CN1982460A - Separation of tropical candiyeast strain and production of xylitol - Google Patents
Separation of tropical candiyeast strain and production of xylitol Download PDFInfo
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- CN1982460A CN1982460A CN 200510037580 CN200510037580A CN1982460A CN 1982460 A CN1982460 A CN 1982460A CN 200510037580 CN200510037580 CN 200510037580 CN 200510037580 A CN200510037580 A CN 200510037580A CN 1982460 A CN1982460 A CN 1982460A
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Abstract
Separation of tropical moniliasis strain and production of xylitol are disclosed. The process is carried out by amplification culturing to obtain seed liquid, preparing hemicellulose hydrolyte culture medium, circulating and reutilizing. It has excellent sugar-tolerance and mithridatism. It could be used above time times.
Description
Technical field
The invention belongs to biological technical field, particularly separate the yeast strain that obtains, and, be used further to the hemicellulose hydrolysate that ferments, the wood sugar in the hydrolyzate is transformed the production method that generates Xylitol its expansion from nature.
Background technology
Xylitol is a kind of five-carbon sugar alcohol, has high sugariness, and anti-dental caries is functional, and metabolic process does not need Regular Insulin to participate in, and the big oral cavity that can make of solution heat produces refrigerant especially characteristics such as sensation, makes it have important application in foodstuffs industry.
At present, the suitability for industrialized production Xylitol is mainly with the technology of chemical method reduction wood sugar, its primary process is: at first with the hemicellulose part in the dilute acid hydrolysis lignocellulosic material, be purified into wood sugar then from the hemicellulose hydrolysate that is rich in wood sugar, last catalytic hydrogenation wood sugar generates Xylitol.Owing to yet comprise the polymkeric substance of other carbohydrate in the lignocellulosic material, chemical technology needs a series of deep purification procedures to remove these by products from wood sugar and Xylitol.Complicated purification step has improved the production cost of Xylitol, has limited the range of application of Xylitol.
Utilize the yeast fermentation hemicellulose hydrolysate produce Xylitol be considered to one always can substituted chemistry technology, reduce the operational path of Xylitol production cost, because the yeast conversion wood sugar generates in the process of Xylitol, other carbohydrate that is present in the hydrolyzate mainly is consumed in yeast cell growth and metabolisable energy is provided, and can not generate corresponding sugar alcohol.Because the process of xylitol zymolysis production also is the biologically pure process of wood sugar and Xylitol simultaneously, so it does not need the requisite wood sugar purification step of chemical method, can also effectively simplify the separation of Xylitol.In addition, yeast cell is as catalyzer, and its power consumption is also relatively low.
For suitability for industrialized production, product generating rate (Xylitol g/l.h), product yield (Xylitol g/ wood sugar g) and this three big fermentation parameter of production concentration (Xylitol g/l) the index that can realize, be the deciding factor whether xylitol fermentation technology has industrial application value.
Utilize the report of yeast fermentation explained hereafter Xylitol a lot, people such as Parajo once made detailed summary (Parajo J C to correlative study, Dominguez H, Dominguez J M.Biotechnological production of xylitol.part1:Interest of xylitol and fundamentals of its biosynthesis.Bioresource Technology.1998, (65) 191-201.Part 2:Operation in culture media made with commercial sugars.BioresourceTechnology.1998, (65) 203-212.Part 3:Operation in culture media made from lignocellulosehydrolysates.Bioresource Technology.1998, (66) 25-40).Obtained reasonable effect (Sang Yong Kim though utilize the commercial grade wood sugar as the xylitol fermentation of matrix, Diok Kun Oh, Soo Ryung:Fermentationprocess for preparing xlitol using Candida tropicalis.United States Patent, Patent Number:5998181; Sang Yong Kim, Deok Kun Oh, Jin Hwan Choi et al.:Fermentation process for preparingxylitol using mutant cells; United States Patent, 5,686,277; Teak-Bum Kim, Yong-Joo Lee, Pil Kim, Increased xylitol production rate during long-term cell recycle fermentation of Candida tropicalis, Biotechnology Letters, 2004,26 (8): 623-627).But the price of pure wood sugar was high originally, had in fact offset most of advantage of zymotechnique as fermented substrate with it.And utilizing existing technology and strain fermentation hemicellulose hydrolysate to produce Xylitol, its product generating rate and production concentration are in very low level always.Following pertinent literature has embodied this point.
Jiangning, He Peng, Lu main forces etc., the free cell recycling repeatedly transforms the method for preparing Xylitol; Chinese patent, ZL01110778.2.
Some previous work reports of inventor Zhang Hourui.As Zhang Hourui, Ceng Jianzhi, Cai Aihua etc. " yeast fermentation bagasse hemicellulose hydrolyzate is produced Xylitol ", biotechnology journal, 2002,18 (6): 725-728.
Mussatto S I，Roberto I C.Optimal Experimental Condition for Hemicellulosic HydrolyzateTreatment with Activated Charcoal for Xylitol Production.Biotechnol Prog.2004，(20)，134-139.
Canilha L，Silva J B A，Felipe M G A.，et al，Batch xylitol production from wheat strawhemicellulosic hydrolysate using Candida guilliermondii in a stired tank reactor，BiotechnologyLetters，2003(25)1811-1814.
Rivas B，Dominguez J M，Doming H，et al.Bioconversion of posthydrolysed autohydrolysisliquors：an alternative for xylitol production from corn cobs.Enzyme and Microbial Technology，2002，(31)431-438.
Inventor Zhang Hourui is at " biotechnology journal " 2000,16 (3): 304-307, propose in " Xylitol is produced in the hemicellulose hydrolysate bio-transformation ": effectively improve production concentration, the product generating rate, and keep high product yield, be that to carry out xylitol fermentation be very important to matrix for the industrialized utilization hemicellulose hydrolysate.Utilize hemicellulose hydrolysate to carry out xylitol fermentation, its product generating rate and the too low major cause of production concentration, be to generate many microbial metabolism inhibitions in the hydrolytic process simultaneously, especially, some toxic substance is nonvolatile, and concentrated hydrolyzate improves its xylose concentration, has also just improved the concentration of toxic substance in the hydrolyzate simultaneously, when concentration of poisons surpasses certain limit, yeast can't be grown at all.Therefore, adopt some physics and chemistry measure to handle hemicellulose hydrolysate, to reduce toxic substance, the leavening property that improves hydrolyzate is absolutely necessary.
But,, also must improve the fermentative production cost of Xylitol if the detoxification process that adopts is too complicated.
Existingly experiment showed, that there is notable difference in different yeast strains to the tolerance of toxic substance in the hydrolyzate.Obviously, if can obtain the Xylitol high yield yeast strain of more anti-poison, utilize so and separate thing without complicated especially treating process treated water and ferment, also can transform wood sugar equally expeditiously and generate Xylitol, thereby effectively alleviate the burden of detoxification process, give full play of the advantage of zymotechnique.
In addition, the hemicellulose hydrolysate preparation method, the composition of other nutritive ingredient of hemicellulose hydrolysate substratum, can fermentation condition control etc. be related to yeast strain equally and bring into play maximum production potential, and the final production cost of product.
Summary of the invention
The objective of the invention is to utilize hemicellulose to carry out in the xylitol fermentation process technology in order to solve effectively, whole production efficient that bring is low excessively because production concentration is low, and the product generating rate is low, is unsuitable for the problem of suitability for industrialized production in the past.
The whole process of the present invention comprises candida tropicalis (Candida tropicalis) bacterial strain that utilizes a strain new, with the mode of the recycle cell hemicellulose hydrolysate that ferments, and the wood sugar in the hydrolyzate is transformed the process that generates Xylitol.
The present invention includes: the contriver utilizes biological means, it is good to be separated to a strain sugar tolerance from wood sugar factory environment waste, anti-poisonous substance ability is strong, the bacterial strain that the wood sugar transformation efficiency is high: i.e. yeast strain 1-18, according to the bacterium colony outward appearance, cellular form, physio-biochemical characteristics and molecular biological characteristics, the contriver determines that the classification position of 1-18 belongs to candida tropicalis; Cultivated candida tropicalis 1-18 cell, prepared the hemicellulose hydrolysate that is fit to fermentation, and utilized the mode of cell with recirculation repeatedly, in batches with the wood sugar of hemicellulose hydrolysate middle and high concentration (〉=100g/L) transform and generate Xylitol; As the hemicellulose hydrolysate of fermented substrate, the nutritive substance that adds is a feature with rice sugar water cooking liquid or wheat bran water cooking liquid.
Belonging to Xylitol superior strain candida tropicalis 1-18 of the present invention, is to screen acquisition as follows.
The strain separating process wherein contains (g/L): wood sugar 20g, yeast extract paste 0.5g, (NH with assimilation wood sugar substratum
4)
2HPO
43g, MgSO
4.7H
2O 0.1g, KH
2PO
40.15g.Wood sugar separates with other composition, and 110 ℃ of sterilization 15min mix during use.If make flat board, add agar 20g/L as solid agent.
Fermentation screening process xylitol fermentation substratum.The composition of fermention medium, except the yeast extract paste consumption is increased to 10g/L, xylose concentration is brought up to outside the 150g/L, and all the other compositions are identical with assimilation wood sugar substratum.
From the discarded wood sugar liquid of wood sugar factory environment collection, waste water, mud are made sample separation, and by sources difference inserts respectively in the liquid assimilation wood sugar substratum.250ml triangular flask loading amount 25ml inserts waste water or the about 1g of mud sample separation, and 30C, 200rpm shake a bottle enrichment culture 36h.Get the line on assimilation wood sugar culture medium flat plate of this nutrient solution and separate, cultivated 4 days for 30 ℃, select the type of preponderating on the flat board, forward slant culture to and enlarge.
Culture is transferred in the fresh xylitol fermentation substratum on the inclined-plane, carries out xylitol fermentation.And, behind the 200rpm shake-flask culture 50h, detect the Xylitol concentration of fermented liquid through 30 ℃.Select the high bacterial strain of Xylitol concentration.
According to the method described above,, surplus totally 5 batches 50 in the duplicate samples, select one and belong to of the present invention, produce the isolate of Xylitol excellent performance, laboratory numbering 1-18 successively from coming from 3 wood sugar factories.
Aforesaid method separates the bacterial strain that obtains and refrigerates or the lyophilization preservation with 4 ℃.
According to Institute of Microorganism, Academia Sinica: " the culture presevation handbook (Science Press. Beijing .1980, the p368-370) method that provides of a book, the physiology series feature of isolate is as follows:
| Carbon source | The result | Carbon source | The result | Carbon source | The result |
| The red bright alcohol of glucose L-sorbose maltose trehalose melibiose melezitose soluble starch Arabinose D-ribose ethanol | ++, be weak++ 0++ 0-+- | Sweet and pure D-glucitol salicin butanedioic acid inositol galactolipin sucrose cellobiose lactose raffinose synanthrin | -+++-+++, weak--- | The D-xylose D-R L-rhamnose glycerine ribitol D-MANNOSE Alpha-Methyl-anti-sodium chloride of D-Glucose glucoside DL-LACTIC ACID citric acid assimilation potassium nitrate | +--+0++++ negative 13-14% |
All " (Candida tropicalis) is identical for the described candida tropicalis of culture presevation handbook (Science Press, Beijing .1980) with document for the above-mentioned form that 1-18 showed, physiological and biochemical property.
With primer 5 '-GCATATCAAAAGCGGAGGAAAAG-3 ' and 5 '-GGTCCGTGTTTCAAGACGG-3 ', (method is with reference to Kurtzman C P for the 26S rDNA D1/D2 zone nucleotide sequence of polymerase chain reaction (PCR) amplification 1-18 bacterial strain, Four new Candida species from geographically diverselocations.Antonie van Leeuwenhoek, 2001,79:353-361).
The nucleotide sequence result who measures with amplified production is:
1 gccttagtag cggcgagtga agcggcaaaa gctcaaattt gaaatctggc tctttcagag
61 tccgagttgt aatttgaaga aggtatcttt gggtctggct cttgtctatg tttcttggaa
121 cagaacgtca cagagggtga gaatcccgtg cgatgagatg atccaggcct atgtaaagtt
181 ccttcgaaga gtcgagttgt ttgggaatgc agctctaagt gggtggtaaa ttccatctaa
241 agctaaatat tggcgagaga ccgatagcga acaagtacag tgatggaaag atgaaaagaa
301 ctttgaaaag agagtgaaaa agtacgtgaa attgttgaaa gggaagggct tgagatcaga
361 cttggtattt tgtatgttac ttcttcgggg gtggcctcta cagtttatcg ggccagcatc
421 agtttgggcg gtaggagaat tgcgttggaa tgtggcacgg cttcggttgt gtgttatagc
481 cttcgtcgat actgccagcc tagactgagg actgcggttt atacctagga tgttggcata
541 atgatcttaa gtcgcccgtc ttgaaacacg gacca
In GenBank nucleic acid sequence data storehouse, carry out homologous sequence search (Nucleotide-nucleotide BLAST) with this sequence.Search Results shows, the nucleotide sequence in the 26S rDNA D1/D2 zone of 1-18 all reaches more than 99% with the existing Candida tropiclis the same area nucleic acid sequence homology of GenBank.
According to can determining of above-mentioned bacterium colony outward appearance, cellular form, Physiology and biochemistry and molecular biological characteristics, the classification position of 1-18 belongs to candida tropicalis (Candida tropicalis).
Candida tropicalis 1-18 (Candida tropiclis1-18) has been preserved in (Chinese Wuhan City, Hubei Province, Chinese typical culture collection center on June 15th, 2005, Wuhan University), protect a surname's numbering: CCTCC NO:M205067, the viability of this culture detected on June 20th, 2005.Storage conditions: lyophilize or 4 ℃ of a surnames guarantor.
Candida tropicalis CCTCC:M205067 strain expanded culture of the present invention carries out as follows.
Seed culture medium is formed: wood sugar 10-20g/L, glucose 10-30g/L, yeast extract paste 5-10g/L, KH
2PO
4, 1.5-5g/L, NH
4H
2PO
4, 0.3-3g/L, MgSO
4.7H
2O, 0.1g/L, pH5-6,115C sterilization 15 minutes.Get the culture inoculation of CCTCC:M205067 test tube slant, shaking table activation culture 15 hours.Get the seed culture fluid after the activation, change fresh seed culture medium over to, continue shake-flask culture, to obtain the q.s liquid seeds by the inoculum size of 5-10% (v/v).The shake-flask culture temperature generally is controlled at 28-35 ℃, and the liquid loading amount generally is controlled at triangular flask volumetrical 10-20%.
Preparing of the hemicellulose hydrolysate substratum that the present invention is used by following method:
To be rich in the lignocellulosic material of hemicellulose such as bagasse, corn cob, straw etc. are the rare sulfuric acid of 0.3-3% (w/w) with the massfraction, or hydrochloric acid, and hydrolysis 10min-2.5h under 100--140 ℃ of condition collects liquid portion, and is referred to as hydrolyzate.
This hydrolyzate is regulated pH1-pH4 with Ca (OH) 2 or CaCO3, filters and removes precipitation, collects clear soln, and liquid is then by macroporous resin adsorption column chromatography (macro-reticular resin), to remove the wherein deleterious material of part.Collect effluent liquid under the post.The macroporous resin that is used for the hydrolyzate processing refers to that mainly with vinylbenzene, divinylbenzene be raw material, at the condition synthetic porousness cross-linked polymer of pore-creating agent existence.Macroporous resin is not with ion-exchange group, but can be divided into nonpolar, low-pole and polarity three major types.What be applicable to that hemicellulose hydrolysate handles mainly belongs to low-pole or polar macroporous resin.The trade mark of the resin of this type of that can obtain on the market such as DM130, HPD500, NKA9, AB-8, S-8 etc.
The hydrolyzate that macroporous resin treatment is crossed is regulated pH to 5-7 with alkali, vacuum concentration to soluble solid content to 20-60%, promptly get hemicellulose hydrolysate.The alkali that this stage uses can be KOH, NaOH, Ca (OH)
2, NH
4OH or CaCO
3, the precipitation of regulating the generation of pH process should remove by filter.
Hemicellulose hydrolysate is added the hot water extract of rice bran or wheat bran, and regulate after the pH to 5-7, just become the hemicellulose hydrolysate substratum that is used for xylitol fermentation.The consumption of rice bran or wheat bran hot water extract in the substratum is about as much as adding 20-150g rice bran or wheat bran in every liter of substratum.Xylose concentration in the hydrolyzate substratum generally is adjusted between the wood sugar 100-300g/L, and optimum concentration is wood sugar 150-200g/L.
Belong to the continuous hemicellulose hydrolysate of CCTCC:M205067 of the present invention and produce the Xylitol process, under following method and condition, realize.
1. the fresh seeds liquid with gained behind the CCTCC:M205067 strain expanded culture inserts fresh hydrolyzate substratum, in the fs, regulate air flow and stirring velocity, the dissolved oxygen of fermentation liquid horizontal dimension is held in more than 20% (with respect to the oxygen in the atmosphere), when the yeast cell of fermentation system is bred to higher density (after the stem cell＞10g/L), reduce rotating speed and air flow, make the dissolved oxygen of fermentation system be controlled at the low dissolved oxygen level of 0.1-5% (to the oxygen in the atmosphere) always.Wood sugar in matrix is exhausted, and finishes fermentation.
2. first fermentation ends is separated fermented liq with the CCTCC:M205067 yeast cell.The fermented liquid of removing cell is used to prepare crystalline xyhose alcohol, and whole cells of separated and collected then join in the fresh hydrolyzate substratum, begin second batch fermentation.Xylose concentration in the substratum, the prescription of substratum, the liquid amount in the fermentor tank, should with preceding a collection of being consistent.And from second batch, all utilize the fermentation of reclaiming yeast cell, and the dissolved oxygen level of substratum is controlled between the 0.01-5%.
3. after the conversion of the wood sugar in the substratum is exhausted, isolate yeast cell, join in the fresh hemicellulose hydrolysate substratum, the fermentation of a beginning new round, circulation can not utilize until yeast cell so repeatedly.
4. during the fermentation, after the glucose in the hydrolyzate nutrient solution exhausted, reply nutrient solution continuous flow added glucose.Press the volumeter of nutrient solution, the stream rate of acceleration is glucose 0.5-5g/L.h.
5. the temperature of fermenting process is controlled at 30-36 ℃ (the suitableeest 32-34 ℃), and the pH value of substratum is controlled between the 4.0-7.0.
The present invention compared with prior art, outstanding substantive distinguishing features and the obvious improvement that are had are:
At first, the present invention can directly utilize cheap hemicellulose hydrolysate as fermented substrate, with very high product generating rate (Xylitol 5.0g/L.h), reaches very high production concentration (Xylitol 180g/L).These two important index levels that super prior art reached far away.
Secondly, in the hemicellulose hydrolysate substratum of the present invention, except that hydrolyzate, it is big to replace in the existing research usage quantity with the rice bran of cheapness or hot water extract, and expensive yeast extract paste, this has not only significantly reduced the cost of substratum, and fermention medium lacks the very dark yeast extract paste of color and luster, and tunning Xylitol decolorizing and refining process also becomes relatively easy.
The 3rd,, and realize under the condition of high production concentration and high product generating rate that the present invention still can maintain the characteristics of product yield height (Xylitol 0.9g/ wood sugar g) at above-mentioned simplification substratum.This also is that existing technology institute is irrealizable.
The 4th, because the cell of used yeast bacterial strain of the present invention has good resistance to poison, in special purified hemicellulose hydrolysate, yeast cell can utilize 10 batch fermentation continuously, time length reaches under hundreds of hours the condition, and the formation efficiency of tunning Xylitol does not subtract.This is that prior art institute is irrealizable equally.
The 5th, because yeast cell of the present invention can be recycled for a long time, the actual cultivation cost of yeast cell is very low.And, except the first time cell cultivation process need the aseptic technique, in over a long time the process of continuously fermenting after this, yeast cell is in absolute high-density advantage state all the time, so the hydrolyzate substratum need not sterilization, whole process also need not aseptic technique, has saved the sterilization cost of substratum and the aseptic technique cost of fermenting process significantly.
Description of drawings
Description of drawings the bacterial strain grown form of this aspect.
Fig. 1 is the typical yeast cell form that 1-18 bacterial strain of the present invention is had;
Fig. 2 is that the 1-18 bacterial strain is adding the synoptic diagram that forms a large amount of pseudohyphas on the Corn Meal Agar substratum of cover glass.
Shown in Figure 1, under the Liquid Culture condition, 1-18 cell outward appearance is circular to oval, belongs to typical yeast cells form; The breeding of sprouting belongs to typical yeast cells divisional mode. Culture medium contains: wood sugar 100g/L, yeast extract 10g/L, KH2PO
4，1.5g/L，NH
4H
2PO
4，3g/L，MgSO
4.7H
2O, 0.1g/L, pH5-6,115C sterilization 15 minutes. After fluid nutrient medium inserted the 1-18 slant strains, 30 ℃, shaking flask was cultivated 15h. Centrifugal collection thalline is in slide coating and be scattered in the distilled water photomicrograph. (400 *)
Can see among Fig. 2 that 1-18 forms a large amount of pseudohyphas at the corn meal agar culture medium that is adding cover glass, on the mycelia with blastoconidium. Corn flour 50g/L, agar 20g/L makes flat board after the sterilization. Plate streaking inoculation 1-18 cell covers a cover glass in streak inoculation place thereupon, cultivates 3 days for 30 ℃. Take off cover glass, to being attached to the pseudohypha photomicrograph on the cover glass. (400 *).
Embodiment
Following example is for specific implementation process of the present invention is described.
Embodiment 1
Seed culture medium is formed: wood sugar 15g/L, glucose 15g/L, yeast extract paste 10g/L, KH
2PO
4, 1.5g/L, NH
4H
2PO
4, 3g/L, MgSO
4.7H
2O, 0.1g/L, pH5-6,115C sterilization 15 minutes.Wood sugar separates sterilization with other composition, 250ml triangular flask loading amount 30ml.Get the culture inoculation of CCTCC:M205067 test tube slant, 15 hours activated seeds of 30 ℃ of shake-flask culture.Get the seed culture fluid after the activation, change fresh seed culture medium over to, continue shake-flask culture 15h, obtain the liquid seeds of about 60g fresh cell/L by the inoculum size of 5% (v/v).
Embodiment 2
Dry bagasse 40kg throws in the 500L enamel hydrolytic decomposition pot, adds 2.5% sulfuric acid (w/v) 300L, directly feeds steam, is heated to 120 ℃ of hydrolysis 2.5h.Emit a jar interior hydrolyzed solution, washing residue once.Merge solution, the CaCO3 neutralisation of sulphuric acid, the elimination calcium sulfate precipitation, solution decompression is concentrated into soluble solid content 10%, by the HPD500 macroporous adsorbent resin column chromatography, and through activated carbon treatment, at last again with solution concentration, must can be used for fermentative preparation Xylitol, the hydrolyzate syrup of the about 300g/L of wood sugar content.The macroporous adsorbent resin column chromatography poison-removing method, (biotechnology journal, 2002,18 (6): 725-728) introduction is arranged in the article that the inventor has delivered.
Embodiment 3
Exsiccant corn cob 1000g is ground into the particle of the about 1-5mm of diameter, adds water 10kg, boils 30min, and the elimination water cooking liquid repeats poach once.The corn cob of elimination water cooking liquid adds 2.5% (w/v) sulfuric acid 8L, 120 ℃ of hydrolysis 2.5h..Collect hydrolyzed solution, residue with poach once.Merge solution twice, CaCO
3Neutralisation of sulphuric acid, elimination calcium sulfate precipitation, solution decompression are concentrated into soluble solid content 10%, by the HPD500 macroporous adsorbent resin column chromatography, and through activated carbon treatment, are evaporated to wood sugar content at last and reach 300g/L.This hydrolyzed solution is directly used in the fermentative preparation Xylitol.
Embodiment 4
Bagasse hydrolyzate with embodiment 2-1 preparation mixes with the rice bran water cooking liquid.The consumption of rice bran water cooking liquid is equivalent to rice bran 50g/L, the about 200g/L of mixed xylose concentration.250ml triangle loading amount 60ml,, insert CCTCC:M205067 seed liquor 3.0ml, 33 ℃, the 200rpm shaking table is cultured to wood sugar and is exhausted.Centrifugation thalline and fermented liquid.Measure the Xylitol concentration in the fermented liquid, collected thalline then is transferred in the fresh substratum of 60ml.So constantly play circulation.Continuous 10 crowdes fermentation result shows (table 1), and total fermentation time reaches 413.5h, cell density, Xylitol generating rate, the equal kept stable of the transformation efficiency of Xylitol.10 batch fermentations drop into wood sugar 120g altogether, generate Xylitol 97.7g, average tree sugar alcohol generating rate 4.0 (g/L.h), and transformation efficiency Xylitol 0.814g/ wood sugar g, the Xylitol weight that every gram cell generates reaches more than the 81.9g.
| Parameter | Lot number | |||||||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
| Fermentation time (h) | 58.5 | 45 | 44 | 42 | 39 | 40.5 | 37 | 37.5 | 34 | 36 |
| Cell density (g/L) | 10.25 | 18.18 | 19.80 | 20.50 | 21.00 | 21.00 | 21.5 | 22.1 | 22.1 | 22.1 |
| Xylitol (g/L) | 156.5 | 158.0 | 160.5 | 163.5 | 162.5 | 163.0 | 163.5 | 163.0 | 168.3 | 167.3 |
| Transformation efficiency (g/g) | 0.78 | 0.79 | 0.80 | 0.82 | 0.81 | 0.82 | 0.82 | 0.82 | 0.84 | 0.84 |
| Generating rate (g/L.h) | 2.68 | 3.51 | 3.65 | 3.89 | 4.16 | 4.02 | 4.42 | 4.34 | 4.95 | 4.65 |
Embodiment 5
The bagasse hydrolyzed solution of embodiment 2-1 preparation also mixes with the rice bran water cooking liquid, regulate xylose concentration to 200g/L, be added in German Bei Lang (B.Bruan.Biotech International) fermentor tank 40L fermentor tank loading amount 27L, inoculation CCTCC:M205067 seed liquor 2L.In the postvaccinal 9h, stir speed (S.S.) progressively is increased to 600r/min by 200r/min, and air flow progressively is increased to 1.5vvm by 1vvm, keeps dissolved oxygen level at (with respect to the oxygen in the atmosphere) more than 20%.After fresh cell density reaches 80g/L, reduce rotating speed to 300r/min, air flow is reduced to 0.5vvm, keeps dissolved oxygen level between 0.01-0.1%, starts charge pump simultaneously, regulates revolution speed, by the speed of 0.5g/L.h at the uniform velocity in the fermentor tank stream add glucose.Regulate with weak ammonia and to keep substratum PH5, the cumulative volume of seed solution, ammonia soln and glucose solution about 3L, the xylose concentration that makes the substratum terminal point about 200/L, cumulative volume 30L.
Fermentation ends leaves standstill 4h naturally, extracts supernatant liquor (about 26-27L) out with pump and is used for detecting and the preparation crystalline xyhose alcohol.
After leaving standstill, yeast cell is sunken to the fermentor tank bottom, the fresh culture (lacking about 1 liter) that adds the amount of the fermented liquid that is slightly less than extraction than the fermented liquid of extracting out, air flow remains at 0,5vvm, mixing speed is 300r/min all the time, and the dissolved oxygen level in the fermentation system about about 0.5% (with respect to atmosphere) greatly, weak ammonia is regulated substratum PH5.Behind the 10h by the speed of 0.5g/L.h at the uniform velocity in the fermentor tank stream add glucose to the wood sugar in the substratum and exhaust, begin the fermentation of a new round then.
Continuous 5 batches fermentation result such as table 2.As seen, because the cell density in the fermentor tank increases, the product generating rate of fermentation also significantly improves.Especially, owing to add glucose, the Xylitol transformation efficiency of fermentation has all reached more than 0.90.
Table 2
| Parameter | Lot number | ||||
| 1 | 2 | 3 | 4 | 5 | |
| Fermentation time (h) | 48 | 39 | 36 | 36 | 36 |
| Cell density (g/L) | 28.5 | 28.4 | 28.4 | 26.78 | 28.8 |
| Xylitol (g/L) | 170.1 | 176.6 | 178.4 | 180.3 | 180.1 |
| Transformation efficiency (g/g) | 0.85 | 0.88 | 0.89 | 0.90 | 0.90 |
| Generating rate (g/L.h) | 3.54 | 4.52 | 4.95 | 5.01 | 5.00 |
Embodiment 6
Adopt the corn cob hydrolyzate of the preparation among the embodiment 2 to mix with the wheat bran water cooking liquid, the consumption of wheat bran water cooking liquid is equivalent to wheat bran 50g/L substratum, mixes the about 200g/L of xylose concentration of back substratum.250ml triangle loading amount 60ml,, insert seed liquor 3.0ml, 33 ℃, the 200rpm shaking table is cultured to wood sugar and is exhausted.Centrifugation thalline and fermented liquid.Measure the Xylitol concentration in the fermented liquid, collected thalline then is transferred to 60ml, in the fresh substratum.All the other circulating fermentation conditions are with the invention process 3, and continuous 10 crowdes fermentation result shows.
Table 3
| Parameter | Lot number | |||||||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
| Fermentation time (h) | 60 | 43 | 48 | 48 | 48 | 48 | 50 | 52 | 52 | 51 |
| Cell density (g/L) | 15.54 | 15.26 | 18.08 | 19.25 | 20.19 | 20.8 | 21.5 | 20.9 | 21.8 | 21.5 |
| Xylitol (g/L) | 143.7 | 152.7 | 158.47 | 158.59 | 160.3 | 160.7 | 164.3 | 166.3 | 165.4 | 165.2 |
| Transformation efficiency (g/g) | 0.72 | 0.76 | 0.79 | 0.79 | 0.80 | 0.80 | 0.82 | 0.83 | 0.83 | 0.83 |
| Generating rate (g/L.h) | 2.40 | 3.55 | 3.30 | 3.30 | 3.34 | 3.35 | 3.29 | 3.20 | 3.18 | 3.24 |
Claims (5)
1, a kind of production bacterium of the hemicellulose hydrolysate substratum production Xylitol that can ferment, it is characterized in that: it is the candida tropicalis bacterium, latin name is: Candida tropicalis, from wood sugar factory environment waste, gather to separate and obtain, lyophil preservation or 4 ℃ of refrigerations, it is the Chinese typical culture center that is positioned at Chinese Wuhan City, Hubei Province that bacterial strain send depositary institution, protects a surname CCTCC:M 205067.
2, a kind of production bacterium of the hemicellulose hydrolysate substratum production Xylitol that can ferment, it is characterized in that: the nucleic acid sequence identity that its 26S rDNAD1/D2 zone is had all reaches more than 99% with the existing Candida tropiclis the same area nucleic acid sequence homology of GenBank.
3, the method for cultivation of the candida tropicalis bacterium of production Xylitol as claimed in claim 1, it is characterized in that: the test tube slant culture of heat-obtaining band candidiasis CCTCC:M 205067 inserts and the general liquid nutrient medium of candida tropicalis, shaking table is cultivated and was obtained activated seed liquid in 10-15 hour, the consumption of content 5-10% inoculum size by volume, activated seed liquid is changed in the fresh seed culture medium, continue shake-flask culture and promptly obtain being used for the yeast starter that Xylitol is produced.
4, the method for production bacterium production Xylitol as claimed in claim 1, it is characterized in that: the process that strain fermentation is produced Xylitol is: the fresh hydrolyzate substratum of fresh seeds liquid access of gained behind the CCTCC:M205067 strain expanded culture is fermented, regulate air flow and stirring velocity, leavening temperature 30-36 ℃, pH is controlled at 4.5-7, the dissolved oxygen level control 0.1-5% of fermentation system, the wood sugar in matrix is exhausted, and finishes fermentation; After first fermentation ends, fermented liq is separated with the CCTCC:M205067 yeast cell, the fermented liquid of removing cell is used to prepare crystalline xyhose alcohol, and the cell of separated and collected then joins in the fresh hydrolyzate substratum, begins second batch fermentation, after the conversion of the wood sugar in the substratum is exhausted, isolate yeast cell, join in the fresh hemicellulose hydrolysate substratum, the fermentation of a beginning new round, circulation can not utilize until yeast cell so repeatedly.
5, the method for production bacterium production Xylitol according to claim 2, it is characterized in that: the hydrolyzate substratum that strain fermentation is produced Xylitol is to be basal component with the hemicellulose hydrolysate, is additive with rice bran or wheat bran hot water extract;
The preparation of the wood fibre hydrolysis thing of described hemicellulose is, adding massfraction in lignocellulosic material is dilute sulphuric acid or the hydrochloric acid of 0.3-3%, hydrolysis 15min-2.5h under 100-140 ℃ of condition, obtain hydrolyzate, under the condition of pH1-7, pass through macroporous resin, collect effluent liquid under the post, vacuum concentration to soluble solid content to more than the 20-60%, promptly get hemicellulose hydrolysate; The hot water extract that adds rice bran or wheat bran in the hemicellulose hydrolysate obtains being used for the hemicellulose hydrolysate substratum that xylitol fermentation is produced; Described rice bran or wheat bran hot water extract consumption are the whole hot water leachables that add 20-150g rice bran or wheat bran in every liter of substratum.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101497904B (en) * | 2008-02-01 | 2011-11-09 | 唐传生物科技（厦门）有限公司 | Method for producing xylitol and arabinose at the same time |
| CN101497903B (en) * | 2008-02-01 | 2011-12-07 | 唐传生物科技（厦门）有限公司 | Method for selectively converting and shunting biological products |
| US20120021467A1 (en) * | 2010-07-22 | 2012-01-26 | Thomson Biotech (Xiamen) PTE. LTD. | Method of producing xylitol and arabinose at same time from hemicellulose hydrolysates |
| CN102943050A (en) * | 2012-11-13 | 2013-02-27 | 北京理工大学 | Method for producing xylitol by recombined candida tropicalis |
| CN101914591B (en) * | 2009-01-16 | 2013-05-01 | 唐传生物科技（厦门）有限公司 | Application of candida tropicalis to preparation of xylitol and application of high-purity xylitol product to pharmacy and health care |
| CN107022579A (en) * | 2017-05-08 | 2017-08-08 | 浙江工业大学 | The method that xylitol is prepared using bamboo shoot process discarded object |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101497904B (en) * | 2008-02-01 | 2011-11-09 | 唐传生物科技（厦门）有限公司 | Method for producing xylitol and arabinose at the same time |
| CN101497903B (en) * | 2008-02-01 | 2011-12-07 | 唐传生物科技（厦门）有限公司 | Method for selectively converting and shunting biological products |
| CN101914591B (en) * | 2009-01-16 | 2013-05-01 | 唐传生物科技（厦门）有限公司 | Application of candida tropicalis to preparation of xylitol and application of high-purity xylitol product to pharmacy and health care |
| US20120021467A1 (en) * | 2010-07-22 | 2012-01-26 | Thomson Biotech (Xiamen) PTE. LTD. | Method of producing xylitol and arabinose at same time from hemicellulose hydrolysates |
| CN102943050A (en) * | 2012-11-13 | 2013-02-27 | 北京理工大学 | Method for producing xylitol by recombined candida tropicalis |
| CN107022579A (en) * | 2017-05-08 | 2017-08-08 | 浙江工业大学 | The method that xylitol is prepared using bamboo shoot process discarded object |
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