ES2642867T3 - Improved beer brewing process - Google Patents

Abstract

Method for the production of beer comprising fermenting a must in the presence of a specific proline protease, followed by a maturation phase and stabilization phase, in which the stabilization phase lasts less than 5 days.

Description

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DESCRIPTION

Improved beer brewing process Field of the invention

The present invention relates to a method of brewing. In particular, it refers to an accelerated method of brewing that involves a specific proline protease.

Background of the invention

In the traditional brewing process, the must obtained by extracting malt is inoculated with brewer's yeast and the fermentation is allowed to proceed for approximately 7 days with a maximum temperature between 8-15 ° C depending on whether a fermentation process is used In fno or hot. This fermentation is also known as primary fermentation. Once the fermentation is complete and the mayonnaise of the fermentable sugars have been converted into alcohol, the yeasts settle. In the case of low fermentation beer, yeasts are collected at the bottom of the fermentation, in other beers they are collected at the top of the fermentation. The "green" beer resulting from this primary fermentation still contains some non-sedimented yeasts, in addition to relatively high levels of undesirable aroma components, in particular diketones, such as diacetyl and acetyl aldehyde.

The conversion of these last undesirable aroma components into bland taste compounds is an important aspect of the subsequent maturation phase of beer. Especially the reduction of diacetyl, a compound with a strange buttery taste, in insorid acetorna is a time-consuming process, but of utmost importance. In the conventional beer process, this stage of maturation takes place for about a week. In the subsequent phase of brewing, the so-called stabilization phase, beer is retained to promote the formation of protema-polyphenol aggregates, followed by the removal of precipitated aggregates. These protema-polyphenol aggregates consist of proline-rich polyphenols and proteins, the so-called turbid, malt proteins. The addition and subsequent precipitation of these aggregates is facilitated during the stabilization phase by cooling the beer to 0 or even -2 ° C for approximately 7 to 10 days. According to Narziss (Narziss, L. 1990 Ferment. 3, 54-62), the last period of stabilization in fno is indispensable. After removing the precipitated matter, the polyphenols and / or proteins that activate the non-precipitated turbidity are normally eliminated to prevent the formation of a "turbidity in fno" in the packaged product. For this purpose, residual non-precipitated polyphenols can be removed by absorption to polyvinylpolypyrrolidone (PVPP) and / or the remaining turbidity activating proteins can be removed by silica gel absorption. Later, the PVPP and / or silica gel, which respectively comprise the polyphenols and / or proteins that activate the absorbed turbidity, can be eliminated together with the elimination of any residual yeast, by filtration in diatomaceous earth.

In general, the conventional beer production process described above lasts approximately 20 days. The resulting product is completely clear and stable so that no visible changes occur during its commercial shelf stability.

To increase the flexibility of the brewery and to save capital costs, a lot of work has been done over the years to minimize the fermentation, maturation and stabilization phases in the brewing process (Narziss, L. 1990 Ferment, 3 , 54-62). For example, in the "pressure fermentation" process, fermentation and maturation are accelerated by increasing fermentation and maturation temperatures: normally at temperatures of approximately 14-18 ° C. In this approach, the more ripening fermentation is completed within a period of 7 days. The entire production process, that is, which includes stabilization, is considerably shortened to slightly more than 2 weeks. However, the resulting beers may develop a slightly yeast flavor and in general a less favorable aroma classification.

In the popular alternative, the so-called "hot-ripening fermentation" process, beers with a superior flavor are obtained that adhere to the conventional low temperature fermentation process. The shortest processing time desired is achieved by raising the temperature of the ripening phase to 18 to 22 ° C. As a result, the green flavor of fresh beer is exceeded within a period of 2 to 3 days. The stabilization of beer is achieved by conventional methods. Although the beers obtained have excellent quality, the energy costs of this process are relatively high.

In yet another approach to accelerate beer maturation, green beer is thoroughly centrifuged to remove yeasts and then passed through bioreactors containing high densities of immobilized yeasts. In combination with an anaerobic heating stage at 90 ° C, all diacetyl is reduced to acetorne within a period of 1-2 hours. This rapidly matured beer is also stabilized with the usual storage period and treatment with PVPP or silica gel. This bioreactor approach is, however, prone to generate foreign yeast aromas and capital expenditures are likely to be high because advanced technological equipment is required.

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In addition, the introduction of newly developed enzymes that are added during the beer fermentation phase is beginning to have an impact on beer manufacturing. In a very recent approach, a proline specific endoprotease is used as an alternative to treatment with PVPP or silica gel to prevent the formation of turbidity in fno (EP 1 326 957). During the fermentation of beer, the enzyme selectively hydrolyzes proline-rich proteins that activate turbidity, thus preventing the precipitation of protema-polyphenol complexes. Informed advantages of this method are the fact that PVPp or silica gel is redundant because the turbidity in fno is effectively prevented by the enzyme. In addition, the method gives a considerably simplified processing stage during this final stage and the most vulnerable in beer processing, but has no significant effect on the duration of the brewing process. In practice, the reactions between PVPP and polyphenols and / or between silica gel and the proteins that activate turbidity are almost instantaneous. The elimination of treatment with PVPP or silica gel, therefore, does not give a shorter brewing process. In another enzymatic approach, an additional enzyme such as a carboxypeptidase that cleaves proline and / or an endoprotease that cleaves glycine can be added to a beer production process, in addition to specific proline endoprotease to further decrease turbidity formation (see document WO3 / 104382).

In yet another enzymatic approach, the enzyme alpha-acetolactate decarboxylase is added to the must with grounds. This addition prevents the formation of diacetyl during the fermentation of beer so that the maturation period, which is primarily intended to reduce diacetyl levels, can be reduced 2 to 3 days (see documents US 5,108,925 and US 4,708,875) .

Although there are many reports on the shortening of the maturation phase of beer production, to date no reports are available on viable methods to accelerate the stabilization phase of beer. However, this stabilization period normally lasts approximately one week at temperatures of approximately 0 ° C, interrupting considerable capacity and requiring considerable energy input. WO2005 / 027953, for example, discloses a process for the production of beer in which a specific prolyl endoprotease is added to reduce the levels of gluten epitopes. The fermentation of the beer was followed by a maturation in fno of 5 days.

Obviously, it will be of great economic importance to shorten and further simplify the beer production process. Such a shortened and simplified production process adds to the flexibility of the brewing plants, thus reducing the fixed costs, in addition to the labor costs.

Summary of the invention

According to the invention, the use of a proline specific protease is provided for the acceleration of a brewing process, preferably shortening the low fermentation period.

The invention also provides:

- a method of accelerating a brewing process comprising preparing a beer in the presence of a proline specific protease, in which the low fermentation period is shortened; Y

- a method for the production of beer comprising fermenting a must in the presence of a proline specific protease, followed by a maturation phase and stabilization phase, in which the stabilization phase lasts less than 5 days.

In addition, the invention provides a beer obtainable by a method of the invention and a bottle, barrel or can comprising such a beer.

Brief description of the drawings

Fig. 1 shows the cumulative normalized dispersion intensity as a function of the particle size measured by PCS in ten-month beers of the IFBM pilot plant.

Detailed description of the invention

The present invention relates to the use of a specific proline protease to accelerate the brewing process. In particular, it refers to the use of a specific proline protease to accelerate the brewing process by shortening and simplifying the stabilization phase of beer production.

The stabilization phase can also be carried out at a higher temperature than in conventional brewing processes. Indeed, therefore, the invention relates to a process in which the post-fermentation phase of the brewing process is shortened in comparison with the state of the art processes and / or at a temperature higher than the processes of the state of the art. Accordingly, the invention also relates to a method of accelerating the brewing process which comprises carrying out the process in the presence of a proline specific protease. In such a process, the phase of

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Stabilization can be shorter and / or carried out at a higher temperature compared to conventional brewing processes.

A wide variety of brewing processes are used in the world. Normally, however, all these processes basically comprise at least the following stages, or stages corresponding thereto:

• Fermentation

• Maturation

• Stabilization

• Filtration (however, some processes do not use filtration)

The process optionally comprises an additional stage or stages to further increase stability, for example shelf stability. In this regard, any known technique can be used, for example, the use of a polyvinylpolypyrrolidone (PVPP) and / or silica gel treatment. Normally, such an additional stage can be carried out between the stabilization and final filtration phase, although such a stage could be carried out in some other part of the process.

The details of each process stage may differ considerably in the different processes, and when carrying out a brewing process, the person skilled in the art will have his own definitions for each phase. To avoid any lack of clarity, in the context of the present invention, the terms fermentation, maturation, stabilization, treatment with PVPP and with silica gel and filtration are intended to be the same as those disclosed in the description. Note that in some processes fermentation is also known as primary fermentation. The fermentation phase is the phase in the brewing intended to ferment the sugars available in alcohol by added yeasts. The ripening phase is also known as secondary fermentation and is intended to convert undesirable aroma components such as diketones into better flavor components. The stabilization phase aims to promote the formation of polyphenol-protein aggregates and allow their precipitation. The treatment with PVPP and / or with silica gel, if used, is intended to eliminate respectively polyphenols and protections that activate non-precipitated turbidity to convert the packaged product into a more stable shelf. Finally, the filtration stage, if used, is intended to eliminate polyphenols and protections that activate precipitated turbidity, any residual yeast, PVPp and / or silica gel before packaging.

Generally, several parameters can be checked during brewing. The end of the fermentation phase can, for example, be determined by measuring the density of the brewed beer, to determine the reduction in the fermentable extract, such as the amount of glucose. The end of the fermentation phase is generally considered the beginning of the maturation phase. The end of the ripening phase, and thus the beginning of the stabilization phase, can usually be determined by measuring the amount of diacetyl present in the beer. However, in practice, this may vary depending on the type of beer desired. For example, in some breweries the maturation phase is considered to be finished once the level of neighborhood diketones is below about 0.10 mg / l and in others once the level of neighborhood diketones is below about 0.05 mg / l Therefore, an expert will be able to define the end of the maturation phase and the beginning of the stabilization phase according to a desired level of neighborhood diketones.

In the context of the present invention, the beginning of the stabilization phase can be defined as the moment where diacetyl levels have been reduced to less than about 0.10 mg / liter if measured according to the EBC method 9.24.1 Dicetons Neighborhoods in beer: Spectrophotometric method. In the context of the present invention, the end of the stabilization period is defined as the moment where the beer is subjected to its final filtration stage, or in the event that a treatment with PVPP and / or with silica gel is part of the brewing process, once the beer is contacted with PVPP and / or silica gel. If the process is one in which no filtration is carried out, the end of the stabilization period is defined as the moment at which the beer is packaged.

According to an embodiment of the present invention, the time needed for stabilization can be shortened to less than about 5 days. Preferably, it is less than 4 or 3 days. More preferably, it is shortened to less than 2 or 1 days. Most preferably, it is shortened to a degree that on the scale of production, the duration of the stabilization is reduced to the equivalent of the period required to cool the beer from the ripening phase to the desired temperature, for example the desired temperature for filtration and / or beer packaging. This period is conventionally called the cooling period. Shortening the stabilization period to the time necessary to cool the beer to the desired temperature is highly advantageous, since then the duration of the stabilization phase only depends on the available cooling capacity.

The beer of the ripening phase is traditionally cooled at a temperature of about -2 ° C to and including about 0 ° C. Surprisingly, it has been found that, after the use of a specific proline protease according to the invention, the stabilization phase can not only be shortened, but also

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It can be performed at a higher temperature than conventionally used. Traditionally, there are stabilization processes that are carried out at higher temperatures, but these processes need much more time, for example 6 weeks and more. Therefore, in another embodiment of the invention, the stabilization period, which may normally be a shortened stabilization period as defined above, is performed in a beer preferably cooled to a maximum of about 2 ° C, more preferably to a maximum about 3 ° C, 4 ° C, 5 ° C or 6 ° C or even more preferably at a maximum of about 7 ° C or about 8 ° C, and most preferably at the desired temperature used for packaging, that is, for the filling of bottles or barrels.

The desired temperature for packaging can be differentiated from process to process, but is preferably carried out at a temperature up to and including 8 ° C, preferably about 7 ° C, in order to obtain the desired level of carbon dioxide dissolved in the beer. In a process where beer is only cooled to the temperature required for packaging, considerably less energy is required than in processes known in the art.

In a preferred embodiment according to the invention, the stabilization phase is reduced to the period required to cool the beer from the temperature at the end of the ripening phase to the desired temperature for packaging, thereby omitting the conventional cold stabilization period.

According to the invention, therefore, the beer can be cooled to a maximum of about 2 ° C, more preferably to a maximum of about 3 ° C, 4 ° C, 5 ° C or 6 ° C, even more preferably at a maximum of about 7 ° C or 8 ° C and most preferably at the desired temperature used for packaging. The beer can then be packaged directly, that is, in this embodiment, the beer is not maintained for any period of time at the temperature at which it has cooled.

In the invention, additional processing steps can be carried out to achieve additional clarification and / or stability, if required. In fact, if a shortened stabilization phase is used that corresponds to the period required to cool the beer to the desired temperature for packaging, it may be desired to treat the beer with PVPP and / or silica gel, for example. This can help ensure prolonged shelf stability.

The beer stabilized according to the present invention can be further processed to achieve a level of clarification and stability that makes the product acceptable for sale. This is generally achieved using an additional treatment stage. A filtration stage can also be used. This is normally the final stage in the brewing process before packaging.

Any known technique in this regard can be used. For example, such clarification treatment and / or additional stability can be achieved using a suitable adsorbent treatment, such as treatment with PVPP, silica gel, galotannins or immobilized crosslinked insoluble agarose. The use of these treatments and their application to brewing is well known to those skilled in the art. PVPP is usually added at an amount of about 10 to about 70 g / hl. The silica gel is usually added at an amount of about 10 to about 70 g / hl.

Examples of immobilized crosslinked insoluble agarose include the combined stabilization systems described in WO97 / 43401 and US 6,001,406 and the combined stabilization system resin manufactured by GE Health Care Bioscience AB (see Taylor et al. 2006, Use of the Combined Stabilization System and its impact on beer composition, Proceedings of the Institute of Brewing and Distilling, Asia Pacific Section, Hobart, Australia). Alternatively, or in addition, an enzymatic treatment, such as papama treatment, could be used to achieve additional stability and / or clarity.

The treatments described above can be used in an immobilized form, for example immobilized PVPP particles.

All additional treatments explained above can be used in conjunction with the method of stabilization of the invention. A combination of the additional treatments may be used, for example two, three, four or all of such additional treatments, in conjunction with the method of stabilizing the invention.

Conveniently, such clarification and / or additional stability treatment can be carried out once a beer has been stabilized according to the invention. The invention also refers, however, to processes in which an additional clarification and / or stability treatment is carried out at some other point in the process or simultaneously with stabilization according to the invention.

In a preferred embodiment of the invention, proline specific endoprotease is used as disclosed in document EPA-1326957, thus optionally making the treatment with PVPP and silica superfluous and making the inherent costs for equipment for PVPP regeneration and redundant. the labor costs associated with the operation of this equipment. Additionally, the natural antioxidant potential of beer is increased and the

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Marginal colloidal stability of many beers treated with PVPP or silica gel. In addition, the risk of exposure to oxygen in the beverage is drastically reduced and residual currents are minimized.

In a preferred embodiment of the present invention, this shortening of the stabilization phase is combined with a shortening of the maturation phase (the phase that primarily intends to convert diacetyl to acetoma). In the context of the present invention, the beginning of the maturation phase is hereinafter defined as the moment where the degree of apparent attenuation reaches from about 70% to about 90%, more preferably from about 77 to about 84%, as estimated using a beer density measurement. The beer density can be measured by a pycnometer or a density meter (EBC methods 8.2.1 and 8.2.2 respectively) and the attenuation limit is determined according to the EBC method 8.6.1 or 8.6.2 (Fermentability, limit of attenuation of the must). In the context of the present invention, the end of the maturation phase can be defined as the moment where diacetyl levels have been reduced to less than about 0.10 mg / liter if measured according to the EBC method 9.24.1 Dicetons Neighborhoods in beer: Spectrophotometric method. The maturation phase can be shortened by methods known in the art. For example, maturation can be accelerated using high temperatures as described for the processes of "pressure fermentation" and "fermentation in hot maturing" (Narziss, L. 1990 Ferment, 3, 54-62). Maturation can also be shortened using immobilized yeasts in a bioreactor approach or using acetolactate decarboxylase activity (ALDC: EC 4.1.15; see, for example, US 5,108,925 or US 4,708,875). Using one or a combination of these methods, maturation can be limited to two days, one day or even less than one day as feasible using a bioreactor operated at a high temperature.

In the context of the present invention, the phase that incorporates both the maturation and stabilization phases of beer production is called the low fermentation period.

Accordingly, the invention provides the use of a proline specific protease to accelerate a brewing process. Normally, the acceleration occurs as a shortening of the post-fermentation period, for example by shortening the duration of the low fermentation period, preferably reducing the stabilization period.

The terms "accelerate" and "shorten" in this context are intended to indicate a brewing process that needs less time to be carried out compared to an equivalent process where a specific proline protease is not used. Acceleration generally occurs as a consequence of a shortened post-fermentation period, that is, that period can be carried out for less time if a specific proline protease is used compared to an equivalent process that does not use such an enzyme.

If the use of the invention is combined with measures to shorten the maturation phase, the entire low fermentation period may be restricted to less than 8, 7 or 6 days. Preferably, it is restricted to less than 5, 4 or 3 days. More preferably, it is restricted to less than 2 days. Even more preferably, the entire low fermentation period is restricted to less than 24 hours.

In a preferred embodiment of the present invention, a low fermentation period that is restricted to 4 days or less can be achieved by combining a proline specific protease with a decarboxylase acetolactate, for example an alpha-acetolactate decarboxylase.

In a preferred embodiment, the proline specific protease and, optionally, the acetolactate decarboxylase, are added to the primary fermentation. After this primary fermentation, the green beer can undergo a rapid maturation procedure and then, after the diacetyl levels have been reduced to less than about 0.10 mg / liter, the beer can be immediately cooled to about 2 ° C, preferably at about 5 ° C, more preferably at about 7 ° C, and most preferably at a suitable temperature for packaging and then, optionally, filtered to obtain a clarified beer, ready for bottling. In this way, an acceleration of the post-fermentation phase, and also of the global brewing process, can be achieved.

The advantages of the process according to the invention apply to fermented low-fermented beers in the upper part, in addition to those fermented in the lower part. The fermented beers on the top ferment and ripen very quickly but, like the fermented beers on the bottom, they require long periods of cold storage to eliminate polyphenol-protema complexes. The advantages of the process according to the invention apply particularly to fermented beers in the lower fermentation. The use and method of the invention can also be applied to beers in which spontaneous fermentation has taken place.

In another aspect, the present invention relates to a method in which the proline specific protease is used according to the invention. That is, the invention provides a method of accelerating a brewing process, preferably reducing the duration of the low fermentation period (such as reducing the duration of the stabilization period), a method comprising carrying out said brewing process. beer in the presence of a proline specific protease. The low fermentation phase, for example the stabilization phase, can be carried out at a temperature higher than that normally used in the brewing process.

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conventional. Normally, the duration of the stabilization phase will be the time required to cool the beer from the ripening phase to the packaging temperature.

In the context of the present invention, the words peptide and proteme are used interchangeably. In the context of the present invention, the words "fog", "opacity" and "turbidity" are also used interchangeably.

To determine the moment where the apparent degree of attenuation reaches approximately 77 to approximately 84%, the beer density measurement is used. The EBC method 9.24.1 Neighborhood Dicetonas in beer is used: Spectrophotometric method to quantify diacetyl levels. Note that the EBC 9.24.1 method is designed to measure diketones in general, however, as diacetyl is by far the main component of the diketones, therefore in the context of the present invention the result of this measurement method is considered Indicative of the level of diacetyl. The diacetyl level can be measured alternatively with a gas chromatogram using the EBC method 9.24.2 Neighborhood dicetons in beer. To quantify the amount of turbidity in a beverage, a turbidimeter can be used. In a turbidimeter the amount of light that is dispersed at a prescribed angle with respect to the direction of the incident light beam is measured. It is known that turbidity measurements are very suitable for turbidity measurement formed as a result of protema-polyphenol interactions.

A polyphenol is defined as a compound that has a chemical structure whose structure contains at least two aromatic rings substituted with at least one hydroxyl group or that has a chemical structure containing at least one aromatic ring substituted with at least two hydroxyl groups. Examples of polyphenols are tannins and flavonoids, which include, for example, catechins, flavonols and anthocyanins.

The term "beer", as used herein, is intended to cover at least the beer prepared from mixtures prepared from non-malted cereals, in addition to all mixtures prepared from malted cereals, and all mixtures prepared from a mixture of malted and non-malted cereals. The term "beer" covers fermented beers in the lower part, but also fermented beers in the upper part, in addition to beers prepared with auxiliaries, and beers with all possible alcohol contents.

The amount of proline specific protease added during fermentation can vary depending on the levels of malt used and the type of fermentation carried out. In a preferred embodiment of the use and / or method according to the invention, about 7.5 to about 15 units (PPU) are added, for example about 10 to about 12.5 units (PPU) of proline specific endoprotease activity , per hectolitre of a 100% malt beer at, for example, approximately 12 degrees Plate. A maximum amount of proline specific protease activity to be added cannot be specified. The maximum amount is, for example, dependent on the desired amount of reduction or prevention of turbidity, the composition of the beer at the pH at which the protease has its maximum activity. The definition of enzyme unit is provided in the Materials and methods section of the present application.

The proline specific protease can be added at different stages during the preparation of a beer. The addition of the enzyme at the beginning of the fermentation gives the best possible results. However, the enzyme can be added to a mixture or to a fermented beer before turbidity has formed.

In this text, the terms prolyl specific protease, proline specific protease, proline specific endoprotease, proline specific endopeptidase and protease having a specific prolyl activity or similar expressions are used interchangeably.

The proline specific protease can be used in the invention in an isolated or purified form. By "isolated" or "purified" is provided a specific proline protease removed from its native environment. For example, the recombinantly produced proline specific protease expressed in host cells is considered isolated for the purpose of the invention, since they are native or recombinant polypeptides that have been substantially purified by any suitable technique such as, for example, the purification method of a single stage unveiled in Smith and Johnson, Gene 67: 31-40 (1988).

A specific proline protease suitable for use in the invention can be recovered from recombinant cell cultures by methods well known to those skilled in the art, including, for example, precipitation with ammonium or ethanol sulfate, acid extraction and chromatographic methods such as high performance liquid chromatography (HPLC).

A specific proline protease suitable for use in the invention may be a naturally purified product, a product or a chemical synthesis, a product produced by a recombinant technique from a prokaryotic or eukaryotic host, which includes, for example, bacterial cells. , of yeast, fungic, of superior plants, of insect and of mairnfero.

It will be understood, however, that the protease can be mixed with vegetables or diluents that will not interfere with the intended purpose of the enzyme and will still be considered as isolated. A specific proline protease suitable for use in the invention may also be in a more substantially purified form. Therefore, the

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Proline specific protease may be comprised in a preparation in which more than 70%, for example more than 80%, 90%, 95%, 98% or 99%, of the proteins in the preparation is a specific proline protease.

Typically, the proline specific protease may be in a form free of or substantially free of any other protease.

A specific proline protease suitable for use in the invention can be used in an immobilized form so that large amounts of protein containing liquids can be treated. Forms for selecting appropriate support materials and suitable immobilization methods have been widely described in the literature, for example in "Immobilization of Enzymes and Cells" (ed. Gordon F. Bickerstaff; ISBN 0-89603-386-4).

In the context of the present invention, a proline specific protease is defined as a protease that cuts off proteins or peptides at sites where the protein or peptide contains a proline residue in its chain. Preferably, a proline specific protease is an endoprotease that "cuts" (hydrolyzes) proteins or peptides at sites where the protein or peptide contains a proline residue. In the method according to the invention, a proline specific endoprotease is preferably used which hydrolyzes the peptide bond at the carboxy terminus of proline moieties. Examples of such enzymes are prolyl oligopeptidases (EC 3.4.21.26), in addition to the prolyl endoprotease derived from Aspergillus niger reported in J. Agric Food Chem, Vol 53 (20), 7950-7957, 2005) and dipeptidyl peptidases specific to proline such as DPP IV (EC 3.4.14.5). A specific proline endoprotease that cuts proline residues at its NH2 end is described, for example, in a Nature publication dated January 15, 1998, Vol. 391, p. 301-304.

As is typical for enzyme activities, the activity of proline-specific endoproteases depends on the pH. Normally, a proline specific endoprotease having a maximum prolyl specific activity at a pH corresponding to the pH of the beer (or mixture or must, etc.) to which it is added is used in the invention. In a preferred embodiment of the method according to the invention, a protease with an optimal acidic pH, that is, with an optimum pH of 6.0 or lower - for example pH 5, 4, or 3 - is added to the fermentation Primary beer. In a more preferred embodiment of the use and / or method according to the invention, a protease with an optimal acid pH is added and is actively secreted by a food grade microorganism in the fermentation broth to the fermentation of beer.

Proline specific proteases were widely found in animals and plants, but their presence in microorganisms seems to be limited. To date, specific proline proteases have been identified in Aspergillus species (EP 0 522 428), Flavobacterium (EP 0 967 285) and Aeromonas (J. Biochem, 113, 790-796), Xanthomonas and Bacteroides. Although the specific proline enzymes of the mayone of these organisms are active at approximately pH 8, the Aspergillus enzyme is optimally active at approximately pH 5. The proline specific protease of the invention can be isolated from one of the aforementioned microbial species, particularly of a species of Aspergillus. Preferably, the proline specific endoprotease is isolated from a strain of Aspergillus niger. More preferably, the proline specific endoprotease is isolated from an Aspergillus niger host manipulated to express excessively a gene encoding a proline specific endoprotease, although other hosts, such as E. coli, are suitable expression vectors. For example, cloning and excess production of the specific proline endoprotease derived from Flavobacterium in, among others, E. coli, has provided certain specific proline endoproteases in a pure form. An example of such an excess production construction is provided in the World Journal of Microbiology & Biotechnology, Vol 11, pp 209-212. An Aspergillus niger host is preferably used to produce a non-recombinant self-construct that uses A. niger promoters to drive the expression of a gene encoding a specific proline endoprotease of A. niger.

Most preferably, the proline specific endoprotease is the endoprotease as disclosed in EPA-1326957, a protease incorporated herein by reference. In EPA-1326957, the use of proline specific endoprotease in brewing is also disclosed. In that document, however, only the use to reduce turbidity in beverages is disclosed. It has not been disclosed that the proline specific endoprotease could be used to accelerate the brewing process as such. In addition, for the person skilled in the art, treatment with PVPP and / or with silica gel is known to be instantaneous. No methods have been disclosed in which the low fermentation period or the stabilization period was shortened. If a conventional process is used without an appropriate stabilization phase, it will be apparent to the person skilled in the art that this produces beers of poor quality, for example with respect to colloidal stability and clarity. It was therefore surprising that, after the use of a specific proline endoprotease in brewing, the process could be shortened without detrimental effects on the quality of the beer.

The activity of acetolactate decarboxylase added during fermentation may vary between limits known to those skilled in the art. An indication of the required activities is provided in a document by Hannemann (MBAA TQ, Vol 39, no 3, 2002, 149-155).

Modern breweries strive to eliminate all bulk powders such as PVPP, silica gel or diatomaceous earth from the brewing process. In this approach, diatomaceous earth filtration

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Final is replaced by a membrane filtration or cross flow filtration. According to another embodiment of the invention, the beer production process according to the invention can be carried out without the use of any bulk powder such as PVPP or hydrogel of silica or diatomaceous earth for filtration. Accordingly, the beer prepared according to the use and / or method of the invention may be free of, or substantially free of, PVPP and / or hydrogel of silica and / or diatomaceous earth.

Using the method according to the invention, beer can be clarified using filtration techniques such as cross flow filtration. Accordingly, the invention covers a process in which a beer is prepared using a proline specific protease in combination with a cross flow membrane filter.

The present invention relates to a method of brewing, by which a proline specific protease is used, whereby the stabilization period can be shortened with respect to a brewing process in which no protease is used. proline specific, while at least the same properties are maintained with respect to the visual clarity of the beer according to EBC 9.29 terminology for visual determination.

The invention also refers to beer obtained by the method according to the invention. Normally, the clarity of beers can be judged in various ways. The EBC 9.29 method describes how turbidity is measured in beers. EBC units are measured in turbidimeters with an angle of 90 degrees. Alternatively, visual evaluation can be done. According to the method of EBC 9.29, that is, when measuring the units of EBC with a dispersion angle of 90 degrees, the beer having a turbidity of less than 0.5 units of EBC will be visually evaluated as "bright". In the event that the turbidity of the beer is between approximately 0.5 and 1.0 units of EBC, the beer will be evaluated visually as "very clear", between 2 and 1 units of EBC "very slightly cloudy", between 2 and 4 units of "turbid" EBC and above 8 units of "very cloudy" EBC. Of course, exact equivalence between different turbidity scales or between visual observations made in different laboratories should not be expected, but a great discrepancy cannot be expected. In addition, finally, visual inspection as done by the consumer will be important from a commercial point of view. Therefore, it is generally accepted that a beer must be (almost) bright to be commercially acceptable, that is, have a value of units of EBC measured with dispersion at 90 degrees of maximum 1.

Surprisingly, it has been found that for the beer prepared with the method according to the invention, the value of EBC units did not correspond to the visual evaluation by a trained panel. It was assumed that higher amounts of protein and polyphenols still present in beer produce dispersion values at 90 degrees higher, but cannot be detected with the naked eye. As shown in the examples, it has been found possible to have a visual evaluation of "bright" for a beer that has a value of EBC units greater than 1 and also a visual evaluation of "almost bright" for a beer that has a value of units of EBC greater than 2, ranging from even to 8. That is, the beer obtained according to the method of the invention has a chemical (physical) composition other than conventionally brewed beers.

This has been confirmed using beer particle analysis stabilized by different methods. This showed that the beers stabilized according to the invention comprise particles that are smaller on average than those identified in beers stabilized using PVPP or silica hydrogel.

Therefore, another aspect of the invention is a beer having a turbidity greater than 1 unit of EBC, preferably equal to or greater than 1.25, and most preferably equal to or greater than 1.5, while visual evaluation by a Panel trained using EBC terminology will classify the beer as "bright" immediately after brewing and measured at 1 ° C. In another embodiment, the invention relates to a beer having a turbidity greater than 2 units of EBC, preferably greater than 3, more preferably greater than 4, even more preferably greater than 6, and most preferably greater than 8 units of EBC , while visual evaluation by a panel trained using EBC terminology will classify the beer as "almost bright" measured at 1 ° C after at least 3 months of storage of the beer at room temperature, preferably at least 6 months of storage of the beer or most preferably 9 months of storage of the beer at room temperature.

The beer obtained according to the use and / or method of the invention will normally be packaged. Any suitable container can be used, for example a bottle, a barrel or a can. The beer prepared according to the use and / or method of the invention can also be packaged in a bulk tank. Accordingly, the invention provides a container comprising the beer obtained according to the use and / or method of the invention, for example a bottle, a barrel or a can comprising such a beer.

Hereinafter, the invention is illustrated by the following non-limiting examples.

MATERIALS AND METHODS

Measurements of proline specific enzyme activity

The activity of proline-specific endoproteases that have optimal pH below pH 6.0 in the synthetic Z-Gly-Pro-pNA peptide at 37 ° C in a sodium citrate / diphosphate buffer at pH 4.6 is tested. The activity of

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Proline specific endoproteases that have optimal neutral pH are tested in the synthetic Z-Gly-PropNA peptide at 37 ° C in a phosphate buffer at pH 7.0.

The activity of dipeptidyl dipeptidases having an optimum pH below pH 6.0 is tested in the synthetic Gly-Pro-pNA peptide at 37 ° C in a sodium citrate / diphosphate buffer at pH 4.6.

The activity of dipeptidyl dipeptidases having optimum pH below pH 6.0 are tested in the synthetic Gly-Pro-pNA peptide at 37 ° C in a phosphate buffer at pH 7.0.

The reaction products of all proline specific proteases are monitored spectrophotometrically at 405 nM. One unit (1 PPU) is defined as the amount of enzyme that releases 1 pmol of p-nitroanilide per minute under these test conditions.

Analysis during the brewing process

The various analyzes were carried out according to the 2004 edition of "Analytica - EBC" (Fachverlag Hans Carl, Nuremberg, Germany), unless otherwise indicated. Malt analyzes were carried out according to the following EBC methods 4-2, 4-5-1, 4-9-1, 4-3-1, 4-18, 8-13-2, 4-8 , 4-15 and 4-14. The maceration was monitored by measuring the rate of saccharification and the filtration of malt mass. The musts were analyzed according to EBC 4-5-1, 4-72, 4-11, in addition to free amino nitrogen (EBC method 8.10), total nitrogen (either by EBC method 8.9.1 - Kjeldahl - or by 8.9.2 - Dumas combustion method), pH and total must polyphenol (EBC method 8.12). The various fermentation experiments were followed by temperature, decrease in extract and pressure. The population of yeast was determined in the inoculation, first day of fermentation and before the filtration of the beer. Diacetyl was measured by gas chromatography during fermentation, maturation and just before filtration. Beer filtration was monitored by yeast counts before and after filtration, by relevant and low pressure drops. Dissolved oxygen was measured in carbonated water, in beer the inlet and outlet of filtration and in bottled beer. The various beer analyzes included the methods of 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, Ross & Clark and NIBEM (9.42) for retention values of head, trans-2-nonenal, and reducing power. Turbidity development was followed using various turbidity measurements at various temperatures (see Example 2) at 25 and 90 degree dispersion angles using the VOS ROTA 90/25 turbidity meter (Haffmans, Venlo, The Netherlands). Expiration tests were carried out according to the EBC 9.30 predictive shelf stability test.

The brewing process

All the brewing experiments were carried out on a 20 hl scale with 300 kg of Pilsen malt (Heineken A type) and 950 I of water for maceration. The brewing incorporated a process of maceration with the following temperature regime: 45 ° C for 20 minutes, 45 to 64 ° C in 20 minutes, at 64 ° C for 15 minutes, from 64 to 76 ° C in 12 minutes, at 76 ° C for 25 minutes and finally from 76 to 78 ° C in 5 minutes, followed by filtration of the malt mass. Hot water was used for bubbling. The cooking was for 90 minutes and hops were added in the form of hops granules. Cold stabilization was carried out under conditions as specified in the individual examples. The beer was filtered using a plate filter coated with a thick and a fine grade precoating. The fine-grade precoating was also used as a filter aid. For bottling a Carbofill packing machine was used. The bottles were pasteurized for 15 minutes at 60 ° C.

EXAMPLES

Example 1 (comparative)

Pilot scale beer production

In the 20-hl semi-industrial pilot brewery at IFBM (Institute Frangais de la Brasserie et de la Malterie, Vandoeuvre-les-Nancy, France) two pure malt musts were made under identical conditions. During fermentation, one of these musts was added a specific proline protease (Brewers Clarex of DSM Food Specialties, Delft, The Netherlands, which contains 5 PPU / gram of product) in a concentration of 10 PPU / hectolitre of must, in addition to acetolactate decarboxylase (Maturex L from NOVO, Bagsvaerd, Denmark, which contains 1,500 ADU / gram of product) in a concentration of 4500 ADU / hectolitre of must at the beginning of fermentation. This fermentation was designated "test fermentation." The other must was fermented as such, and "control fermentation" was designated.

Brewing

The beers were produced from 300 kg of barley malt and hops granules. The maceration conditions were a ratio of water: grinding of 4: 1 (vol / weight), pH 5.6. The maceration diagram included a first stage at 45 ° C for 20 minutes, a second stage at 64 ° C for 15 minutes, a third stage at 74 ° C for 30 minutes, and finally a transfer of the must to the tank at 78 ° C for 5 minutes. The heating rates were 1 ° C / minute each time. After the transfer of the must to the tank, the malt mixture is

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filter in a filtration tank; first, must recirculation and hot bubbling (78 ° C) at pH 5.6 were applied. The resulting musts were cooked for 90 minutes, after which good separations of precipitates were made with a whirlpool.

Fermentation

The fermentations were carried out with the yeast strain of the lower part Rh as verified by VLB (Berlin, Germany) by inoculating with 17,106 viable cells / ml of the 12 ° C must plate. The fermentation process was carried out at 12 ° C to 5 ° C Dish (3 days) and then continued at 14 ° C until the end of the fermentation (3 days).

Post-fermentation

At the end of the fermentation, the control fermentation was maintained at 14 ° C for three more days to reduce diacetyl levels. After this maturation period, the beer was cooled to 0 ° C in one day and the beer stabilized for 5 days at 0 ° C before treatment with single-use PVPP at a dosage of 30 g / hl, beer filtration with diatomaceous earth and bottling.

For the test fermentation, however, no ripening period was applied. In addition, the stabilization period was restricted to the period required to cool the beer to 4 ° C. Directly after the end of the fermentation, the beer was cooled to 4 ° C for a period of 4 days, following a linear temperature decrease. Because Brewers Clarex was used during fermentation, no beer stabilizing agents (such as PVPP or silica gel) were added. Instead, after cooling the beers, they were filtered directly with diatomaceous earth, and subsequently bottled. Standard beer parameters (for example, pH, color, bitterness, ethanol, etc.) were tested according to the methods established by the European Beer Convention (EBC), which are described in its "Analytica - EBC".

Results

Generally, the analysis of standard beers indicates that the beer parameters were similar for both control and test fermentation. Dissolved oxygen levels were comparable in both trials and less than 0.2 mg / l. The initial turbidity levels were comparable for both beers, while the test beer has significantly lower values in the alcohol cold turbidity test according to L. Chapon (EBC method 9.41). Additionally, during the beer expiration test "Beer shelf stability prediction" (EBC method 9.30), the test beer yielded better than the control fermentation beer.

The level of neighborhood diketones of beers from both the test and control fermentations was below 0.10 mg / liter (measured with the EBC method 9.24.1 Neighborhood dicetonas in beer: Spectrophotometric method). Sensory analysis was performed by a trained panel (consisting of eight people) in IFBM (Vandoeuvre-les-Nancy, France) in a standardized procedure, in which after testing the sample, the experts assigned an intensity score for the flavor attributes. The results were collected to form a sensory profile of the sample. The tests were made according to the guidelines for sensory analysis, described in Chapter 13 of the 2004 edition "Analytica - EBC". No significant taste differences could be observed for the taste panel between the control and test fermentation beer.

Conclusion

This example demonstrates that the application of a specific proline protease and acetolactate decarboxylase could produce a beer that has standard beer parameters (measured with methods established by the European Beer Convention (EBC)) that are similar to beer beer parameters. reference and that does not show significant taste differences compared to the reference beer. In addition, the use according to the invention leads to a time-saving process, because a low fermentation period of 9 days (control) could be reduced to 4 days. In addition, significant energy savings could be achieved by cooling to 4 ° C for 4 days, instead of cooling to 0 ° C in one day and maintaining this temperature for another 5 days.

Example 2

Shortening of the cold stabilization period and its effects on the clarity of stabilized enzyme, 100% malt beers

Brewing trials were carried out to assess the effect of a specific proline endoprotease in 100% malt beers after the use of shortened stabilization periods. For this purpose, six different musts were prepared and fermented according to exactly the same protocol (see Materials and methods). At five fermenters, the specific proline protease of Aspergillus niger was added at a concentration of 0.125 PPU / liter (see Materials and methods for defining activity) and in the five fermentations, the enzyme was added to the must, before the inoculation. The sixth fermentation served as a reference and no specific proline enzyme was added. All fermenters were inoculated with fresh low fermentation yeast (approx. 17 million cells / ml of must) and the fermentation took place at 12 ° C up to 5 plate followed

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from a temperature rise to 14 ° C for the elimination of neighborhood diketones such as diacetyl. At the end of maturation (in this case nine days after the beginning of fermentation), the beer cooled to minus 1 ° C. The matured beer of all fermenters with the enzyme added was maintained for various periods at less than 1 ° C and then filtered through diatomaceous earth. Four batches to which the proline specific enzyme had been added were filtered as such. The fifth batch with specific proline protease added was treated in line with single use PVPP (40 g / hl; injected into the beer filter) and then filtered.

The matured beer of the fermenter without added enzyme was also cooled to less than 1 ° C and was subjected to a 'classical' stabilization protocol, that is, the beer was kept at less 1 ° C for 9 days followed by the same treatment in line with 40 g / hl of single use PVPP and subsequently filtered with diatomaceous earth. After packaging, the bottles were pasteurized at 15 PU and stored at 20 ° C.

After a storage period of six weeks, the various beers were subjected to turbidity measurements at 20 ° C and 1 ° C. For this purpose, the beer was pre-incubated for 24 hours at the measurement temperature, that is, either 1 or 20 ° C and then the turbidity of the beer was measured at this temperature. Turbidity readings were recorded using a turbidimeter that mediates a dispersion at 90 and 25 degrees. The dispersion data at 90 and 25 degrees obtained with this turbidimeter (a Haffmans VOS ROTA 90/25) are shown in Table 1 as values of "H90" and "H25", respectively, together with the relevant measurement temperature. Before these turbidity measurements, the bottles were shaken to homogenize and left enough time for the gas bubbles to disappear. Multiple measurements were carried out to ensure that gas bubbles did not influence the reading. Apart from these turbidity measurements, visual and flavor evaluations were carried out using a trained panel (n = 5).

Table 1.

 Comparative Lot 1 Lot 2 Lot 3 Lot 4 Comparative Lot 5 Comparative Reference

 Number of stabilization days at -1 ° C

 0 1 3 5 0 9

 Prol Enzyme Spec

 sf sf sf sf sf no

 PVPP (40 g / hl)

 no no no no sf sf

 Actual turbidity (H25) at 20 ° C

 0.30 0.31 0.32 0.28 0.28 0.27

 Actual turbidity (H25) at 1 ° C

 0.34 0.34 0.37 0.32 0.30 0.28

 Actual turbidity (H90) at 20 ° C

 0.48 0.59 1.06 0.83 0.48 0.48

 Actual turbidity (H90) at 1 ° C

 0.52 0.66 1.17 0.90 0.53 0.49

 Expiration test according to EBC 9.30 (H25)

 0.70 0.35 0.33 0.27 0.22 0.22

 Expiration test EBC 9.30 (H90)

 1.0 0.75 1.26 0.92 0.52 0.48

 Visual evaluation at 8 ° C at six weeks after bottling (EBC 9.29 terminology)

 bright bright bright bright bright bright

 Flavor (3 weeks after bottling)

 good good good good good good

According to a visual inspection, all bottled beers, that is, which include beer treated with enzymes that did not undergo any cold stabilization period, were completely clear after cooling for 24 hours at 8 ° C after a period of storage of six weeks at 20 ° C.

Surprisingly enough, these visual turbidity assessments were not completely in line with the different turbidity readings obtained. According to the EBC standard, beers measured according to the EBC 9.29 method (measured at a 90 degree dispersion angle) and giving values between 1.0 and 2.0 EBC are "very slightly cloudy." Above 2.0 EBC, beers begin to be "slightly cloudy." Dispersion readings at 90 degrees gave values of up to 1.26 EBC (see Table 1), and all of these beers were visually evaluated as "bright." This discrepancy is discussed in Example 3.

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It is also considerable that, according to the expert taste panel, none of the beers produced showed an abnormal flavor profile.

Example 3 (comparative)

Turbidity measurements and turbidity formation

Three 20 hl fermentations were performed according to the protocol described in Example 2 for a 100% malt must of 12 Plato. The first fermentation (reference) was carried out without the addition of a specific proline protease during fermentation. At the two other fermentations, the specific proline protease of A. niger was added in the inoculation at concentrations of 0.125 and 0.20 PPU / I respectively. At the end of maturation (in this case nine days after the beginning of the fermentation), the three beers stabilized in fno for 5 days at 1 ° C, after which the yeast and the final precipitates were removed. In no case was a PVPp or silica gel treatment applied. The three beers were filtered on a diatomaceous earth filter, which had a thick and a fine grade precoat. Finally, the beers were bottled and pasteurized at 15 PU and then stored at room temperature for shelf stability studies.

After various storage periods, the beer was pre-incubated for 24 hours at 1 ° C and then the turbidity of the beer was measured at this temperature at the 90 degree dispersion angle. Before measuring, the bottles were shaken to homogenize and left enough time for the gas bubbles to disappear. Multiple measurements were carried out to ensure that gas bubbles did not influence the reading. The data obtained are summarized in Table 2.

Table 2.

 Storage months at 20 ° C

 0 3 6 9

 H90 (EBC) values measured at 1 ° C

 Reference (without enzyme)

 7.2 24.8 20.7 21.8

 Fermentation + enzyme (0.125 PPU / I)

 1.5 4.6 7.5 6.9

 Fermentation + enzyme (0.20 PPU / I)

 1.3 4.0 6.9 5.2

Along with the turbidity measurements, the beers were visually inspected by a trained panel (n = 5) and judged using EBC 9.29 terminology.

As expected, reference beers not treated with enzyme showed high values of EBC H90. In line with these high values of H90, visual inspection classified all samples at t = 0, 3, 6 and 9 months as "very cloudy" (the classifications used the terminology specified in the EBC method 9.29).

Of the beers treated with enzyme, only fresh samples (t = 0) showed low H90 values (1.5 and 1.3 EBC respectively). Unlike the inventors' expectations, the visual panel readings for fresh enzyme-treated beers (t = 0 months) were "bright" while, based on the H90 values as obtained, a classification of "was expected" very slightly cloudy. "

Enzyme treated samples stored for longer periods showed all H90 values of 4 EBC or higher. Surprisingly, the visual classifications obtained from the two beers treated with enzyme after 3, 6 and 9 months of storage were classified as "almost bright" instead of "cloudy", as expected from their H90 values. .

Therefore, the present inventors conclude that, as a result of the use of proline specific protease in the beer fermentation process, the instrumental H90 dispersion values are not indicative of the perception of visual turbidity, nor of the colloidal stability of the beer packaged according to the invention. In addition, the present inventors conclude that the beer according to the invention is different from the beers known in the art.

Example 4 (comparative)

Particle size analysis of beers stabilized by different methods

In the IFBM semi-industrial pilot brewery (Institute Frangais de la Brasserie et de la Malterie, Vandoeuvre-les-Nancy, France), four fermentations were carried out according to the protocol described in Example 2 for a must 100 malt of 12 Plate. To one of the fermentations the specific proline protease of A. niger was added in the inoculation at a concentration of 0.125 PPU / I. To the other three fermentations no specific proline protease was added. At the end of maturation (in this case nine days after the beginning of fermentation),

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All beers were stabilized in fno for 5 days at 1 ° C, after the yeast and fine precipitates were removed. One of the three beers that did not stabilize with the proline specific protease was then stabilized with 30 g / hl of single use PVPP (injected into the beer filtration line). Another beer to which no specific proline protease was added was stabilized with 30 g / hl of silica hydrogel (injected into the beer filtration line). Beer treated with the specific proline protease of A. niger and the remaining "untreated" control beer were filtered as such, that is, without the addition of PVPP or silica hydrogel. The four beers were filtered on a diatomaceous earth filter, which had a coarse grade and a fine grade precoating, and a diatomaceous earth filter aid was added during filtration in all cases. Finally, the beers were bottled and pasteurized at 15 PU and then stored at room temperature. During the process, oxygen levels were carefully minimized, and were found to be comparable for all four beers.

After approximately 10 months of storage at room temperature, the distribution of the particle size of the four beers was studied in Brewing Research International (BRI, Nutfield, United Kingdom) using photonic correlation spectroscopy (PCS) at 6 ° C. This technique does not count particles as such, but provides a light scattering reading and is capable of attributing proportions of that dispersion to classes of specific size particles, allowing for this document the comparison of the distributions of the particle size of the particles. Four samples of beer.

The cumulative normalized dispersion intensity is represented in Figure 1. Figure 1 indicates that in the "untreated" control beer all the scattered light results from the smallest particles of 2000 nanometers. For beers stabilized with PVPP and silica hydrogel, all light scattering particles are smaller than approximately 1250 and 750 nm, respectively. However, the particles in beer treated with proline specific protease are much smaller on average: no larger particles of 385 nm were found. For comparison, the graphs in Figure 1 were quantified by two variables:

• d50, the particle diameter at which 50% of the light scattering is of smaller particles than the diameter, and:

• d90, the diameter of the particle at which 90% of the light scattering is of smaller particles than the diameter.

The latest data is provided in Table 3.

Table 3: d50 and d90 values (see text for explanation) for IFBM beer samples with different stabilization. NT: Untreated, PVPP: polyvinylpyrrolidone polymer, SHG: silica hydrogel,

PSP: proline specific protease.

 Treatment

 Temperature (° C) d50 (nm) dgo (nm)

 NT-control

 6 895 1073

 30 g / hl of PVPP

 6 556 1141

 30 g / hl SHG

 6 537 629

 0.125 PPU / I PSP

 6 325 361

Both of the values of d50 and d90 in Table 3 indicate that the particles that generate dispersion in beer with the proline specific protease are substantially smaller than in beers stabilized with PVPP or silica hydrogel. This is in line with the hypothesis of the present inventors, that the hydrolysis of the proteins that activate turbidity by the proline specific protease prevents or reduces the formation of protein-polyphenol complexes during aging. The present inventors conclude that, as a result of the action of the proline specific protease, the turbine activating proteins are hydrolyzed so that the complexes between proteins and polyphenols remain smaller during storage, thus leading to the discrepancy observed between the evaluation of visual turbidity by expert panels and the dispersion readings at 90 relatively high instrumental degrees. Again, the present inventors conclude that the beer obtainable using the method of the invention is different from the beers known in the art.

Example 5

Shortening of the cold stabilization period and its effects on the development of turbidity in 100% malt beers after storage of 4 and 6 months at room temperature

This example provides data on the long-term stability of the beers described in Example 2. Bottled and pasteurized beers were stored at room temperature (approximately 20 ° C)

over a period of up to 6 months. After 4 and 6 months of storage, the beers (Lots 1, 2, 3, 4 and 5 plus the Reference as listed in Table 1) were analyzed using turbidity measurements and the visual evaluation was also described in Example 2 The data obtained are presented in Table 4.

Table 4

 Comparative Lot 1 Lot 2 Lot 3 Lot 4 Comparative Lot 5 Comparative Reference

 Number of stabilization days at -1 ° C

 0 1 3 5 0 9

 Prol Enzyme Spec

 sf sf sf sf yes no

 PVPP (40 g / hl)

 no no no no yes sf

 Data obtained after 4 months of storage at room temperature

 Actual turbidity (H25) at 20 ° C

 0.60 0.58 0.51 0.38 0.36 0.36

 Actual turbidity (H25) at 1 ° C

 1.69 1.25 0.87 0.61 0.57 0.55

 Actual turbidity (H90) at 20 ° C

 0.82 0.87 1.32 0.96 0.65 0.51

 Actual turbidity (H90) at 1 ° C

 2.76 1.96 2.17 1.44 0.68 0.69

 Visual evaluation at 8 ° C

 Very slightly cloudy Very slightly cloudy bright bright bright bright

 Visual evaluation at 1 ° C

 Very slightly cloudy Very slightly cloudy Very slightly cloudy Very slightly cloudy bright Bright

 Data obtained after 6 months of storage at room temperature

 Actual turbidity (H25) at 20 ° C

 1.78 1.64 1.56 1.40 1.34 1.35

 Actual turbidity (H25) at 1 ° C

 2.17 1.74 1.38 1.12 1.10 1.04

 Actual turbidity (H90) at 20 ° C

 1.59 1.49 2.11 1.52 1.07 1.04

 Actual turbidity (H90) at 1 ° C

 4.24 3.10 2.97 1.94 1.11 1.00

 Visual evaluation at 8 ° C

 Very slightly cloudy Very slightly cloudy Very slightly cloudy Very slightly cloudy bright bright

 Visual evaluation at 1 ° C

 Very slightly cloudy Very slightly cloudy Very slightly cloudy Very slightly cloudy bright bright

5

The data provided in Table 1 of Example 2 shows that after 6 weeks of storage at room temperature, after visual evaluation according to EBC 9.29, all bottled and pasteurized beers are classified as "bright". According to these data, the use of the specific proline protease guarantees excellent beers, although the stabilization period in fno is reduced to the period required to cool the matured beer to minus 1 ° C, that is, essentially without a period of Maintenance at this low temperature. However, as shown in Table 4, the turbidity stability of the beers changes when the beers are stored for longer periods. After four months of room storage,

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All beers treated with enzymes that receive stabilization at least 0 to 5 days develop a very mild turbidity. Therefore, the present inventors conclude that if the proline specific protease is used in combination with a minimum stabilization period, 100% malt beers remain visually stable for a period of at least up to 6 weeks. The beer in Lot 5, treated with the specific proline protease plus PVPP, is still "bright" even when it underwent the period of zero sub-zero stabilization. Thus, the last finding indicates that using a specific proline protease in combination with PVPP allows a spectacular shortening of the stabilization period in fno without any detrimental effect on the long-term turbidity stability of the beer. The fact that the present inventors are dealing with 100% malt beers makes this finding even more surprising.

Example 6

Combination of enzymatic treatment with short stabilization periods at elevated temperatures

Apart from the duration of the stabilization period in fno, the energy required to cool the beer to below 0 ° C also adds very significant cost to the beer production process. Therefore, the benefits of using a specific proline protease should ideally not be limited to much shorter periods of stabilization, but should make the sub-zero stabilization temperatures redundant. To test the last option, the present inventors initiated a new set of pilot brewing experiments. Six trials of brewing were carried out on a 20 hl scale, essentially under the conditions described in the Materials and Methods section and in Example 2 of the present application. Again, the effects of use of a specific proline protease were compared with the use of PVPP and silica. Various fno stabilization protocols were used ranging from a few hours to 4 days at 0 and 7 ° C. Because the proline specific protease plus PVPP appears to have a potent beer stabilizer combination, the conditions of Lot 5 of the experiment described in Example 2 were repeated. However, in the present experiment, the beer did not cool to less than 1 ° C but at 7 ° C only. The last temperature provides a convenient option, since it represents a maximum temperature normally used for bottle or barrel packaging.

In the first 20 hl fermentation, the matured beer was subjected to a period of stabilization in fno which involved 1 day of cooling at 0 ° C, followed by a storage period of 4 days at 0 ° C. Then, the beer was filtered with diatomaceous earth and divided into 2 parts during the beer filtration: the first 7 hl were treated by PVPP 30 g / hl mixed with diatomaceous earth (filter aid) and the last 7 hl were treated with Siligel S (Spindal, Armainvilliers, France; 40 g / hl) also mixed with diatomaceous earth (filter aid). After filtration, the beers were bottled and pasteurized. In Table 5, these two products are called Lot 1 (with PVPP) and Lot 2 (with silica gel).

The second 20 hl fermentation was carried out in exactly the same way as the first fermentation. However, in this case, the beer underwent a period of stabilization in fno which involved 1 day of cooling at 7 ° C followed by a storage period of 4 days at 7 ° C. These two products are called Lot 3 (with PVPP) and Lot 4 (with silica gel).

Both the third and fourth fermentation of 20 hl were carried out by adding the specific proline endoprotease derived from A. niger in the must before inoculation at a concentration of 0.125 PPU / I. The beer resulting from this third fermentation was subjected to a period of stabilization in fno which implies 1 day of cooling at 0 ° C followed by a storage period of 4 days at 0 ° C, followed by filtration in diatomaceous earth (without addition of neither PVPP nor silica gel), bottling and pasteurization (Lot 5). The beer from the fourth fermentation underwent a stabilization period in fno which involved 1 day of cooling at 7 ° C followed by a storage period of 4 days at 7 ° C. Again followed by filtration in diatomaceous earth (without addition of PVPP or silica gel), bottling and pasteurization (Lot 6).

In the fifth fermentation, stabilization in fno was carried out precisely by cooling the matured beer to 0 ° C (lasting less than a day), after the beer was filtered with diatomaceous earth. Then, the resulting beer was again divided into 2 parts during the filtration of the beer: the first 7 hl were treated with PVPP 30 g / hl mixed with diatomaceous earth (Lot 7). The last 7 hl of this beer were treated with Siligel S 40 g / hl, also mixed with diatomaceous earth (filter aid), bottled and pasteurized (Lot 8).

In the sixth fermentation, the specific proline endoprotease derived from A. niger in the must was added again, prior to inoculation at a concentration of (0.125 PPU / l). After maturation, the beer was cooled directly to 7 ° C and treated with PVPP 30 g / hl mixed with diatomaceous earth. After filtration, this beer is also bottled and pasteurized directly. In Table 5 the resulting beer is called Lot 9.

Shortly after bottling, all beers underwent a large number of analyzes of standard beers such as alcohol, bitterness, diacetyl, polyphenol and nonenal levels. In addition, head retention values were determined (NIBEM and Ross & Clark). The data obtained showed that the qualities of the different beers obtained are similar and unexpected differences cannot be shown. Sensory analyzes also showed only minor differences. As expected, the biggest differences were found in the performance of beers in various expiration tests. For example, expiration data according to EBC 9.30 and

5

10

fifteen

twenty

Final turbidity after an accelerated aging test at 60 ° C for 6 days was determined shortly after bottling the various beers. The results of these measurements are shown in Table 5 (see "Expiration" in addition to "6 days 60 ° C.) Table 5 also provides the various H90 turbidity data and visual evaluations of bottled beer as recorded after storage period of 8 weeks at room temperature Measurement of this H90 data was carried out as described in Example 2.

Table 5

 Comparative Lot 1 Comparative Lot 2 Comparative Lot 3 Comparative Lot 4 Comparative Lot 5 Comparative Lot 6 Comparative Lot 7 Comparative Lot 8 Comparative Lot 9

 Days at 0 ° C

 4 4 4 Cool alone 0 Cool alone 0

 Days at 7 ° C

     4 4 4 Cool only 0

 + prol enzyme

 No no no no yes Yes no no yes

 + PVPP

 30 g / hl no 30 g / hl no no No 30 g / hl no 30 g / hl

 + Silice

 No 40 g / hl no 40 g / hl no No no 40 g / hl no

 Expiration test: EBC 9.30 Final turbidity at 1 ° C (H90)

 1.70 5.07 3.57 11.4 1.62 1.58 1.85 6.30 0.68

 Expiration test: 6 days 60 ° C 24 h at 1 ° C (H90)

 7.41 14.15 11.48 38.82 3.22 4.02 7.60 15.18 2.09

 Data obtained after 8 weeks of storage at room temperature

 (H90) at 1 ° C

 0.62 2.97 1.13 6.71 0.97 1.74 1.14 3.31 0.63

 (H90) at 20 ° C

 0.69 0.71 0.50 0.58 0.82 1.50 1.08 0.60 0.54

 Visual at 8 ° C

 Brill. Brill. Brill. Vsh Brill. Brill. Vsh Vsh Brill.

 Visual at 1 ° C

 Brill. Sh Vsh Turbias Brill. Brill. Vsh Sh Brill.

 "Brill" .: Bright, "Vsh": Very slightly cloudy, "Sh": Slightly cloudy. All after visual evaluation according to EBC 9.29 after storage for 24 hours. "Cool only 0" refers to a cooling to the indicated temperature without a maintenance period after that temperature.

The data provided in Table 5 demonstrates once again that the use of proline specific protease alone allows periods of stabilization in fn dramatically shortened (see Lot 5). Additionally, the data in Table 5 demonstrate that such a short stabilization period is also feasible at elevated temperatures (see Lot 6). As such, this finding is economically highly relevant for a specific category of brewers. The data presented in Example 5 in combination with the relatively high EBC 9.30 expiration values for stabilized beers without using the proline specific protease as shown in Table 5 of this example also suggest that these stabilization conditions may not be suitable to ensure visually bright beers after prolonged storage periods. If long-term shelf periods are anticipated, the data presented here (Lot 9) suggest that a "Cool to bottling temperature alone" protocol, which is a rapid cooling to approximately 7 ° C, is adequate on condition that a specific proline protease is combined with a PVPP treatment. Thus, the most surprising finding of the present example is that combining the proline specific protease (optionally in combination with PVPP if long-term shelf periods are expected) seems to allow a complete omission of the stabilization period at fno. The implication is a very significant processing shortcut: from maturation directly to bottle filling and even generating visually bright beers stable on shelf.

Claims (9)

1. Method for the production of beer comprising fermenting a must in the presence of a proline specific protease, followed by a maturation phase and stabilization phase, in which the stabilization phase lasts less than 5 days. 5 2. Method according to claim 1, wherein the duration of the stabilization phase is reduced to the time required to cool the beer to the desired final temperature for filtration and / or packaging. 3. Method according to claims 1 or 2, whereby the stabilization phase takes place at a temperature suitable for beer packaging, preferably at about 7 ° C. 4. Method according to any one of claims 1 to 3, wherein an alpha-acetolactate decarboxylase 10 is also present during fermentation. 5. Method according to any one of claims 1 to 4, wherein the duration of the low fermentation period is less than 8 days. 6. Method according to any one of claims 1 to 5, wherein PVPP is used. 7. Method according to any one of claims 1 to 6, comprising a filtration step, wherein a cross flow membrane filter is used. 8. Method according to any one of the preceding claims, wherein the proline specific protease is a specific proline protease with an optimum acidic pH. 9. Beer obtainable by the method according to any one of claims 1 to 8. 10. A bottle, can or barrel comprising beer according to claim 9. twenty