CN101490235A

CN101490235A - Improved brewing process

Info

Publication number: CN101490235A
Application number: CNA2007800265513A
Authority: CN
Inventors: 明-达姆·源; 路泊·埃登斯; 叶罗恩·路易斯·洛恩·范
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2006-07-13
Filing date: 2007-06-22
Publication date: 2009-07-22
Also published as: CA2906891A1; CA2906891C; ES2642867T3; BRPI0713856A2; DK2041256T3; PT2402425T

Abstract

The present invention relates to the use of a proline-specific protease for accelerating the beer brewing process. In particular, it relates to the use of a proline-specific protease for accelerating the beer brewing process by shortening and simplifying the stabilisation phase of beer production. The stabilisation period as such can be omitted from the beer making process hereby saving significant costs and adding to the flexibility of beer producing plants.

Description

Improved fermentation process

Technical field

The present invention relates to the method for beer fermentation.Particularly, the present invention relates to quicken the method for fermentation beer, described method relates to proline specific protease.

Background technology

In the traditional beer manufacture method, with the wort of cereuisiae fermentum inoculation, and under the top temperature between 8-15 ℃, carry out about 7 days fermentation by the acquisition of extraction Fructus Hordei Germinatus, top temperature depends on uses the still warm fermentation process of cold fermentation method.This fermentation is also referred to as primary fermentation.In case fermentation is finished, most of fermentable sugar changes when having turned to alcohol, is settled out yeast.Under the situation of storage (larger) beer, yeast is collected in the fermentation bottom, and in other beer, they are collected at the fermentation top." prematurity " beer that derives from this primary fermentation still contains some unprecipitated yeast and the high-caliber relatively odour component of not expecting, particularly diketone such as diacetyl and ethanoyl aldehyde.

These odour components of not expecting afterwards are the importance in beer stage of maturity subsequently to tasteless conversion of compounds.Especially, with diacetyl (compound) with butter peculiar smell but being reduced to the 3-oxobutanol is time-consuming very important technology.In traditional beer technology, this maturing step carries out an about week.In the brewage stage (so-called steady stage) subsequently, keep beer to promote the formation of protein polyphenol aggregate, remove sedimentary aggregate then.These protein polyphenol aggregates are formed by polyphenol with from proline rich (so-called muddy active (haze the active)) protein of Fructus Hordei Germinatus.In the steady stage, by beer is cooled to 0 or even-2 ℃ about 7 to 10 days so that these aggregates are assembled and with postprecipitation.(54-62), back this cold stationary stage is indispensable for Narziss, ferment.3 L.1990 according to Narziss.After removing sedimentable matter, remove unprecipitated polyphenol and/or muddy active protein usually, to prevent in packaged products, forming " ice-cold muddiness ".For reaching this purpose, can remove residual not precipitation polyphenol on the polyvinylpolypyrrolidone (PVPP) by being adsorbed on, and/or can remove residual muddy active protein on the silica gel by being adsorbed on.Can pass through diatomite filtration afterwards, come along and remove containing the PVPP of the polyphenol of absorption and/or muddy active protein and/or silica gel respectively, and remove the yeast of any remnants.

In a word, above-mentioned traditional beer manufacture method is about 20 days consuming time.The product that obtains is fully limpid and stable, does not change thereby visible can not take place during its quality guaranteed period.

For the adaptability that improves distillery with save cost of capital, carried out extensive work in recent years so that the fermentation in the brewing method, maturation and steady stage the shortest (Narziss, L.1990ferment.3,54-62).For example in " pressure fermentation " method, be increased to about 14-18 ℃ by fermenting usually with maturing temperature and quicken fermentation and ripe.In the method, fermentation adds and ripe finished in 7 day period.Whole manufacturing process (promptly comprising stable) significantly foreshortens to and was slightly larger than for 2 weeks.Yet the beer that obtains may develop and slight yeast taste and the relatively poor taste grade of taste generally.

In popular alternative approach (so-called " cold fermentation-Wen maturation " method), obtain taste beer preferably by adhering to traditional low temperature fermentation process.Obtain desired shorter process period by the temperature in stage of maturity being brought up to 18 to 22 ℃.As a result, the prematurity taste of young beer was overcome in 2 to 3 day time period.Obtain beer stability by traditional method.Although the beer that obtains has splendid quality, the energy expenditure of this method is higher relatively.

In quickening the sophisticated other method of beer, immature beer is carried out centrifugal completely, to remove yeast, make it then through containing the highdensity zymic bio-reactor that is fixed.The combination anaerobism is heated to 90 ℃ step, and diacetyl all in 1-2 hour time period all are reduced to the 3-oxobutanol.In addition, also stablize the beer of this fast-ripenin with common cold shelf time and PVPP or silica gel treatment.Yet this bioreactor process is easy to produce the yeast peculiar smell, and owing to used sophisticated technologies equipment, the assets cost is higher probably.

In addition, introduce enzyme newly developed (it added in the beer fermentation stage) and just beginning that manufacturing exerts an influence to beer.In nearest method, use the proline specific endo-protease to prevent the formation (EP 1 326 957) of freezing muddiness as the substitute of PVPP or silica gel treatment.During beer fermentation, the protein of the muddy activity of this enzyme selectivity ground hydrolysis, proline rich, thereby the precipitation of prevention protein-polyphenol complex.It is unnecessary that the advantage of having reported of this method is that PVPP or silica gel become, because this enzyme can effectively prevent freezing muddiness.In addition, the stage of this method this final sum most fragile in beer processing has produced the procedure of processing of significantly being simplified, and the length of brewage process is had no significant effect.In fact, between PVPP and the polyphenol and/or the reaction between silica gel and the muddy active protein almost be instantaneous.Therefore, remove PVPP or silica gel treatment and can not cause producing shorter brewage process.In another enzymatic means, in (pitched) wort that tilts, add alpha-acetolactate decarboxylase.This interpolation prevents to form diacetyl in beer fermentation, thereby the ripening stage (its main purpose is to reduce the diacetyl level) can be reduced 2 to 3 days (with reference to US 5,108,925 and US 4,708,875).

Although exist many about shortening the report in the ripening stage that beer makes, up to now not about the report of the feasible method of acceleration beer stationary phase.Yet this stationary phase that relates to the considerable energy input of considerable turnout and needs is usually 0 ℃ of week consuming time.Further shorten and simplify the beer manufacturing processed and obviously have great economics importance.The manufacturing processed that this class shortens and simplifies will be strengthened the adaptability of fermentation plant, thereby reduce fixed cost and labor cost.

Summary of the invention

Be used for the purposes that (preferably by shortening shelf time) quickens the brewage process according to the invention provides proline specific protease.

The present invention also provides:

-being used to quicken the method for brewage process, described method comprises makes beer when having proline specific protease, and wherein shelf time is shortened; With

-being used to make the method for beer, described method comprises fermenting wort when having proline specific protease, is stage of maturity and steady stage afterwards, wherein the steady stage continuity is less than 7 days.

In addition, the invention provides beer that can obtain by the inventive method and the bottle that contains described beer, bucket or jar.

Summary of drawings

Fig. 1 has shown by PCS and has disperseed the function of intensity to granular size at the accumulation stdn of measuring from the beer in age in October of IFBM pilot plant.

Detailed Description Of The Invention

The present invention relates to the purposes that proline specific protease is used for accelerating Process of Beer Brewing. Particularly, the present invention relates to proline specific protease and accelerate the purposes of Process of Beer Brewing by the stabilization sub stage of simplifying Brewage.

Stabilization sub stage also can carry out under the temperature higher than traditional fermentation method. Therefore the present invention relates to a kind of method effectively, and wherein the fermentation after-stage of brewing process is compared with art methods and is shortened and/or carries out under the temperature higher than art methods. Therefore, the invention still further relates to for the method for accelerating Process of Beer Brewing, this comprises and carries out the method when having proline specific protease. Compare with the traditional fermentation method, the stabilization sub stage in the method can be shorter and/or carries out under higher temperature.

There is the lot of beer brewing method to be used in the world. All these methods comprise following steps basically at least, or the stage corresponding with it yet typically:

Fermentation

Ripe

Stable

Filter (yet certain methods is not used filtration)

Described method randomly comprises the further additional step that improves stability (as the quality guaranteed period).Any known technology can be used for this purpose, for example uses polyvinylpolypyrrolidone (PVPP) and/or silica gel treatment.Typically, this class additional step can carry out between stable and final filtration stage, although this class step can be carried out in some other parts of described method.

The details of each procedure of processing is may be quite different in different methods, and, carry out the brewage process aspect, those skilled in the art have its oneself definition to each stage.Obscure for fear of any, in the context of the invention, term fermentation, ripe, stable, PVPP and silica gel treatment and filtration be intended to specification sheets in disclosed identical.Attention: in certain methods, fermentation is also referred to as primary fermentation.Fermentation stage is to be intended in the brewage by the yeast that adds obtainable sugar-fermenting is the pure stage.Stage of maturity is also referred to as secondary fermentation, and its odour component (as diketone) that is intended to not expect is converted into the better composition of taste.Stable phase is intended to promote the formation of polyphenol-protein aggregate and makes their precipitations.If use PVPP and/or silica gel treatment, then it is intended to remove respectively unprecipitated polyphenol and muddy active protein, so that packaged shelf life of products is more stable.At last, if use filtration step, then it is intended to remove yeast, PVPP and/or the silica gel of sedimentary polyphenol and muddy active protein, any remnants before packing.

Usually, during brewage, can detect quantity of parameters.Can be by for example measuring the density of the beer that is preparing, the end of fermentation stage is determined in the minimizing (as amount of glucose) of definite extract that can ferment.The end of fermentation stage is considered to the beginning in stage of maturity usually.Typically, can determine the end in stage of maturity and the beginning of steady stage thus by measuring the diacetyl quantity that exists in the beer.Yet this can change according to the type of required beer in the practice.For example, in some are brewageed, think that in case ortho position diketone level is lower than about 0.10mg/l the stage of maturity finishes, and in other is brewageed, think that promptly the stage of maturity finishes in case ortho position diketone level is lower than about 0.05mg/l.Therefore, the technician can define the end in stage of maturity and the beginning of steady stage according to desired ortho position diketone level.

In the context of the present invention, the beginning of steady stage can be defined as: when measuring according to EBC method 9.24.1 Vinical Diketones in Beer:Spectrophotometric Method, ortho position diacetyl level is lowered to and is less than the moment that about 0.01mg/ rises.In the context of the invention, the end of stationary phase is defined as: beer is carried out moment of its final filtration step, perhaps, and when PVPP and/or silica gel treatment are brewing method a part of, the moment that beer is contacted with PVPP and/or silica gel.If this method is not carry out filtering method, then the end of stationary phase is defined as the packaged point of beer.

According to one embodiment of the invention, the stable time that needs can be shortened to being less than about 7 days.Preferred its is less than 6,5,4 or 3 days.More preferably, it is shortened to being less than 2 or 1 days.Most preferably, it is shortened to following degree: in the manufacturing scale, the stable time length is reduced to the period that equals beer was cooled to from the stage of maturity temperature required (for example beer filtration and/or pack required temperature).Be called cooling period this period traditionally.It is highly favourable will foreshortening to stationary stage beer is cooled to the temperature required time, because the time length of steady stage is only depended on obtainable cooling power like this.

Traditionally, will be cooled to from the beer in ripe stage from-2 ℃ high about 0 ℃ approximately to (comprising).We surprisingly find: when using according to proline specific protease of the present invention, the steady stage can not only be shortened, and it also can carry out under the temperature that is higher than use traditionally.The stabilising method of carrying out are arranged traditionally, but these methods to expend the longer time under higher temperature, for example 6 weeks or more.Therefore, in another embodiment of the present invention, carry out preferably being cooled on about 2 ℃ at most beer stationary phase (it can typically be as defined above the stationary phase that shortens), more preferably at most about 3 ℃, 4 ℃, 5 ℃ or 6 ℃ or even more preferably to about 7 ℃ or about 8 ℃ at most, most preferably to the preferred temperature that is used for packing (i.e. bottling or barrelling).

Pack required temperature can be between method difference, but preferably at the most and comprise 8 ℃, preferably about 7 ℃, carry out, thereby obtain the carbon dioxide dissolved of the level wanted in the beer.Only be cooled in the temperature required method of packing at beer, compare the energy that needs much less with means known in the art.

In a kind of embodiment preferred according to the present invention, the temperature the when steady stage is reduced to beer finished from the stage of maturity is cooled to packing temperature required required period, thereby has omitted traditional cold stationary phase.

Therefore according to the present invention, beer can be cooled to about 2 ℃ at most, more preferably to about 3 ℃, 4 ℃, 5 ℃ or 6 ℃ at most or even more preferably to about 7 ℃ or about 8 ℃ at most, most preferably be cooled to the preferred temperature that is used to pack.Then can be directly beer packed, in this embodiment promptly, it is no longer kept section any time the temperature that has been cooled at beer.

In the present invention, if necessary, can carry out other procedure of processing to reach extra clarity and/or stability.In fact, if use, then can need with for example PVPP and/or silica gel treatment beer corresponding to the stationary phase that beer is cooled to the shortening of packing temperature required needed period.This can assist in ensuring that the quality guaranteed period stability of prolongation.

The industrial application of this embodiment is: can be omitted stationary phase from method for preparing beer, thereby significantly save cost, and strengthen the adaptability of beer manufacturing works.

The beer stable according to the present invention can be further processed, to reach clarity and the level of stability that product can be sold.This uses extra treatment step to finish usually.Also can use filtration step.This normally packs last preceding step in the brewing process.

Any known technology can be used for this purpose.For example, described extra clarification and/or stabilizing treatment can be finished by using suitable adsorption treatment, the processing of for example using PVPP, silica gel, Nutgalls tannin (gallotannins) or the crosslinked insoluble agarose of fixed to carry out.The purposes of these processing and their those skilled in the art that base on practicality in brewageing know.PVPP is usually to add from about quantity of 10 to about 70g/hl.Silica gel is usually to add from about quantity of 10 to about 70g/hl.

The example of the crosslinked insoluble agarose of fixed comprises WO97/43401 and US 6,001,406 described combinative stability systems, (consult Tayler etal. with GE Health Care Bioscience AB, 2006, Use of the Combined Stabilisation System and its impact on beercomposition, Proceedings of the Institute of Brewing and Distilling, Asia PacificSection, Hobart, Australia) the combinative stability system resin of Zhi Zaoing.Perhaps, or in addition, can use enzyme to handle (as handling) to reach further stable and/or clarification with papoid.

Above-mentioned processing can be used with consolidated form (as fixed PVPP particle).

All above listed extra process can be united use with stabilization process of the present invention.The combination of extra process (as two, three, four or all these class extra process) can be united use with stabilization process of the present invention.

In case beer is stabilized according to the present invention, can carry out extra clarification of this class and/or stabilizing treatment expediently.Yet the invention still further relates to following method: wherein extra clarification and/or stabilizing treatment other point in the method carries out, or carries out synchronously with of the present invention stablizing.

In a kind of embodiment preferred of the present invention, use as the disclosed proline specific endo-protease of EP-A-1326957, thereby it is unnecessary to make that PVPP and silica treatment become, and it is unnecessary to make that the intrinsic cost be used for the PVPP reclaim equiment and the labor cost relevant with this device of operation become.In addition, self resistance of oxidation of beer is enhanced, and the gluey stability in the edge of the beer of many PVPP or silica gel treatment (marginal colloidal stability) has obtained promotion.In addition, the oxygen exposure of this beverage is thoroughly reduced, and waste liquid stream is minimized.

In a kind of embodiment preferred of the present invention, the shortening of steady stage or even omission and the shortening in stage of maturity (this stage main purpose is diacetyl is converted into 3-hydroxyl butanols) be used in combination.In the context of the invention, the beginning in stage of maturity is defined as below: about 70% to about 90% of assessed value the moment, more preferably from about 77% to about 84% the moment when apparent attenuation degree reaches use beer density measurement.Beer density can be measured by specific gravity flask or densometer (being respectively EBC method 8.2.1 and 8.2.2), and the decay limit is determined according to EBC method 8.6.1 or 8.6.2 (Fermentability, Attenuation of wort).In the context of the invention, the end in stage of maturity can be defined as: when measuring according to EBC method 9.24.1 (Vicinal Diketones inBeer:Spectrophotometric Method), the diacetyl level is reduced to and is less than the moment that about 0.01mg/ rises.Can shorten the stage of maturity by means known in the art.For example, can use the high temperature accelerates maturing, as described in " pressurization fermentation " and " cold fermentation-Wen maturation " method (Narziss, Ferment L.1990,3,54-62).Also can be by using the yeast that is fixed in the bio-reactor, or by using the acetolactate decarboxylase activity to shorten maturation (ALDC:EC 4.1.1.5; Consult for example US 5,108,925 or US 4,708,875).Use one of these methods or its combination maturation can be restricted to two days, one day or even less than one day (feasible) as the bio-reactor that uses operation at high temperature.

In the context of the invention, the maturation of integration beer manufacturing and the stage of steady stage are called shelf time.

Therefore, the invention provides the purposes that proline specific protease is used to quicken the brewage process.Typically, quicken to finish period, for example finish, preferably finish by shortening stationary phase by shortening the storage time length by shortening the fermentation back.

Term " acceleration " and " shortening " are intended to represent in this paper context compare with the equivalent method that does not use proline specific protease and expend the beer-brewing method that shorter time is finished.Quicken usually to take place as the result in period after the fermentation of shortening (promptly when the use proline specific protease, compare, can expend this period still less the time finishes) with the equivalent method that does not use this enzyme.

If purposes of the present invention is used in combination with the means that shorten the ripening stage, whole shelf time can be restricted to and be less than 8,7 or 6 days.Preferred its is restricted to and is less than 5,4 or 3 days.More preferably, it is restricted to and is less than 2 days.Even more preferably, whole shelf time is restricted to and is less than 24 hours.

In a kind of embodiment preferred of the present invention, can be by combination proline specific protease and acetolactate decarboxylase (as the α acetolactate decarboxylase), shelf life is made as 4 days or still less.

In a kind of embodiment preferred, in primary fermentation, add proline specific protease and optional acetolactate decarboxylase.Behind this primary fermentation, can carry out the fast-ripenin step to immature beer, and can be subsequently reduce to be less than and after about 0.10mg/ rises beer is cooled to about 2 ℃ at once in the diacetyl level, preferably to about 5 ℃, more preferably to about 7 ℃, most preferably, randomly filter then to obtain clarifying beer in order to bottling to the temperature that is suitable for packing.Can realize thus to the fermentation latter stage and to the acceleration of whole brewing process.

The advantage of the inventive method is applied to the lager beer of top and submerged fermentation.The beer fermentation of top fermentation and ripe very fast, but identical with Lager, they all need the very long cold storage phase to remove polyphenol-protein complexes.The advantage of the inventive method is specially adapted to the submerged fermentation lager beer.Purposes of the present invention and method also can be applicable to carry out the beer of spontaneous fermentation.

The present invention relates to the method for using proline specific protease of the present invention on the other hand.That is to say, the invention provides the method that is used to quicken the brewage process, this is preferably undertaken by the time length (for example by reducing the time length of stationary phase) that reduces shelf time, and described method is included in carries out described brewage process when having proline specific protease.Storage (as the steady stage) can carry out under the temperature that the temperature of typical case's use is high in than traditional beer brewing method.Typically, the time length of steady stage will be that beer is cooled to the required time of packaging temp from the stage of maturity.

In the context of the invention, word " peptide " and " protein " commutative use.In the context of the invention, word " muddiness ", " muddy dark (cloudiness) " and " muddiness " are used interchangeably.

In order to determine that apparent attenuation degree has reached for about 77% to about 84% the moment, use the quantitative diacetyl level of EBC method 9.24.1 Vinical Diketones in Beer:Spectrophotometric Method.Attention: EBC method 9.24.1 is designed to measure general diketone level, however since so far diacetyl be the main component of diketone, therefore the result of this measuring method is considered to represent the diacetyl level in the context of the invention.Perhaps can use gas-chromatography in beer, to measure the diacetyl level with EBC method 9.24.1 VinicalDiketones.For quantitative in addition, can use muddy survey meter (turbidmeter) to the beverage opacity.In muddy survey meter, measure with respect to the amount of light of incident light wave with the specified angle scattering.Opacity is measured the known muddiness formation that protein-polyphenol interacts and causes that is highly suitable for measuring.

Polyphenol is defined as having the compound of following chemical structure, and described chemical structure contains at least two aromatic nucleus that replaced by at least one hydroxyl, or contains the aromatic nucleus that at least one is replaced by at least two hydroxyls.The example of polyphenol is tannin or flavonoid, and it comprises for example catechin, flavonol and cyanidin(e).

Term used herein " beer " is used for containing at least from beer of being made by the converted mash of the cereal preparation of not germinateing and the beer from being made by all converted mashs of germinated ceral preparation, and from the beer that germinates and all converted mashs of the not mixture preparation of germinated ceral are made.This paper use term " beer " that contain submerged fermentation with the beer top fermentation, and with the beer of additive preparation with contain the beer of all possible pure content.

During fermentation the amount of the proline specific protease of Tian Jiaing can change according to the gentle fermented type that carries out of malt water that uses.In a kind of embodiment preferred of the present invention and/or method, add from about 7.5 to about 15 units (PPU), for example from about 10 proline-specific activity in 100% malt beer of the about 12 degree Plato of every hectolitre to about 12.5 units (PPU).The active maximum of proline specific protease to be added can not specificly be pointed out.Maximum depends on: for example, desired muddiness reduces or prevention, have the composition of beer under the pH of its maximum activity at proteolytic enzyme.The unit definition of enzyme provides in " material and method " part of the application.

Proline specific protease can add in the beer preparation different periods.When the fermentation beginning, add enzyme and obtain possible best result.Yet enzyme can add in the mash or before forming muddiness and add in the beer of fermentation.

In this article, term prolyl specific protease, proline specific protease, proline specific endo-protease, proline specific inscribe peptase and have the proteolytic enzyme of prolyl specific activity or similarly express and be used interchangeably.

Proline specific protease can be used for the present invention with separated or purified form." separated " or " purified " is meant the proline specific protease that is removed from its natural surroundings.For example, with regard to the object of the invention, the proline specific protease that the reorganization of expressing in host cell produces is considered to separated, natural or recombinant polypeptide by the basic purifying of any appropriate technology (for example Smith andJohnson, disclosed single step purification among the Gene 67:31-40 (1988)) also is considered to separated.

Can be applicable to that proline specific protease of the present invention, described method comprise for example ammonium sulfate or ethanol sedimentation, sour extracting and chromatography such as high performance liquid chromatography (HPLC) by well known to a person skilled in the art that method reclaims from the cell culture of reorganization.

Be applicable to proline specific protease of the present invention can be the product, chemosynthesis product of natural purifying, by the product that recombinant technology is made from protokaryon or eucaryon host, described host comprises for example bacterium, yeast, fungi, higher plant, insect and mammalian cell.

Although it is to be understood that proteolytic enzyme can with do not influence active carrier of enzyme purpose or mixing diluents, it still can think separated.Be applicable to that proline specific protease of the present invention can also be by the form of more abundant purifying.Therefore, proline specific protease can be included in the following preparation, more than 70%, is proline specific protease more than 80%, 90%, 95%, 98% or 99% protein for example in the described preparation.

Typically, proline specific protease can be the form that does not contain or be substantially free of any other proteolytic enzyme.

Be applicable to that proline specific protease of the present invention can use with the fixed form, thereby can handle a large amount of proteinaceous liquid.Select the approach of suitable support material and suitable stationary method that extensively description, for example " Immobilization of Enzumes and Cells were arranged in the literature " (ed.Gordon F.Bickerstaff; ISBN 0-89603-386-4).

In the context of the invention, proline specific protease is defined as following proteolytic enzyme, and described proteolytic enzyme contains the position incision protein or the peptide of proline residue in protein or peptide chain.Preferably, proline specific protease is to contain position " incision " (hydrolysis) protein of proline residue or the endo-protease of peptide at protein or peptide.In the method for the invention, the preferred proline specific endo-protease that uses at proline residue carboxyl terminal hydrolysising peptide key.This zymoid example is prolyl oligopeptidase (EC3.4.21.26), and J.Agic Food Chem, Vol 53 (20), 7950-7957,2005) the prolyl endo-protease in described Aspergillus niger source and the dipeptidyl peptidase such as the DPP IV (EC 3.4.14.5) of proline specific.At proline residue NH ₂-the terminal proline specific endo-protease that cuts proline residue is as for example 15 January 1998, and Vol.391 is described in the Nature disclosure p.301-304.

With like that typical at enzymic activity, the activity of proline specific endo-protease depends on pH.Typically, use following proline specific endo-protease in the present invention, described proteolytic enzyme has maximum prolyl specific activity under the pH of the beer that is added corresponding to it.In a kind of preferred embodiment of the inventive method, in elementary beer fermentation, add have acid pH just when (promptly have 6.0 or lower-as pH5,4 or 3 pH just when) proteolytic enzyme.In a kind of preferred embodiment of purposes of the present invention and/or method, in beer fermentation, add following proteolytic enzyme, described proteolytic enzyme have acid pH just when, and secrete actively in the ferment substratum of setting out by food rank microorganism.

Proline specific protease extensively is found in the animal and plant, but to exist in microorganism be limited for they.Up to now, in Aspergillus (EP 0 552 428), (J.Biochem.113 790-796), has identified proline specific protease in Xanthomonas and the Bacteroides kind for Flavobacterium (EP 0 967 285) and Aeromonas.Although from most proline specific enzyme in these biologies activity is arranged near pH8, the Aspergillus enzyme is active best near pH5.Proline specific protease of the present invention can separate from one of mentioned microorganism species, particularly from Aspergillus.Preferably, the proline specific endo-protease separates from Aspergillus niger.More preferably, the proline specific endo-protease separates from following A spergillus niger host, and described host was designed to express the gene of coding proline-specific, although other host such as E.coli also are suitable expression vector.For example, clone and the excess production of proline specific endo-protease in E.coli etc. that is derived from Flavbacterium makes some proline specific endo-protease to obtain with respective pure form.The example that this class excess is produced construct is provided in World Journal of Microbiology ﹠amp; Biotechnology, Vol 11, pp 209-212.Preferred use Aspergillus niger host to make nonrecombinant following self construct (self-construct), described self construct uses the genetic expression of A.niger promotor control coding A.niger proline specific endo-protease.

Most preferably, the proline specific endo-protease is a disclosed endo-protease among the EP-A-1326957, and described proteolytic enzyme is incorporated this paper by reference into.The purposes of proline specific endo-protease in brewage also disclosed in EP-A-1326957.Reduce purposes muddy in the beverage yet in this document, only disclose.Openly proline-specific not can be used for quickening the brewage process equally.In addition, known PVPP of those skilled in the art and/or silica gel treatment are instantaneous.Are shortened unexposed shelf time or stationary phase method.Obviously know those skilled in the art, use under the situation of the traditional method that does not have the suitable steady stage, can obtain the beer of quality (for example colloid is stable and clarification) difference.Therefore surprisingly, do not have detrimentally affect by in brewage, using proline specific endo-protease, this process to be shortened to beer quality.

The acetolactate decarboxylase activity that adds in the fermentation can change in limit well known by persons skilled in the art.To required active indication by Hannemann (MBAA TQ, Vol 39, no 3,2002,149-155) provide in the literature.

Modern distillery makes great efforts to remove all main powder such as PVPP, silica gel or diatomite from the brewage process.In this process, final diatomite filtration filters replaced by membrane filtration or cross flow (crossflow).According to another embodiment of the present invention, finishing of beer-brewing method of the present invention can not use any main powder (as PVPP or silica hydrogel or diatomite) to filter.Therefore, the beer of purposes and/or method preparation can not contain or be substantially free of PVPP and/or silica hydrogel and/or diatomite according to the present invention.

Use method of the present invention, for example can using, the filtering filtering technique of cross flow comes clarifying beer.Therefore, the present invention includes following method, in described method, use proline specific protease to prepare beer in conjunction with the cross flow membrane filtration.

The present invention relates to beer-brewing method, wherein use proline specific protease, for the brewing method that does not use proline specific protease, be shortened stationary phase thus, keeps and estimate according to EBC 9.29 the identical character of beer range estimation clarity of terminology states simultaneously at least.

The invention still further relates to the beer that obtains by the inventive method.Usually can judge the clarity of beer with number of ways.EBC method 9.29 has been described the muddiness of how measuring in the beer.EBC unit measures under an angle of 90 degrees in muddy survey meter.Perhaps can estimate.According to EBC method 9.29 (when promptly measuring EBC unit under 90 degree scattering angle), having the beer that is lower than 0.5EBC unit can be " vivid " by range estimation.The beer opacity about 0.5 and 1.0EBC unit between the time, beer can be by range estimation for " very transparent ", 2 and 1EBC unit between the time be " very light muddiness ", 2 and 4EBC unit between be " muddiness " and be " very muddiness " more than 8EBC unit.Certainly can not be expected at different muddy scale or the different visual observations made the different experiments chamber and accurately equate, but great difference can not occur.In addition, the range estimation of finally being undertaken by the human consumer is important from commercial point of view.Therefore, accepting beer usually should (almost) be vivid (promptly measure to have under 90 degree scatterings and mostly be 1 EBC unit most), so that commercial being accepted.

Surprisingly, find that the EBC value does not also correspond to the visually rank of skilled group for for the beer of the inventive method preparation.Protein and the polyphenol of supposing higher quantity still are present in the scattering value that causes being higher than 90 degree in the beer, but naked eyes can't detect.As shown in embodiment, discovery may be made the visually rank of " vivid " to having the beer that is higher than 1 EBC unit value,, and also may make the visually rank of " vivid substantially " to beer with the EBC unit value that is higher than 2 (even up to 8).That is to say that the beer that obtains according to the inventive method has (physics) chemical constitution different with the beer of traditional fermentation.

Used this being verified by the grain size analysis of the stable beer of different methods.Its demonstration: stable beer comprises following particle according to the present invention, and described particle is on average less than using the particle through identifying in the stable beer of PVPP or silica hydrogel.

Therefore, another aspect of the invention is and have the beer that is higher than 1EBC unit's opacity, preferably be equal to or higher than 1.25, most preferably be equal to or higher than 1.5, and after brewageing, use the EBC terminology this beer can be classified as " vivid " at once at 1 ℃ of visually rank of measuring down by skilled group.In another embodiment, the present invention relates to have the beer that is higher than 2EBC unit's opacity, preferably be higher than 3, more preferably be higher than 4, even more preferably be higher than 6 and most preferably be higher than 8EBC unit, and after beer being stored in the envrionment temperature at least March, preferably beer being stored at least after June or most preferably beer is stored in the envrionment temperature after at least 9 months, use the EBC terminology this beer can be classified as " almost vivid " by skilled group at 1 ℃ of visually rank of measuring down.

The beer that purposes and/or method obtain according to the present invention will typically be packed.Can use any suitable packing, as bottle, bucket or jar.The beer of purposes and/or method preparation also can be packaged in the cylinder (bulktank) according to the present invention.Therefore, the invention provides the packing that comprises the beer that according to the present invention purposes and/or method obtain, for example comprise bottle, bucket or the jar of this beer.

Hereinafter set forth the present invention by following non-limiting example.

Material and method

The proline specific protease activity measurement

Under 37 ℃, in Citrate trianion/Sodium phosphate dibasic damping fluid (pH4.6),, measure the activity of proline specific endo-protease with the optimal pH that is lower than pH6.0 at synthetic peptide Z-Gly-Pro-pNA.

Under 37 ℃, in the phosphate buffered saline buffer (pH7.0),, measure the activity of proline specific endo-protease with neutral optimal pH at synthetic peptide Z-Gly-Pro-pNA.

Under 37 ℃, in Citrate trianion/Sodium phosphate dibasic damping fluid (pH4.6),, measure the activity of dipeptidyl peptidase with the optimal pH that is lower than pH6.0 at synthetic peptide Gly-Pro-pNA.

Under 37 ℃, in the phosphate buffered saline buffer (pH7.0),, measure the activity of dipeptidyl peptidase with the optimal pH that is lower than pH6.0 at synthetic peptide Gly-Pro-pNA.

Monitor the reaction product of all proline specific proteases with spectrophotometer at the 405nM place.A unit (1PPU) is defined in the enzyme quantity that per minute under this testing conditions discharges 1 micromole's N-methyl-p-nitroaniline.

Analysis during the brewing process

Except as otherwise noted, according to 2004 editions " Analytica-EBC " (Fachverlag Hans Carl, Nurnberg Germany) carries out multiple analysis.Carry out the Fructus Hordei Germinatus analysis according to following EBC method 4-2,4-5-1,4-9-1,4-3-1,4-18,8-13-2,4-8,4-15 and 4-14.By measuring conversion coefficient and wort filtration monitoring mashing.Analyze wort according to EBC 4-5-1,4-7-2,8-13-2,4-8,4-15 and 4-14, and free amino nitrogen (EBC method 8.10), total nitrogen (by EBC method 8.9.1-Kjeldahl-or 8.9.2-Dumas combustion method), pH and total wort polyphenol (EBC method 8.12).Carry out multiple fermentation process according to temperature, extract minimizing and pressure.Before reinforced, fermentation first day and beer filtration, determine the yeast population.By gas-chromatography in fermentation, ripe neutralization measures di-acetyl before filtering.Count yeast by corresponding pressure reduction and flow velocity before and after filtering, with the monitoring beer filtration.In filling the water of carbonic acid, (enter in the beer that filters and leach and in the bottled beer) measure dissolved oxygen.Multiple beer analysis comprises EBC 9.4,9.6,9.7,9.8,9.11,9.24.1,9.24.2,9.29,9.30,9.35,9.37, the Ross ﹠amp that is used for head retention (headretention), trans-2-nonenal and reducing power; Clark and NIBEM (9.42) method.(Haffmans, Venlo TheNetherlands) under 25 and 90 degree scattering angle, use multiple muddy method of masurement to measure muddy development down in differing temps (consulting embodiment 2) to use VOS ROTA 90/25 muddy survey meter.Applied voltage test is carried out in prophesy quality guaranteed period detection according to EBC9.30.

Brewing method

All brewage experiment is carried out with the 20hl scale, wherein uses 300kg pilsen Fructus Hordei Germinatus (Heineken A type) and is used for 9501 water of wort.Brewage and comprise mashing process with following temperature program(me): 45 ℃ 20 minutes, in 20 minutes from 45 ℃ to 64 ℃, 64 ℃ 15 minutes, in 12 minutes from 64 ℃ to 76 ℃, 76 ℃ 25 minutes and finally in 5 minutes from 76 ℃ to 78 ℃, be wort filtration then.Spray uses hot water.Boiled 90 minutes, and add hops with the form of hops bead.Under the condition described in individual embodiment, carry out cold stable.Use is surrounded by coarse and the filter plate filtered beer fine-grained filter material layer.The filter material layer of fine-grained is also as body feed.Carbofill filter paper is used in bottling.Bottle carried out pasteurization in following 15 minutes at 60 ℃.

Embodiment

Embodiment 1 pilot scale beer is made

(Institute Francais de la Brasserie et de la Malterie, Vandoeuvre-les-Nancy France) in 20hl half industry test of brew-house, brewages two kinds of pure Fructus Hordei Germinatus under the same conditions at IFBM.In fermentation, add proline specific protease (Brewers Clarex from DSM Food Specialities with the concentration of 10PPU/ hectolitre Fructus Hordei Germinatus to one of these Fructus Hordei Germinatus, Delft, TheNetherlands, contain 5PPU/ gram product), and the concentration with 4500 ADU/ hectolitre Fructus Hordei Germinatus adds acetolactate decarboxylase (Maturex L from NOVO, Bagscaerd, Denmark when the fermentation beginning, contain 1,500 ADU/ gram product).This fermentation is called " test fermentation ".Another Fructus Hordei Germinatus ferments equally, and is called " control fermentation ".

Brewage

Make brewing material from 300kg barley germ and hops bead.The mashing condition is the water of 1:4: Fructus Hordei Germinatus ratio (vol/wt), pH5.6.Mashing figure comprises 45 ℃ of the first steps of 20 minutes, 64 ℃ of second steps of 15 minutes, 74 ℃ of the 3rd steps of 30 minutes and final 78 ℃ of 5 minutes end mashings.Rate of heating is each 1 ℃/minute.After stopping mashing, wort filtration is advanced in the Lanter fat; Carry out Fructus Hordei Germinatus circulation and heat (78 ℃) spray under pH5.6 subsequently earlier.The Fructus Hordei Germinatus that obtains boiled 90 minutes, used whirlpool to carry out good trub afterwards and separated.

Fermentation

Fermentation use available from VLB (Berlin, bottom yeast strain Rh Germany) carries out, with the Fructus Hordei Germinatus of 12 ℃ of Plato with 17.10 ⁶Viable cell/ml inoculation.Fermenting process carries out at 12 ℃, up to 5 ℃ of Plato (3 days), then 14 ℃ of continuation, until fermentation ends (3 days).

After the fermentation

During fermentation ends, control fermentation was being kept three days 14 ℃ more, to reduce the diacetyl level.After this ripening stage, beer at one day internal cooling to 0 ℃, and is stablized beer 5 days at 0 ℃, use the PVPP of 30g/Hl dosage to handle afterwards separately, with diatomite filtration beer and boil.

Yet, for the test fermentation, do not use the ripening stage.In addition, be restricted to stationary phase beer is cooled to 4 ℃ of required periods.After the fermentation ends, directly reduce, in 4 days period, beer is cooled to 4 ℃ according to linear temperature.Because in fermentation, use Brewers Clarex, so do not add beer stablizer (for example PVPP or silica gel).Instead, after the cooled beer, use the diatomite direct filtration, and subsequent bottling.The method bioassay standard beer parameter (for example pH, color, bitter taste, ethanol etc.) definite according to European Brewery Convention (EBC), described method is described among their " Analytica-EBC ".

The result

In general, to point out to contrast and test the beer parameter of fermentation similar for the standard beer analysis.Dissolved oxygen is on close level in two groups of tests, and is lower than 0.2mg/l.The initial muddiness of two kinds of beer is on close level, but in the pure ice-cold muddy test according to L Chapon (EBC method 9.41), test beer has significantly lower value.In addition, in beer applied voltage test " Prediction of the Shelf-life of Beer " (EBC method 9.30), test beer is better than the beer performance that derives from control fermentation.

The adjacent diketone level of the beer that obtains from test and control fermentation all is lower than 0.10mg/ liter (with EBC method 9.24.1 Vivinal Diketones in Beer:Spectrophotometric Method measurement).By IFBM (Vandoeuvre-les-Nancy, skilled group France) (being made up of 8 people) carries out sensory analysis in standardized program, after tasting sample, the evaluator declares with the intensity score value to flavouring quality.Collect the result, form the sensation spectrum.This test is finished according to the guidance of sensory analysis, is described in the 13rd chapter of 2004 editions " Analytica-EBC ".Taste panel is not being observed remarkable taste difference from the bierstube of contrast and test fermentation.

Conclusion

This embodiment explanation: by using proline specific protease and acetolactate decarboxylase, can produce and have standard beer parameter the beer of (measuring) with the method that European Brewery Convention (EBC) is definite, it is with similar with reference to the parameter of beer, and compares with reference beer and not show significant taste difference.In addition, use the present invention can obtain the timesaving method, because 9 days shelf time (contrast) can be reduced to 4 days.In addition, by internal cooling to 4 ℃ replacement in 4 days at one day internal cooling to 0 ℃ and this temperature was kept 5 days again, can reach significant energy saving.

Embodiment 2 shortens cold stationary phases and to enzyme influence stable, 100% malt beer clarity

Brewage test, by using the cooling stage that shortens, proline specific protease is to the influence of 100% malt beer with assessment.For this reason, prepare six kinds of different worts, and (consult that " material and method ") are fermented according to identical flow process.In five fermentor tanks, add 0.125PPU/ liter (consulting the activity definition in " material and method ") concentration from Aspergillus niger proline specific protease, and in all five fermentations, before inoculation, enzyme is added in the cold wort.The 6th fermentation be with for referencial use, and do not add proline specific protease.All fermentor tanks are all used fresh storage yeast (about 1,700 ten thousand cells/ml wort) inoculation, and fermentation is carried out up to 5plato at 12 ℃, and temperature rises up to 14 ℃ then, is used to remove adjacent diketone, as diacetyl.(be the fermentation beginning back Ninth Heaven in this case) during ripe the end beer is cooled to negative 1 ℃.One-tenth pasteurized beer from all fermentor tanks that added enzyme keeps the different time periods down at negative 1 ℃, uses diatomite filtration then.Having added four batches of proline specific protease filters too.With special purpose PVPP (40g/hl; Being injected into the beer filter) inter-process contains the 5th batch of proline specific protease of interpolation, filters then.

Also be cooled to negative 1 ℃ and carry out " classics " and stablize flow process from the one-tenth pasteurized beer of the fermentor tank that does not add enzyme, promptly beer was negative 1 ℃ of maintenance 9 days, and the special purpose PVPP with 40g/hl carries out identical inter-process then, and uses diatomite filtration subsequently.After the filling with bottle pasteurization and be stored at 20 ℃ under 15PU.

Behind the shelf time in six weeks, at 20 ℃ and 1 ℃ multiple beer is carried out muddiness and measure.Beer is measured the following preincubate of temperature (promptly 1 ℃ or 20 ℃) 24 hours for this reason, under this temperature, measuring the beer muddiness then.Use muddy survey meter under 90 and 25 degree scatterings, to measure, write down muddy reading.90 and the 25 degree scattering datas that obtain with this muddiness survey meter (Haffmans VOS ROTA 90/25) are shown as " H90 " and " H25 " value respectively with the measurement of correlation temperature in table 1.Before muddy the measurement bottle shaken and makes it even, and place chien shih bubble collapse when sufficiently long.Take multiple measurements to guarantee that bubble does not influence reading.Except that these muddy measurements, also use skilled group (n=5) to estimate and assess with taste.

Table 1

	The 1st batch	The 2nd batch	The 3rd batch	The 4th batch	The 5th batch	Reference
	The 1st batch	The 2nd batch	The 3rd batch	The 4th batch	The 5th batch	Reference	-1 ℃ of stable fate	0	1	3	5	0	9
Proline specific protease	Be	Be	Be	Be	Be	Not	-1 ℃ of stable fate	0	1	3	5	0	9
Proline specific protease	Be	Be	Be	Be	Be	Not	PVPP(40g/hl)	Not	Not	Not	Not	Be	Be

20 ℃ actual muddy (H25)	0.30	0.31	0.32	0.28	0.28	0.27
20 ℃ actual muddy (H25)	0.30	0.31	0.32	0.28	0.28	0.27	1 ℃ actual muddy (H25)	0.34	0.34	0.37	0.32	0.30	0.28
20 ℃ actual muddy (H90)	0.48	0.59	1.06	0.83	0.48	0.48	1 ℃ actual muddy (H25)	0.34	0.34	0.37	0.32	0.30	0.28
20 ℃ actual muddy (H90)	0.48	0.59	1.06	0.83	0.48	0.48	1 ℃ actual muddy (H90)	0.52	0.66	1.17	0.90	0.53	0.49
Applied voltage test (H25) according to EBC9.30	0.70	0.35	0.33	0.27	0.22	0.22	1 ℃ actual muddy (H90)	0.52	0.66	1.17	0.90	0.53	0.49
Applied voltage test (H25) according to EBC9.30	0.70	0.35	0.33	0.27	0.22	0.22	The EBC9.30 applied voltage test	1.0	0.75	1.26	0.92	0.52	0.48
Bottling six weeks of back are in 8 ℃ of visual assessments (terminology EBC 9.29)	Vivid	Vivid	Vivid	Vivid	Vivid	Vivid	The EBC9.30 applied voltage test	1.0	0.75	1.26	0.92	0.52	0.48
	Vivid	Vivid	Vivid	Vivid	Vivid	Vivid	Taste (bottling 3 weeks of back)	Good	Good	Good	Good	Good	Good

According to visual observation, all bottled beer (promptly comprising the beer that does not carry out any cold stationary phase) all clarifications fully after the shelf time postcooling 24 in 20 ℃ of six week disappears to 8 ℃ through the enzyme processing.

Very surprisingly, muddy assessment of these visions and the different muddy reading that obtains and not quite identical.According to the EBC standard, be " very slight muddy " according to the value between EBC 9.29 methods (measuring under the 90 degree scattering angle) beer of measuring and 1.0 and the 2.0EBC of generation.2.0EBC the mountain, beer begins to be called " slight muddy ".The 90 degree scatter read numerical value height that produce are to 1.26EBC (consulting table 1), yet all these beer visions are rated " vivid ".This contradiction is explained in embodiment 3 to some extent.

It should be noted that also that according to professional taste panel the beer that is produced is the taste of display abnormality spectrum not all.

Embodiment 3 opacities are measured and muddy formation

According to the flow process of describing among the embodiment 2 100% wort of 12Plato is carried out three kinds of 20Hl fermentations.Do not add proline specific protease in first kind of fermentation (reference) fermentation.When inoculation in two kinds of other fermentations respectively with 0.125 and the proline specific protease from A.niger of 0.20PPU/l concentration.(be back 9 days of fermentation beginning in this case) during ripe the end, all three kinds of beer are removed yeast and cold precipitation afterwards all 1 ℃ of cold stablizing 5 days.Do not carry out PVPP and handle, do not carry out silica gel treatment yet.All three kinds of beer filter on diatomaceous earth filter, and described filter has coarse and filter material layer fine-grained.At last with beer bottling and under 15PU pasteurization, storage is used for quality guaranteed period research at ambient temperature then.

Behind the multiple shelf time with beer 1 ℃ of preincubate 24 hours, under this temperature and 90 degree scattering angle, measure the beer muddiness then.Shaking the bottle before the measurement makes it even, and chien shih bubble collapse when placing sufficiently long.Take multiple measurements to guarantee that bubble does not influence reading.The data that obtain are summarized in the table 2.

Table 2

Measure with opacity,, and use EBC 9.29 terminology evaluations by skilled group (n=5) visual observation beer.

The same as expected, the reference beer of handling without enzyme shows higher H 90EBC value.Consistent with these higher H 90 values, visual observation with all t=0,3,6 and 9 months sample deciding grade and level be " very muddy " (term described in the deciding grade and level use EBC method 9.29).

In the beer that enzyme is handled, only fresh (t=0) sample shows lower H90 value (be respectively 1.5 and 1.3EBC).The beer (the t=0 month) that opposite with our expectation, vision group will this be fresh, handle through enzyme reads to be " vivid ", and according to the H90 value that obtains, expection is the grading of " very slight muddiness ".

The sample of the standing storage of handling through enzyme all shows 4EBC or higher H90 value.Surprisingly, store 3,6 and derive from two kinds of visions of handling beer through enzyme after 9 months and be rated " almost vivid ", rather than according to their H90 value desired " muddiness ".

Therefore, we infer: as the result who uses proline specific protease in the beer fermentation method, the H90 scattering value of instrument is not indicated the muddiness sensation of vision, does not indicate of the present invention through beer packed gluey stability yet.In addition, we infer: beer according to the present invention is different with beer known in the art.

Embodiment 4 granular size analyses are by the stable beer of different methods

At IFBM (Institute Francais de la Brasserie et de la Malterie, Vandoeuvre-les-Nancy, France) in the half industry test brew-house, carry out four fermentations according to the flow process that is used for 12Plato 100 worts described in the embodiment 2.When inoculation, in one of them fermentation, add the proline specific protease from A.niger of 0.125PPU/l concentration.When maturation finishes, (be the fermentation beginning back Ninth Heaven in this case),, remove yeast and cold precipitation afterwards all beer cold stablizing 5 days under 1 ℃.Use 30g/Hl single-use PVPP (injecting beer filtration) stable then not with one of three kinds of stable beer of proline specific protease.Another beer that does not add proline specific protease is stable with 30g/Hl silica hydrogel (injecting beer filtration).Be used for not contrasting beer with remaining " processed " with spline filter, promptly do not add PVPP or silica hydrogel from the beer that the proline specific protease of A.niger is handled.Filter all four kinds of beer on diatomaceous earth filter, described diatomaceous earth filter has coarse and filter material layer fine-grained, and adds the diatomite body feed when filtering in all situations.At last with beer bottling and under 15PU pasteurization, be stored under the envrionment temperature then.In this process, minimum oxygen level carefully, and find that it is suitable for all four kinds of beer.

After storing about 10 months at ambient temperature, at Brewing Research International (BRI, Nutfield, United Kingdom) use Photon Correlation Spectroscopy (PCS) 6 ℃ of particle size dispersion of studying four kinds of beer down.This technology is not counted particle equally, but provides the scattering of light reading, and this scatter read can be characterized by specific granular size kind, thereby allows the relatively particle size dispersion of four kinds of beer samples.

The scholar is in Fig. 1 for accumulation stdn scattering strength.Fig. 1 shows: in " untreated " contrast beer, all scattered lights derive from the particle less than 2000 nanometers.For with PVPP and the stable beer of silica hydrogel, all particles that make light scatter are respectively less than about 1250 and 750nm.Yet average is littler in the beer that proline specific protease was handled: do not have the particle of discovery greater than 385nm.Be the chart in the comparison diagram 1, come quantitatively with two variablees:

D50, particle diameter, 50% scattering of light from less than the particle of this diameter and;

D90, particle diameter, 90% scattering of light is from the particle less than this diameter.

Back one data are provided in the table 3.

Table 3: through the d50 and the d90 value (consulting text interpretation) of the stable iFBM beer sample of difference.NT: be untreated PVPP: polyvinylpyrrolidone polymer, SHG: silica hydrogel, PSP: proline specific protease.

Handle	Temperature (℃)	d ₅₀(nm)	d ₉₀(nm)
Handle	Temperature (℃)	d ₅₀(nm)	d ₉₀(nm)	The NT-contrast	6	895	1073
30g/hl?PVPP	6	556	1141	The NT-contrast	6	895	1073
30g/hl?PVPP	6	556	1141	30g/hl?SHG	6	537	629
0.125PPU/l?PSP	6	325	361	30g/hl?SHG	6	537	629

D50 in the table 3 and d90 value all show: the particle that produces scattering in the beer of proline specific protease is arranged in fact less than the particle in the beer stable with PVPP or silica hydrogel.This hypothesis (proline specific protease is to the hydrolysis of muddy activated protein prevention or reduced the formation of protein-polyphenol complex in the maturation) with us is consistent.We infer: as the exercising result of proline specific protease, muddy activated protein is hydrolyzed, thereby protein and the mixture between the polyphenol in the storage keep lessly, have therefore caused muddy assessment of professional group's vision and high relatively instrument 90 to spend viewed difference between the scatter reads.We infer once more: use the obtainable beer of the inventive method different with beer known in the art.

Embodiment 5 shortens cold stationary phase and 100% malt beer is stored 4 and 6 at ambient temperature Muddy Influence and Development after individual month

The permanent stability data of embodiment 2 described beer are provided in this embodiment.Bottled and be stored under the envrionment temperature (about 20 ℃) as many as 6 months through the beer of pasteurization.Storage 4 and after 6 months uses the muddiness that also is described in embodiment 2 to measure and visual assessment analysis beer (the 1st, 2,3,4 and 5 batch adds the listed reference of table 1).The data that obtain are listed in the table 4.

Table 4

The data presentation that table 1 provides among the embodiment 2: after storing for 6 weeks at ambient temperature, according to the visual assessment according to EBC 9.29, all are bottled and all be rated " vivid " through the beer of pasteurization.According to these data, use proline specific protease to guarantee fabulous beer separately, even if be shortened into cold stationary phase sophisticated beer is cooled to negative 1 ℃ of required period, promptly under this low temperature, do not keep for some time.Yet as shown in table 4, when beer was stored the longer time, the cloud-stability of beer changed.After in environment, storing four months, all beer of handling through enzyme are carried out producing very faint muddiness from 0 day cold stable to as many as 5 days.Therefore we infer: if proline specific protease was used in combination with the shortest cold stationary phase, then 100% malt beer can keep visual stable the period in 6 weeks of as many as at least.Although add that with proline specific protease the 5th batch of beer that PVPP handles has carried out the shortest subzero stationary phase, it still remains " vivid ".Therefore back one is found to show: proline specific protease and PVPP be used in combination allow significantly to shorten cold stationary phase, and to the long-term cloud-stability of beer without any harmful effect.We use is that the fact of 100% malt beer makes that this discovery is more astonishing.

Embodiment 6 handles enzyme and makes up through the short stationary phase of improving under the temperature

Except that cold stationary phase length, beer is cooled to energy required below 0 ℃ has added significant cost to method for preparing beer.Therefore, use the advantage of proline specific protease should not only limit to shorter cold stationary phase ideally, it is unnecessary to make that also subzero equilibrium temperature becomes.In order to test back one selection, we have begun one group of new pilot scale and have brewageed experiment.Carry out six with the scale of 20hl and brewage test, adopt the condition described in the application's " material and method " part and the embodiment 2 substantially.To use the effect of proline specific protease to compare again with the effect of using PVPP and silicon-dioxide.Use multiple cold stable flow process, described flow process under 0 and 7 ℃ in several hours to 4 days scope.Because as if proline specific protease add PVPP and represented effectively stable combination, repeat the condition of the 5th batch of experiment described in the embodiment 2.Yet in this experiment, beer is not cooled to negative 1 ℃ and only be cooled to 7 ℃.Back one temperature provides a kind of and has selected easily, because it has represented bottling or the normally used top temperature of barrelling.

In first 20hl fermentation, sophisticated beer carries out cold stationary phase (relate to 1 day, be cooled to 0 ℃) and carries out 4 day shelf lives at 0 ℃ then.Use diatomite filtration beer then and with its separated into two parts in beer filtration: 7hl the earliest handles (body feed) with having mixed diatomaceous PVPP with 30g/hl, last 7hl is with also having mixed diatomaceous silica gel S (Spindal, Armainvilliers, France; 40g/hl) handle (body feed).After the filtration with beer bottling and pasteurization.In table 5, these two kinds of products are called the 1st batch (usefulness PVPP's) and the 2nd batch (usefulness silica gel).

Second 20hl fermentation carried out in the mode identical with first fermentation finished thoroughly1.Yet beer relates to the cold stationary phase that was cooled to 7 ℃ in 1 day in this case, carries out 4 day shelf lives at 7 ℃ then.These two kinds of products are called the 3rd batch (usefulness PVPP's) and the 4th batch (usefulness silica gel).

Third and fourth 20hl fermentation all undertaken by the proline specific endo-protease that adds from A.niger with the concentration of 0.125PPU/I in the cold wort of inoculation forward direction.The beer that derives from this third fermentation relates to the cold stationary phase that was cooled to 0 ℃ in 1 day, then 0 ℃ of shelf lives of carrying out 4 days, then diatomite filtration (not adding PVPP or silica gel), the bottling and pasteurization (the 5th batch).Beer from the 4th kind of fermentation relates to the cold stationary phase that was cooled to 7 ℃ in 1 day, carries out 4 days shelf lives then under 7 ℃.Also use diatomite filtration (not adding PVPP or silica gel), bottling and pasteurization (the 6th batch) then.

In the 5th kind of fermentation, cold stable finish by only sophisticated beer being cooled to 0 ℃ (consuming time be less than 1 day) used diatomite filtration beer then.And then the beer that obtains is divided into two parts in beer filtration: 7hl the earliest handles (the 7th batch) with having mixed diatomaceous PVPP with 30g/hl.Last 7hl beer is with also having mixed diatomaceous silica gel S with 40g/hl (Spindal, Armainvilliers, France; 40g/hl) handle (body feed), bottling and pasteurization (the 8th batch).

In the 6th kind of fermentation, also before inoculation, in cold wort, use proline-specific from A.niger with the concentration of (0.125PPU/l).After the maturation beer directly is cooled to 7 ℃, and handles with 30g/hl with having mixed diatomaceous PPVP.Also beer is directly bottled after the filtration and pasteurization.The beer that obtains in table 5 is called the 9th batch.

Soon, all beer is carried out a large amount of standard beer analysis after the bottling, for example alcohol, bitter taste, diacetyl, polyphenol and aldehyde C-9 (nonenal) level.Also determine head retention (NIBEM andRoss﹠amp; Clark).The multiple quality of beer that the data presentation that obtains obtains is similarly, does not show the difference of not expecting.The organoleptic analysis also only shows less difference.As expected, find that maximum difference is the performance of beer in multiple pressurization detects.For example, behind multiple beer bottling, measure soon according to the pressurization data of EBC 9.29 and the final muddiness of 6 days, 60 ℃ accelerates maturing tests.These measurements the results are shown in table 5 (consulting " pressurization " and " 60 ℃ 6 days ").Table 5 also provides multiple H90 opacity data and the visual assessment of the bottled beer that writes down after 8 shelf liveves in week at ambient temperature.Measurement to these H90 data is carried out as described in embodiment 2.

Table 5

All storing 24 as a child based on visual assessment according to EBC 9.29." only cool off 0 " and be meant and be cooled to the indication temperature and do not keep subsequently in described temperature.

The data that provide in the table 5 show once more: use proline specific protease to allow the cold steady time (consulting the 5th batch) that significantly shortens separately.In addition, this class of data declaration short stationary phase also is feasible (consulting the 6th batch) under the temperature that improves in the table 5.Equally, this discovery is relevant with economic height for brewer's specific category.The data that occur among the embodiment 5 are not in conjunction with using the higher relatively EBC.9.30 pressurization value of the stable beer of proline specific protease to point out as shown in the table 5 of present embodiment yet, and these stable conditions can be not enough to guarantee vivid beer on the shelf lives after-vision that prolongs.If can expect the secular quality guaranteed period, then herein (the 9th batch) Notes of Key Data of occurring the flow process (promptly being quickly cooled to about 7 ℃) of " only being cooled to the bottling temperature " be enough, as long as with proline specific protease and PVPP treatment combination.Therefore as if, the most surprising discovery of present embodiment is: proline specific protease combination (if expect long-term shelf-lives, then randomly making up with PVPP) is allowed to omit fully cold stationary phase.This has hinted the processing shortcut of highly significant: directly to bottling, and produce shelf stable, vivid beer visually from maturation.

Claims

1. proline specific protease is used to quicken the purposes of brewage process, preferably quickens the brewage process by shortening shelf time.

2. purposes as claimed in claim 1, the acceleration of the described brewage process of its squadron was finished by the time length that reduces the steady stage.

3. purposes as claimed in claim 2, the wherein said steady stage carries out under the beer packed independent temperature being suitable for, preferably about 7 ℃.

4. purposes as claimed in claim 2, the time length of wherein said steady stage is reduced to beer is cooled to and is used to required time of expectation outlet temperature of filtering and/or packing.

5. as each described purposes of above-mentioned claim, be used in combination with alpha-acetolactate decarboxylase.

6. as each described purposes of above-mentioned claim, (PVPP) is used in combination with polyvinylpyrrolidone.

7. as each described purposes of above-mentioned claim, be used in combination with the cross flow membrane filter.

8. be used to quicken the method for brewage process, described method comprises: prepare beer when having proline specific protease, wherein shelf time is shortened.

9. be used to make the method for beer, described method comprises: fermenting wort when having proline specific protease, carry out stage of maturity and steady stage then, and wherein the steady stage has and is less than 7 days time length.

10. method as claimed in claim 8 or 9, the time length of wherein said steady stage is reduced to beer is cooled to and is used to required time of expectation outlet temperature of filtering and/or packing.

11. method as claimed in claim 8 or 9, the wherein said steady stage is carried out preferred about 7 ℃ being applicable under the beer packed temperature.

12., wherein also have alpha-acetolactate decarboxylase between yeast phase as each described method in the claim 8 to 11.

13. as each described method in the claim 8 to 12, the time length of wherein said shelf time is less than 8 days.

14., wherein use PVPP as each described method in the claim 8 to 14.

15. as each described method in the claim 8 to 14, it comprises filtration step, uses the cross flow membrane filter in the described filtration step.

16. the method according to any one of the preceding claims or purposes, wherein proline specific protease be have acid pH just when proline specific protease.

17. can be by the beer that obtains as each method in the claim 8 to 16.

18. comprise bottle as beer as described in the claim 17, bucket or jar.