beyond expectation, and yeast phase yeast vigor keeps about 90% (graphic representations 6.70).

The proportion of alcohol concn and fermented liquid during graphic representation 6.71 provided continuously ferment in 3 months.After initial in batches soon (about 180 hours) alcohol concn be stable at 70 grams per liters, proportion reaches the about 2.2 ° of P of Schwellenwert.The unexpected increase of above-mentioned yeast flora is not reflected in the reduction of alcohol concn.The most logical explanation that this yeast flora increases is, the major part of yeast flora has entered the propagation phase and the multiplication that produces yeast concn.The reduction meeting of having anticipated alcohol concn conforms to the increase of yeast concentration, but this obviously is not because ethanol maintains in the whole process of continuously fermenting on its quasi-stable state value 70 grams per liters.Concentration of saccharide changes curve (graphic representation 6.72) the announcement conclusion identical with ethanol and specific gravity curve with fermentation time.Fermentation this time reaches its quasi-stable state about 250 hours the time continuously fermenting.

Graphic representation 6.73 provides di-acetyl and 2, the 3-diacetylmethane concentration curve and the time relation of continuously fermenting.As the result of κ-carrageenan gel and LCC290 ortho position diketone, at initial after date di-acetyl and 2 in batches, 3-diacetylmethane concentration raises and reaches quasi-stable state value 225 and 400 micrograms per litre respectively.

The operating parameters of Fig. 6 .69LCC3021 fermentation and the relation of fermentation time

The relation of the saccharomycetic total cell concn of Fig. 6 .70LCC3021 and viability and fermentation time.

The alcohol concn that Fig. 6 .71LCC3021 yeast continuously ferments and the relation of proportion and fermentation time.

The carbohydrate that Fig. 6 .72LCC3021 yeast continuously ferments changes the relation with fermentation time.

The ortho position diketone concentration that Fig. 6 .73LCC3021 yeast continuously ferments and the relation of fermentation time.

6.5.4 the comparison of various carriers

6.5.1 super flocculating yeast bacterium of κ-carrageenan gel beads, LCC290 as immobilization matrix and the leavening property of the medium flocculating yeast bacterium of LCC3021 are provided in all joints of～6.5.3.Think that all three kinds of carriers all are adapted at fermentation just continuously in 50 liters of through-flow tubular type biological reactors of experimental scale gas lift.All three kinds of situations all realized liquid hold-up 24 hours.It is more faster than the medium flocculating yeast bacterium of LCC3021 and κ-carrageenan fixed yeast system to adopt the super flocculating yeast fermentation of LCC290 to reach quasi-stable state.LCC290 fermentation reaches its highest alcohol concn 70 grams per liters proceeding to about 250 hours.The LCC3021 fermentation reaches its stable state alcohol concn 70 grams per liters about 600 hours.Alcohol concn tended towards stability in two different time points of fermenting process when κ-carrageenan continuously fermented, and reached 45 grams per liters for the first time between 200～500 hours, rises to 70 grams per liters then and keep this concentration up to the experiment end about 575 hours.As if three kinds of fermentation systems all reached about every milliliter 1,000,000,000 cell of the highest free yeast cell concn.The inconsistent alcohol production rate to κ-carrageenan system of yeast cell concentration has negative impact (to compare initial quasi-stable state alcohol concn lower with LCC290 yeast system).The yeast concentration of each system reaches peak value at interval at different time.For the LCC290 fermentation, alcohol concn reaches its maximum value between 500-1000 hour of fermentation; And the LCC3021 fermentation reached the maximum cell number between the 1500-2000 that continuously ferments hour.κ-carrageenan cure system reaches high cell concentration continuously fermenting between 700-1000 hour.The di-acetyl and 2 of three kinds of super flocculating yeast bacterium of immobilized cell fermentation-LCC290, the medium flocculating yeast bacterium of LCC3021 and κ-carrageenan immobilized yeasts, 3-diacetylmethane quasi-stable state concentration is also dissimilar.For the LCC290 fermentation, di-acetyl is 375 micrograms per litre; And the LCC3021 fermentation level is stabilized in about 225 micrograms per litre.With regard to κ-carrageenan fermentation, di-acetyl concentration reaches 500 micrograms per litre continuously fermenting interim, and level is reduced to about 200 micrograms per litre gradually in 500 hours.In three kinds of fermentations, 2,3-diacetylmethane concentration has reflected di-acetyl concentration.Between whole LCC290 and LCC3021 yeast phase, 2,3-diacetylmethane concentration is higher than di-acetyl.κ-carrageenan fermentation has shown different mode, and two two acyls are higher than 2 during the quasi-stable state for the first time at it, the 3-diacetylmethane, and after this di-acetyl concentration falls to and is lower than 2,3-diacetylmethane concentration.This yeast concentration data and ethanol production data are also pointed out, and have reached the quasi-stable state of the uniqueness of opening in twice minute between κ-carrageenan yeast phase.

Relatively that is a kind of better for different fermentations system and assessment, when the advantage of this system may become complicated during based on an above standard.For example single with total alcohol production rate during as the criterion of success, all three kinds of test macro evaluations equate, because all reach production 70 grams per liter ethanol in 24 hour residence time of 50 liters of retort volumes.It is more much higher than the requirement of simple production alcoholic acid to produce vendible beer.Should estimate the ability that its production can be accepted beer (packing alcohol production rate and di-acetyl level) according to the potentiality expense increase of carrier, according to the utilizability of carrier, operation difficulty or ease, environment protection (abandoning), the stability of carrier and the handiness of carrier system of system to the fermentation system that is proposed as carrier.In order to estimate these many-sided performances, business circles adopt a kind of being called the no dimension analytical procedure of " balance scoring card Balanced Scorecard " (Kaplan and Norton, 1996).The first step comprises the necessary standard of this system of characterization and evaluation.Give each standard scoring (1-5 branch) then, taste was the poorest in 1 minute, and taste was best in 5 minutes.When analyze finishing, add up each option total points, what have best result is optimal selection under this situation.

That table 6.2 provides that the 50 liters of through-flow tubular type production of experimental scale gas lift retort being considered as can be used for fermenting may options, with the analytical results of the balance scoring card of fixation support.Estimated 6 kinds of carriers:

The chitosan pearl,

The diatomite pearl,

Granulated glass sphere, κ-carrageenan gel beads, medium flocculation LCC3021 yeast and super flocculation LCC290 yeast, main purpose is to produce vendible beer.Estimated each carrier system with above-mentioned size.In a word, the super flocculating yeast bacterium of LCC290 performance the best is medium flocculating yeast bacterium LCC3021 then, and other four kinds of carrier scorings are between 16-20, and the 3rd is κ-carrageenan system, can sell beer because it has been produced with testing apparatus.The further effort that the assessment of these carriers points out exploitation to continuously ferment system strongly should be assembled self as the immobilization pattern.The utilizability of the carrier that this Class Options provides (being not difficult to obtain), expense (cost is low because do not need miscellaneous equipment), (can be contained in factory's existing utility) easy to operate have surpassed the latent instability of yeast throw out in the mixing system.May utilize self aggregate that the susceptibility of shearing force is controlled the size of throw out in the fermenting process, and may reach the further raising that increases mass transfer thereby reach the biological reactor volumetric productivity.

Table 6.2 is used for the comparison of several fixation supports of fermentation beer just of air lift type biological respinse can system

6.6 with gas lift draft tube technology production North America type lager beer

North America (NA) the type lager beer of producing pure taste has proposed many challenges to the EnologistWinemaker.NA type lager beer feature is that look shallow, and taste is not quite bitter, residual sugar low (light refreshing), and no advantage fragrance, thereby do not have lingering fragrance relatively.Because these natural characteristicss, the EnologistWinemaker may can not hear few fragrance defective.High-caliber di-acetyl (butter flavor), acetaldehyde (granny smith flavor) and Sulfur flavor (empyreumatic rubber, skunk flavor, rotten-egg odour, the ripe vegetables flavor of burning) are modal problems of bothering contemporary EnologistWinemaker.Though the bacterial contamination of fermention medium also may be a kind of reason of these unpleasant odors of beer, the improper more normal generation of fermenting process control is higher than the unpleasant odor of expectation.

The enforcement test of continuously fermenting is the part of this paper, and whole test has been controlled pollution level in wort and the fermenting container by diligent enforcement Aseptic technique.The fermentation test of all three kinds of carriers has continued some months, does not show the pollution (with the monitoring of the 4th chapter reported method) of the level of can measuring.The di-acetyl (target is to be lower than 30 micrograms per litre) and the acetaldehyde (target is to be lower than 10 mg/litre) that are higher than desired level make the product that just ferments continuously horrible, but this level is not because bacterial contamination.These find to report that with document (Pajunen etc., 2000, Kronlof etc., 2000) do not have difference.Belgian EnologistWinemaker is transformed into tool with the beer of the contained high levels acetaldehyde of its continuous fermentation process production and sells characteristic, the product of putting on market be apple flavor undertint beer (Andries etc., 1996b).

The high-load di-acetyl in back that just ferments in the wine-making industry also is normal.Some wine brewing merchant carries out a kind of what is called " temperature freely raises (temperature free rise) " method to help to reduce di-acetyl after finishing its fermentation just.Other people select to make ortho position diketone (di-acetyl and 2,3-diacetylmethane) be reduced to desired level their product simple insulation long period in weathering process.In the another kind method, several research groups have developed the described rapid ageing technology of the 2nd chapter and have handled the di-acetyl high-content.Though this method is very effective, the another kind of complicacy that it increases for total wine-making technology might be difficult to accept.Yet early stage at the wine-making industry continuous fermentation process, the economy of rapid ageing technology is quite clear, and reduce the complicacy of technology as far as possible and transfer to continuous production to promote traditional batch fermentation, may be wise.The back is adopted and is incubated to control the diacetyl content in the last beer in batches because this reason, decision research are just fermented in 50 liters of experimental scale gas lift systems continuously.Not predicting when this doctor's problem begins to increase this procedure of processing, yet must carry out this mensuration to compare the contrast beer of quantity-produced beer and batch production.

Be incubated after the fermentation just 6.6.1 adopt continuously in batches

Determine that the completed key parameter of first fermentation is the di-acetyl level in the whole last fermented liquid.It is rate-limiting step (graphic representation 3.5) in the di-acetyl approach that the precursor α-acetylactis of di-acetyl is transformed into di-acetyl.Last reaction property is chemical reaction and depends on temperature.If " life " beer is that di-acetyl advances into cold aging processing in α-acetylactis chemical transformation, the di-acetyl level may surpass taste threshold 20 micrograms per litre in the beer of generation, unless prolong cold digestion time its precursor is slowly changed.6.5 joints described three kinds continuously ferment in the retort di-acetyl level of emitting be higher than required target value 30 micrograms per litre in the undiluted beer.If remove yeast at this stage filtering fermentating liquid, di-acetyl will keep high level, therefore, adopt warm in batches for some time that the di-acetyl value is reduced to and can accept under the limit.

Collection is continuously fermented the beer insulation that produces in 40 liters of stainless steel beer cans of 21 ℃.Liquid small sample in the timing extraction jar (100 milliliters), and analyze di-acetyl, ethanol, proportion, ester and fusel.Graphic representation 6.74 shows and continuously ferments as immobilization matrix with the LCC290 yeast that a collection of beer di-acetyl that produces reduces and the relation of soaking time.Can effectively make the di-acetyl level regard " reducing (pre-drop) in advance " boundary as in the wine-making industry from the soaking time that about 600 micrograms per litre are reduced to below 30 micrograms per litre.

Another experimental study soak mix the effect that di-acetyl is reduced.In soak, keep liquid to stir the bottom that carbonic acid gas jet (0.14 cubic metre/hour) imports the beer holding tank by 1.27 centimetres of stainless steel tubes.Graphic representation 6.75 is this result of experiment, it seems CO ₂The jet stirring that provides is to almost not influence of di-acetyl changing down in this secondary insulation jar.This possibility of result shows, CO ₂Therefore the undercompounding that gas mixture provides can not improve the speed of reaction of the 1st chemical reaction (α-acetylactis changes di-acetyl into), maybe can not improve the rate of mass transfer that makes the 2nd reaction (yeast changes di-acetyl into the 3-oxobutanol) faster generation.Might not have the jar cell suspension of stirring abundant yet, can become the 3-hydroxyl fourth acetone that does not have fragrance by further reduction of diacetyl when (the 1st step) in the chemical conversion that speed limit takes place.

Just this in batches insulation the in fermentation back seen graphic representation 6.76 and 6.77 respectively to the ester of pot liquid and the influence of fusel concentration and alcohol concn and proportion continuously.It seems that from these results soak does not almost act on (graphic representation 6.76) to the concentration of acetaldehyde, ethyl acetate, propyl alcohol, isopropylcarbinol, primary isoamyl alcohol, Isoamyl Acetate FCC.The proportion of liquid is reduced to 2.0 ° of P soak just 12 hours from 2.7 ° of P in the insulation jar, then this than low value about.Alcohol concn was stable at 70 mg/litre in 65 hour testing period, these results show that soak mainly influences di-acetyl and 2, the concentration of 3-diacetylmethane and very little to ester, fusel and alcoholic acid influence.

Fig. 6 .74 LCC290 yeast continuous relation of just fermenting back ortho position diketone concentration and being incubated in batches in the gas lift system.

Fig. 6 .75 LCC290 yeast continuous relation of just fermenting back di-acetyl concentration and being incubated in batches in the gas lift system.

Sample stirs with this sample of the jet feeding of carbonic acid gas, and another sample leaves standstill and do not stir.

Fig. 6 .76 LCC290 yeast continuous relation of just fermenting back ester and fusel concentration and being incubated in batches in the gas lift system.

Just fermentation back di-acetyl concentration and proportion are incubated (relation) to Fig. 6 .77 with thermosol continuously in the gas lift system with the LCC290 yeast.

Carried out thermal-insulating scheme in batches for the super flocculating yeast bacterium of LCC290, the medium flocculating yeast bacterium of LCC3021 or the κ-carrageenan immobilized yeast liquid that produces that in 50 liters of gas lift biological reactors, continuously ferments.Graphic representation 6.78 provides the di-acetyl of these three kinds of tests to reduce graphic representation.Di-acetyl all successfully is reduced to its target value 30 micrograms per litre under all three kinds of situations.Yet reach three kinds of situation differences of this reduction required time.Realized reducing to 30 micrograms per litre in about 48 o'clock during with LCC290, only needed about 24 and 40 hours and LCC3021 fermentation and κ-the carrageenan fermentation reaches this target value from 600 micrograms per litre.Infer that the initial initial value of this difference and di-acetyl is relevant and irrelevant with the kind of used immobilization matrix.

Graphic representation 6.79 explanation comes to the same thing to LCC3021 and κ-when time calibration is carried out in carrageenan fermentation and the di-acetyl of Fig. 6 .78.The original di-acetyl of back two kinds of fermentations is reduced curve to be moved on the di-acetyl minimizing curve that the initial value that makes them drops on the super flocculating yeast bacterium generation of LCC290.As if through this conversion, the di-acetyl of all three kinds of systems reduces curve and drops on the same line.With Table Curve 2D software, these results' curve meets first _ order kinetics equation (Levenspiel, 1972) (graphic representation 6.80), can calculate 6.79 calibrated experimental data from following formula:

[di-acetyl]=648.54e ^(-0.0426t)(6.1)

Relation conefficient is 0.96.This result supports all three kinds of immobilization systems to show identical this theory of di-acetyl reducing power strongly, should not wonder this result, because di-acetyl reduction normal relevant with yeast strain (Nakatani etc., 1984).κ-carrageenan system is fixed with the variant that LCC3021 yeast, LCC290 yeast are a kind of selected LCC3021 bacterium in its gel structure.

In case the di-acetyl level is lower than target level 30 micrograms per litre,, carry out last product preparation (filtering, dilute carbonating gas and packing) then at cold place's storage (2 ℃) 7 angel's beer agings.Table 6.3 brief summary the analysis of the beer in 50 liters of experimental scale systems, produced as immobilization matrix with LCC290 yeast, LCC3021 yeast or the immobilized yeast of κ-carrageenan.Graphic representation 6.81 is these beer of quantity-produced and the ester of the contrast beer produced of industrialization and radiation (radar) figure of fusel in batches.Compare quantity-produced beer to contain ester (ethyl acetate, Isoamyl Acetate FCC) lower with the beer in batches (contrast) that industrialization is produced, and propyl alcohol is higher, and isopropylcarbinol, premary amyl alcohol and primary isoamyl alcohol are lower.The acetaldehyde level of continuously fermenting in the product is higher than contrast.Foaming level, initial cold muddiness, heat muddiness, dimethyl sulphide, carbonic acid gas, sulfurous gas and air level are up to specification.

Yeast continuous fermentation just back di-acetyl concentration and the relation that in batches is incubated in the gas lift system that graphic representation 6.78 usefulness LCC290 yeast, LCC3021 yeast and carrageenan are gel immobilized.

Yeast continuous fermentation just back di-acetyl concentration and the relation that in batches is incubated in the gas lift system that graphic representation 6.79 usefulness LCC290 yeast, LCC3021 yeast and carrageenan are gel immobilized.The value of LCC3021 fermentation and carrageenan fermentation is identical with the LCC290 fermentation to starting point through time calibration.

The yeast of graphic representation 6.80 usefulness LCC290 yeast, LCC3021 yeast and carrageenan gel sets is continuous fermentation just back di-acetyl concentration and the relation that in batches is incubated in the gas lift system.The value of LCC3021 fermentation and carrageenan fermentation is identical with the LCC290 fermentation to starting point through time calibration.

Other Several Parameters of influence (apparent extract, true extract, the original extract of calculating, color, bitter taste) is different with contrast closely to be subjected to that product is diluted to ultimate density 5.0%v/v from its original alcohol concn, because the product that continuously ferments just can reach required alcohol concn because its original alcohol concn higher (70 grams per liters, and batch process is 60 grams per liters) need use water as more dilutions.Graphic representation 6.82 is radiograms of the contained alcohol of above-mentioned same liquid, di-acetyl, pH color and bitter taste.Alcohol content, di-acetyl and pH are in target zone, and color and bitter taste have exceeded specification.Color is relatively poor to need the height dilution relevant with the beer of producing that continuously ferments, and this can correct by the color that improves wort nutrition charging.Bitter taste also is owing to same dilution error, and also available wort charging is corrected.

Table 6.3 brief summary of the beer analysis of gas lift system production

Fig. 6 .81 continuously ferments with LCC290 yeast, LCC3021 yeast or the LCC3021 yeast that is fixed on the carrageenan gel beads in the gas lift system and produces the radiogram of beer.This figure provides the ester and the fusel situation of final beer.All products stand to be incubated at the initial fermentation after date in batches.

Fig. 6 .82 continuously ferments with LCC290 yeast, LCC3021 yeast or the LCC3021 yeast that is fixed on the carrageenan gel beads in the gas lift system and produces the radiogram of beer.This figure provides the ester and the fusel situation of final beer.All products are just standing to be incubated behind the yeast phase in batches.

6.6.2 best jet selection

The great majority wine brewing is not difficult to obtain carbonic acid gas in the room, because it is yeast-leavened natural byproduct.The CO that produces is collected in the wine brewing room ₂, wash this air-flow then and remove the small amount of impurities (being generally sulfocompound) that wherein may bring in the collection gas.Then the gas compression of this purifying is become liquid and store the room of waiting to make wine and use (99.95% is pure).In continuously fermenting, use CO ₂See seemingly a kind of logical selection as jet from the operation viewpoint.Factory can utilize their gathering system to reclaim the CO that leaves in the jar that continuously ferments ₂And adopt other gas only can increase the another kind of complicacy of this existing factory operation.

Yet continuously ferment to replace present batch production process practically, must produce the product that matches closely with current brand beer.If believe the biology/biochemical action of yeast contact is reduced as far as possible, perhaps may realize this product that matches.Known in fermentation just CO ₂Metabolism of yeasts there is detrimentally affect.(this system's inherent outlet pressure suppresses the CO in the liquid nutrient medium in high conical jar ₂Freely discharging) this effect is exaggerated.These situation tendencies produce the beer that contains low ester class and higher fusel.Carried out successful trial by regularly injecting some this CO that assist gas is removed fermentation from conical pot bottom ₂This CO ₂The effect that suppresses be it seems and reduced, contains less fusel and higher ester class in the products obtained therefrom.

Because the increase of this knowledge has been studied in the gas lift system with nitrogen replaced C O ₂As jet.Collected several fermented liquids in 50 liters of gas lift retort and in Labatt wine brewing laboratory, processed with following step.Collect retort (24 hour residence time) liquid after 14 hours, separate out " unpasteurized beer " from the yeast updip, make it through the insulation of 48 hours room temperatures (21 ℃), allow di-acetyl and acetaldehyde reach the technical specification (di-acetyl＜30 micrograms per litre, acetaldehyde＜10 mg/litre) of Labatt.Make cold aging 7 days of this liquid then and use the processing of stdn industry rules.

Table 6.4 will be with the LCC290 yeast at CO ₂Jet system and N ₂Continuously ferment in the jet system result of the final beer that obtains and the beer (contrast) of standard industry production compares.The beer flavor of spray nitrogen is better than the beer liquid that industrialization is produced.In this wine of analysis revealed the 1-propyl alcohol up to 2 times approximately low three times of dimethyl sulphide concentration, color and bitter taste score value it seems and be higher than industrial wine that foaming power is also like this with the NIBEM test determination.CO ₂The beer of jet generation compares N ₂Low and the 1-propyl alcohol content height of the ester class of jet generation (ethyl acetate, Isoamyl Acetate FCC) content.Contrast beer, N have been calculated ₂Jet beer and CO ₂The ratio of ester of jet beer (ethyl acetate, Isoamyl Acetate FCC, acetic ester, ethyl octylate, ethyl decylate) and fusel (1-propyl alcohol, isopropylcarbinol, primary isoamyl alcohol) is found to be respectively 0.30,0.27,0.15.

The several prods chemical analysis brief summary that table 6.4 continuously ferments and produces with the gas lift system that is mounted with the super flocculating yeast bacterium of LCC290

Fig. 6 .83 provides the radiogram of ester class, fusel and the acetaldehyde concentration of these beer.The very approaching contrast beer of pattern of spray nitrogen beer except propyl alcohol content is higher.CO ₂Jet fermentation show much lower ester class and with the unmatched fusel of contrast.The di-acetyl of two kinds of beer that continuously ferment and acetaldehyde level are lower than the labatt technical specification.

Find that more than prompting spray nitrogen has improved ester class output, its level is similar to commodity beer, and spray CO ₂The beer ester class concentration that produces is lower.These results suggest are at CO ₂Metabolism of yeasts changes in the jet environment.No matter spray what gas, the propyl alcohol content in the beer that continuously ferments is more much higher than industrial fermentation contrast beer.Though propyl alcohol concentration is lower than taste threshold 100mg/L, notice that comparing these difference with batch fermentation may be a kind of index that slight variation has taken place in metabolism in continuously fermenting.May higher propyl alcohol content be owing to the Threonine that constantly provides also, it can produce propyl alcohol by the oxoacid degradation pathway.

Fig. 6 .83 spray nitrogen and spray CO ₂The radiogram that continuous gas lift fermentation beer is compared with the ester class and the fusel of suitability for industrialized production beer.

Chapter 7, conclusion

Can draw from the research that this paper did to draw a conclusion.Carry out continuously just fermentation with 50 liters of through-flow tubular type biological reactors of experimental scale gas lift, be incubated with the control di-acetyl through 2 days more in batches, can produce can received no important taste defective beer.24 hours minimum residence times or secondary response tankage turnover every day 1 are attainable to heavy wort juice (17.5 ° of P) fermentation is become final fermented liquid (2.5 ° of P).Super flocculating yeast bacterium LCC290 medium flocculating yeast bacterium LCC3021 and κ-carrageenan immobilized yeast all are the appropriate carrier that can be used for this gas lift system.Heavier previously prepared carrier such as siran granulated glass sphere and Celite diatomite pearl are not surrogates practical in the gas lift draft tube system.Can realize that κ-carrageenan gel beads continuously fermented 2 months at least, LCC290 and LCC3021 continuously fermented 3 months and do not have any experimental microbial contamination or retort unstable properties at least.In addition, this system of continuously fermenting can deal with contingent variation in the supply of industrialization wort in the long-term operation process.

Continuously ferment with super flocculating yeast bacterium LCC290 and spray nitrogen, carry out insulation in batches in 2 days then, produced the beer that taste and industrial production contrast beer mates the most.It is effectively that design was incubated the high density di-acetyl of handling from the liquid of fermentor tank discharge just continuously with 2 days in batches, though be not the control method of optimum.The ability of the system reducing di-acetyl that continuously ferments of three kinds of tests is very similar, and as previous suspection, this performance is attributable to the bacterial strain type.Soak does not influence the ester class and the fusel concentration of beer in the soak in batches.Containing 3% oxygen in jet provides competent oxygen to wort, and the yeast flora viability maintains more than 90% in the whole fermentation process as a result, and the beer of producing has the taste that can be people's acceptance.Continuously ferment than reaching quasi-stable state quickly as immobilization matrix with yeast LCC290 and LCC3021 with κ-carrageenan gel beads system.The unstable of the gel immobilized fermentation of κ-carrageenan may be that fermentation is lower than target level because the increase of fixed yeast flora causes product.For the ideal continuous production, this phenomenon is very unfavorable, because increased reaction and restarted required time in the extension startup of calamity failure back.The prolongation starting period also requires the longer phase of continuously fermenting with attractive.

This continuous gel beads production technique has been produced the pearl that is used for the desired number tested in the experimental scale retort.Yet, must further optimize this pearl production technique to produce the narrower pearl of size distribution.Also need further to study this technology to determine its plant-scale feasibility.Except having studied the Kenis type in this research, for making this selection practical, must find and test by increasing diameter just increase the motionless mixer number, and a kind of novel motionless mixer that realization expands the scale of production.

Adopted sour pulse tracer technology herein, made us be assessed during the super flocculating yeast bacterium of LCC290, the medium flocculating yeast bacterium of LCC3021 and the κ-carrageenan immobilized yeast real attenuation mixing time and speed of circulation in 50 liters of experimental scale biological reactors.This blended data conforms to the sinusoidal function of decay, can calculate mixing time and speed of circulation by this function.

Carry out short mix in this gas lift draft tube system, the mixing time that all three kinds of fixation supports are calculated is less than 200 seconds.Mixing time slightly reduced when the surface gas flow velocity of all three kinds of test macros improved.When the surface gas flow velocity (2～6 mm/second) of all tests, the LCC290 system shows the fastest mixing time (100～120 seconds).No matter the surface gas flow velocity, the liquid circulation time of all three kinds of carriers is closely similar, and they are linear decline the with the corresponding increase of gas flow rate also.The fixation support of all tests is taken turns 3～6 and has been finished liquid mixing (to 98% reaction of pulsatile once) in the retort circulation.50 liters of experimental scale systems of these confirmatory tests as a result provide sufficient mixing for continuously fermenting.The mixing time I dead angle can occur and be unfavorable to beer fermentation.

This doctor research work proves that clearly the scale that further enlarges design in Labatt wine brewing laboratory, structure and this production system is feasible.Recommend to adopt the gas lift draft tube biological reactor (tentatively) that super flocculating yeast bacterium of LCC290 and spray nitrogen are housed to continuously ferment, be incubated as first-selected system in 21 ℃ then in batches.

Detailed Description Of The Invention---second section

The 4th chapter material and method

4.1 yeast strain and feature

Adopt storage ageing brewer yeast bacterial strain: Labatt Cultrue Collerction (LCC) 3021 in the whole work.(Saccharomyces uvarum Beijerinck varcarlsbergensis Kudryavtsev, 1960 is synonym (Kurtyman, 1998) for S. cervisiae and Sucus Vitis viniferae sugar yeast Ka Ersibai mutation.LCC3021 does not grow in the time of 37 ℃.This helps to differentiate that LCC3021 stores old yeast and most of light color (beer) yeast, and the latter can grow under 37 ℃ and higher temperature.LCC3021 is a kind of sedimentary fermentation bacterial strain, and the old yeast of most of storage is also like this, but exception is arranged.Also have this bacterial strain energy glucose fermentation, semi-lactosi, sucrose, maltose, raffinose and melibiose, but non-fermenting starch.The ability of fermentation melibiose also is that the taxonomist differentiates it and the used a kind of method of light yeast.The same with great majority wine brewing bacterial strain, LCC3021 is a polyploid, breeds by mitotic division.The old yeast of storage is not by reduction division propagation under normal wine brewing condition, and this advantage makes the wine brewing bacterial strain stable in heredity, exchanges (Kreger-van Rij, 1984) because genetic material is unlikely.

4.2 yeast inoculum preparation

Get-80 ℃ of refrigerators frozen yeast alive at peptone-yeast extract paste nutrition (PYN) agar (peptone 3.5g/L, yeast extract paste 3g/L, KH ₂PO ₄2g/L, MgSO ₄7H ₂O, 1.0g/L, (NH ₄) ₂SO ₄1.0g/L, glucose 20.0g/L, agar 20.0g/L, water-soluble) and streak inoculation on the growth medium, obtain to separate good bacterium colony, from 3-4 days yeast flat board of growth, choose an asepsis ring (containing several bacterium colonies) and be seeded in the face 10ml wort test tube.Be called " overnight culture " in 21 ℃ of overnight incubation, be added to then in the wort of relatively large (often for 200ml) to increase the yeast amount.Several days afterwards, this mixture is added in the wort of another more volume, obtain required yeast amount up to breeding.Usually estimate that every liter of wort can produce the old yeast of about 20 gram storages.In order to prepare the yeast inoculum, in 4 ℃ with the centrifugal culture of 10000rpm (radius 0.06m) 10 minutes.Centrifugal back tipping liquid, the yeast that obtains suitable weight in wet base from precipitation is for inoculation.

4.3 attenuate substratum

Canada Labatt zythepsary provides the distiller's wort of 17.5 ° of P of proportion.Concentration, proportion and the free amino nitrogen of the fermentable saccharide of this wort that is used for this research fermentation have been provided in the appendix A 2.1.Dale etc. is seen in other detailed description that this wort is formed, 1986, and Hoekstra, 1975, Hough etc., 1982, Klopper, 1974 and Taylor 1989.

Batch fermentation: wort is through 100 ℃ of 45 minutes heat sterilizations, and cooling then is then with immobilized cell pearl or the free yeast inoculation that suspends

Continuously ferment: the wort that is used to continuously ferment is used moment pasteur's method sterilization (Fisher Plate Heat Exchanger, combi-flow Type Eurocal 5FH) before adding the gas lift biological reactor.Regularly as the bacterial contamination of monitoring wort as described in 4.6.Pollution abandons immediately and collect new wort from this factory if measure in wort.Moment, the volumetric flow rate of pasteur sterilizer was 0.8 cubic metre/hour.One duct type moist closet is arranged, and wort remaines in 85 ℃ of medial temperatures therein, and minimum temperature is 80 ℃.Moist closet volume 1.13 * 10 ^-2Cubic meter, 51 seconds residence times in the chamber.It is 2 ℃ when leaving that heating back wort cools off rapidly therein.

4.4 process for fixation

κ-carrageenan gel X-0909 is given by Copenhagen Pectin A/S.Such as (1996) such as Neufeld detailed description, with κ-carrageenan gel beads that old yeast cell is store in the embedding of motionless mixer explained hereafter, initial cell load is every milliliter of gel 10 ⁷-10 ⁸Individual cell, each time experiment all describes in detail.The foundation of motionless mixer technology is that (Cole-Parmer Instrument Co. USA) has inoculated mutually between saccharomycetic κ-carrageenan (3%v/v is dissolved in 0.2%w/v KCl solution) solution in non-water continuous phase vegetables oil (Mazola Corn Oil) and water-dispersion and forms emulsion with online polyacetal motionless mixer as described in Figure 15.Yeast mixes rapidly with carrageenan solution in the heating chamber of graphic extension, forms emulsion in the time of 37 ℃.In ice bath, cool off the κ-carrageenan drop gelling of inducing in the emulsion fast, bathe hardening in (22 grams per liter) at KCl then.Produce yeast and carrageenan mixture with 24 member motionless mixers (6.4 millimeters of diameters).Mixing tank with 42 12.7 mm dias of second cover produces emulsion.The pearl footpath of using in this research experiment is greater than 0.5 millimeter, less than 2 millimeters.

4.5 comprise the granular size cumulative distribution of the κ-carrageenan gel beads of fixed yeast mycetocyte

In 30 liters of gel beads production processes, take κ-carrageenan gel beads sample to calculate size-grade distribution at random based on the material weight in wet base.The all about 500 gram weight in wet bases of each sampling adopt screen method to determine that the bead degree distributes.Make pearl pass through a series of screen clothes of 2.0,1.7,1.4,1.18,1.0 and 0.5 millimeters of mesh.Accelerating each pearl sample with the KCl liquid of 4.5 liter of 22 grams per liter sieves.Suppose that κ-carrageenan gel beads is complete sphere, so color sieve diameter is made particle diameter.Suppose that also pellet density is even, irrelevant with granularity.

4.6 yeast cell counting and viability are measured

Free saccharomycetic viability and the cell concn that suspends, adopt U.S.'s wine brewing international methylene blue staining technology of chemist association (ASBC) (Technical Committee and Editoriod Committee of ASBC, 1992) to measure the viability of yeast cell.It is that work is extremely to be the ability that becomes colorless form according to this dyestuff of viable cell oxidation that this staining is measured yeast flora.Dead cell lacks the ability of this dyestuff of oxidation thereby dyes blueness.The method for preparing Fink-Kuhles methylenum coeruleum damping fluid is with 500ml A liquid (0.1g methylenum coeruleum/500ml dH ₂O) and 500ml B liquid (13.6gKH ₂PO ₄/ 500ml dH ₂O 498.65ml and 2.5g Na ₂HPO ₄12H ₂O/100ml dH ₂O 1.25ml) mixes, produce the final methylenum coeruleum damping fluid of pH4.6.

The yeast liquid of mixed diluting and methylenum coeruleum liquid become suspension in test tube, make the suspension that 100 yeast cell are arranged in the field of microscope approximately.Get the suspension that a droplet mixes and place on the microslide covered.Counting indigo plant is dyed and is not had a cytochrome contact 1-5 minute with dye liquor after.Viable cell per-cent is reported as the per-cent of total cell count.Measure cell concn with opticmicroscope and hematimeter (Hauser Scientific Company).

The viability of immobilized cell and cell concn: make fermented liquid isolate gel beads also with 10 ml distilled water drip washing from fermented liquid by aseptic screen cloth (mesh is through 500 microns), saccharomycetic 1 milliliter of gel beads that will contain embedding adds in the 50ml sampling receptacle of the sterilization that 19 ml distilled waters are housed.Use then Device (Brinkmann Instruments) destroys pearl, discharges cell from gel.As above-mentioned free suspension cell, measure cell viability and concentration.

4.7 microbiologic analysis

Liquid phase analysis: at least once get the sample that continuously ferments weekly and do microbiological analysis.Before being conveyed in the biological reactor, the used wort that continuously ferments also detects pollution wherein.Whether there to be aerobic and anerobe in order measuring, sample to be seeded on Universal Beer agar (UBA, the Difco Laboratories) flat board that is added with the 10mg/L cycloheximide, cultivated 10 days for 28 ℃.To be used to detect flat board that anerobe pollutes places and has (Oxoid) in the anaerobic jar of chuck, this chuck can be drawn oxygen remaining in the jar and produce anaerobic environment.Become peach anaerobism indicator (Oxoid) when there is oxygen in employing and verify anaerobic state in the jar.Sample is seeded in adds CUsO ₄(0.4g/L) r microzyme culture medium (YM agar, Difco Laborafories) was being trained 7 days for last 25 ℃, detected the wild yeast fungi pollution.Adopting peptone yeast extract paste nutrient agar medium noted earlier (PYN) to cultivate 7 days old yeast of the non-storage in the screening sample in 37 ℃ pollutes.37 ℃ of yeast of not growing on PYN show (beer) yeast that do not have light color or do not have the 37 ℃ of contaminated bacterias that can grow.

Gel phase is analyzed: our development in laboratory a kind of test guarantee that the immobilized cell pearl that is used to ferment does not have polluted bacteria before dropping into biological reactor.Main concern avoids by the beer spoilage bacterium such as Pediococcus belongs to and Lactobacillus belongs to bacterium or the wild yeast bacterium pollutes.3 milliliters of carrageenan gel beads are inoculated in several following several different choice liquid nutrient mediums of 100ml, be placed in the 250ml flask, 100rpm shaking culture in cultivating shaking table at 25 ℃.NBB meat soup (Nachweis von Bierschadlichen Bacterien) (BBL cat#98139, NBB Broth, Base 0.02g/L cycloheximide) is a kind of half selective medium that is used to detect the beer spoilage bacterium.Copper sulfate meat soup (16g/L YM broth, Difco; 0.4g/L CuSO ₄) be a kind of half selective medium that detects the wild yeast fungi pollution.At last, standard method (STA) adds cycloheximide meat soup (16g/L " StandardMethods " broth, Difco; 0.02g/L cycloheximide) be used for detecting the bacterium (Power and McCuen, 1988) that water, waste water, milk-product and food are found.Select this to select substratum, can in three days, detect and identify potential beer spoilage bacterium.Sample has muddiness to show that sample has polluted and makes the pollutent identity and infer.

Respiratory deficiency (RD) type yeast cell detection method: triphenyltetrazolium chloride (TTC) soverlay technique: differentiate respiratory-deficient yeast and all the other yeast with this method, the principle of its foundation is that TTC is a kind of colourless salt, forms red precipitate during reduction.When TTC being covered at yeast extract paste-peptone-dextran (YPD) agar (yeast extract paste 10g/L, peptone 20g/L, dextran 20g/L, agar 20g/L dH ₂O joins) when going up the yeast colony of growth, to breathe competent yeast and will reduce TTC, these bacterium colonies become wind rose or red, however the respiratory-deficient yeast can not reduce this dyestuff, and keep its original color.

Serial dilution culture to bacterium is proper concn (about 100 cell/0.2 milliliter), carries out plating, cultivates dull and stereotyped about 3 days of YPD up to see yeast colony in aerobic environment for 21 ℃ then.Each dull and stereotyped TTC that covers 20 milliliters 50 ℃ covers agar.After each solution is cooled to 50 ℃, mix A liquid (12.6g/L NaH ₂PO ₄11.6g/LNa ₂HPO ₄: 30.0g/L agar, at dH ₂Among the O, 121 ℃ of sterilizations in 15 minutes) cover agar with B liquid (2.0g/ L chlorination 2,3, the aqueous solution of 5-triphenyltetrazolium chloride, 121 ℃ of sterilizations in 15 minutes) preparation TTC.Room temperature is cultivated after 3 hours and is read plate.RD per-cent is reported as the per-cent of observing the bacterium colony that is unstained in the sum.

4.8 the scanning electron microscopic observation (SEM) of immobilized yeast in κ-carrageenan gel beads

From biological reactor, take out the κ-carrageenan gel beads that contains immobilized yeast by thief hole, place 10ml threaded cap glass phial, pearl is immersed in a small amount of fermented liquid.Be transported to the SEM place in the thermally insulated container with icing the cover glass phial and being contained in immediately.κ-carrageenan the gel beads that fixedly contains immobilized yeast with 2% (v/v) glutaraldehyde of the Sorensen phosphoric acid buffer (Hayat 1972) of 0.07M pH6.8 preparation, 1% (w/v) perosmic anhydride of joining with same damping fluid is fixed then, by serial gradient: 50, the dehydration of 70,80,90,95,100% (v/v) spirituous solution is each 15 minutes, changes liquid three times with 100% alcohol then.Pass through CO ₂Carry out critical point drying (LaddResearch Industries Burlinton, VT) before some pearls are freezed in liquid nitrogen, broken, be collected in 100% alcohol.Freezing crushing comes out the inner face of pearl but is out of shape minimum.Behind the critical point drying with gold/palladium (Polaron SC500sputter Coater of sample sputter coating 30 nanometers, Fison Instruments England), uses Hitachi S-4500 field-emission scanning Electronic Speculum (Nissei Sangyo then, Tokyo, Japan) scanning.

4.9 biological reactor sampling method

Biological reactor thief hole (Scandi-Brew Type T Membrane Sample Valve) reservoir is filled 70% (v/v) ethanolic soln to keep parameatal sterile state between each sub-sampling.Be sampling, take off the stopper of ethanol storage tank bottom, thief hole is opened in emptying and with the thorough drip washing of ethanol.With sample collection in a undercut phial or a threaded lid the jar in, volume 5-60ml does not wait, and depends on required analysis.For detecting microbial contamination, make the 10ml fermented liquid pass through an aseptic membrane filter with vacuum pump.0.45 micron of this membrane pore size is placed in the suitable selectivity substratum as described in 4.6 joints.

For carrying out chemical analysis, from 100ml sealing undercut phial, extract the 60ml sample by barrier film, by Schleicher and Schull, the double-deck syringe filter membrane system of FP-050 carries out syringe and filters, and this filter membrane aperture is 5 microns and 0.45 micron.Then volume required sample branch is packed in the phial of 20ml head space, a Teflon barrier film and an aluminium lid arranged in this phial.The sample volume that needs is listed in the table 4.1

The sample volume that the various chemical analyses of table 4.1 are required

4.10 dissolved oxygen is measured

The scope that doctor's Thiedig Digox 5 dissolved oxygen analysers detect dissolved oxygen in wort, fermenting wort and the beer is 0.001-19.99mg/L (Anon, 1998).Vilacha and Uhlig (1985) have tested many instruments of measuring the beer dissolved oxygen, find that the Digox analyser draws true valuable exact value.Digox 5 used electrochemical determinations are measured three electrode arrangements of electric current based on electrothermal meter.Detecting cell is made up of a mensuration electrode (negative electrode) and counter electrode (anode).These electrodes exposed are in the liquid of wherein oxygen concn to be determined.After fixing the mensuration electromotive force of a regulation, the place reacts at the mensuration electrode.Measure electrode place oxygen molecule at big silver and be reduced into hydroxyl ion.Two water moleculess and an oxygen molecule reaction absorb 4 hydroxyl ions of 4 electron production simultaneously in the reaction 4.1

O ₂+2H ₂O+4e ^-→4OH ^- (4.1)

4 electronics that stainless steel anode absorbs negative electrode release flow through to guarantee electric current.In the reaction formula 4.2, the electric current I of mensuration and oxygen concn C _{L, O}Be directly proportional

I＝K×C _L，O (4.2)

Wherein constant K is subjected to the influence of electronic number, cathodic surface area and the mensuration electrode surface frictional belt degree of depth of Faraday's number, per molecule conversion.Constant characteristic measurement electromotive force is the selectivity (to oxygen) of mensuration and the key factor of accuracy.By one not the reference electrode of load current make measuring voltage stable.With the electrostatic potential that electron feedback is provided, provide constant to measure electromotive force like this.The surface of measuring electrode links to each other with the reference electrode electrolysis by a film.

According to the measurement range of final oxyty, error is ± 3% (Anon 1998).Proofread and correct the dissolved oxygen analyser with Thiedig ActiveCalibration, Digox 5 produces the oxygen (0.5mg/L) of a specified amount according to Faraday's law among the Thiedig Active Calibration, then the measured value of itself and matrix is crosschecked.This makes this instrument can be in 1 minute proofread and correct according to pressure, temperature with corresponding to the mobility status of these (parameter values) measured, because the exchange of molecule is a kind of diffusion process in the transmitter, temperature influence causes speed of response and the rising of measuring electric current faster.Therefore Digox 5 has also installed a transmitter and has measured temperature and the fluctuation of equalising temp automatically.

Digox 5 has some advantage above the oxygen sensor based on film.Because Digox 5 does not adopt electrolytic solution, loss of sensitivity is quite slow, measures seldom deposition only takes place on the electrode.Also have, can determine sensitivity by active correction any time.Just this instrumental method is easy for cleaning and re-graduation.Silver chloride is deposited on the negative electrode and the electrolytic solution variation can cause reading to reduce gradually in most of film sensors.Recommend to change film and electrolytic solution and proofread and correct again every several weeks for this reason.This is the task of a tediously long effort.Carry out when calbrating film transmitter, laboratory are everlasting oxygen saturation level, this may produce tangible error, particularly when wort and beer matrix oxygen level are very low.Temperature has triple influences to film sensors: membrane permeability changes, and oxygen partial pressure can change, and the oxygen solubility in the electrolytic solution can change.It is very difficult that these three kinds of factors of film sensors are carried out temperature compensation.

Dissolved oxygen in the shelf lives wort is measured: with flexible

Food grade pipeline (1/4 inch of internal diameter) be positioned near thief hatch aseptic link to each other (see 4.2.1 save) of wort storage tank T1 or the T2 awl top, the end.The peristaltic pump of an adjustable speed provide 11 liters/hour volumetric flow rate by dissolved oxygen analyser district (

L/S ^TMDigitalStandard Drive, Cole-Parmer cat#P-07523-50).Record wort dissolved oxygen measured value after 4-5 minute.

Dissolved oxygen in the biological reactor is measured.Cleaning Digox 5 analyser districts before the dissolved oxygen mensuration sterilize the connection of transmitter inlet in carrying out biological reactor

Food grade pipeline (1/4 inch of internal diameter) is gone into 70% (v/v) ethanol liquid pump by this analyser 15 minutes with about 10 liters/hour volumetric flow rate.The dissolved oxygen analyser is connected to the laboratory tap water, makes hot water (70 ℃) pass through transmitter at least 2 hours.Adopt this method but not vapor sterilization be because analyser district material can not tolerate temperature more than 70 ℃.After two hours cleanings, the pipe of clamping the gangway is to keep aseptic in the analyser.The pipe that to just sterilize in one deck stream stink cupboard is connected with the gangway of analyser.With the stainless steel hole of the aseptic connection of the free end of this pipe (using clip) 1/4 inch internal diameter on the biological reactor top board, measure then.When without this hole of biological reactor, with the sterilization of one section weak point

The food grade pipe sealing.Collect fermented liquid, the dissolved oxygen in the on-line determination gas lift biological reactor by a hole that is positioned at the biological reactor top board.Fermented liquid flows out by a stainless steel filter (seeing the 4.1.2 joint) that is connected in the 1/4 inch stainless steel tube that passes the biological reactor top board.Fermented liquid is flowed through flexible then

Food grade pipe (1/4 inch internal diameter), this pipe be connected to an adjustable speed peristaltic pump (

L/S ^TMDigital Standard Drive, Cole-Parmer cat#P-07523-50), the volumetric flow rate that provides 11 liters/hour is by dissolved oxygen analyser district.Fermented liquid is by passing second 1/4 inch stainless steel mouth recirculation of biological reactor top board.Adopt

Food grade pipe (Cole-Parmer, 1999) connects transmitter and biological reactor, because the supplier illustrates that it has 30cm ³Mm/ (scm ²CmHg) * 10 ^-10Low oxygen-permeability.Measure after circulation 4-5 minute.

4.11 chemical analysis

Proofread and correct with suitable standard reagent.All reagent purities that are used for this analysis are greater than 99%.Be further purified by distillation when needing.

4.11.1 ethanol

Adopt internal standard gas-chromatography (GC) method (1992) of U.S. wine brewing chemist Technical Committee of association and editor volume person meeting to measure alcohol concn.Degassing sample is directly handled with mark 5% (v/v) in the Virahol, injects the Perkin Elmer8500 gas chromatograph that flame ionization detector (FID) and Dynatech self-actuated sampler are housed.Adopt Chromosorb 102,80-100 order post, with helium as carrier gas.Chromatographic condition: flow velocity 20ml/ minute, 175 ℃ of injector temperature, 250 ℃ of detector temperatures, 185 ℃ of column temperatures.

4.11.2 sugared summary

With cationic exchange coloum (Bio-Rad Aminex is housed, HPX-87K) and Spectra-Physics (SP8100XR) high performance liquid chromatograph (HPLC) of RI-detector (Spectra-Physics SP6040XR), measure glucose, fructose, maltose, DP3 (trisaccharide maltose), DP4 (maltotetrose), polysaccharide peak 1 and glycerol concentration in the fermented sample.Moving phase is dipotassium hydrogen phosphate 0.01M.This system is equipped with Spectra-Physics (SP8110) self-actuated sampler, and instrument is operated with back-pressure 800psi.Sample and eluent flow rate by post are 0.6ml/ minute, 85 ℃ of column temperatures, 40 ℃ of detector temperatures, volume injected 10 microlitres.

4.11.3 proportion

This research is described the proportion of wort and fermention medium with true extract (Plato degree, ° P), and it is the generally acknowledged unit that is used for Brewing industry.As filtration fermented sample as described in 4.8 joints, vibration mixes then with digital density meter (AntonPaar DMA-58 densometer) assay determination wort proportion (° P).Fermented sample is injected glass U-shaped pipe, and electronic oscillation is measured proportion, directly draws a ° P.The numerical value of aqueous sucrose solution per-cent (w/v) when the Plato degree refers to 20 ℃, its proportion is identical with described wort.Because the calibration of Plato degree and the solution proportion and the solute concentration correlation table of gained are based on aqueous sucrose solution, it is the approximation of extract quantity.This speech of extract is meant, the soluble substance total amount (Hardwick, 1995) that exists in the wine brewing material and/or may obtain by processing is as carbohydrate, protein, tannin etc.At present extract is that the proportion of unit is expressed with the Plato degree still in Brewing industry, in default of with the better relevant more suitable reference value of variation of the wort component of different sources.

4.11.4 total di-acetyl amount

Total di-acetyl in beer and the fermented sample (2.3-dimethyl diketone) amount is the headspace analysis thing sampling technique on basis with the method for Technical Committee of ASBC and editorial board (1992), separates (Hewlett-Packard5890) and electron capture detection (ECD) by Capillary GC then.This method is measured " total di-acetyl ", because this method mensuration is di-acetyl and precursor α-acetolactic amount thereof.Carrier gas is the argon gas (1.0ml/ minute) that contains 5% methane, adopts J﹠amp; W DB-Wax post.Splitting ratio 2: 1, assist gas are helium, 60ml/ minute.105 ℃ of injector temperature, 120 ℃ of detector temperatures.This system is equipped with Hewlett Packard 7694E head space self-actuated sampler, and with 2, the 3-hexanedione is as interior mark.40 minutes cycling time of sample, the phial starting time is 65 ℃, 30 minutes, and 2 minutes clamping times of 4.8psig, ring loading time 0.2 minute, ring starting time 0.1 minute, 0.27 minute inject time.18.8psig is pressed in carrier gas, 95 ℃ of transport pipe temperature, 65 ℃ of ring temperature.

4.11.5 the volatile matter of beer

Volatile matter with interior mark (n-butanols) GC (Hewlett Packard 5890) head space method and flame ionization detector (FID) mensuration beer comprises acetaldehyde, ethyl acetate, isopropylcarbinol, 1-propyl alcohol, Isoamyl Acetate FCC, primary isoamyl alcohol, ethyl hexanoate and ethyl octylate.Carrier gas is a helium, and 6.0ml/ minute, GC was equipped with Hewlett Packard 7694 headspace sampler.200 ℃ of GC injector temperature, 200 ℃ of detector temperatures, the furnace temperature change curve: 40 ℃ 5 minutes, 40-200 ℃ (10 ℃/minute), 200-220 ℃ (50 ℃/minute, 220 ℃ 5 minutes).FID gas comprises carrier gas 6.0ml/ minute, consists of helium 30ml/ minute and 28psig hydrogen 50ml/ minute and 25psig, air 30ml/ minute and 35psig.With flow velocity 0.8ml/ minute cleaning membrane.Pressure head 4.0psig.When self-actuated sampler linked to each other by the syringe needle in the injection port, phial pressure was 15.9psig, and nebulizer gas pressure is 7.1psig, and capital is pressed 4psig, divided flow velocity 18ml/ minute, column flow rate 6ml/ minute.District's band temperature: 70 ℃ of phials, encircle 80 ℃, 150 ℃ of transport pipes.40 minutes cycling time of GC, phial starting time 35 minutes, 0.25 minute clamping time, ring loading time 0.1 minute, ring starting time 0.1 minute, 3 minutes inject time, sample loop volume 1ml.

4.11.6 free amino nitrogen (FAN)

The free amino nitrogen world method (1992) of employing Technical Committee of ASBC and editorial board is measured the free amine group nitrogen concentration in the fermented sample, use Perkin Elmer LS50B spectrophotometer, this spectrophotometry show in triketohydrindene hydrate and the sample the color reaction between nitrogenous.Absorbance is directly related with the free amino nitrogen amount of existence.

A) colouring reagents 19.83g Na ₂HPO ₄

30.00g KH ₂PO ₄

A 2.78g ninidrine

1.50g fructose

B) dilution reagent 2.00g KIO ₃

596ml distillatory deionized water

404ml 95% (v/v) ethanol

Be stored in the refrigerator and in room-temperature applications

C) glycine storage liquid 0.1072g/100ml distillatory deionized water

D) glycine reference liquid should be store liquid and be diluted to 1: 100 (v/v) with the distillatory deionized water.This reference liquid contains 2mg/L FAN.

With distilled water diluting sample to 100: 1, the sample of 2ml dilution is moved in 3 test tubes each.Three test tubes are respectively adorned 2ml distillatory deionized water as blank in addition.Preparation simultaneously respectively contains three test tubes of glycine reference liquid 2ml.Add the 1ml colouring reagents to all samples, place 100 ℃ of water-baths accurately to place 1 minute then.Then test tube was cooled off 20 minutes with 20 ℃ of water-baths.In each test tube, add the agent of 5ml extent of dilution, thorough mixing then.Allow sample leave standstill then 10～15 minutes.With the absorbance of spectrophotometric instrumentation 570 nanometers, with the FAN amount of formula 4.3 calculation samples

FAN(mg/L)＝(A _P-A _B-A _F)2d/As (4.3)

Wherein FAN is the amount mg/L of free amino nitrogen in the sample.A _PBe the mean light absorbency value of test fluid, A _BBe the mean light absorbency value of blank solution, A _FFor proofreading and correct the mean light absorbency value that black malt juice and beer are used, the 2nd, the FAN amount in the glycine reference liquid, d is the dilution of sample multiple, A _SIt is the mean light absorbency value of glycine reference liquid.

Chapter 5, continuously ferments with gas lift biological respinse can system

The continuous beer fermentative production selects for use the through-flow tubular type biological reactor of gas lift system to be because its good mass transfer (liquid-solid) and mixed characteristic.Particularly importantly liquid-solid mass transfer carries nutrition to give solid phase immobilized cell biological catalyst because of it relates to from liquid phase, and for the yeast of embedding provides fermentation substrate, these biological reactors also provide good aeration, and energy consumption is low, and simple in structure.This make the air lift type biological reactor to large-scale operation as be used for the commercialization wastewater treatment very attractive (Driessen etc., 1997, Heijnen1993).

5.1 tubular type biological reactor explanation that gas lift is through-flow

This joint describes the used air lift type retort of this work in detail

5.1.1 biological reactor body

For 13 liters of through-flow tubular type biological reactors of (8 liters of working volumes) gas lift of this work design are a kind of three-phase fluidized bed (Gu liquid// gas), wherein driving the pot liquid circulation by the spray carbon dioxide gas makes immobilized cell keep the (Heijnen that suspends, 1996), see Figure 16.Biological respinse can container photo is seen Figure 17.The detailed sketch of attached detailed dimensions is seen Figure 18.Carbonic acid gas and air are by 0.11 meter of a length, the sintered stainless steel atomizer (CO that external diameter is 0.013 meter ₂Purgernozzle, Part#9222, Hagedom ﹠amp; Gannon USA) enters the biological reactor tapered bottom.Use CO ₂As fluidizing agent, use air to the yeast cell oxygen supply.One draft tube is positioned at biological reactor column body central authorities, and function is the riser in this fluidised bed system and outside annular space plays the downtake effect.Inner draft tube is outstanding down from the cylindrical particle separator, is arranged on three stainless steel fins of the top area that biological reactor enlarges.Keep this draft tube and particle separator parts to be positioned at biological reactor, at utmost reduced danger from the microbial contamination of external environment.Original this retort exit has a screen cloth to be used to separate immobilized cell and liquid.Yet easily stopping up, this screen cloth uses stainless steel column, when the immobilized cell pearl separates them from the draft tube top with liquid phase when annular space moves down.Particle will clash into cylinder and in the liquid phase that falls back and can overflow not leave retort.Have a zonule not have the particle of immobilized cell like this near the retort outlet, this retort top enlarges has also increased the isolating surface-area of gas bubbles.

5.1.2 biological reactor top board

Figure 19 is this biological reactor top board mode chart.It is minimum to reduce risk of pollution that roof hole keeps, and the hole directly is welded on the top board or adopts the compression tube stub This top board is equipped with inoculation hole, thermowell and a thermometer, and the barrier film and the dissolved oxygen that are used for gas sampling are measured liquid outlet and return aperture.One temperature sensor is inserted in and feeds back to temperature controlling system in the thermowell.Temperature controller feeds back to magnetic valve.This valve can open and close the ethylene glycol supply to the biological reactor insulation jacket.(Cole-Parmer Waterproof ThermocoupleThermometer is #90610-20) with the T type probe monitors temperature that is welded in the retort top board with thermometer.Dissolved oxygen measure to adopt dissolved oxygen analyser (doctor's Theidig Digox 5), for accurate oxygen reading, require liquid meat soup flow velocity by the analyser zone be the 9-11 liter/hour.Making dissolved oxygen by pipe imbitition from retort that stretches into 1/4 inch of the internal diameter of fermented liquid through top board measures.As shown in figure 20, the tip of this pipe can be removed larger particles with a filter from liquid when liquid pumps into the dissolved oxygen analyser, and liquid returns biological reactor by another the 1/4 inch hole in the top board then.

5.1.3 the sterile sampling cleaning of valve

This biological reactor is equipped with the film sampling valve (Scandi-that is welded in the tank skin

).Designing this valve is used for taking a sample under aseptic condition.This film is directly with fermented liquid sealing, makes the valve can be with steam and alcohol by two outlets sterilize fully (Figure 18).There is a little alcohol reservoir chamber to keep aseptic between several sub-samplings near this film outside.This kind valve is used for all biological reactor samplings, and the liquid composition of supposition sampling point there is no significantly different with the effusive liquid composition in retort exit.Described in this paper material and method chapter, from being positioned at the valve sampling of tank wall.For verify jar composition of the effusive liquid in exit with from the liquid phase of tank body sampling with this hypothesis, carried out mixing time research.

Measure the mixing time (Chistie, 1989) of air lift type biological reactor with pulse chase method.1ml 10N HCl is injected the biological reactor annular space rapidly, and record pH over time.Wherein time t=0 second is inject time.Injecting 10N NaOH makes pH return its raw value.PH electrode (Cole-Parmer, cat.#P-05990-90) 277 millimeters of length, 3.5 millimeters of diameters.With Ingold 2300Process type pH transmitter monitoring pH.Carry out 2 pH corrections with standard buffer solution Beckman pH7.0 green cushion liquid Part#566002 and the red damping fluid Part#566000 of Beckman pH4.0 through calibrating.With Cheryl Hudson and John Beltrano design in 1994 (University of Western Ontairo, London is Ontario) with 30 seconds the data of frequency record of 3750Hz through the improved software program of Norm Meusour1999.

(Jandel Scientific Software, Labtronics Guelph Ontario) smooth this pH data to use Savitsky-Golay algorithm among the TableCurve 2D then.The Savitzky-Golay algorithm is a kind of smoothing time domain method (Anon, 1996) according to the least squares quartic polynomial fitting process that passes moving window in the pH data.Then with the smoothing data normalization and produce the figure of Δ pH and time.When pH reach equilibrium value～95% the time, get by near one minute as mixing time.Carbon dioxide gas with three kinds of different volumes flow velocitys: 283cm ³/ minute, 472cm ³/ minute (volumetric flow rate that whole this work is used) and 661cm ³/ minute, measure mixing time.Under all three kinds of situations, the pH in the retort reaches balance (end in～95%) in less than two minutes, see appendix 1.It is believed that mixing time is enough short, verified the hypothesis of good mixing in our the initial reaction jar.This make we supposed from the reaction tank skin adopt liquid sample its form with from export effusive jar average band stay liquid ingredient after 24 hours significantly different.From the visible clear and definite liquid recirculation (Chisti, 1989) that disperses to be superimposed upon in the typical mixing of gas lift biological reactor of accompanying drawing.

5.2 the schema of continuous beer fermentation system

Figure 21 brewages the schema of the continuous beer fermentation system in the small-sized wine brewing pilot plant of company limited (London, state, ontario), its detailed components illustrated in table 5.1 for being placed in Labatt.In brief, collect the wine brewing wort,, deposit in the big basin (T-1 and T-2) with moment pasteur sterilizer (Fisher Plate Heat Exchanger, Combi-flow typeEurocul 5FH) sterilization from London Labatt factory.During continuously fermenting, wort is conveyed in the air lift type biological reactor (BR-1) that fixed yeast cell is housed with the flow velocity of controlling.The fermented liquid overflow is left retort and is collected in the receiving tank (T-3).Below each joint the operation of the continuous beer fermentation of Figure 21 system is described in detail in detail.

5.2.1 wort is collected and is stored

Collect wort to the 1600 liter conical basin of not oxygenation of the usefulness of continuously fermenting through pipeline from Labatt London factory, it is few that this basin cleans the oxygen that wort is absorbed with carbon dioxide gas in advance.The jar of all this scales comprises that wort basin T-1 and T-2 are by Labatt rules cleaning and disinfection before use.Wort with moment pasteur's method sterilization, is transferred among ready wort basin T-1 and the T-2 (also cleaning with carbonic acid gas in advance) in 2 ℃ then.Wort can preserve for 2 weeks in 2 ℃ in these jars, offer the biological reactor BR-1 that continuously ferments.Two weeks, the charging of retort was changed to from second wort basin (new sweet wort is housed) charging when finishing.Stoppage time when adopting two identical wort basin T-1 and T-2 as far as possible to reduce to change new sweet wort.Under all situations, wort was added retort (BR-1) preceding at least two days, measuring wort has pollution-free.Then abandon it as polluting, collect new sweet wort and sterilization immediately.

Lay up period reduces the oxyty of wort as far as possiblePurpose is not make the wort oxygen level of storage be in the constant minimum level under low temperature does not freeze the condition of wort.Require it can prevent wort and oxygen generation chemical reaction and cause undesirable aging reaction (Narzib etc., 1993),, and at utmost reduce the danger of wort at contaminating microorganisms storage period so that continue to provide wort to biological reactor.Be used to store big (only) 1600 liters conical jar (T-1 and the T-2) that continuously ferment with wort and originally be designed for batch fermentation but not the wort basin.Therefore cool off these jars and can not fully keep wort, preserves after three days the wort temperature variation between jar interior different zones up to 15 ℃ (table 5.2) in 2 ℃.Warm district in jar has increased the risk of microbial reproduction, therefore needs to stir to guarantee the even low temperature in whole jar, because these reasons are installed in each wort basin (T-1 and T-2) awl with tube type blow head at the end.Experimentize with the preferred plan of determining to give canned full wort and keeping constant low-level dissolved oxygen.For the first time in the experiment, fill the not oxygenation of collection and through the wort of moment pasteur's method sterilization to basin.In case basin is filled 1600 liters of worts, spray into 0.113 cubic metre/hour carbonic acid gas at the bottom of the jar.When testing for the second time, also collect oxygenation not and with the wort of moment pasteur's method sterilization.This time before malt charge juice, cleaned basin 3 hours, spray into carbon dioxide gas (0.113 cubic metre/hour) in a small amount in the time of in wort input jar continuously with carbonic acid gas (0.85 cubic metre/hour).The wort of this low flow velocity carbonic acid gas continuous bubbling by storing in the jar provides wort to the jar that continuously ferments simultaneously.The oxyty of two experiments periodic monitoring wort in a storage period in week.

Graphic representation 5.7 has shown the relation of oxyty and wort period of storage.When basin during without carbon dioxide gas pre-washing the air in the tank deck head space make wort take in some oxygen.Therefore purge tank in advance not will make dissolved oxygen in the wort reach minimum constant level and will spend the much longer time.The wort oxyty keeps constant low-level in whole storage period when purge tank in advance.Therefore can take to clean in advance wort basin (T-1 and T-2) and provide the carbonic acid gas little airflow by the slight positive pressure of wort continuously, store the integral part of rules as all worts that continuously ferment with the maintenance jar at lay up period.

Also compared with or carbonic acid gas that need not 0.113 cubic metre/hour temperature variations in basin when jet, water rather than wort carry out this experiment, adopt the T type temp probe (Cole-ParmerWaterproof Thermocouple Thermometer cat.#90610-20) that has connected thermometer.Collect tap water 1600 and be raised in the wort basin, the water temperature of three days records of balance basin different zones.Spray into water 24 hours in the jar with 0.113 cubic metre/hour carbonic acid gas, also write down water temperature.Write down the room temperature of each situation, temperature is set to 2 ℃ in the basin.

See Table 5.2, jet with carbonic acid gas, more uniform temperature in the basin is measured regional temperature between 0.1～4.1 ℃, and jar content is not freezing.This lower temperature helps to prevent not wish in the wort storage period microbial reproduction found.The wort basin discharges gas by the aseptic gas strainer that is positioned at tank deck, usefulness adjustable speed peristaltic pump (P-1) (

L/S ^TMDigital Standard Drive, ColeParmer cat#07523-50) warp

Food grade L/S16 flexible tube is with the inlet of wort 8 liters of biological reactors of input (BR-L).

5.2.2 system continuously ferments with the through-flow tubular type biological reactor of gas lift

Wort is imported near the awl bottom of retort BR-1 by 1/4 inch hole, by agglomerating stainless steel jet thrust use filter (Millipore,

-FG50,0.2 μ m filter unit) during the air of degerming and carbonic acid gas (99.99% is pure) mixed gas flow into jar.With a gyrator under meter (R-3) control carbonic acid gas flow velocity in STP, with precalibrated substance flow controller (M-1) control air flow velocity in STP.Fermented liquid leaves retort as overflow, and the reinforcement pvc pipe of 1 inch of the internal diameter of flowing through enters 30 liters of stainless steel holding tanks (T-3), and this holding tank cools off with outside ethylene glycol coil pipe, keeps 4 ℃ of temperature.

5.2.3 product is collected

Product holding tank (T-3) has a big ingate (1 inch internal diameter), and this hole is designed so that fermented liquid can flow down along tank skin and reduces bubble as far as possible and produce.This jar also have an aseptic gas filter (Millipore,

-FG50,0.2 μ m filter unit) discharge gas for retort (BR-1) and this holding tank (T-3).With 2 inches one 1/4 inch valve (V-12) emptying regularly on being positioned at the bottom of the holding tank.

5.2.4 glycol-cooled coil pipe

The ethylene glycol of temperature-23 ℃ and pressure 45psig is transported to small-sized wine brewing pilot plant from the London company of brewageing, and the cooling jacket by wort basin (T-1 and T-2), gas lift biological reactor (BR-1) and product holding tank (T-3) circulates.Two wort basins and biological reactor are equipped with the liquidus temperature probe, and it provides and feeds back to temperature regulator, thereby control refrigerating ethylene glycol flows in the chuck of jar.The wort basin store wort in 2 ℃ and in the biological reactor temperature be controlled at 12-22 ℃, depend on concrete experiment.The product holding tank does not have the control of automatization temperature, but manually actuated control ethylene glycol flow keeps jar temperature in about 4 ℃.The temperature that does not need precisely control product holding tank (T-3) is not because the only simple clearancen of the liquid in the jar performs an analysis and further processing.Ethylene glycol also flow through chuck and cooling from wort jar (T-1 and T-2) to the wort line of pipes of biological reactor (BR-1).In case ethylene glycol circulates through chuck, it gets back to the main pipeline of small-sized wine brewing pilot plant, is back to London factory then, and general temperature is-15 ℃, pressure 40psig.

5.3 the sterilization scheme of biological reactor

Biological reactor BR-1 is filled 2% (v/v's) CX/A (Diversey Lever Canada) solution (a kind of detergent with sterilizing), and jet body soaked overnight.The emptying retort is also used cold water flush.Cleaning liquor and water flushing, circulation repeats secondary.For preparing to allow the biological reactor vapor sterilization, wort and jet pipe are disconnected.Steam pipeline is connected in the biological reactor inlet, open then with lower valve: retort inlet valve and wash-out valve (V-6, V-7), jet imported valve (V-17), products export valve (V-9, V-11), film sample cock (V-8, V-10) and holding tank outlet orifice (V-12).Slowly open the steam valve of factory then, the conditioned reaction tank valve can be observed the thread of steam to the exit of each outside opening.Be exposed to steam after 60 minutes, all external valves on the off-response jar (V17, V8, V10, V12) are except wort bypass valve (V6).The wort bypass valve is also closed when closing steam valve, and at this moment, sterile filters is connected to holding tank non-sterile air when preventing to cool off and enters this system and cause pollution.Along with factory's steam pipeline is closed the biological reactor gas pipeline and also is communicated with malleation when keeping this system cools at valve V17 place again.

5.4 fermentation system is started working

To make wine and collect 20 liters of stainless steel pressure jars from factory with wort, 100 ℃ were heated 45 minutes in the sterilization cabinet.Will be in the aseptic input refrigerative of the fixed cell wort (40%v/v), the jar of sealing is transported to the small-sized wine brewing pilot plant that the biological respinse can system is housed.These 20 liters of jars be connected in be equipped with one 3/8 inches strengthen pvc pipes (Cole-Parmer, quick connection fittings USA) (Cornelius Anoka, Mn, USA).The other end of this pvc pipe is connected in the film sample cock (V-8) on the biological respinse tank skin.The carbon dioxide gas of Sterile Filtration is added to 20 liters of jars with 10psig, opens the film thief hole and makes the immobilized cell mixture from this jar input biological reactor, does not allow inoculum contact the extraneous air environment.Remove the internal component of " connecting fast " accessory of 20 liters of jars, be immobilized cell when preventing to import biological reactor and stop up.The granularity cumulative distribution (undersized) of κ-carrageenan gel beads is seen graphic representation 5.8.The particle arithmetic average diameter D that calculates _PamBe 1.252mm, Sauter median size D _PsmBe 1.17mm.The particle median diameter is 1.255mm.Experimental data and average diameter of particles calculate sees appendix 1.

Behind the inoculation immobilized cell, reach target value up to sugar and di-acetyl concentration, with regard to proportion, be lower than 3 ° of P, with regard to di-acetyl, be lower than 100 μ g/L with the batch mode biological reactor.Being ready to this system then continuously ferments.For with hot water drip washing with vapor sterilization wort transport pipe, open valve V-2 (or V-4 of T-2), V-5 and V-6, close V-1 (or V-3 of T-2) and V-7 simultaneously, separately the wort stream.Provide about 80 ℃ water flushing wort transport pipe through V-2 (or V-4 of T-2) then.Connect factory's steam pipeline, vapor sterilization wort transport pipe at least 30 minutes at same position behind the hot water injection.When closing steam pipeline, also close bypass valve (V-6).Close V-2 (or V-4 of T-2) after this system cools, disconnect steam pipeline.Open wort tank valve V-1 (or V-3 of T-2) and bypass valve (V-6), open wort transferpump P-1.V-6 delivers to Sewage outlet with wort by bypass valve, and the phlegma in pipeline is replaced by fresh cold wort.Close bypass valve and open retort inlet valve V-6 this moment, and processing begins to continuously ferment.

Per two all two basins (T-1 and T-2) alternate supplies worts.T-1 supply wort stops continuously feeding pump P-1 after two weeks, closes the valve V-5 of biological reactor ingress, then the wort transport pipe is connected in the 2nd basin T-2.The pipeline that washes as described in the previous paragraph and sterilize recovers to continuously ferment after stopping less than one hour of short duration.

Each parts of schema shown in table 5.1 Fig. 5 .6 describe in detail: PTFE, tetrafluoroethylene; SS, stainless steel

The used symbol of Figure 21

The flexible pipe connecting and the head of living

Gas filter

Pressure-regulator

Pump

Valve

Fig. 5 .7 wort oxyty and period of storage in the wort hold tank (T-1 or T-2) under different tinning conditions

Table 5.2 wort hold tank (T-1 or T-2) does not spray CO ₂After the gas balance 3 days, and the CO that sprays 0.113 cubic metre/hour ₂Behind the gas 24 hours, water temperature situation in jar

Fig. 5 .8 contains the accumulation particle size dispersion of the κ-carrageenan gel beads of fixed yeast mycetocyte

The storage ageing brewer yeast bacterium of the 6th chapter κ-carrageenan gel sets

Scientists has been studied the various matrix of physical property embedding intact cell, comprises Protanal TXF 200 (Bejar etc., 1992; Curin etc., 1987; Masschlein and Ramos-Jeunehomme, 1985; Nedovic etc., 1996; Shindo etc., 1994; White and Portno 1978), agarose (Hooijmans etc., 1990; Lundberg andKuchel1997) and carrageenan gel (Norton etc., 1995; Wang etc., 1982).Carrageenan is a kind of food grade materials, and its advantage is that its physical strength surpasses other gel (Buyukgungor, 1992) when being used for cell embedding.

Yeast cell is settled down in κ-carrageenan gel beads monitor the repeated batch fermentation three-wheel in this chapter first part after.Immobilized cell and the viability that is released into the cell of liquid phase have been checked.Monitored the fermentation parameter in the whole repeated batch fermentation process, comprised ethanol, maltose, trisaccharide maltose, fructose and glucose, and compared with the contrast of the yeast cell fermentation that suspends with dissociating following of same nutritional condition.Seldom (Virkajarviand Kronlof, 1998) of announcing so far about the data of the physical influence of pair cell behind extraneous stress of extended immobilizationization and Continuous Contact and the tunning.This chapter second section has detected and prolonged viability, cell mass distribution and the physical appearance that the time of continuously fermenting is fixed on the yeast cell in the carrageenan gel beads in the air lift type biological reactor.Also detected the saccharomycetic relative percentage of respiratory-deficient of immobilization and free suspension cell group in the long-time internal reaction jar.

Carrageenan is made up of multiple 3-6 Anhydrogalactose unit, and the difference of various carrageenans is the different of sulfate group number and position on the repeated galactose units.Figure 22 is seen in the graphic extension of carrageenan gelation mechanism.When carrageenan was in collosol state, its polysaccharide chain was ball of string configuration at random.When providing crosslinked when the enough spirals of formation and for contiguous network, gelationization takes place.Owing to form more spirals or because the spiralization aggregate, the gel more firm rigidity (Rees1,972) that more has that can become.

Three kinds of common carrageenan types are λ, ι and κ type.See Fig. 6 .2 explanation, they are different on sulfuric ester content, and the sulfuric ester amount has influence on the solvability of polysaccharide chain.The λ carrageenan is the height sulphating, lacks the ability (Marrs, 1998) that forms gel.The ι carrageenan forms elastomeric weak gel in the presence of calcium ion, do not show tangible synersis.The tendency that further forms spiral or aggregate when gel is strong, network is shunk and causes synersis (Rees, 1972) takes place when liquid " oozes out ".The moderate sulphating of κ-carrageenan forms more firm rigid gel when having potassium ion, will experience certain synersis.The gel that the intensity that produces κ-carrageenan improves makes it become the ideal material of the whole yeast cell of immobilization.

The chemical structure of Fig. 6 .2 λ, ι and κ-carrageenan

A key character of carrageenan is its reversibly hot gelationization performance.Viscosity increases and the generation gelationization when carrageenan solution cools off.When solution heated, viscosity reduced, and carrageenan returns collosol state.By controlling the composition of short gelationization cationic solution, can change the temperature of carrageenan when colloidal sol is transformed into gel, κ-carrageenan gelatinization temperature increases with the solution chlorination potassium concn and improves.Can utilize this phenomenon to design the method for cell fixation, because can avoid serious temperature fluctuation (Neufeld etc., 1996).May command carrageenan gelatinization temperature makes it enough high and keep gel at fermentation condition, also can enough hang down yeast cell can be mixed with the carrageenan of collosol state and before the pearl gelationization its viability not to be had deleterious effect.

Shown that immobilization has further research of needs to yeast cell metabolism and physiological many influence factors in the gel matrix.Microenvironment and the free cell in the liquid phase that immobilized cell stands are different, because before substrate can be transported to its surface, the barrier that other has gel matrix and other embedding yeast cell to produce need overcome (Figure 23).The mass transfer velocity in the gel matrix many researchs (Estape etc., 1992 have been done; Hannoun andStephanopoulos, 1986; Korgel etc., 1992; Kurosawa etc., 1989; Merchant etc., 1987;

Deng, 1992; Venancio and Tiexiera, 1997) obtained immobilized cell is carried out the better understanding that the nutrition restriction may have potential negative effect to leavening property.The effective diffusion coefficient of small molecules in the carrageenan gel is suitable with its spread coefficient in water, and this gel allows small molecules, and for example glucose and ethanol carry out molecular diffusion.Yet in typical immobilized cell fermentation, nutrition is mainly given immobilized cell pearl (Hannoun and Stephanopoulos, 1986) by the convection current rapid transport except that molecular diffusion.But when nutriment entered pearl, transhipment slowed down because main by molecular diffusion. the yeast cell that this means the gel beads edge may have the unique nutritional advantages that is better than the pearl centrocyte.

The several months is carried out in the also necessary aging of considering immobilized yeast along with continuously fermenting and at the quasi-stable state condition bottom fermentation of stipulating, the cell of embedding takes place old and feeble.Yet, during batch fermentation, the environment time to time change of yeast cell, cell only is recycled and reused for the fermentation of limited number of times, abandons then.More the multiplex (MUX) studies the long term that continuously ferments to the yeast cell viability, and this relates to its leavening property.

This chapter A has checked that partly yeast in the repeated batch fermentation three-wheel settles down the kinetics in κ-carrageenan gel beads.Monitored immobilization and free zymic viability and the cell concn that suspends, and ethanol, ° P and sucrose concentration.B has partly checked the influence of fermentation time to cell position in the gel beads and distribution and yeast cell form.Four different times have been checked with scanning electron microscope (SEM): 1) produce behind the pearl immediately; 2) batch fermentation is after two days; 3) after experimental scale air lift type biological reactor continuously fermented two months; 4) κ in the gel beads different zones-carrageenan fixed yeast cell after experimental scale gas lift biological reactor continuously fermented 6 months.The viability and the concentration of the yeast cell in immobilization and the liquid phase have also been measured.Also detected the relative percentage of respiratory-deficient yeast after in the air lift type retort, continuously fermenting 5 months (free cell in immobilization or the liquid phase), and compared with traditional batch beer fermentation finding per-cent.Adopt the old yeast strain of storage on producing in the whole research.

6.1 experimental arrangement

κ-carrageenan gel beads is produced: κ-carrageenan gel X-0909 is that Copenhagen Pectin AS gives.κ-carrageenan gel beads contains the old yeast cell of storage of embedding, uses the motionless mixer explained hereafter, and the initiator cell load is 2.6 * 10 ⁷Cell/ml gel (patent application 2133789 (Neufeld etc.), 1996), pearl footpath 0.5-2.0mm.

Fermention medium: the wine brewing wort that Canadian Labatt wine brewing room provides, 17.5 ° of P of proportion see in the materials and methods and describe in detail.

The repeated batch fermentation kinetics of A part κ-carrageenan gel beads immobilized yeast

Ferment in 2 liters of erlenmeyer flasks in 21 ℃, with the 150rpm jolting.The carrier load of immobilized cell pearl is 40% (v/v), 1 liter of fermentation cumulative volume.Each fermentation continues 7 days, drops into fresh fixed cell pearl in the wort of R1, makes mixture pass through aseptic stainless steel sift (500 microns of screen sizes) during fermentation ends and separates from fermented liquid and obtain these pearls.These pearls are dropped in the fresh sterilization wort of R2 with same ratio again.(R3) batch fermentation then for the third time.Each fermentation is taken a sample 2 times three day every day, and the 4th, 5 day once a day.Ferment in duplicate or in triplicate.All fermentations are carried out under the same conditions with the control fermentation of the suspension cell that dissociates, and the just free cell of latter's exception is with the ratio input fermentation of 4 grams per liters.Viability, cell concn, liquid phase carbohydrate and the alcohol concn of free and immobilized cell in the analytic sample.But make the total glucose fermentation of substrate produce the ethanol yield factor (yield factor) Y with formula 3.20 calculating immobilized cell fermentation three-wheels and free cell control fermentation _P/SFor all fermentations, calculate from fermentation and begin the productive rate factor when maltose runs out.

Calculate R1, R2 and R3 and free cell contrast from beginning to ferment when maltose exhausts the amount of alcohol that each total work volume per unit fermentation time of biological reactor produces, i.e. alcohol production rate V with formula 3.25 _EthanolWith regard to efficiency factor and alcohol yied, the mutual and indistinction of the contribution of immobilization and free suspension yeast cell.

Calculate the numerous growth velocity of local high specific and the cell doubling time of average free cell contrast with formula 3.3 and 3.4.

Viability and the morphological specificity of B partial fixing yeast when prolonging fermentation

The batch fermentation condition: batch fermentation carries out oscillation frequency 150rpm in 21 ℃ in 2 liters of Erlenmeyer flasks.Carrier load is 40% (v/v) the volume 1L that always ferments.

The condition of continuously fermenting: continuously ferment and adopt the through-flow tubular type biological reactor of experimental scale gas lift.All data derive from 8 liters of working volume retort, and the scanning electron microscope result during except 2 months derives from 50 liters of retort that adopt identical fermention medium and process for fixation.With air and CO ₂Gas mixture make fixed cell pearl (40%v/v) fluidisation in the retort, under 12,17 and 22 ℃ of temperature controlled fermentation conditions, operate biological reactor, residence time 0.9-1.8 days.The highest alcohol concn of six months experimental session gas lift retort reaches 73 kilograms/cubic metre, average 58 kilograms/cubic metre.

Microbiologic analysis:At least take a sample once from the liquid phase of gas lift biological reactor weekly,, comprise wild yeast, the old yeast of non-storage, and aerobic and anaerobic beer spoilage bacterium to check pollution condition.After 5 months, get the female mycetocyte of liquid phase alcohol and check that with double its respiratory-deficient becomes strain.

Scanning electron microscope (SEM):At following four different times: 1) produce behind the immobilized cell pearl and before being inoculated into fermention medium, 2) batch fermentation is after 2 days, 3) in the through-flow tubular type biological reactor of experimental scale gas lift, continuously fermented 2 months after, 4) in the through-flow tubular type biological reactor of experimental scale gas lift, continuously fermented 6 months after, gather and to contain immobilization and store the κ of old yeast cell-carrageenan gel beads (1.0-1.5mm diameter) sample and make SEM and check.The method that SEM checks is seen 4.7 joint descriptions with relevant specimen preparation.At SEM simultaneously with the concentration and the viability of the described method of 4.6 joints assessment yeast cell (immobilization and freely suspend).

6.2 result and discussion

The A partial fixing is saccharomycetic repeated batch fermentation kinetics in κ-carrageenan gel beads

Immobilized cell is dropped into the new sweet wort secondary fermentation time at every turn again and greatly reduces, see graphic representation 6.4 (a) (b) (c) repeated batch fermentation three-wheel, the relation of maltose, trisaccharide maltose, glucose, fructose and ethanol and fermentation time are described.From these figure as can be seen, the time that sucrose exhausts fully, the first round (R1) is 64 hours, and second to take turns (R2) be 44 hours, and third round (R3) is 26 hours.The free suspension cell fermentation of the contrast sucrose that does not contain the fixed cell pearl exhausts has fully spent 82 hours, sees graphic representation 6.5.The highest from the third round ethanol ultimate density of graphic representation 6.4 also visible three-wheel repeated batch immobilized cell fermentation.Because κ-carrageenan is a kind of hydrogel, some ethanol band is in pearl when dropping into new sweet wort again.As a result, compare, in the zero-time of R2 and R3 fermented liquid, had some ethanol with the fermentation of contrast free cell, and the starting point concentration of glucose, maltose, trisaccharide maltose and fructose lower in immobilized cell fermentation (graphic representation 6.4 and graphic representation 6.5).Calculate the productive rate factor of each time fermentation and can determine on basis relatively that every consumption 1 gram sucrose produces a few gram alcoholic acid output.

Graphic representation 6.4 (a) utilizes the maltose in the old yeast cell repeated batch fermentation first round of storage (R1) be fixed in κ-carrageenan gel beads, the relation of trisaccharide maltose, glucose, fructose and alcohol concn and fermentation time.

Graphic representation 6.4 (b) utilization is fixed on the maltose that the old yeast cell repeated batch fermentation second of storage in κ-carrageenan gel beads is taken turns in (R2), the relation of trisaccharide maltose, glucose, fructose and alcohol concn and fermentation time.

Graphic representation 6.4 (c) utilizes the maltose in the old yeast cell repeated batch fermentation of the storage third round (R3) be fixed in κ-carrageenan gel beads, the relation of trisaccharide maltose, glucose, fructose and alcohol concn and fermentation time.

Maltose, trisaccharide maltose, glucose, fructose and the alcohol concn of the old saccharomycetes to make fermentation of storage (no immobilized cell) that graphic representation 6.5 contrasts freely suspend and the relation of fermentation time.

Graphic representation 6.6 (a) and (b) when having compared R1, R2 and R3 maltose and alcohol concn respectively with the relation of fermentation time.In R1 repeatedly, yeast cell always absorbs maltose immediately after dropping into new sweet wort.Alcohol concn early peaks when R1, also reaches higher concentration than two batch fermentation.Shown in graphic representation 6.6 (b), the ethanol production during R1 lags behind at first, lags behind sharply to reduce when these immobilized cells drop into R2 again, further reduces when dropping into R3 again.

Graphic representation 6.6 (a) adopts old yeast cell repeated batch fermentation R1, the R2 of the storage that is fixed in κ-carrageenan gel beads and the maltose concentration of R3 and the relation of fermentation time.

Graphic representation 6.6 (b) adopts old yeast cell repeated batch fermentation R1, the R2 of the storage that is fixed in κ-carrageenan gel beads and the alcohol concn of R3 and the relation of fermentation time.

The immobilized cell concentration of biological reactor per unit cubic capacity and the relation of fermentation time when graphic representation 6.7 (a) shows R1, R2 and R3.Fig. 6 .7 (b) shows the relation that the free cell that discharges from the fixation cell cytoplasmic matrix enters liquid phase and fermentation time in these fermentations.Fig. 6 .7 (c) shows immobilization and the free yeast cell sum in the retort per unit cubic capacity in three batch fermentations.When Fig. 6 .7 (a) shows R1 in κ-carrageenan gel immobilized cell cell concn after wort is gone in initial inoculation constantly increase.Cell continues to breed in the glue pearl when the glue pearl is dropped into the new sweet wort of multiple R2 again.For the third time, when the cell of embedding dropped into new sweet wort again, immobilized cell concentration gathered way and slows down.The change in concentration situation that discharges into free cell, immobilized cell and total cell of liquid phase during the R1 fermentation from κ-carrageenan gel matrix is seen graphic representation 6.8.

The relation that old yeast cell mean concns and fermentation time are store in the immobilization of retort per unit cubic capacity when graphic representation 6.7 (a) R1, R2 and R3 fermentation.The error bar is represented experimental data bound (n=2).

The old barm cell concentration of the storage that is released into liquid phase of retort per unit cubic capacity and the relation of fermentation time when graphic representation 6.7 (b) R1, R2 and R3 fermentation.The error bar is represented experimental data bound (n=2).

Total (in immobilization and the liquid phase) the old barm cell concentration of storage of retort per unit cubic capacity and the relation of fermentation time during graphic representation 6.7 (c) R1, R2 and R3 fermentation.The error bar is represented experimental data bound (n=2).

Graphic representation 6.8 usefulness are fixed on three repeated batch of the old yeast cell of storage fermentation R1 immobilized for the first time in κ-carrageenan gel beads, and liquid phase and total (immobilization adds liquid phase) cell concn are to the variation diagram of fermentation time.

The concentration of immobilized cell increases in κ during R1-carrageenan gel beads, and speed is similar to the control fermentation that only contains the liquid phase cell.Average growth curve by the fermentation of comparison diagram 6.9 contrast free cells and graphic representation 6.10 are fixed on that the similar growth curve of cell is confirmed in the carrageenan.Gel beads is not settled down by yeast as yet fully during the R1, and gel matrix be it seems to the yeast cell unrestraint effect of growing in the pearl.When the R2, matrix be it seems the propagation that has limited the pearl inner cell, takes turns so that cell count increases less shown between yeast phase.This may be the character owing to gel, or yeast cell is crowded in the pearl, or cell lacks due to the nutrition supply.

Graphic representation 6.9 biological reactor per unit cubic capacitys freely suspend the storage average cell concentration (n=3) of old yeast control fermentation and the relation of fermentation time.There is not immobilized cell between these yeast phases.

The relation that old yeast concentration and fermentation time are store in the immobilization of every milliliter of gel when graphic representation 6.10R1, R2 and R3 fermentation.The error bar is represented the bound (n=2) of experimental data.

The fermentable sugars substrate is transformed into alcoholic acid output Y when having shown three batch fermentations and contrast in the table 6.1 _P/SGive the biological reactor ethanol volume productive rate that goes out with table 6.1 data computation in the table 6.2.The ethanol production that sugar-fermenting produces each other or with the no significant difference of contrast.Output all be higher than Guy-Lussae formula prediction theoretical yield 0.51 90%.As discussed previously, other by product that biomass produces and yeast cell forms has stoped its efficient to reach more than 95% of theoretical value (Hardwick, 1995).In the fermentation of the three batches of repeated batch ethanol volume productive rate of biological reactor criticize with criticize between significantly different.Along with whenever adding a wheel load subdivision batch fermentation, alcohol yied improves, and immobilized cell comparison ferment approved for distribution is more voluminous during to R3.Produce during the not obvious R1 of being higher than of the ethanol total amount that produces during R2, but fermentation time is less than half of R1 and control fermentation.Have many factors to improve the fermenting speed of the every batch of immobilized cell that repeats to ferment contribution is arranged, adapted to fermentation condition and cell concn improves gradually as yeast cell, the total cell count of retort per unit volumetrical is much higher than contrast during to R3.In the graphic representation 6.7 (b), the relation of free suspension cell (discharging from gel matrix) concentration and fermentation time proves that the cell count that discharges increases with every batch fermentation in the liquid phase from gel beads.When gel beads is filled yeast cell fully, it seems to discharge more cell in liquid phase.Husken etc. (1996) have carried out some researchs and have detected the expansion of bacterial cell colony and send/discharge from κ-carrageenan gel sheet.Vives etc. (1993) report that the yeast cell maximum concentration that they reach is every gram gel 10 in κ-carrageenan gel beads ⁹Individual cell, the concentration that reaches in the gel particle when this is R2.Between B partial continuous yeast phase, seen similarly high cell concentration.Yet the highest cell lifting capacity depends on the composition and the other factors of initial cell lifting capacity, gel in the gel matrix.

Table 6.2 is compared with free suspension cell batch fermentation, the alcohol yied [V of biological reactor immobilized cell batch fermentation (R1, R2 and R3) _EthanolThe ethanol kilogram number of=generation/per hour, every cubic metre of retort volume]

Fermentation	V Ethanol(kg/m 3h)*
		The contrast of R1 R2 R3 free cell	0.470 0.668 1.246 0.805

* the calculated value when wort is ingested fully

Have influence on ferment another factor of being seen retort unit volume gain in yield of every batch of repeated batch and relate to the adaptability of yeast cell.During to first fermentation ends, the metabolic mechanism of yeast cell has adapted to the described fermentation condition of fermentation, and the shortening of lag-phase when batch fermentation began after this can cause improves fermenting speed.All control fermentation old yeast of storage of prepared fresh in this research.The contrast yeast that interesting is drops into fresh free suspension again with drop into immobilized cell come together further the to detect influence of its relative pair cell mass action again.

Fig. 6 .11 shows with methylene blue method and makes indicator its viability low (＜50%) when immobilized cell drops in the wort of R1 at first, but after fermenting 48 hours, the immobilized cell viability surpasses 90%.To settle down in pearl its viability fast still high for yeast cell during the R3.Viability lowers slightly during then to the R3 fermentation ends, yet, between three repeated batch yeast phases, be released into free cell viability in the liquid nutrient medium than its immobilized cell height.Immobilization matrix may have negative impact to yeast cell viability (mass transfer limit and/or space constraint).Perhaps the yeast cell of Huoing may preferentially be released in the liquid nutrient medium from immobilization matrix than non-viable cell.

Adopt the average data of three suspension yeast control fermentation gained that independently dissociate in the appendix 1 to draw ln (X/X _o) with the graph of a relation of fermentation time, see graphic representation 6.12.Its slope equal 21 ℃ with 150rpm vibration, cell reaches the height ratio appreciation rate in part in distiller's wort.The height ratio proliferation rate in this saccharomycetic part is 0.096/ hour, and cell doubling time is 7.22 hours.The μ that obtains in this work _MaxBe defined as local μ _Max, because, just can reach the true μ that uses in the Monod formula as having only as described in the theoretical chapters and sections as S during significantly greater than Monod constant K s _MaxNeed make the Monod constant K s that multiplex (MUX) more makes to estimate restricted substrate in these fermentations, with confirm as the Monod formula definite, the local μ of calculating _MaxBe real the highest.

The relation that old yeast cell viability (methylene blue staining method) and fermentation time are store in immobilization in Fig. 6 .11R1, R2 and the R3 fermentation.

The interim Ln of exponential growth (X/Xo) of the free suspension yeast control fermentation that Fig. 6 .12 is average and batch fermentation time relation, the cell concn when wherein X is t.Cell concn when Xo is t=0 (n=3).

The viability of immobilized yeast and morphological feature when B partly prolongs fermentation time.Before the gel beads contact fermentation culture and with after the motionless mixer processing immobilized cell pearl, the cell concn of gel beads is 2.6 * 10 ⁷Cell/ml (value in the table 6.3 is the mean value of two samples).The SEM photography shows that individual cells is evenly distributed in (Figure 24) in the whole gel beads.

Freely in the whole yeast phase of table 6.3 suspend and be embedded in viability (methylene blue method) and the concentration that old yeast cell is store in the immobilization in κ-carrageenan gel beads

* according to a sample

Batch fermentation is viability＞90% after two days, and cell concn increases by 10 times (table 6.3) in the gel beads.Cell (＞90% is alive) also begins to be released into from gel in the liquid phase of fermentation, produces every milliliters of liquid 10 ⁷Cell concn.Form little yeast colony in the gel beads, many bud traces have been arranged on the individual cells, seen Figure 25.

Fixed yeast cell viability decline (table 6.3) after in the air lift type biological reactor, continuously fermenting 2 months, but cell keeps high vigor (＞90%) in the liquid phase, and the difference several times in experimental scale air lift type biological reactor is continuously fermented and supported this discovery.The SEM of Figure 26 is presented at the pearl periphery and has formed result (Bancel and Hu, 1996, Godia etc., 1987 that two months big yeast bacterium colonies have confirmed other scholars; Wada etc., 1979, Wang etc., 1982).Photograph with SEM and to have done the comparison of immobilized cell pearl outer rim yeast form and gel beads center yeast form to several duplicate samples, the cell that is positioned at the pearl periphery is avette, smooth, many bud traces (Figure 27) is arranged, and shows that yeast is in propagation (Smart, 1995).It seems that the cell photo (Figure 28) at pearl center distortion has taken place, and seldom has the bud trace to form, and lacks the bud trace and may show that the nutrition of pearl center such as oxygen supply are limited.The yeast surface imperfection also may show cell aging (Barker and Smart, 1996 among Figure 28; Smart, 1999).

After the air lift type biological reactor continuously fermented 6 months, immobilized yeast viability dropped to below 50% (table 6.3) in the carrageenan gel.Though the immobilized cell concentration and the viability of collecting in the time of should noting 6 months have only a data point, five months data are similar, and fixed cell concentration is every milliliter of gel 1.14 * 10 ⁹Individual cell, viability＜50%.Progressively reduce in time though see the immobilized cell viability, the cell viability in the liquid phase is still high really.In addition, even total in the pearl immobilized cell viability say lowlyer, but retort has still been produced the beer of fermentation finished thoroughly1 in 6th month continuously ferments.The possible cause of these discoveries comprises the obvious contribution of the high free suspension yeast cell of vigor to fermentation.Being positioned at the high immobilized cell of the vigor of pearl periphery and pearl centrocyte in addition, to compare the mass transfer barrier less, also has contribution.It is unclear that whether immobilized cell can redistribute or these cells are still stayed the place that it is settled down first in gel matrix.Cell concn after continuously fermenting 6 months in these pearls reaches 10 of maximum ⁹The cells/ml gel beads.

Figure 29 is the image with the whole pearl of SEM shooting.Almost half is hollow for many pearls of checking.Hollow cavity may be the result of carrageenan gel structure degraded, and the SEM preparation method may further promote its degraded.In fresh pearl goods, do not observe this hole.The previous work of other people (Bancel etc., 1996) shows that proliferative cell can cause the gel network weakness.Audet etc. (1988) report adds κ-carrageenan with locust bean gum, has improved the physical strength of the gel beads of fixation of bacteria.

At whole 6 months beer fermentation experimental sessions, test the pollution condition of gas lift retort at least once in a week.Do not measure bacterial contamination in any time of experiment.Latter two month, the black yeast bacteria concentration that records fluctuates in 1-5cfu/ml.This yeast can be grown in the PYN substratum at 37 ℃, does not go up aerobic and anaerobic growth at DUBA substratum (the bacterium selectivity is arranged), and the nonfermented dextrin is at CuSO ₄Do not grow on the substratum (wild yeast bacterium selectivity is arranged).

Respiratory-deficient yeast cell average percent is 7% after five months, the mean value 2% of normal findings when being higher than in the industrialization batch fermentation with this bacterial strain.Other scholar has reported similar discovery (Norton and D ' A more, 1995).The respiratory-deficient yeast that sudden change the produces glucose of can not degrading becomes CO ₂And water.The mitochondrial activity of these yeast has permanent damage, normally because due to the Mitochondrial DNA Mutation (Hardwick, 1995).The artefact that the SEM specimen preparation causes may cause and obscures.Adopted nucleus magnetic resonance (NMR) spectroscopic analysis (Fernandez, 1996) and Laser Scanning Confocal Microscope technology such as (Bancel and Hu, 1996) to come Noninvasive also to detect immobilized cell.The NMR camera work make investigators can study cell and biochemical in the microbial film transhipment, flow and spatial distribution.Some scholars (Bancel and Hu, 1996) also prove can adopt confocal laser scanning microscope, CLSM, observes fixed cell in the porous gelatin microcarrier by a series of optical sections.

Though adopt methylene blue method to deposit the standard indicator of viability as cell in wine-making industry, this method has many shortcomings (Mochaba etc., 1998).It is that the foundation of living or not living is the ability that this dyestuff of viable cell oxidation becomes colorless form that methylene blue method is measured yeast flora.Non-viable cell lacks ability thereby painted (O ' Connor-Cox etc., 1997) of this dyestuff of oxidation.Plate count and slide culture technique are grown on agar plate according to cell and are produced macrocolony, or produce the ability (ASBC Technical Committee and editorial board, 1992) of microcolony on substratum topped on the microslide.In Labatt, detecting the work of yeast viability in the immobilization matrix after long-time, not only adopting methylenum coeruleum, and adopting aforesaid method and carry out the Laser Scanning Confocal Microscope technology with active coloring.Except measuring the viability of cell, further work also must focus on the viability of immobilized cell.When with viability explanation cell growth and replication, vigor has also detected the ability (Smart etc., 1999) that saccharomycetic leavening property, activity or yeast recover down from stress.

Chapter 7, the fragrance product in the continuous beer fermentation of the air lift type system

7.1 fermenting procedure

Adopt immobilized cell to continuously ferment to produce beer and other application differs widely, because products therefrom weighs with a kind of interested composition such as ethanol incessantly, but the balance of many chemical ingredientss, they must balance just can produce qualified beer.Checked continuous just fermentation and then in batches between soak oxygen to the metabolic effect of yeast fragrance matter.The residence time of also having detected two kinds of levels is to the metabolic influence of fragrance matter.At last, commercialization alpha-acetolactate decarboxylase preparation is joined in the wort that continuously ferments the total di-acetyl concentration of monitoring liquid phase.

7.1.1 the relative content of air is to the influence of metabolism of yeasts in continuously first yeast phase biological reactor fluidizing agent.

Change the air content in the biological reactor fluid gas, thereby changed oxygen level, keep residence time, temperature and all other controllable process variables constant simultaneously.Keep the volume of gas constant flow rate in 472ml/ branch (STP), 15 ℃ of temperature.Adopt in the whole test to contain the saccharomycetic κ of immobilization LCC3021-carrageenan gel beads, the initiator cell load is every milliliter of gel 1 * 10 ⁸Individual cell.Adopted four kinds of volume of air flow velocitys (table 7.1) in the test, biological reactor average retention time Rt is 1.18 days.

Continuously ferment in the phase volumetric flow rate of the air that provides to biological reactor by shower nozzle of table 7.1.Total volume flow rate 472ml/ minute STP.Rest part is CO in the gas ₂

Volume of air flow velocity (ml/ minute)	Air per-cent (%v/v) in the fluidizing agent	Initial (my god)	Finish (my god)	Total time (my god)
					94 354 34 0	19.9 75.20 7.2 0	10 27 41 59	26 40 58 66	17 14 18 8

Experimental session carries out following analysis repeatedly: barm cell concentration and viability in amino nitrogen (PAN), total fermentable saccharide (as glucose), ethanol, total di-acetyl, beer volatile matter (ester of selecting and alcohol) and the liquid phase.The pollution condition of weekly at least assaying reaction jar also.When inferring that every kind of volume of air flow velocity continuously ferments, be in quasi-stable state and measure oxyty in the biological reactor liquid phase when (third-order reaction jar turnover time at least).

7.1.2 soak in batches after the fermentation: contact oxygen is to the influence of metabolism of yeasts

Even the oxygen level of biological reactor fluidizing agent lower (34 ml/min STP), Zong the experimental session acetaldehyde of 7.1.2 joint and the height of di-acetyl concentration also are that lager beer market, North America is unacceptable.Therefore adopted a kind of novel method, under 21 ℃ of slightly high temperature, be incubated continuously first fermented liquid 48 hours, in batches to reduce this two kinds of compound concentrations.The result of prosthomere 7.1.2 shows that the air content in the fluidizing agent has a significant effect to the survey perfume compound in addition.Therefore the aerobic and anaerobic condition that has detected first fermentation downstream process (carrying out secondary insulation in batches) is to the metabolic influence of yeast spices.Adopt the height flocculation variant of LCC3021 yeast strain in 50 liters of air lift type retort, to carry out continuously fermentation just in this test, because the sample volume that this institute needs is to 8 liters of retort capacity and Yan Taida.Operational condition is CO in the fluidizing agent ₂1180ml/ minute, air 189ml/ minute STP, retort average retention time Rt were 1.0 days, 15 ℃ of temperature, the storage ageing wine wort of 17.5 ° of P of high specific gravity.Take 4 duplicate samples (phial of 100ml undercut), two parts of operations under anaerobic altogether, in addition two parts of contact aerobic environments.

The anaerobism sampling procedure is as follows: with two 10ml and 6 25ml undercut phial autoclavings, be placed on then the anaerobism box (Labmaster100, mbraun, USA) in.This box uses argon gas as purge gas.Allow 100ml phial balance 45 minutes, use then aluminium lid and

Diaphragm seal.With 70% (v/v) ethanol liquid disinfectant the 50ml syringe of 3 inches No. 16 syringe needles is housed, takes a sample from retort by the barrier film of poking sample cock, and sample is injected the anaerobism phial that 100ml cleaned in advance.Must provide an outlet of this undercut phial by another asepsis injector syringe needle, the pressure when packing sample liquid in the phial with release.Aerobic sample, is drawn into from retort in the unencapsulated 100ml sample bottle by opening the film sampling valve fully without syringe and syringe needle, the contact atmosphere.

Room temperature left standstill liquid sample 2 hours, allowed the yeast sedimentation, remaining cell concn about 10 in the liquid ⁶Cell/ml.In case sedimentation is gone into the liquid pouring in each 100ml phial in 3 25ml phials.In the anaerobism box, handle the anaerobism sample, absorb to reduce oxygen as far as possible, and aerobic sample is handled in laminar flow is liked the wind cupboard.Sample in every part of 100ml phial is sub-packed in 3 less 25ml phials, so that can not analyze sample because of sampling changes fermenting process.When aerobic sample transfer in less phial the time, is not added a cover at 21 ℃ and cultivated.In anaerobism sample transfer to 3 a less phial, with aluminium lid and

Diaphragm seal.For avoiding in the phial because of emitting CO ₂And generation pressure prevents sample to contact with outside aerobic environment simultaneously, with this barrier film of syringe needle puncture.An end that syringe needle is contacted external environment is immersed in (pressure head is less than 1 centimetre) in the ethanol, prevents that air from backflowing in the sample.At 2,24 and 48 hours collection analysis samples.Also directly sampling and analysis immediately from retort, the fermentation state during with the assessment fermentation in the retort.Total fermentable saccharide (as glucose), ethanol, total di-acetyl and beer volatile matter (ester of selecting and alcohol) in the analytic sample.

7.1.3 the liquid hold-up time was to the influence of metabolism of yeasts thing when beer just fermented continuously

For the tracer liquid residence time to the active influence of metabolism of yeasts, test, comprise changing with being immobilized onto the wort volumetric flow rate that LCC3021 yeast cell in κ-carrageenan gel beads carries out continuous beer retort when just fermenting.In the whole test temperature of retort keep 17 ℃ constant, the gas volume flow velocity of supply response jar also keeps being constant at 472ml/ minute STP.Gas is air (11ml/ minute STP) and CO ₂The gas mixture of (461ml/ minute STP).The initial concentration of yeast cell is 2.6 * 10 in κ-carrageenan gel ⁷Cells/ml gel beads, biological reactor contain the pearl of 40% (v/v).Carry out following analysis during the whole test repeatedly: carbohydrate, free amino nitrogen (FAN), total fermentable saccharide (as glucose), ethanol, total di-acetyl, beer volatile matter (ester of selecting and alcohol), liquid phase barm cell concentration and viability.The pollution condition of weekly at least assaying reaction jar.

7.1.4 employing commercialization alpha-acetolactate decarboxylase reduces the total di-acetyl in the fermentation just of continuous beer

Most of North Americas wine brewing merchant thinks that the di-acetyl high density is a kind of not optimum taste defective in their beer.Total di-acetyl concentration is higher than the threshold value (70-150 micrograms per litre) of North America lager beer traditional batch fermentation always in the fermentation of carrying out so far just continuously.Di-acetyl reduces and to occur in the fermentation later stage when no longer having oxygen and not importing other sugar during batch fermentation.In the system of continuously fermenting, low-level by the oxygen maintenance of shower nozzle supply response jar, and the continuous new sweet wort of supply response jar.Therefore explored the New Policy that adopts di-acetyl concentration in the commercialization enzyme preparation control successive reaction jar.

During attenuate, a kind of Xie Ansuan synthetic intermediate product, α-acetylactis, oxidized decarboxylation outside yeast cell and formed di-acetyl.Yeast cell is reuptaked di-acetyl then and is converted into the relatively poor 3-oxobutanol of fragrance.When the wort batch fermentation, it is rate-limiting step that α-acetylactis oxidative decarboxylation base forms di-acetyl.During continuously fermenting, high total di-acetyl concentration that biological reactor produces is unacceptable (300-400 micrograms per litre).The commercial enzyme alpha-acetolactate decarboxylase (ALDC) of Novo-Nordisk A/S can directly change α-acetylactis into the 3-oxobutanol, thereby avoids producing undesired di-acetyl intermediate product (graphic representation 7.1) (Jepsen, 1993).

The effect of graphic representation 7.1 alpha-acetolactate decarboxylases (ALDC)

Alpha-acetolactate decarboxylase is added in the wort of input biological reactor, detect its clean effect total di-acetyl concentration.Other method that reduces di-acetyl comprises and is incubated 48 hours after the fermentation and immobilization secondary fermentation system (this is the technology (Anon, 1997) of Alpha-Laval) has also done exploration in batches.Other this two kinds of strategies can successfully reduce the level of fermentation back di-acetyl, but all can not (being the retort exit) influence the di-acetyl level from the source.By in wort, adding the di-acetyl concentration that ALDC has reduced the retort liquid effluent, can farthest reduce or eliminate this fermentation aftertreatment phase.

ALDC has optimum activity during in pH6.0 in the old wort of 10 ℃ storage.And the industrialization wort is generally pH5.0, at this moment, and ALDC activity in the time of 35 ℃ the highest (Anon, 1994).Under typical lesser temps of beer fermentation and pH condition, the ALDC activity is not best.Canadian food and medicine rules (SOR/97-81) have been revised by Her Majesty the Queen in right of Canada as represented by the minister of Healt in 1997 thereby allowing to use in alcoholic beverage ALDC to use in Canadian wine-making industry for it has opened the gate.Carry coding and can produce the ALDC enzyme from the subtilis of ALDC (E.C.4.1.1.5) gene of bacillus brevis.Because ALDC is a kind of enzyme that is produced by genetically modified microorganism (GMO), in commerical prod, should allow the public know before this enzyme of use.These experiments are with storing old yeast LCC3021.The storage ageing wine wort of 17.5 ° of P of high specific gravity is provided by London Labatt wine brewing room.Cell concn in monitoring ethanol, total fermentable saccharide (as glucose), total di-acetyl and the liquid phase.Be fixed in κ-carrageenan gel beads as yeast cell as described in the 4th chapter.Biological reactor is able to three conversions before supposition reaches quasi-stable state.The used di-acetyl method of this work is called " total two acyls " as previously mentioned, because this method mensuration is di-acetyl and precursor α-acetolactic amount thereof.Observing total di-acetyl reduction during current the experiment is because enzyme directly is transformed into 3-oxobutanol and the combined action of its derivative di-acetyl concentration reduction thereafter with α-acetylactis.

The alpha-acetolactate decarboxylase that is used to test (ALDC) is Novo Nordisk A/S, Denmark

Give.This enzymic activity is 1500ADU/g, and ADU causes that under standard conditions α-acetylactis decarboxylation per minute produces the enzyme amount of 1 micromole 3-oxobutanol, sees described in the Novo Nordisk Method AF27 (Anon, 1994).

The condition of continuously fermenting: in 8 liters of gas lift tubular type biological reactors, drop into 40% (v/v) and contain immobilization and store the κ of old yeast cell-carrageenan gel beads and continuously ferment.Use CO ₂The gas mixture of (438ml/ minute STP) and air (34ml/ minute STP) sprays into retort.The duration of test leavening temperature is controlled at 15 ℃.The retort residence time, Rf was 1.5 days, monitored the total di-acetyl concentration under these conditions, reached the average quasi-stable state of control di-acetyl concentration.The variation that then ALDC is added total di-acetyl concentration in the wort monitoring reaction jar with 72 micrograms per litre concentration (108ADU/ liter).

Experiment 1 is collected the wort of fermenting house in 20 liters of stainless cylinder of steels, and 100 ℃ of autoclaving heating 45 minutes are kept at wort in 2 ℃ of temperature controlled water bath during the charging retort.In case reached the total di-acetyl concentration of quasi-stable state in the retort, in the wort of 20 liters of jars, add the ALDC of 72 micrograms per litre (108ADU/ liter), the biomass load in initial κ-carrageenan gel beads is every milliliter of gel 3 * 10 ⁷Cell.

Experiment 2 is polluted in order at utmost to reduce, and as described in the 4th chapter sealing and other more senior sealing is made by this system outlet place.As test 1, the wort of fermenting house is collected in 20 liters of stainless cylinder of steels 100 ℃ of autoclavings 45 minutes.During the charging retort wort is kept in 2 ℃ of temperature controlled water bath.Biological load amount in initial κ-carrageenan gel beads is every milliliter of gel 3 * 10 ⁷Cell.In case reached the total di-acetyl concentration of quasi-stable state in the retort, in the wort of 20 liters of jars, added the ALDC of 72 micrograms per litre (108ADU/ liter).

Experiment 3 is collected in 17.5 ° of P distiller's worts (1400 liters) of oxygenation not in the Fructus Hordei Germinatus juice hold tank (T-1) of pilot plant, through moment pasteur's method sterilization, uses CO ₂Jet storage sees that to keep constant oxyty＜010mg/L the 5th chapter is described.From then on jar is imported biological reactor until reaching the total di-acetyl concentration of quasi-stable state with wort.Testing aseptic adding remaining time ALDC (72 micrograms per litre) in wort then.Measure the amount of residue wort in the hold tank, calculating makes enzyme concn reach the required enzyme amount of 72 micrograms per litre (108ADU/ liter), adds ALDC then.The enzyme amount that is fit to is dissolved in 10 liters of sterilization worts, transfers in 20 liters of stainless steel pressure jars, thief hole is connected with wort basin (T-1) by aseptic pipeline.Use aseptic CO ₂Gas pushes into ALDC solution in the wort basin, in order to ensure wort thorough mixing in ALDC liquid and the basin, sprays into a jar interior CO ₂Flow velocity increased to 4720ml/ minute STP totally 1 hour, returned its normal flow then.What the basin maintenance was enough contains the ALDC wort to finishing experiment.Initial biomass load is every milliliter of gel 10 in κ-carrageenan gel beads ⁸Cell.

7.2 result and discussion

7.2.1 biological reactor fluidizing agent hollow gas relative quantity is to the influence of metabolism of yeasts thing when just fermenting continuously

Graphic representation 7.2-7.11 is yeast viability and cell concn, free amino nitrogen (FAN), total fermentable saccharide (as glucose), an ethanol in the liquid phase, always di-acetyl, acetaldehyde, ethyl acetate, 1-propyl alcohol, isopropylcarbinol, Isoamyl Acetate FCC, primary isoamyl alcohol, ethyl hexanoate and ethyl octylate concentration were mapped to the time of continuously fermenting.The constant air per-cent in retort is jet of all biological reactor operational conditions maintenances is directly pressed shown in the figure in the whole proposal.Table 7.2 brief summary the mean value of (minimum third-order reaction jar conversion sheet) each analyte during quasi-stable state.

Show 7.2. in the residence time, enter of the influence of the volume of air flow velocity of retort by jet thrust when Rt is 1,18 day, be the quasi-stable state value the concentration of the important metabolite of yeast in liquid phase yeast and the biological anti-detailed jar.

* the last 4 days mean value of each operational condition

Graphic representation 7.2 and 7.3 is presented at that the yeast flora in the liquid phase does not reach zero in this experiment.The perfume compound of this work sutdy is produced jointly by free and fixed yeast, can not determine the Relative Contribution in each source.The free suspension yeast cell of this work has more than one source: biomass propagation and be discharged into cell the liquid nutrient medium from gel beads.Composite model with cell release and propagation studies show that, even retort still has cell mass liquid to be discharged into (Karamanev, 1991) in the liquid with the operation of highly diluted rate when cell discharges from microbial film.

Yeast cell concentration and the relative time relation of continuously fermenting in graphic representation 7.2 liquid phases.The air STP volumetric flow rate that provides to biological reactor by jet is provided this figure.Remaining gas is CO ₂Experimental session volume of gas constant flow rate was at 472ml/ minute STP.

Yeast viability and the relative time relation of continuously fermenting in graphic representation 7.3 liquid phases.The air STP volumetric flow rate that provides to biological reactor by jet is provided this figure.Remaining gas is CO ₂Experimental session volume of gas constant flow rate was at 472ml/ minute STP.

The concentration that graphic representation 7.4 is followed the tracks of free amino nitrogen (FAN) in the liquid phase.Interesting is to notice minimum FAN concentration takes place when 34ml/ minute STP air.This and maximum concentration of ethanol or minimum total fermentable sugars (as glucose) concentration are not overlapping.Volume of air flow velocity in jet is when 94 are increased to 354ml/ minute, and the alcohol concn in the retort liquid phase reduces simultaneously total fermentable sugars (as glucose) and increases, and sees graphic representation 7.5.This may show opposite with fermentation, because available oxygen increases, more many cells breathing takes place.When volumetric flow rate when STP354ml/ minute reduces to 34ml/ minute, alcohol concn raises once more, yet can not reach the concentration of STP flow velocity in the time of 94ml/ minute.Be difficult to the comparison STP34ml/ minute accurate concentration of alcoholic acid with STP94ml/ minute the time because when also having cell aging, STP354ml/ minute due to the influence of the oxygen of Continuous Contact high level, immobilized cell group's the variation other factors to the influence of this system.White and Portno (1978) mention the variation of the different time yeast fragrance thing metabolite concentrations that continuously ferment in their tower-type fermentor in the graphic representation 2.4.

Free amino nitrogen concentration and the relative time relation of continuously fermenting in graphic representation 7.4 worts.The volume of air STP flow velocity that provides to biological reactor by jet is provided this figure.Remaining gas is CO ₂Experimental session volume of gas constant flow rate was at 472ml/ minute STP.

Ethanol and total fermentable saccharide (as glucose) concentration and the relative time relation of continuously fermenting in graphic representation 7.5 liquid phases.The air STP volumetric flow rate that provides to biological reactor by jet is provided this figure.Remaining gas is CO ₂Experimental session volume of gas constant flow rate was at 472ml/ minute.

Visible oxygen is to the remarkably influenced of the generation of total di-acetyl in the graphic representation 7.6.Because it is generally acknowledged di-acetyl is undesirable perfume compound in the beer, a main reason optimizing the oxygen level in the biological reactor is the level of this perfume compound of control.With behind 354ml/ minute spray air, flow velocity was reduced to STP34ml/ minute, and total di-acetyl reduces.The known oxygen increase can cause the precursor of this di-acetyl of α-acetylactis to form during batch fermentation increases (Kunze, 1996).

Graphic representation 7.7 shows the definite relation between jet middle air content and the acetaldehyde concentration.Along with the increase of air per-cent in jet, the acetaldehyde amount also increases.Acetaldehyde is given beer granny smith flavor, and the amount that exists usually in the commodity beer is less than the 20mg/ liter.

Total di-acetyl concentration and the relative time relation of continuously fermenting in graphic representation 7.6 liquid phases.The air STP volumetric flow rate that provides to biological reactor by jet is provided this figure.Remaining gas is CO ₂Experimental session volume of gas constant flow rate was at 472ml/ minute.

Acetaldehyde concentration and the relative time relation of continuously fermenting in graphic representation 7.7 liquid phases.The air STP volumetric flow rate that provides to biological reactor by jet is provided this figure.Remaining gas is CO ₂Experimental session volume of gas constant flow rate was at 472ml/ minute.

Table 7.2 and graphic representation 7.8-7.9 show ethyl acetate, Isoamyl Acetate FCC, ethyl hexanoate and ethyl octylate quasi-stable state concentration and continuously ferment time relation.For the ester of all mensuration, ventilation speed changes to 354ml/ minute STP from 94 and causes its concentration to reduce.When ventilation speed drops to 34ml/ minute STP from 354, Isoamyl Acetate FCC, ethyl hexanoate and ethyl octylate concentration raise, however being seen level when not being increased to 94ml/ minute ventilation speed.The reaction pattern of these compounds conforms to each other closely, and ethyl hexanoate is bigger with the relative fluctuation ratio Isoamyl Acetate FCC that ethyl octylate shows.Ethyl acetate concentration is actual during to 34ml/ minute STP further descends when the air volume flow prompt drop.For all esters that this research is measured, when the air in the fluidizing agent was eliminated fully, concentration showed rising.Along with liquid phase cell concn in the retort reduces rapidly, each ester concentration raises and reduces then.

Graphic representation 7.8 ethyl acetate concentrations and the relative time relation of continuously fermenting.The air STP volumetric flow rate that provides to biological reactor by jet is provided this figure.Remaining gas is CO ₂Experimental session volume of gas constant flow rate was at 472ml/ minute.

Isoamyl Acetate FCC, ethyl hexanoate and ethyl octylate concentration and the relative time relation of continuously fermenting in graphic representation 7.9 liquid phases.The air STP volumetric flow rate that provides to biological reactor by jet is provided this figure.Remaining gas is CO ₂Experimental session volume of gas constant flow rate was at 472ml/ minute.

Graphic representation 7.10 and 7.11 has provided higher alcohols primary isoamyl alcohol, isopropylcarbinol and 1-propyl alcohol to the time relation of continuously fermenting, and for the alcohol of all mensuration, its concentration increase is because ventilation was increased to 354ml/ minute from STP 94ml/ minute.Isopropylcarbinol shows maximum relative fluctuation when ventilation speed changes.1-propyl alcohol concentration is lower than the Low Flavour Threshold that 600-800mg/ rises, yet its concentration is higher than the beer of common commercialization batch production in the experiment of continuously fermenting, and the latter is usually less than the 16mg/ liter.This be in normal range primary isoamyl alcohol and isopropylcarbinol different.Think that it is (Gee and Ramirez, 1994) due to propionic acid reduces that compound 1-propyl alcohol raises.Other people (Hough etc., 1982; Yamauchi etc., 1995) also the formation of 1-propyl alcohol is belonged to amino amino butyric acid and Threonine and be respectively ketobutyric acid and the corresponding oxoacid and aldehyde metabolism of propionic aldehyde due to.

Primary isoamyl alcohol and isopropylcarbinol concentration and the relative time relation of continuously fermenting in graphic representation 7.10 liquid phases.The air STP volumetric flow rate that provides to biological reactor by jet is provided this figure.Remaining gas is CO ₂Experimental session volume of gas constant flow rate was at 472ml/ minute STP.

1-propyl alcohol concentration is to the time relation of continuously fermenting relatively in graphic representation 7.11 liquid phases.The air STP volumetric flow rate that provides to biological reactor by jet is provided this figure.Remaining gas is CO ₂Experimental session volume of gas constant flow rate was at 472ml/ minute STP.

Because do not wish that di-acetyl in the beer, acetaldehyde and fusel are excessive, control oxygen is very important with the generation that limits them.Discuss as literature review, when the oxygen of yeast cell supplies to increase, amino acid precursor increase thereby higher alcohols, oxoacid and di-acetyl surplus that anabolism forms.Because Transacetylase catalysis ester forms, and is known to the increase that can utilize oxygen, ester concentration reduces.Transacetylase is subjected to the inhibition (Norton and D ' Amore, 1994) of unsaturated fatty acids and ergosterol (they can increase) when having oxygen.

For the biological reactor condition of this experiment usefulness, quasi-stable state in the retort liquid phase of mensuration (minimum third-order reaction jar turnover) oxyty approaches zero (less than the 0.03mg/ liter).

This experiment does not have directly air STP in the fluidizing agent relatively 94ml/ minute and 34ml/ minute data, because upper air current speed (354ml/ minute) is separated them.This is that immobilization matrix and the time of continuously fermenting may also cause other change that fragrance produces because yeast is exposed to the physiological status after the previous retort condition.

Pollution is not all measured in any position in the experimental session biological reactor.For the balance yeast to the demand of oxygen to keep the yeast viability and to require at utmost to reduce oxygen to obtain beer with desired flavor, can explore other method, as add nutrition such as zinc, magnesium, or provide yeast cell to keep other required xenobiontics of viability.These admixtures can further reduce the demand of yeast to oxygen. and another kind may be to operate with low-down oxygen concn the most of the time, and regularly giving yeast pulse oxygen keeps cell viability.

7.2.2 soak in batches after the fermentation: contact oxygen is to the effect of metabolism of yeasts

Because just total di-acetyl takes several method to reduce this compound concentrations to acceptable level not in the normal range of commercialization beer during fermentation ends.A kind of method is to adopt insulation to store at once after the fermentation just continuously.Graphic representation 7.12-7.21 shows the total fermentable saccharide of liquid phase (as glucose), ethanol, total di-acetyl, acetaldehyde, ethyl acetate, 1-propyl alcohol, isopropylcarbinol, Isoamyl Acetate FCC, primary isoamyl alcohol and the ethyl hexanoate concentration graph of a relation to the back soaking time of fermenting.Shown in each figure, with the quasi-stable state collected continuously just fermented sample leave in aerobic or anaerobic condition under.

Graphic representation 7.12 shows that total fermentable saccharide (as glucose) descended rapidly in the first two hour in aerobic and the anaerobism sample, descends slower at all the other soaks.The reason that this phenomenon is possible is that sugar concentration is higher when having more yeast and soak to begin before the first two hour introversion goes out.But the picked-up no significant difference of glucose fermentation between aerobic and the anaerobism sample, though notice that some difference is arranged at first.

Alcohol concn rises rapidly when beginning insulation in the graphic representation 7.13, and the alcohol concn of anaerobism and aerobic sample increases in time with parallel mode almost then.Anaerobism sample alcoholic acid initial concentration increases the time overlaid with most of Sugar intake.Alcohol concn is higher in the sample of insulation end of term anaerobic treatment.

But the continuous fermentation just of graphic representation 7.12 gas lift biological reactors back is aerobic and the relation of the average glucose fermentation concentration of anaerobic treatment sample and fermentation back soaking time.The error bar is represented the bound of experimental data.(n＝2)

The continuous fermentation just of graphic representation 7.13 gas lift biological reactors back aerobic and the average ethanol concentration of anaerobic treatment sample and the relation of fermentation back soaking time.The error bar is represented the bound of experimental data.(n＝2)

Acetaldehyde increases in early days graphic representation 7.14 aerobic samples are exposed to aerobic condition outside biological reactor after, soluble this result of combined action that aerobic condition and sugar consumption and ethanol produce.Acetaldehyde concentration rose from 17mg/ and drops to the 9mg/ liter to 48 hours insulation end of term anaerobism samples, within the specification of quality 10mg/ that makes its strength of fluid reduce to the North America lager beer rises.

Graphic representation 7.15 has provided the relation of total di-acetyl concentration and soaking time.The result shows that soak system's elimination from then on oxygen provides more advantageous conditions of reduction di-acetyl.The shape of total di-acetyl curve may with free amino nitrogen consumption and subsequently in the cell generation Xie Ansuan relevant, di-acetyl is its byproduct (Nakatani etc., 1984a; Nakatani etc., 1984b).Just the last anxious di-acetyl concentration of fermentation is 326 μ g/ liters continuously, and the anaerobic heat-preservation end of term is 33 μ g/ liters, is lower than the taste threshold letter of commodity beer.

The continuous fermentation just of graphic representation 7.14 gas lift biological reactors back aerobic and the average acetaldehyde concentration of anaerobic treatment sample and the relation of fermentation back soaking time.The error bar is represented the bound of experimental data.(n＝2)

Aerobic and the average total di-acetyl concentration of anaerobic treatment sample and the relation of the back soaking time of fermenting after graphic representation 7.15 gas lift biological reactors just ferment continuously.The error bar is represented the bound of experimental data.(n＝2)

Graphic representation 7.16-7.18 shows the concentration of ethyl acetate, Isoamyl Acetate FCC and ethyl hexanoate and the relation of fermentation back soaking time.The observed pattern of all esters is aerobic identical with the anaerobism sample.Ester concentration does not have differently from the insulation later stage between the aerobic and anaerobism sample, and this moment, the ester concentration of aerobic sample descended and the rising of anaerobism sample.Because compare the ester concentration that continuously ferments with the ester concentration of commodity beer low slightly, therefore need to select to help the condition that ester produces.

The continuous fermentation just of graphic representation 7.16 gas lift biological reactors back aerobic and the average ethyl acetate concentration of anaerobic treatment sample and the relation of fermentation back soaking time.The error bar is represented the bound of experimental data.(n＝2)

The continuous fermentation just of graphic representation 7.17 gas lift biological reactors back aerobic and the average Isoamyl Acetate FCC concentration of anaerobic treatment sample and the relation of fermentation back soaking time.The error bar is represented the bound of experimental data.(n＝2)

The continuous fermentation just of graphic representation 7.18 gas lift biological reactors back aerobic and the average ethyl hexanoate concentration of anaerobic treatment sample and the relation of fermentation back soaking time.The error bar is represented the bound of experimental data.(n＝2)

The relation of soaking time after graphic representation 7.19-7.21 shows primary isoamyl alcohol, 1-propyl alcohol and isopropylcarbinol concentration and ferments.These alcohol are not seen notable difference between 48 hours insulation aerobic and anaerobic treatment of the end of term.Yet 24 hours sample shows that all occasions concentration of aerobic treatment is higher.

The continuous fermentation just of graphic representation 7.19 gas lift biological reactors back aerobic and the average primary isoamyl alcohol concentration of anaerobic treatment sample and the relation of fermentation back soaking time.The error bar is represented the bound of experimental data.(n＝2)

The continuous fermentation just of graphic representation 7.20 gas lift biological reactors back aerobic and the average 1-propyl alcohol concentration of anaerobic treatment sample and the relation of fermentation back soaking time.The error bar is represented the bound of experimental data.(n＝2)

The continuous fermentation just of graphic representation 7.21 gas lift biological reactors back aerobic and the average isopropylcarbinol concentration of anaerobic treatment sample and the relation of fermentation back soaking time.The error bar is represented the bound of experimental data.(n＝2)

Fig. 7 .22 is used for 48 hours aerobic and some perfume compounds of anaerobic heat-preservation after date and commodity beer radiograms relatively.Radiogram is that the wine brewing industry is commonly used to characteristic with various different beer and is placed on and investigates among the figure and compare (Sharpe, 1988).The most approaching and the common sale beer of the beer that continuously ferments of from then on scheming visible anaerobic heat-preservation matches.As seen except that the 1-propyl alcohol is significantly higher than batch fermentation beer, anaerobism beer is all in the normal range of selling beer from appendix 6.All to observe the 1-propyl alcohol higher for all products that continuously ferment in this work.

Just yeast phase forms 1-propyl alcohol and significantly reduction of soak continuously, no matter take aerobic or anaerobic condition.Kunze (1996) says higher alcohols such as 1-propyl alcohol when following factor will increase batch fermentation: mix, the wort brute force is ventilated and is added new sweet wort repeatedly and gives existing yeast.Finally, ideal situation is to fully phase out afterwards soak by optimizing condition in the first fermentation reaction jar.Yet can be by optimizing holding temperature (yeast is removed di-acetyl and depended on very much temperature), remain in fermentable sugars amount in the liquid when soak begins, optimize the zymic concentration that exists, the hydrokinetics of insulation jar (by improving the removal that yeast and contacting of beer are improved di-acetyl) and take further step to eliminate the oxygen in this stage and further acquisition improvement.

Provided the beer volumetric productivity that calculates in the appendix 3.This saves the biological reactor operate continuously, and 24 hours residence times, insulation in batches in 48 hours then shows that productivity is 1.8 times of present industrialization batch process.One of industrialization batch process circulation faster 7.5 days, beer volumetric productivity be 0.093 cubic metre/(a cubic meter tankage is taken advantage of fate), and continuous processing beer production rate described herein is 0.165 cubic metre.If further research can foreshorten to 24 hours with soak in batches, the beer production rate will be higher 2.3 times than industrialization batch processes.If continuous processing need not be incubated in 24 hours in batches, the beer volumetric productivity will be 7.5 times of batch processes.Except improving volumetric productivity, other is that as shortening the preceding time of listing, reducing wine brewing room volume and the less yeast propagation that needs, these must balance each other with anatomizing of relative running cost from the advantage that makes continuous processing in batches.

Other scholars (Kronlof and Virkajarvi, 1996; Nakanishi etc., 1993; Yamauchi etc., 1995) be devoted to develop the multistage and continuously ferment, wherein the fs continuously ferment (aerobic) only cause the wort fermentable sugars partly to consume.Though this method has shown some success, this system very complex on the fragrance deposits yields.In addition, this system has produced the environment (high sugared concentration, temperature and oxygen, and low alcohol concn) that more is subject to microbial contamination first aerobic stage.Originally be operated in air lift type biological respinse can system and carry out, retort fermentable sugars concentration is low, pH is low, alcohol concn is high, oxygen concn is low, makes its environment be not suitable for taking place potential and pollutes.

The radiogram of the normalization method concentration that graphic representation 7.22 gas lift biological reactors obtain behind the aerobic and anaerobic heat-preservation of back 48 hours of fermentation continuously just.Normalization data is according to the mean value of duplicate sample, and the data of commodity beer are taken from the mid point of listing data in the appendix 6.

7.2.3 the liquid residence time is to the influence of the important metabolite of yeast between the first yeast phase of continuous beer.

Graphic representation 7.23-7.28 shows the analytical results that obtains from the biological reactor liquid phase.The mean concns and the flow velocity of analyte when table 7.3 has been listed this quasi-stable state of testing used two kinds of liquid hold-up timings (at least through the turnover of 3 secondary response jars).When though the flow velocity of wort input retort increases in the liquid phase yeast viability do not have considerable change, yeast concentration has changed, and sees graphic representation 7.23.

Table 7.3 (a) brief summary the biological reactor residence time to the liquid phase yeast that is in quasi-stable state and the influence of the important metabolite concentration of yeast (mean value); (b) brief summary the biological reactor residence time retort outlet is in the influence of the liquid phase yeast and the important metabolite flow velocity of yeast (mean value) of quasi-stable state.

Yeast concentration and the relative time relation of continuously fermenting in Fig. 7 .23 liquid phase, the influence of liquid hold-up time in the retort.Rt is a liquid hold-up fate in the retort.

Fig. 7 .24 and 7.25 show when the liquid hold-up time when 1.8 days are reduced to 0.9 day wort substrate free amino nitrogen (FAN) and always fermentable sugars (as glucose) concentration all increase.As seen be accompanied by the retort residence time from the material balance of table 7.4 and reduce, the spending rate of total fermentable sugars (as glucose) increases and the free amino nitrogen spending rate reduces and follows the liquid hold-up time decreased.Produce ethanol yield factor Y from the fermentable sugars fermentation _P/S, be increased to 0.5 from 0.3.Because go into this system with air and carbon dioxide gas are jet, ethanol has than small loss in the gas phase, and this may influence productive rate factor Y by influencing the ethanol balance _P/SThe research that BudacandMargaritis (1999) cooperation is carried out has adopted gas chromatography-mass spectrum technology (GC-MS) to prove quantitatively, and gas lift biological reactor top board detects the beer flavorful volatiles and comprises ethanol, acetaldehyde, acetyl acid esters and Isoamyl Acetate FCC when continuously fermenting.

But ethanol and glucose fermentation concentration is to the relative time relation of continuously fermenting in graphic representation 7.24 liquid phases, the effect of liquid hold-up time in the retort.Rt is a liquid hold-up fate in the retort.

Free amino nitrogen and 1-propyl alcohol concentration and the relative time relation of continuously fermenting, the effect of liquid hold-up time in the retort in graphic representation 7.25 liquid phases.Rt is a liquid hold-up fate in the retort.

Table 7.4 is according to the average data of table 7.3, to the material balance of free amino nitrogen and total fermentable saccharide (as glucose), the effect of residence time.

Take from the entrance concentration of appendix 1

The tunning concentration of ethanol reduces with the variation of liquid hold-up time in the liquid phase.Yet this system can produce more ethanol in the shorter liquid hold-up time according to mass rate as a whole.Because this work purpose is not just produced ethanol separately, but produces many composition equilibrated beer, the highest alcohol production rate must balance each other with other factors.But commercialization beer just during fermentation ends overwhelming majority's glucose fermentation substrate must exhaust.

Graphic representation 7.26 shows acetaldehyde and the reaction of total di-acetyl concentration to the wort change in flow.Analyte concentration and gain in yield thereof when the liquid hold-up time decreased.Initial several days yeast that ferment when the beer batch fermentation are discharged acetaldehyde (kunze, 1996).

Total di-acetyl and acetaldehyde concentration and the relative time relation of continuously fermenting, the effect of liquid hold-up time in the biological reactor in graphic representation 7.26 liquid phases.Rt is a retort liquid hold-up fate.

Graphic representation 7.25 and 7.27 shows the influence of minimizing retort residence time to the middle-and-high-ranking pure 1-propyl alcohol of liquid phase, isopropylcarbinol and primary isoamyl alcohol concentration.When the retort residence time reduced, these three kinds of higher alcohols concentration reduced.

Isopropylcarbinol and primary isoamyl alcohol concentration and the relative time relation of continuously fermenting, the influence of liquid hold-up time in the biological reactor in graphic representation 7.27 liquid phases.Rt is a retort liquid hold-up fate.

Table 7.3 (b) provides the mass rate of ethyl acetate and Isoamyl Acetate FCC, and the two all increases when reducing in the liquid hold-up time.Graphic representation 7.28 shows that ethyl acetate concentration reduces and the Isoamyl Acetate FCC rising in the liquid phase.Or not the oxygen supply to this system because this experiment allows cell proliferation in the liquid phase increase, the condition in this moment retort has promoted the ester generation.Hough etc. (1982) say to improve propagation and reduce this conditions favouring of oxygen and produce in ester.

Ethyl acetate and Isoamyl Acetate FCC concentration and the relative time relation of continuously fermenting, the effect of liquid hold-up time in the biological reactor in Fig. 7 .28 liquid phase.Rt is a retort liquid hold-up fate.

7.2.4 adopt the alpha-acetolactate decarboxylase commodity reduction continuous beer total di-acetyl between yeast phase just.

Experiment 1: biological reactor has polluted oxybiontic gram positive coccus before finishing this test.Because it is pollution-free that the wort microbioassay that provides shows, so it is contaminated to be defined as retort itself.This proposes retort and need upgrade to improve resistant to pollution safe class.Yet, close and when ALDC being added in the wort of supplying, find total di-acetyl density loss before this system.Unfortunately from then on data can not draw any conclusion, because retort is polluted the effect of obscuring are arranged.

Experiment 2: since the upgrading of many biological reactors, the not pollution of whole this system of experiment 2 manipulate.Graphic representation 7.29-7.31 has provided the data of this experiment.Table 7.5 brief summary add the quasi-stable state mean concns of total di-acetyls before and after the ALDC.Add ALDC in the wort, total di-acetyl density loss 47%, this makes this enzyme have application prospect (mean value is taken from the turnover of third-order reaction jar) in the future.Total fermentable sugars of this duration of test (as glucose) and cell concn slightly change shown in graphic representation 7.30 and 7.31, and this may be because the wort of adding ALDC front and back supply response jar has slight difference and causes.

In graphic representation 7.29 liquid phases total di-acetyl concentration with continuously ferment the time, add the influence of ALDC in the attenuate substratum.Experiment 2.

Fermentable sugars (as glucose) and alcohol concn and continuously ferment the time in graphic representation 7.30 liquid phases add the influence of ALDC in the attenuate substratum.Experiment 2.

In graphic representation 7.31 liquid phases cell concn with continuously ferment the time, add the influence of ALDC in the attenuate substratum.Experiment 2.

Potentially obscure effect for what eliminate that wort changes, collect a large amount of worts (1400 liters) from the wine brewing room in the experiment 3, when reaching the quasi-stable state baseline, ALDC directly added in the wort in the insulation jar.This further eliminates the carrying out that wort potential heterogeneity may have influence on experiment 2.This wort basin is equipped with the wort dissolved oxygen level that the carbonic acid gas gas jets can make supply and keeps low-level consistently.

Experiment 3: add the influence of ALDC in the wort of supplying with between graphic representation 7.32-7.34 explanation continuous beer yeast phase to total di-acetyl, total fermentable sugars (as glucose), ethanol and free suspension cell concentration.Table 7.6 has also provided and has added the total di-acetyl mean concns of ALDC front and back quasi-stable state (mean value is taken from third-order reaction jar turnover back) in the wort.This experimental session is not all measured pollution any time.Total di-acetyl density loss 45% when adding ALDC.Do not see that ethanol, total fermentable sugars (as glucose) or free suspension cell concentration have notable difference.This batch fermentation finding with AschengreenandJepsen (1992) is consistent.

In graphic representation 7.32 liquid phases total di-acetyl concentration with continuously ferment the time, add the influence of ALDC in the attenuate substratum.Experiment 3.

In graphic representation 7.33 liquid phases ethanol and total fermentable sugars (as glucose) concentration with continuously ferment the time, add the influence of ALDC in the attenuate substratum.Experiment 3.

In graphic representation 7.34 liquid phases cell concn with continuously ferment the time, add the influence of ALDC in the attenuate substratum.Experiment 3.

The result of experiment 2 and 3 shows that ALDC had remarkably influenced to total di-acetyl concentration really when the gas lift biological reactor continuously fermented, and total di-acetyl on average descends 46%.This has the potentiality that reduce or eliminate the secondary processing that the di-acetyl that makes in the continuous gas lift system reduces.The ALDC of higher dosage has been adopted in these tentative experiments, and therefore so technology is adopted quantity, method and the time that this enzyme require optimization gives the ALDC of wort.If available this enzyme has high reactivity under the fermentation condition brewageing, maybe this enzyme immobilization can be reused and can further be saved cost (Dulieu etc., 1996).Another kind of consider it is the enzyme addn of the acceptable genetically modified microorganisms producing of the public.The dosage that the supplier recommends is 2kg/100000L, adds the Cheng Ben $131.05/kg of commercial enzyme, and the fermented material cost increases $0.26/hL.As it is employed to carry out these experiments, and this enzyme dosage is 72 μ g/L (108ADU/L) or 7.2kg/1000hLALDC, increases Cai Liaofeiyong $0.94/hL.The economics that adopts ALDC to reduce di-acetyl during gas lift continuously ferments depends on the optimal dose of enzyme under the biological reactor condition and the time that its use is saved.

Add ALDC in the table 7.5 attenuate substratum to the ferment average quasi-stable state effect of total di-acetyl concentration of continuous beer in the gas lift retort

* average according to quasi-stable state value after the third-order reaction jar turnover time.

According to following standard, above being supported in and adopting immobilized yeast in the through-flow tubular type biological reactor of the gas lift system is to substitute a kind of practicable method that batch fermentation is produced beer with the proposal that relevant free cell continuously ferments:

-reach taste to match,

-biological reactor volumetric productivity is higher,

-complicacy is minimum,

-demonstration operate continuously for a long time,

-by the control of the air (oxygen) in controlling flow oxidizing gases fragrance,

-add alpha-acetolactate decarboxylase as a kind of selection of controlling di-acetyl,

-no bacteria pollution,

-financial the benefit.

Still have many fields to need further research, but this technology is not difficult at large scale test more.The gas lift biological reactor has been used for the wastewater treatment of industry size, and this makes that the scale that enlarges the continuous beer fermentation is technical feasible.The Grolsch wine brewing room of Holland has been reported and has been adopted 230 cubic metres of gas lift retort to handle its waste water (Driessen etc., 1997).Brewage one of biggest obstacle that industry commodity scale continuously ferments and may whether can be accepted a kind of novel process by the EnologistWinemaker of the deep constraint of industry tradition.The collection of the yeast secondary metabolites data that produce between the continuous beer yeast phase that this work is carried out has been emphasized to producing the importance of the oxygen in the beer fragrance controlling flow oxidizing gases.This discovery shows that the air that improves in the retort fluidizing agent causes the rising of acetaldehyde, di-acetyl and higher alcohols (primary isoamyl alcohol and isopropylcarbinol) under given operational condition, ester (Isoamyl Acetate FCC, ethyl hexanoate, ethyl octylate) and alcohol concn descend simultaneously.These Notes of Key Datas might be produced unique product by the composition control beer flavor of control retort fluidizing agent.

When exception is a air in removing fluidizing agent in the retort liquid phase free suspension cell concentration keep greater than 10 ⁸Cell/ml.There is more than one yeast cell group in this system in retort: immobilized yeast and liquid phase suspension yeast.Because a large amount of yeast of living are bred in the liquid phase of retort, might utilize continuous biological reactor as the yeast multiplier (-icator).Just the fermentation back increases by 48 hours in batches continuously.Soak can obtain the taste in commercially available beer scope.Experimental results show that for producing this soak temperature of this taste be 1 ℃, importantly at utmost reduce soak liquid contact oxygen.Yet, except soak make this technology increase by two days and other complicacy, the commercial batch fermentation of this method than colored 7-14 days is still obviously fast.

At last, ideal situation is to eliminate secondary soak by the conditionally complete of optimizing in the continuous elementary biological reactor.Yet by optimize soak temperature (yeast is removed very temperature dependent of di-acetyl), remain in fermentable sugars amount in the liquid when soak begins, the hydrokinetics feature of the zymic concentration of existence, insulation jar (can promote to remove di-acetyl by improving contacting between yeast and the beer) and the measure of taking to eliminate this interim oxygen can further shorten secondary soaking time.

Other scholars (KronlofandVirkajarvi, 1996; Nakanishi, 1993; Yamauchi etc. 1995) be devoted to develop the multistage and continuously ferment, wherein continuously ferment the fs (aerobic) causes part only to consume fermentable saccharide in the wort.Though this strategy has shown some success, these system very complex with regard to taste produces.In addition, aerobic stage of first of this system has produced the microbial contamination environment of susceptible (as high sugared concentration, temperature and oxygen, and low alcohol concn) more.The gas lift biological reactor of this work has low stable state fermentable sugars concentration, low pH, high alcohol concn and low oxygen concentration, and this makes environment be not suitable for the potential pollution.In the of a specified duration wine brewing room of exploitation, reduce complicacy and development machines people antipollution technology is successful important factor as far as possible.In the wort that the confession continuously ferments, add the demonstration of alpha-acetolactate decarboxylase (ALDC) commercial formulation and on average can reduce di-acetyl 46%.Yet because ALDC is a kind of enzyme of microorganism (GMO) generation of genetic modification, this is need be to the thing of public's explanation before this enzyme is used for commodity.In addition, the commercialization enzyme of control di-acetyl does not under fermentation conditions have optimum activity.

Continuously ferment more than 6 months with κ-carrageenan gel sets, free suspension cell keeps the viability more than 90% in the liquid phase, and the viability of immobilized cell is reduced to less than 60%.Scanning electron microscope discloses the form that the cell that is positioned at the gel beads periphery has many places bud trace and rule, and near the cellular form the pearl center is irregular, and the bud trace seldom points out growth impaired.Microscope also points out near the yeast that is positioned at the pearl center to present old and feeble sign.Such as the 5th the joint discussion κ-carrageenan gel have many features to make it become ideal yeast immobilization matrix.Yet also do not make the industrialization method of pearl at present, the processing as pearl in a part of commercial plant of pearl manufacturing process has increased complicacy and cost in the matrix because yeast will be embedded in.Other process for fixation such as self aggregation or flocculation should further be explored.This may eliminate in the environment of plant complicacy of handling pearl, and if can regular destruction yeast throw out, just can guarantee to remove regularly from biological reactor senile cell.

Claims (19)

1. method of producing drinkable alcohol, it is characterized in that, this method adopts utilizes the air lift type biological reactor of internal recycle to continuously ferment the stage, with immobilized flocculating yeast cell the wort that contains fermentable saccharide is carried out fermentation just in this stage under the supply of restriction oxygen, will comprising not then, the ejecta to the small part fermentation of Saccharomyces congloberatus is transported to the processing of secondary fermentation in batches holding stage to finish fermentation.

2. the method for claim 1, the wherein said stage of continuously fermenting is fermentation just.

3. the method for claim 1, wherein said wort be ventilation before introducing biological reactor.

4. the method for claim 1, wherein oxygen is imported into the stage of continuously fermenting as a component of mixed gas.

5. method as claimed in claim 4, wherein said mixed gas comprise air and extra nitrogen.

6. the method for claim 1, wherein said continuously fermenting provides yeast for the batch fermentation in the secondary fermentation processing holding stage in batches.

7. method as claimed in claim 4, wherein said mixed gas contains the oxygen content up to 20%.

8. method as claimed in claim 4, the oxygen content of wherein said mixed gas are 2%-5%.

9. the method for claim 1, it comprises the preliminary batch fermentation stage, the autoagglutination of flocculating yeast cell forms the throw out of the fixed yeast that comprises the stage that is used to continuously ferment in this stage.

10. the method for claim 1, wherein said yeast is high flocculence yeast or super flocculence yeast.

11. the method for claim 1, it comprises cold aging time.

12. the method for claim 1, wherein said secondary fermentation in batches processing holding stage do not have extra oxygen in the presence of carry out.

13. method as claimed in claim 2, wherein fermentation just is in secondary fermentation processing holding stage end in batches.

14. the method for claim 1, wherein said secondary fermentation in batches processing holding stage have reach 48 hours during.

15. the method for claim 1, wherein said drinkable alcohol is beer.

16. the method for claim 1, wherein said drinkable alcohol are light color type beer.

17. the method for claim 1, wherein said drinkable alcohol are North America type beer.

18. the method for claim 1, wherein said drinkable alcohol is lager beer.

19. the method for claim 1, the stage ejecta that wherein will continuously ferment is delivered to one of a plurality of jars of insulation in batches by the road of menifold more than, and the described processing of secondary fermentation in batches holding stage carries out in described insulation jar.

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