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Title:
METHOD OF PRODUCING A GRAIN MALT AND THE MALT PRODUCT OBTAINED IN THIS WAY
Document Type and Number:
WIPO Patent Application WO/2018/093285
Kind Code:
A1
Abstract:
The present invention pertains to the food industry, in particular to the brewing industry, and it can be used in the processing of grain in the process of making malt. More specifically, the present invention pertains to a method of production of grain malt from cereal grain, which may involve: conditioning of the grain at moisture content of the grain in the range of 15-80% and for a time not longer than one day for moisture content in the indicated range; irrigation and/or soaking of the conditioned grain with an aqueous solution of a food enzyme having hydrolase activity; incubating the grains until they germinate with mass transport of the enzyme from the aqueous phase to the grain volume. The method may further comprise that the conditioning and/or the incubating are done until achieving a geometrical relation of channel size between the starch granules in the grain and the hydraulic diameter of the enzyme molecule; drying of the grain to a moisture content level of 6-12%. The present invention also proposes a method of production of grain malt involving: conditioning of the grain at moisture content of the grain in the range of 15-80% and for a time not longer than one day for moisture content in the indicated range; irrigation and/or soaking of the conditioned grain with wort with extractivity of 9 to 18 wt. %, containing previously cultivated microorganisms having hydrolase enzyme activity, and enzymes produced by said microorganisms; incubating the grains until they germinate with mass transport of the enzyme from the wort to the grain volume; wherein the conditioning and/or the incubating are also done until achieving a geometrical relation of channel size between the starch granules in the grain and the hydraulic diameter of the enzyme molecule; drying of the grain to a moisture content level of 6-12%. Furthermore, the invention pertains to a malt product obtained by the indicated methods with improved qualities.

Inventors:
AFONIN, Dmitrii Vladimirovich (ul. Kupchinskaya, 8 korp. 1, kv. 23, St.Petersburg 1, 192281, RU)
MATVEEV, Igor Valerievich (ul. Karbysheva, 4 korp. 1, kv. 15, St.Petersburg 1, 194021, RU)
Application Number:
RU2016/000797
Publication Date:
May 24, 2018
Filing Date:
November 18, 2016
Export Citation:
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Assignee:
BALTIKA BREWERIES - PART OF THE CARLSBERG GROUP (6 Verkhniy per, 3St.Petersburg, 2, 194292, RU)
International Classes:
A23L7/25; C12C1/02; C12C1/027; C12C1/067; C12C1/18; C12C5/00
Domestic Patent References:
WO1998003627A11998-01-29
WO2000024864A12000-05-04
WO1994029430A11994-12-22
WO1999019467A11999-04-22
WO2002038787A22002-05-16
WO2006069289A22006-06-29
WO2011068803A12011-06-09
WO2011127802A12011-10-20
WO2012001139A12012-01-05
WO1988009795A11988-12-15
WO2005052148A22005-06-09
WO2010128140A12010-11-11
WO2010063288A22010-06-10
Foreign References:
GB1316226A1973-05-09
FR2312559A11976-12-24
GB2356869A2001-06-06
RU2249032C12005-03-27
RU2250248C22005-04-20
RU2535870C22014-12-20
US5405624A1995-04-11
US7241462B22007-07-10
US5024941A1991-06-18
US4794175A1988-12-27
US4247637A1981-01-27
US6255084B12001-07-03
US6620924B22003-09-16
US4863864A1989-09-05
US4618579A1986-10-21
Other References:
YOSHIKI YAMASAKI, AGRIC. BIOL. CHEM., vol. 41, no. 5, 1977, pages 755 - 762
SVENSSON ET AL., CARLSBERG RES. COMMUN, vol. 48, 1983, pages 529 - 544
BOEL ET AL., EMBO J., vol. 3, 1984, pages 1097 - 1102
HAYASHIDA ET AL., AGRIC. BIOL. CHEM., vol. 53, 1989, pages 923 - 929
ASHIKARI ET AL., AGRIC. BIOL. CHEM, vol. 50, 1986, pages 957 - 964
ASHIKARI ET AL., APP. MICROBIO. BIOTECH, vol. 32, 1989, pages 129 - 133
HOUGHTON-LARSEN ET AL., APPL. MICROBIOL. BIOTECHNOL, vol. 62, 2003, pages 210 - 217
Attorney, Agent or Firm:
NILOVA, Maria Innokentievna (PATENTICA, BOX 1125St.Petersburg, 190000, RU)
Download PDF:
Claims:
CLAIMS

1. A method of production of malt from cereal grain, comprising the steps of:

• contacting cereal grains with an aqueous solution of one or more enzyme(s) having hydrolase activity

• incubating grains until they germinate

• drying the grains to a moisture content level of 6 to 12 %.

2. The method according to claim 1 , wherein the method comprises the steps of:

a) conditioning the cereal grains to a moisture content of the grain in the range of 15-80%

b) contacting cereal grains with an aqueous solution of one or more enzyme(s) having hydrolase activity

c) incubating grains until they germinate

d) drying the grains to a moisture content level of 6 to 12 %.

3. The method according to claim 2, wherein step a) comprises incubating said cereal grains in an aqueous solution for at the most one day.

4. The method according to any one of claims 2 to 3, wherein step a) comprises incubating said cereal grains in an aqueous solution for in the range of 5 to 10 hours.

5. The method according to any one of claims 2 to 4, wherein step a) comprises conditioning the cereal grains to a moisture content in the range of 24 to 40%.

6. The method according to any one of claims 2 to 5, wherein the cereal grains have an initial moisture content of <18%, and wherein step a) comprises conditioning said grains to a moisture content in the range of 20 to 30%, preferably in the range of 22 to 26%.

7. The method according to any one of the preceding claims, wherein at least one enzyme with hydrolase activity is an a-amylase.

8. The method according to any one of the preceding claims, wherein at least one enzyme with hydrolase activity is an amyloglucosidase.

9. The method according to any one of the preceding claims, wherein at least one enzyme with hydrolase activity is a β-glucanase.

10. The method according to any one of the preceding claims, wherein the cereal grains are contacted with at least two enzyme(s) selected from the group consisting of a-amylases, amyloglucosidase, xylanases, β-glucanases and pullulanases.

11. The method according to any one of the preceding claims, wherein the step of incubating said grains is performed until the grains have a moisture content of at least 28%.

12. The method according to any one of the preceding claims, wherein the step of incubating is performed at a temperature in the range of 12 to 24°C.

13. The method according to any one of the preceding claims, wherein the aqueous solution comprising enzyme(s) has a pH in the range of 5.5 to 8.

14. The method according to any one of the preceding claims, wherein the steps of contacting and incubating are performed for a total of time in the range of 1 to 10 hours, for example for in the range of 1 to 5 hours.

15. The method according to any one of the preceding claims, wherein the step of contacting cereal grains with an aqueous solution comprising enzyme(s) is repeated more than once.

16. The method according to any one of the preceding claims, wherein the steps of conditioning, contacting and incubating are performed for a total of time in the range of 5 to 20 hours, such as in the range of 6 to 15 hours, for example in the range of 7 to 12 hours.

17. A method of production of grain malt from cereal grain, involving:

a) conditioning of the grain to a moisture content of the grain in the range of 15-80% and for a time not longer than one day for moisture content in the indicated range;

b) irrigation and/or soaking of the conditioned grain with an aqueous solution of a food enzyme having hydrolase activity;

c) incubating the grains until they germinate with mass transport of the enzyme from the aqueous phase to the grain volume;

wherein the conditioning and/or the incubating are done until achieving a geometrical relation of channel size between the starch granules in the grain and the hydraulic diameter of the enzyme molecule;

d) drying of the grain to a moisture content level of 6-12%.

18. The method according to any one of claims 2 to 17, characterized in that the temperature range of the water used for the malting in stages (a) and (b) is from 5 to 65° C.

19. The method according to any one of claims 2 to 18, characterized in that the pH of the water used for the malting in stages (a) and (b) is in the range of 4 to 8.7.

20. The method according to any one of claims 2 to 19, characterized in that the use of cofactors is further included in stage (b).

21. The method according to claim 20, characterized in that the cofactors are calcium ions.

22. The method according to claim 15, characterized in that the calcium ions are added to the aqueous solution in the quantity of 80-1200 mg/l.

23. The method according to any one of claims 17 to 22, characterized in that the food enzyme is selected from amyloglucosidase, protease, a-amylase, β-glucanase, xylanase, pullulanase, lipoxygenase, glucose isomerase, lipase, protease or combinations of these.

24. The method according to any one of claims 17 to 23, characterized in that stage (b) of introducing a food enzyme in aqueous solution is done one or more times.

25. The method according to any one of claims 17 to 24, characterized in that stage (c) is done until achieving a mass transport of enzyme from the aqueous solution to the grain volume of at least 80%.

26. The method according to any one of the preceding claims, characterized in that cereal grain is selected from the group consisting of barley, wheat, rye and oats.

27. A method of production of grain malt, involving:

a) conditioning of the grain at moisture content of the grain in the range of 15-80% and for a time not longer than one day for moisture content in the indicated range;

b) irrigation and/or soaking of the conditioned grain with wort with extract content of 9 to 18%, containing previously cultivated microorganisms having hydrolase enzyme activity, and enzymes produced by said microorganisms;

c) incubating the grains until they germinate with mass transport of the enzyme from the wort to the grain volume;

wherein the conditioning and/or the incubating are done until achieving a geometrical relation of channel size between the starch granules in the grain and the hydraulic diameter of the enzyme molecule;

d) drying of the grain to a moisture content level of 6-12%.

28. The method according to claim 27, characterized in that the temperature range of the water used for the malting in stage (a) and the wort in stage (b) is from 5 to 65° C.

29. The method according to claims 27 to 28, characterized in that the pH of the water used for the malting in stage (a) and the wort in stage (b) is in the range of 4 to 8.7.

30. The method according to any one of claims 27 to 29, characterized in that the use of cofactors is further included in stage (b).

31. The method according to claim 30, characterized in that the cofactors are calcium ions.

32. The method according to claim 31 , characterized in that calcium ions are added in a quantity of 80-1200 mg/l.

33. The method according to any one of claims 27 to 32, characterized in that the previously cultivated microorganisms having hydrolase enzyme activity are chosen from Aspergillus niger, Bacillus licheniformis, Talaromyces emersonii, Trichoderma longibrachiatum, Bacillus amyloliquefaciens.

34. The method according to any one of claims 27 to 33, characterized in that said cultures of microorganisms having hydrolase enzyme activity produce enzymes chosen from amyloglucosidase, protease, a-amylase, β-glucanase, xylanase, pullulanase, lipoxygenase or combinations of these.

35. The method according to any one of claims 27 to 34, characterized in that stage (b) of introducing the previously cultivated microorganisms having hydrolase enzyme activity is done one or more times.

36. The method according to any one of claims 27 to 35, characterized in that an inactivation of the microorganisms is done in addition in stage (d).

37. The method according to any one of claims 27 to 36, characterized in that there is additionally included a stage of introducing a food enzyme having hydrolase activity, in aqueous solution, before, after, or together with the introduction of the previously cultivated microorganisms having hydrolase enzyme activity.

38. The method according to claim 37, characterized in that the additionally introduced food enzyme is chosen from amyloglucosidase, protease, a-amylase, β-glucanase, xylanase, pullulanase, lipoxygenase, isomerase, lipase, protease or combinations of these.

39. The method according to any one of claims 27 to 38, characterized in that stage (c) is carried out until achieving a mass transport of enzyme from the aqueous solution and/or the wort to the grain volume of at least 80%.

40. The method according to any one of claims 27 to 39, characterized in that the cereal grain selected from the group consisting of barley, wheat, rye, and oats.

41. The method according to any one of the preceding claims, wherein the malt has an extract content of at least 70%, such as at least 75% of dry matter.

42. Malt obtained by the method according to any one of the preceding claims, characterized by an extract content of at least 75% of dry matter and/or no detectable precursors of dimethylsulfoxide (DMS).

43. Malt product prepared from malt according to claim 42.

44. The malt product according to claim 43, wherein the malt product is a malt based beverage, for example beer.

45. A method for production of a malt based beverage said method comprising the steps of

• Producing malt by the method according to any one of claims 1 to 41 ;

• preparing an aqueous extract of said malt

• optionally fermenting said aqueous extract

thereby producing a malt based beverage.

46. The method according to claim 45, wherein the method further comprises adding one or more additional compounds.

47. The method according to claim 46, wherein said additional compounds may be one or more selected from the group consisting of hops, and CO2.

48. The method according to any one of claims 45 to 47, wherein the malt based beverage is beer.

Description:
METHOD OF PRODUCING A GRAIN MALT AND THE MALT PRODUCT

OBTAINED IN THIS WAY

Field of technology of the invention

The present invention pertains to the food industry, in particular to the brewing industry, and it can be used to process grain in the process of making malt. The improved method of malting makes it possible to shorten the malting time by at least 50% and increase the electricity savings by up to 90% as compared to traditional malting techniques. Furthermore, the present method has no stringent requirements on the temperature and the salt composition of the water used for the malting. In addition, in comparison with the standard malting technology reduced levels of waste products are formed, e.g less sprouts, rinse water with high level of chemical oxygen demand (COD) and biological oxygen demand (BOD). Moreover, the invention pertains to a malt product obtained by the indicated method and characterized by improved quality. Background

Various methods are known for obtaining malt, which is the basic raw material for the production of beer and related beverages. Various characteristics of the resulting malt, such as organoleptic properties (taste, color, odor, etc.) extract content, and the absence of harmful impurities directly affect the quality of the end product. In this respect, much attention has been on improving the technological process of malt production, including, but not limited to providing optimal malting conditions, but also by direct and indirect action on the complex biochemical reactions in order to improve the quality of the end product.

Today there are many methods known for the preparation of malt, including a method in which the grain is disinfected, steeped in water at temperature of 15-25° C, germinated at temperature of 18-21° C and moisture content of 45-50% (RU2249032, IPC C12C1/00, filed 22 Aug 2003, published 27 March 2005). The drawback of this method is the length of the process, which negatively affects the energy and labor costs. There has also been described a method for production of malt in which grain is washed, soaked and germinated, while controlling the biochemical processes in the grains by successively bringing them into contact with aqueous preparations having significantly different acidity acidic pH, and controlling for each of the preparations the temperature, the time of onset and the duration of the contact with the grain (RU 2250248, IPC C12C1/00, C12C1/047, filed 19 April 2002, published 20 April 2005). To minimize raw material losses through growth of roots, the grain is exposed to a relatively short action by an activated aqueous preparation with pH 2-5 after the washing stage. In order to accelerate the processes at the steeping and germinating stages, there is a repeated exposure to an activated preparation with pH 8-1 1. Reduced or even elimination of unwanted growth of roots and sprouts after the optimal germinating period is done by relatively lengthy exposure to an activated aqueous preparation at pH 2-4. The indicated aqueous preparations are obtained by passage through the electrode chamber of a diaphragm electrolyzer, through which an electric current is passed at the same time. The drawback of this method is the labor intensity of the process, the need to use additional equipment, and also the complex exposure techniques. There is a known method for production of malt, which involve washing, disinfecting and soaking of grain, followed by germination of the grain, making use of wheat grain which is steeped in water at a temperature of 19-20° C in two stages, separated by an air intermission; the steeping of the grain is done until a moisture content of 38% is reached, after which the grain is germinated for 68 hours at temperatures decreasing from 17 to 13° C (RU 2535870, IPC C12C1/02, C12C1/027, filed 07 Feb 2013, published 20 Dec 2014). This method makes it possible to obtain malt with improved quality, and also enables the use of grain with a sufficiently high level of protein for the preparation of malt. A notable drawback of this method is the length of the process.

In the U.S. patent US 5405624 (IPC C12C1/00, C1203/02, C12C1/18), filed 05 Nov 1993, published 11 April 1995) there is disclosed a method of producing a brewery product with intense taste, involving the steeping of barley, the processing of the barley with an enzyme, the production of malt and its heating to 70-89° C for 0.5-3 hours while maintaining a moisture content of 30-55 wt. %, drying of the malt, production of wort, and fermentation with yeast. In that method, the starting material is only one cereal crop (barley), which reduces the assortment of malts; moreover, the process is distinguished by notable labor intensity. The closest to the method of the present invention is a method for production of malt from cereal crops involving steeping of the grain, which includes one or more stages at a temperature of 5-30° C at pH 1.5-14 (preferably pH 4-6), until achieving a moisture content of 20-60 wt. %, followed by drying at a temperature of 40-150° C until reaching a moisture content of 2-15 wt. %, and adding in any one of the stages of the process one or more bacterial cultures and/or activated spores to enhance the enzyme activity (US7241462, IPC C12C1/00, filed 23 July 1997, published 10 July 2007). Supplementing the bacterial cultures and/or activated spores substantially improves the quality of the malt and, accordingly, the quality of the beverages made from it. The drawback of this method is that the costs of the resources and energy for the additional technological operations (especially the obtaining of the activated spores) substantially increase the prime cost of the end product; furthermore, the length of the process may be up to 7 days.

The problem which the present invention proposes to solve is an optimization of the process of malting, resulting in improved quality of the malt, a shortening of the malting time, lower energy costs for the processing of the raw material grain, and a lower cost of the overall production process. Summary of the invention

As a result of investigating the malting process the inventors unexpectedly found that an optimization of the biological conditions of the malting of cereals, both with and without husk, provides a substantial improvement in the quality of the malt. Thus the invention provides an effective method for preparing malt involving incubation of cereal grains with an aqueous solution of one or more enzyme(s) having hydrolase activity, followed by germination and drying. The methods may also comprise a step of conditioning cereal grains to a moisture content of in the range of 15 to 80%, preferably in the range of 20 to 60%, more preferably in the range of 20 to 50%, wherein said step typically is performed before the incubation with enzyme(s). Each of these steps as well as useful enzymes having hydrolase activity are described in more detail below.

The invention is based on the surprising finding that incubation of cereal grains with enzyme(s) having hydrolase activity prior to and/or during germination allows for reduced consumption of enzyme(s). Thus, for preparation of malt based beverages it is important to obtain a high extract content of malt. This may be ensured by using conventional malting methods, which however are both time, water and energy consuming. In order to enhance the extract content, it may be useful to add enzyme(s) during mashing o malt and/or grains. The invention has however surprisingly revealed that reduced amounts of enzyme(s) are sufficient if the enzyme(s) are added prior to and/or during germination according to the methods of the invention. Furthermore, the addition of enzyme(s) allows for reduced germination times and water usage.

In one embodiment the methods of the invention involves ensuring a geometrical relation between the channel size separating the starch granules in the grain and the hydraulic diameter of the exogenous enzyme. Not wishing to be restricted to a specific theory, the inventors of the present invention surmise that an optimal regime for malting of grain can be ensured upon achieving a geometrical relation between the channel size separating the starch granules in the grain and the hydraulic diameter of the enzyme at which there is observed an accelerated movement of enzymes from the aqueous solution to the grain volume. This process can substantially shorten the malting time and, accordingly, lower the energy costs for the processing of the raw material grain and make the overall production process more economical.

Thus, in one embodiment, the invention pertains to a method of production of grain malt, involving conditioning of the grain at moisture content of the grain in the range of 15-80% and for a time not longer than one day for moisture content in the indicated range; irrigation and/or soaking of the conditioned grain with an aqueous solution of a food enzyme having hydrolase activity; incubating the grains until they germinate with mass transport of the enzyme from the aqueous phase to the grain volume;

wherein the conditioning and/or the incubating are done until achieving a geometrical relation of channel size between the starch granules in the grain and the hydraulic diameter of the enzyme molecule; drying of the grain to a moisture content level of 6-12%.

In another embodiment, the invention pertains to a method of production of grain malt, involving conditioning of the grain at moisture content of the grain in the range of 15-80% and for a contact time not longer than one day to achieve a moisture content in the indicated range; irrigation and/or soaking of the conditioned grain with wort with extractivity of 9 to 18 wt. %, containing previously cultivated microorganisms having hydrolase enzyme activity, and enzymes produced by said microorganisms; incubating the grains until germination with mass transport of the enzyme from the wort to the grain volume; wherein the conditioning and/or the incubating are done until achieving a geometrical relation of channel size separating the starch granules in the grain and the hydraulic diameter of the enzyme; drying of the grain to a moisture content level of 6-12%.

In yet another embodiment, the indicated method further includes a stage of introducing an aqueous solution comprising a food enzyme having hydrolase activity. In certain embodiments, the indicated method further includes a stage of temperature inactivation of the microorganisms.

In yet another embodiment the invention pertains to a malt product obtained by any one of the above indicated methods, characterized by at least one of the following quantities: an extract content of at least 75% of dry matter, a friability according to the Analytica-EBC method 4.15 of not more than 75%, and also absence of precursors of DMS. Brief description of the drawings

Figure 1. Microscopy of starch granules. There are shown micrographs of starch granules (transverse section of a barley grain) for fast malt (a), classical malt (b) and eco-malt obtained by the method disclosed in the present invention (c). Scale 50 pm.

Figure 2. Measurement of hydraulic radii of various protein molecules. The methods of photon correlation spectrometry (PCS) and dynamic light scattering (DLS) are used to measure the hydraulic radius of enzyme(s). The size in pm is shown on the X-axis (pm is indicated as "mem" in the figure). Data are shown for enzyme preparations of Diazyme® (amyloglucosidase), Termamyl® (a-amylase) and laminex3G (a complex enzyme preparation enriched in β-glucanase). Detailed description of the invention

The present invention proposes a new method of production of grain malt, suitable for malting of cereals with and without husk, whereby the malting is done under conditions ensuring the achievement of a geometrical relation between the size of the enzyme molecules and the internal structural elements of the grain. The present invention also relates to a malt product obtained with the aid of the indicated method.

DEFINITIONS

In the present invention, the term "grain" refers to kernels, often denoted "seeds" of representatives of the family of monocotyledons, specifically the crops used in agriculture. In a preferred embodiment of the invention, the term "grain" refers to seeds of such agricultural crops as wheat, rye, oats, barley, and buckwheat. In the most preferred embodiment of the present invention, barley grain is used. Preferably, the term "grain" refers to ungerminated grains.

The term "cereal grains" as used herein refers to grains of a cereal plant. The cereal plant may for example be selected from the group consisting of barley, rice, sorghum, maize, millet, triticale, rye, oat and wheat. In preferred embodiments of the invention the cereal grains are barley grains. Said barley may be any barley plant including both hulled and non-hulled varieties.

In the present invention the term "malt" means the product obtained upon germination of cereal grains. Malt may in particular be the product obtained upon germination of cereal grains followed by drying (e.g. by kiln drying) of the germinated cereal grains.

The term "malt product" as used herein refers to any product prepared from malt as a main ingredient. Non-limiting examples of malt products include wort or malt based beverages, such as beer.

The term "eco-malt" pertains to a product obtained by the method disclosed in the present invention and having a certain microstructure of the grain and improved characteristics.

The term "malting" pertains to the process of germinating a grain resulting in formation of malt. The molecular and biochemical transformations occurring within the grain in the process of malting take place under natural conditions, but the inventors of the present invention in the course of experimental investigations have shown that by varying certain physical factors (e.g. temperature, moisture content, pH etc.) one can deliberately control this process, and halt the transformations at a certain stage depending on the needed characteristics.

The term "starch granules" as used in the present invention refers to granules of starch of various size which are enclosed in the endosperm cells of the grain. The term "soaking" as used in the present application denotes the stage of preparation of the grain for germination, during which it takes on moisture, specifically: moistening. For the moistening of the grain, the grain can be immersed in an aqueous solution or the grain can be wetted (irrigated).

The term "germinating" as used in the present application refers to the moment of appearance of sprouts. Thus, a cereal grain may be considered to have germinated once a chit is visible. In the course of this stage, the grain undergoes fundamental biochemical changes and to a substantial degree is subjected to the influence of environmental factors (temperature, moisture content, oxygen content, and so on). The term "drying" as used in the present application denotes the stage of bringing the grain, germinated to a certain stage, to a particular moisture content. Preferably this process occurs under the influence of a combination of high temperatures and decreased moisture content, as a result of which enzymes may progressively be inactivated. In a preferred embodiment of the present invention, the drying is done to a moisture content of 6-12%.

The term "food enzyme" as used in the present application refers to a preparation of a biological catalyst of protein origin (enzyme), able to catalyze biochemical reactions and thereby accellerate the technological process. In a preferred embodiment of the invention, the term "food enzyme preparation" or "food enzyme" denotes an enzyme or collection of enzymes having hydrolase activity, which are chosen from the group consisting of amyloglucosidase, protease, a-amylase, β-glucanase, xylanase, pullulanase, lipoxygenase, isomerase (e.g. glucose isomerase), lipase, protease or combinations of these enzymes.

The term "cofactor" as used in the present application refers to auxiliary factors of non-protein origin taking part in enzyme reactions occurring in the embodiment of the methods according to the present invention. In a preferred embodiment, the term "cofactor" refers to calcium ions, which are added to the aqueous solution used for the malting. "Cofactors" may in some instances also be referred to as "coenzymes". The term "previously cultivated microorganisms having hydrolase enzyme activity" as used in the present application denotes cultures of microorganisms, known from the prior art, which are producers of hydrolase enzymes that are used in particular stages of the technological processes in the food industry, especially in brewing. In a preferred embodiment of the invention, the term "previously cultivated microorganisms having hydrolase enzyme activity" pertains to cultures of microorganisms chosen from among Aspergillus niger, Bacillus licheniformis, Talaromyces emersonii, Trichoderma longibrachiatum, Bacillus amyloliquefaciens. In the most preferred embodiment of the invention, the term "previously cultivated microorganisms having hydrolase enzyme activity" pertains to the indicated cultures of microorganisms producing enzymes chosen from the group consisting of amyloglucosidase, protease, a-amylase, β-glucanase, xylanase, pullulanase, lipoxygenase, isomerase, lipase, protease or combinations of these.

The term "wort" as used in the present application pertains to nutritive media for the cultivation of microorganisms, including solutions of sugars, grape or malt wort. In particular, the term "wort" may refer to an aqueous extract of malt prepared by mashing and optionally sparging milled malt. In a preferred embodiment, the extract content of the wort amounts to 5 to 25 wt. %, more preferably 9 to 18 wt. % of dry matter. In particular, the extract content of wort may be in the range of 5 to 25° Plato, more preferably 9 to 18° Plato. In the present invention, the phrase "transport of enzymes to the grain" means that, upon introducing the food enzyme in aqueous solution and/or the previously cultivated microorganisms having enzyme activity by irrigation and/or soaking, a transport of enzymes occurs from the aqueous solution to the grain presumably by ensuring a geometrical relation between the channel size separating the starch granules and the hydraulic diameter of enzyme, for example ensuring a transport of at least 50%, preferably 50 to 90%, more preferably 60 to 95%, more preferably 80% to 99% of the entire content of enzymes from the aqueous solution to the grain volume. Preferably said transport is determined as the coefficient of mass transport of the enzyme as described in Example 3 below. The phrase "geometrical relation between the channel size separating the starch granules and the hydraulic diameter of the enzyme" as used in the present application means the following. With the aid of microscopy or using tomography or other available measurement means, the diameter (A) of the channel separating starch granules is measured. With the help of the photon correlation spectrometry (PCS) and dynamic light scattering (DLS) methods, the hydraulic diameter of the enzyme (B) is calculated, i.e., the diameter of the enzyme in the aqueous solution under defined conditions. Next, by varying certain physical factors (especially the moisture level and optionally the pH of the water), there is obtained a defined value of B/A for which the hydraulic diameter of the enzyme molecule (B) does not exceed the distance (A) separating the starch granules. Preferably, B/A is from 0.8 to 0.95, more preferably B/A < 0.9. At this ratio, conditions are created for an enhanced mass transport of enzyme from the aqueous solution to the grain. In the present invention, the term "DMS-p" means a precursor substance of dimethylsulfide - a compound worsening the organoleptic qualities of beer.

The terms "COD" and "BOD" as used in the present application are some of the most important indicators to describe the level of pollution of waste waters of industries by organic compounds, in particular, "COD" is an indicator of the chemical oxygen demand and "BOD" an indicator of the biological oxygen demand. COD may also be referred to as "Chemical oxygen consumption" or "COC", and BOD may also be referred tp as "biochemical oxygen consumption" or "BOC".

The term "extract' as used in the present application in relation to malt (the extract content of malt) means the weight percentage of an extract of dry matter of malt (% of dry matter), i.e., a quantity which is expressed by the total level of extractable substances which, upon mashing of grain products (mixing of ground grain products with water), passes into solution from the particular malt as a percentage of the malt dry matter. Extract content of malt is preferably determined according to EBC method 4.5.1.

Thus, the extract content of malt may be determined according to the formulae:

P (M + 800)

E [% (m/m)] 100 - P

E. · 100

E 2 [% (m/m)] 100 - M where

Ei = the extract content of sample, in % (m/m)

E 2 = the extract content of dry malt, in % (m/m)

P = the extract content in wort, in % Plato

M = the moisture content of the malt, in % (m/m)

800 = the amount of distilled water added into the mash to 100 g of malt, in ml.

"a.d.s." is an abbreviation for "absolutely dry substance".

The term "approximately" as used herein in relation to numbers means +/-10%, preferably +1-5%, more preferably +/-1 %.

The term "moisture content" of a cereal grain as used herein refers to the % of H2O w/w in said grain. The moisture content of cereal grains may be determined by determining the weight of the cereal grains, followed by drying said cereal grains and determining the weight of the dried cereal grains. The difference in weight between the wet and dry cereal grains is considered to be H2O, and the moisture content is provided as the weight of H 2 0 divided by the total weight of the cereal grains (wet cereal grains).

Detailed description of the invention

Conditioning cereal grains

The methods of the invention may optionally comprise a step of conditioning cereal grains to a moisture content of the grain in the range of 15-80%, preferably in the range of 20 to 60%, more preferably in the range of 20 to 50%. In general this step is denoted step a) herein, however it is also referred to as the first stage.

Typically, the step of conditioning cereal grains to a specific moisture content is performed by either soaking cereal grain in an aqueous solution, e.g. in water or by irrigating the cereal grains with said aqueous solution. The cereal grains may be kept in contact with said aqueous solution until the desired moisture content is obtained. If conditioning is performed by irrigating the cereal grains with an aqueous solution, said irrigation may be repeated one or more times, e.g. as often as required to obtain the desired moisture content. The step of conditioning may typically be performed for at the most one day, preferably for in the range of 5 to 20 hours, such as for in the range of 5 to 10 hours.

In one embodiment the cereal grains are only contacted with sufficient aqueous solution in order to obtain the desired moisture content. In such embodiments essentially the whole of aqueous solution added may be taken up by the cereal grains. Accordingly there will essentially be no waste water. The moisture content after conditioning may depend on several factors including the moisture content of the cereal grains employed. In one embodiment the cereal grains are conditioned to a moisture content of in the range of 20 to 60%, preferably in the range of 20 to 50%, for example in the range of 20 to 45%, such as in the range of 24 to 40%. In one embodiment, the cereal grains have a starting moisture content of <18%, and it such embodiments it may be preferred that the cereal grains are conditioned to a moisture content in the range of 20 to 30%, for example in the range of 22 to 26%, such as to approximately 24%, for example 24%.

In one embodiment, the cereal grains have a starting moisture content of in the range of 10 to 15%, and it such embodiments it may be preferred that the cereal grains are conditioned to a moisture content in the range of 20 to 30%, for example in the range of 22 to 26%, such as to approximately 24%, for example 24%.

In one embodiment the cereal grains have a starting moisture content of <24%, for example a starting moisture content of in the range of 18-24%. In such embodiments, it may be preferred that the cereal grains are conditioned to a moisture content of up to 45%, such as in the range of 30 to 45%, for example in the range of 35 to 45%, such as in the range of 38 to 40%.

In one embodiment the cereal grains are conditioned until a moisture content of at least 20%, preferably at least 24%, more preferably at least 28%. In one embodiment, the aqueous solution used in the step of conditioning may be water. In another embodiment the aqueous solution used in the step of conditioning may be similar to the aqueous solution comprising the enzyme(s), except that it does not comprise enzymes. Such aqueous solutions are described herein below in the section "Contacting with enzyme(s)". For example, the pH of the aqueous solution may be as described in that section, e.g. a pH in the range of 6 to 7, for example approximately 7.4.

The step of conditioning may be performed at any adequate temperature, for example a temperature in the range of of 0 to 50°C, typically at a temperature in the range of 0 to 30°C, such as at a temperature in the range of 5 to 20°C.

Conditioning may also be performed as described herein below in the section "Production of malt by conditioning to a geometrical relation". In particular it may be performed as the first stage of the method described in that section.

Contacting with enzyme(s)

The methods of the present invention in general comprise a step of contacting cereal grains with an aqueous solution comprising one or more enzyme(s) having hydrolase activity. Said aqueous solution may also be referred to as "aqueous solution comprising enzyme(s)" herein. In some embodiments this step is performed subsequent to a step of conditioning e.g. performed as described in the section "Conditioning cereal grains". This step can be repeated one or more times.

As described herein, the methods of the invention may comprise the step of contacting cereal grains with an aqueous solution comprising one or more enzyme(s) (herein also referred to "the step of contacting") and a step of incubating the grains until they germinate (herein also referred to as "the step of incubating"). These steps may be performed sequentially, simultaneously or partly simultaneously. In embodiments wherein the step of contacting is repeated, the step of incubating may be divided into several sub-steps performed between each step of contacting and subsequent to the last step of contacting.

In general, the cereal grains (e.g. the conditioned cereal grains) may be soaked in the aqueous solution comprising enzyme(s) or the cereal grains may be irrigated with the aqueous solution comprising enzyme(s). If the cereal grains are irrigated with the aqueous solution comprising enzyme(s), said irrigation may be repeated one or more times.

In one embodiment the cereal grains are only contacted with a predetermined amount of aqueous solution comprising enzyme(s), which can be taken up by the cereal grains. This may subsequently ensure that there will essentially be no waste water. In addition to enzyme(s), said aqueous solution may comprise one or more additional compounds, e.g. buffer and/or salt. It is in general preferred that the pH of said aqueous solution comprising enzyme(s) is adjusted to allow activity of the enzyme(s) having hydrolase activity. Accordingly, the aqueous solution may comprise one or more buffers. In embodiments of the invention wherein the aqueous solution only comprise one enzyme, then it may be preferred that the pH of the aqueous solution is adjusted to approximately the optimum of said enzyme. For example, if the enzyme is a-amylase the pH of the aqueous solution may be in the range of 6 to 7, such as approximately 6.5.

Frequently, however, the aqueous solution may comprise more than one enzyme. In such cases the pH may be adjusted to a pH where all of the enzyme(s) have at least some activity. For example, the pH of the aqueous solution may be any of the pH described herein below in the section "Production of malt by conditioning to a geometrical relation" in relation to the water used for malting. In some embodiments the pH of the aqueous solution will be in the range of pH 5.5 to 8, such as in the range of pH 5.5 to 7.5, for example approximately pH 7.4, such as approximately pH 6.

The aqueous solution may also comprise salts. Thus, in embodiments of the invention wherein the aqueous solution only comprises one enzyme, then it may be preferred that the conductivity of the aqueous solution is adjusted to approximately the optimum of said enzyme. For example, if the enzyme is a-amylase the conductivity of the aqueous solution may be in the range of 500 to 1500 pS, such as approximately 1000 pS. Said salts may for example be calcium ions. The aqueous solution may comprise calcium ions, e.g. in a concentration of 80-1200 mg/l.

The aqueous solution may also comprise one or more cofactors, e.g. as described in the section "Production of malt by conditioning to a geometrical relation".

However, the aqueous solution may frequently comprise more than one enzyme in which case the conductivity may be adjusted to a conductivity where all the enzyme(s) have at least some activity. For example, the conductivity of the aqueous solution may be in the range of 20 to 1000 pS, for example in the range of 30 to 500 pS, such as in the range of 100 to 200 pS, for example approximately 120 pS.

The enzyme(s) having hydrolase activity may be any useful enzyme(s) having said activity. For example it may be one or more of the food enzymes described herein below in the section "Production of malt by conditioning to a geometrical relation". In particular it is preferred that the aqueous solution comprises at least an a-amylase.

An α-amylase according to the invention is an enzyme capable of catalyzing endohydrolysis of (1→4)-a-D-glucosidic linkages in polysaccharides containing three or more (1→4)-a-linked D-glucose units. In particular, the a-amylases according to the present invention may be α-amylase enzymes classified under EC 3.2.1.1.

A particular α-amylase enzyme to be used in the methods of the invention may be a Bacillus a-amylase. Well-known Bacillus a-amylases include a-amylase derived from a strain of B. licheniformis, B. amyloliquefaciens.or B. stearothermophilus. In one aspect of the present invention, a contemplated Bacillus α-amylase is an α-amylase as defined in WO 99/19467 on page 3, line 18 to page 6, line 27. Another example of an α-amylase to be used with the present invention is the enzyme disclosed as SEQ ID NO: 3 in WO 99/19467 or a functional homologue thereof sharing at least 70%, such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, such as at least 98% sequence identity therewith. The a-amylase may also be an a-amylase sharing at least 70%, such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, such as at least 98% sequence identity with the amino acid sequence disclosed as SEQ ID NO: 3 in WO 99/19467 with the mutations: 1181* + G182* + N193F. Also contemplated is the α-amylase in the enzyme, preparation Termamyl® SC (Novozymes A/S, Denmark). Another particular a -amylase to be used in the methods of the invention may be any fungal α-amylase, e.g., an a -amylase derived from an Aspergillus species, and preferably from a strain of Aspergillus niger. Especially contemplated is the α-amylase shown as SEQ ID NO: 1 in WO 2002/038787 or a functional homologue thereof sharing at least 70%, such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, such as at least 98% sequence identity therewith. In a preferred embodiment, the α-amylase is the polypeptide of SEQ ID NO:3 or a functional homologue thereof sharing at least 70%, such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, such as at least 98% sequence identity therewith. In one embodiment, the aqueous solution comprises at least an amyloglucosidase. Amyloglucosidase is also known as glucan 1 ,4-a-glucosidase or glucoamylase.

An amyloglucosidase according to the invention is an enzyme capable of catalyzing hydrolysis of terminal (1→4)-linked a-D-glucose residues successively from non-reducing ends of the chains with release of β-D-glucose. In particular, the amyloglucosidase according to the present invention may be amyloglucosidase classified under EC 3.2.1.3. One example of an amyloglucosidase is Uniprot: B0CVJ1 , which discloses a polypeptide from Laccaria bicolor. Other examples are the amyloglucosidases from Trametes cingulata described in WO 2006/069289. The amyloglucosidase may also be an amyloglucosidase from the fungus Gloeophyllum, e.g. from G. abietinum, G. sepiarium, or G. trabeum. Such amyloglucosidases may for example be a polypeptide comprising an amino acid sequence having preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18 of WO201 1068803.

The amyloglucosidase may also be amyloglucosidase from Penicillium oxalicum, e.g. those disclosed by Yoshiki YAMASAKI, Agric. Biol. Chem., 41 (5), 755-762, 1977)- The amyloglucosidase may also be a polypeptide comprising an amino acid sequence having preferably at least 61.5%, more preferably at least 63%, more preferably at least 65%, more preferably at least 68%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, 97%, 98%, 99% or 100% sequence identity to the mature polypeptide of SEQ ID NO: 2 of WO201 1127802.

The amyloglucosidase may also be any of the variants of glucoamylase described in WO2012/001139. Thus, for example the amyloglucosidase may be a polypeptide comprising an amino acid sequence having preferably at least 61.5%, more preferably at least 63%, more preferably at least 65%, more preferably at least 68%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, 97%, 98%, 99% or 100% sequence identity to the polypeptide of SEQ ID NO:1 or SEQ ID NO:2 of WO2012/001 139.

In another embodiment the amyloglucosidase may be one of the glucoamylases described in Svensson et al.,Carlsberg Res. Commun. 48: 529-544 (1983); Boel et al., EMBO J. 3: 1097-1102 (1984); Hayashida et al., Agric. Biol. Chem. 53 : 923-929 (1989); U.S. Patent No. 5,024,941 ; U.S.Patent No. 4,794,175, WO 88/09795;U.S. Patent No. 4,247,637; U.S.Patent No. 6,255,084; U.S. Patent No. 6,620,924; Ashikari et al., Agric. Biol. Chem. 50: 957-964 (1986); Ashikari et al., App. Microbio. Biotech. 32: 129-133 (1989), U.S. Patent No. 4,863,864; WO 05/052148, U.S. Patent No. 4,618,579; and Houghton-Larsen et al., Appl. Microbiol. Biotechnol. 62 : 210-217 (2003).

The amyloglucosidase may also be Diazyme® (Dupont), and in particular Diazyme®X4 (Dupont).

In one embodiment, the aqueous solution comprises at least a β-glucanase.

A β-glucanase according to the invention may be an enzyme capable of catalyzing endohydrolysis of (1→3)- or (1→4)-linkages in β-D-glucans when the glucose residue whose reducing group is involved in the linkage to be hydrolysed is itself substituted at C-3. In particular, the β-glucanase according to the present invention may be β-glucancase enzymes classified under EC 3.2.1.6.

The β-glucanase may be comprised in an enzyme complex or an enzyme mixture. Thus, the β-glucanase may be provided in the form of any of the enzyme complexes described in WO2010128140. The β-glucanase may also be provided as Laminex® (Dupont), preferably as Laminex®3G (Dupont).

In one embodiment the aqueous solution comprises at least 2, such as at least 3, for example all of the following enzyme(s):

• α-amylase (e.g. any of the a-amylases described above)

• amyloglucosidase (e.g. any of the amyloglucosidases described above)

• xylanase (e.g. Laminex® (Dupont))

• β-glucanase (e.g. Laminex® (Dupont))

· pullulanase (e.g. enzyme NZ26062 (Novozymes))

The aqueous solution may comprise the enzyme(s) in a purified or partly purified form. Thus, the enzyme(s) may be purified enzyme(s). They may also be in the form of a crude extract of an organism producing the enzyme(s). They may also be partly purified from such a crude extract. As described below, the enzyme(s) may also be provided in the form of microorganisms expressing said enzymes, however this is less preferred.

The step of contacting cereal grains with enzyme(s) may also be performed as described herein below in the section "Production of malt by conditioning to a geometrical relation". In particular it may be performed as the second stage of the method described in that section.

Incubating grains

The methods of the invention comprise a step of incubating cereal grains until they germninate. This step may also be referred to as the "step of incubating" herein. Typically, said step is performed subsequent to the step of contacting cereal grains with the aqueous solution comprising enzyme(s) having hydrolase activity. However the step of incubating may also be performed simultaneously with the step of contacting. If the step of contacting is repeated, the step of incubating may be divided into sub-steps, where each sub-step is performed after a step of contacting.

In embodiments where the cereal grains are soaked in said aqueous solution during the step of contacting, excess aqueous solution may be removed from the cereal grains, and subsequently the cereal grains may then be subjected to the step of incubating.

As described above, the step of contacting may also be performed by irrigating the cereal grains with the aqueous solution, in which case the cereal grains may be held in a suitable container with irrigation one or more times during the step of holding.

Typically, the step of incubating the cereal grains is performed until the cereal grains start to germinate. Thus the step of incubating may be performed for in the range of 1 to 10 hours, for example for in the range of 2 to 8, for example in the range of 1 to 5 hours hours, such as in the range of 4 to 6 hours.

As described above, the steps of contacting and incubating may be performed at least partly simultaneously. Accordingly, the steps of contacting and incubating the cereal grains may be performed for in the range of 1 to 10 hours, for example for in the range of 2 to 8 hours, for example in the range of 1 to 5 hours, such as in the range of 4 to 6 hours.

In one embodiment the steps of conditioning, contacting and incubating are performed for a total of time in the range of 5 to 20 hours, such as in the range of 6 to 15 hours, for example in the range of 7 to 12 hours.

The step may be performed at any temperature allowing germination of the cereal grains, for example by any of the temperatures described below in the section "Production of malt by conditioning to a geometrical relation" with respect to temperature ranges of the water used for the malting. In one embodiment the step of incubating is performed at a temperature in the range of 12 to 24°C, such as at approximate 14°C.

Typically, the moisture content of the cereal grains increases during the step of incubating. It is preferred that the moisture content at the end of the step of incubating is at least 20%, preferably at least 24%, even more preferably at least 28%, for example in the range of 28 to 50%.

The step of incubating may also be performed as described herein below in the section "Production of malt by conditioning to a geometrical relation". In particular it may be performed as the third stage of the method described in that section.

Drying

The methods of the invention may comprise a step of drying germinated cereal grains to a moisture content level of 6 to 12%. This step is also referred to as the "step of drying" herein.

The step of drying may be performed by any conventional method, e.g. by conventional kiln drying. Typically drying is performed at elevated temperatures, e.g. typically temperatures in the range of 50 to 80°C. The kiln drying may be performed at varying temperatures, for example the temperature may be increased during drying. For example the drying may be performed within a temperature range from 50 to 65°C, wherein the temperature is increasing during drying. In another example the drying may be performed within a temperature range from 50 to 80°C, wherein the temperature is increasing during drying. The total time for drying may for example be in the range of 3 to 10 hours, such as from 3 to 7 hours, e.g. from 3.5 to 6 hours. Typically, the drying time may be longer if lower temperatures are employed and shorter if higher temperatures are employed. Interestingly, the enzymes comprised in the malt produced according to the methods of the invention are still active after drying. This is exemplified by the high extract content of the malt. The step of drying may also be performed as described herein below in the section "Production of malt by conditioning to a geometrical relation". In particular it may be performed as the fourth stage of the method described in that section.

Properties of malt

The malt produced by the methods of the invention may herein also be referred to as "eco-malt". The methods of the invention can significantly shorten the time span of malting, thus reducing electrical energy requirements, but in the same time produce malt with high extract content.

Thus, the malt prepared by the methods of the invention preferably has an extract content of at least 70%, preferably of at least 73%, more preferably at least 75%, such as at least 76% (in % of dry matter). Extractivity is preferably determined according to the Analytica-EBC (method 4.5.1).

Beer, wort or malt may contain dimethyl sulfide (DMS) or precursors thereof. A high level of DMS imparts a beer flavor that may be undesirable. Malt prepared according to the present invention frequently comprises a low level of DMS precursors (DMS-p). In some embodiments the malt prepared by the methods of the invention comprise no detectable DMS-p. DMS-p levels may for example be determined as described in Example 6 of WO 2010/063288. Production of malt by conditioning to a geometrical relation

According to one of the embodiments of the present invention, the method of production of grain malt involves the following stages.

In the first stage, the grain is conditioned to a grain moisture content in the range of 15-80% and for a time not more than one day with moisture content in the indicated range. As has been discovered by the inventors of the present invention in the course of their experiments, by a preparatory incubating of the grains at grain moisture content in the range of 10% to 30%, 15 to 45%, 15 to 80%, 20 to 50%, 20 to 70%, 30 to 60%, 45 to 70%, 55 to 80%, 60 to 90%, and also at pH of the water in the range of pH 3.5 to 4.5, pH 4.0 to 6.2, pH 4.0 to 8.7, pH 4.7 to 7.2, pH 5.3 to 8.9, 6.5 to 8.5) there is observed an improved mass transport of enzyme to the grain volume. Not wishing to be bound by a specific theory, the inventors surmise that the improvement in the mass transport of enzyme in the following stages occurs by virtue of ensuring a geometrical relation between the size of the channels in the grain separating the starch granules and the previously measured hydraulic diameter of the enzyme molecule, which can be achieved both in the stage of conditioning and in the stage of introducing the food enzyme and the subsequent incubating of the grain. For example, the size of said channels may be determined as described herein in Example 1. The hydraulic diameter of the enzyme(s) is preferably determined in situ, e.g. during or after the step of conditioning, the step of contacting and/or the step of incubating. The hydraulic diameter may for example be determined as described in Example 1 below. It is understood that once suitable conditions have been established for the step of conditioning, the step of contacting and the step of incubating, resulting in a desired geometrical relation, then the geometrical relation need not be determined for each repetition of the method.

Depending on the size of the enzyme molecule, one will choose conditions for conditioning and/or incubating of the grain at the specified moisture content and pH to achieve the indicated geometrical relation. Frequently, enzyme(s) having hydrolase activity will have a hydraulic diameter in the range of 0.007 to 5 pm, more frequently in the range of 0.001 to 0.1 pm. Accordingly, in some embodiments it may be preferred that the average diameter of the channels separating starch granules is at least 5.5 μιτι, for example it may be preferred that the average diameter of the channels separating starch granules is in the range of 0.001 1 to 0.1 1. Amyloglucosidase may have a hydraulic diameter in the range of 0.007 to 5 pm, for example in the range of 0.007 to 0.02 pm and/or in the range of 2 to 5 μιτι. Accordingly, in some embodiments it may be preferred that the average diameter of the channels separatingstarch granules is at least 5.5 μιτι, or at least 0.022 pm. β-glucanase and/or xylanase may have a hydraulic diameter in the range of 0.007 to 3 μιτι, for example in the range of 0.007 to 0.02 μιτι and/or in the range of 0.3 to 3 pm. Accordingly, in some embodiments it may be preferred that the average diameter of the channels separatingstarch granules is at least 3.3 pm, or at least 0.022 pm. a-amylase may have a hydraulic diameter in the range of 0.04 to 2 pm, for example in the range of 0.04 to 0.1 pm and/or in the range of 0.5 to 2 pm. Accordingly, in some embodiments it may be preferred that the average diameter of the channels separatingstarch granules is at least 2.2 pm or at least 0.1 1 pm.

In embodiments of the invention, particularly where a mixture of enzymes is employed, the indicated geometrical relation may be chosen according to only one of the enzymes of the mixture, according to an average of the hydraulic diameters of the enzyme(s) of the mixture or according to the enzyme of the mixture having the largest hydraulic diameter. Typically, there is chosen an average of the hydraulic diameter(s) of the enzyme(s) of the mixture. Since the size of the channels between the starch granules may change over time, it is also comprised in the invention that the indicated ratio may first be achieved for smaller enzymes, but during the step of incubating (e.g. during the third stage) then an appropriate diameter also for larger enzymes may be obtained. Thus, without being bound by theory it is speculated that some enzymes of a mixture may begin to enter as soon as the ratio between the minimum hydraulic diameter of the enzyme(s) and that of the average channel diameter separating the starch granules reaches 0.9 or less, and entry of enzymes may continue until the ratio between the maximum hydraulic diameter of the enzyme(s) and the average channel diameter reaches 0.9 or less. In the second stage, a food enzyme or a mixture of food enzyme(s) in an aqueous solution is introduced by irrigation and/or soaking of the grain. This stage can be carried out one or more times. In a preferred embodiment of the present invention, the food enzyme is chosen from the group consisting of but not limited to amyloglucosidase, protease, a-amylase, β-glucanase, xylanase, pullulanase, lipoxygenase, isomerase, lipase, protease or combinations of these. Said isomerase may in particular be glucose isomerase.

In the third stage, the grain is preferably held -if necessary until achieving the geometrical relation of the channel size between the starch granules and the previously measured hydraulic diameter of the protein molecule of the enzyme, if this has not already occurred in the conditioning stage, and until it germinates with mass transport of the enzyme from the aqueous phase to the grain volume.

In a preferred embodiment, the coefficient of mass transport of the enzyme from the aqueous solution to the grain volume reaches at least 50%, preferably 50 to 90%, more preferably 60 to 95%, more preferably at least 80%, more preferably 80% to 99%. The coefficient of mass transport may be determined as the ratio of mean extract content of malt prepared according to the method of the invention and the mean extract obtained by mashing barley in the presence of enzymes in the mashing liquid. One useful method for determining the coefficient of mass transport is described in Example 3 below.

Enzymes not active in the process detailed above typically remain bound to the surface of the grain. It is considered likely that this residual enzymatic activity at the grain surface ensures a "closure" of surface pores, thus preventing inactivation of the internal enzymic activity.

In the fourth stage, the grain is dried to a moisture content of 6-12%.

In preferred embodiments of the present invention, the temperature range of the water used for the malting is from 5 to 65° C, from 10 to 40° C, from 20 to 50° C, from 30 to 60° C, from 5 to 30° C, from 10 to 20° C. For example, the third stage may be performed at aforementioned temperatures. In preferred embodiments, the pH of the water used for the malting is in the range of pH 4 to 8.7, pH 3.5 to 4.5, pH 4.0 to 6.2, pH 4.0 to 8.7, pH 4.7 to 7.2, pH 5.3 to 8.9, or pH 6.5 to 8.5. Said water used for malting may be the aqueous solution comprising food enzyme(s). In certain embodiments, cofactors are used in addition. Thus, the aqueous solution may comprise one or more cofactor(s). In a preferred embodiment, calcium ions are used as the cofactors, being added to the aqueous solution for the malting in a concentration of 80-1200 mg/l. In accordance with another variant embodiment of the present invention, the method of production of grain malt involves the following stages.

In the first stage, a conditioning of the grain is done to achieve a moisture content of the grain in the range of 15-80%, and for a time not more than one day at moisture content in the indicated range. Thanks to the indicated moisture content (15-80%), and also the pH of the water (pH 4-8.7), a formation of a grain microstructure may be observed, which may ensure the later stages an improved mass transport of enzyme to the grain volume, which may be based on achieving a geometrical relation between channel size separating the starch granules and the previously measured hydraulic diameter of the enzyme.

In the second stage, previously cultivated microorganisms having enzyme activity may be introduced by irrigation and/or soaking of the grain. This stage can be done one or more times. In a preferred embodiment of the present invention, the previously cultivated microorganisms having enzyme activity are chosen from among Aspergillus niger, Bacillus licheniformis, Talaromyces emersonii, Trichoderma longibrachiatum, Bacillus amyloliquefaciens. In the most preferred embodiment, these cultures of microorganisms produce enzymes chosen from among amyloglucosidase, protease, a-amylase, β-glucanase, xylanase, pullulanase, lipoxygenase, isomerase (e.g. glucose isomerase), lipase, protease or combinations of these. However, the scope of the invention is not limited by the above-cited list of microorganisms and enzymes.

In the third stage, the grain is preferably held if necessary until achieving the geometrical relation of the channel size between the starch granules and the previously measured hydraulic diameter of the protein molecule of the enzyme, and until it germinates with mass transport of the enzyme from the aqueous phase to the grain volume. In a preferred embodiment, the mass transport of the enzyme from the aqueous solution to the grain volume reaches a coefficient of mass transport of at least 80%. Said coefficient may be determined as described herein above. The rest of the enzymes remain on the surface of the grain. This surface activity of the enzymes may ensure a "closure" of pores, preventing an inactivation or evaporation of the internal activity with the water during the drying of the grain to the required moisture content. In the fourth stage, the grain is dried to a moisture content of 6-12%.

In preferred embodiments of the present invention, the temperature range of the water used for the malting is in the range from 5 to 65° C, from 10 to 40° C, from 20 to 50° C, from 30 to 60° C, from 5 to 30° C, or in the range from 10 to 20° C. In preferred embodiments, the pH of the water used for the malting is in the range of pH 4 to 8.7, pH 3.5 to 4.5, pH 4.0 to 6.2, pH 4.0 to 8.7, pH 4.7 to 7.2, pH 5.3 to 8.9, or pH 6.5 to 8.5.

In certain embodiments, cofactors are used in addition. In a preferred embodiment, calcium ions are used as the cofactors, being added to the aqueous solution for the malting in a concentration of 80-1200 mg/l.

In yet another embodiment, the indicated method further includes a stage of introducing a food enzyme having hydrolase activity in an aqueous solution. In a preferred embodiment, said additionally introduced food enzyme is chosen from the group including but not limited to amyloglucosidase, protease, a-amylase, β-glucanase, xylanase, pullulanase, lipoxygenase, isomerase (e.g. glucose isomerase), lipase, protease or combinations of these. As a rule, the additional introduction of the food enzyme is done in the second stage of the process described herein. It may be done before, after, or at the same time as the introduction of the previously cultivated microorganisms having hydrolase enzyme activity.

In certain embodiments, the method further includes a stage of temperature inactivation of the microorganisms. Variant embodiments of the method of production of a grain malt which are disclosed in the present invention are characterized by a short malting time (not more than one day), and also by the fact that no DMS-p is formed in the malting process, being a compound which afterwards impairs the organoleptic qualities of the beer.

According to yet another variant embodiment, the present invention pertains to a malt product obtained by any one of the above indicated methods, characterized by an extract content of at least 75% of dry matter, a friability according to the Analytica-EBC method 4.15 of not more than 75%, and also absence of precursors of DMS. This product may be obtained from grain regardless of the state of the husk, thanks to creating of technological conditions for transport of enzyme from the aqueous solution to the grain volume. The enzyme may be preserved under the thermal conditions of the drying and inactivation of the microorganisms. By controlling the parameters of the medium (e.g. moisture content, pH), the principle of the geometrical relation of the enzymes and the internal, prepared grain structure may be realized. As a result, one obtains eco-malt, a product having improved qualities, without the activation of compounds which are precursors of dimethylsulfide (DMS-p). EXAMPLES

Example 1

FORMATION OF A GEOMETRICAL RELATION BETWEEN CHANNEL SIZE SEPARATING STARCH GRANULES AND HYDRAULIC DIAMETER OF THE ENZYME.

Using microscopy or tomography, the diameter of the channel was measured between the starch granules (A). Figure 1 shows micrographs of the starch granules (transverse section of a barley grain) for fast malt (a), classical malt (b) and eco-malt, obtained by the method disclosed in the present invention (c). The darker areas represent the starch granules, whereas the lighter areas represent the channels between the starch granules. The average diameter of the starch channel may be determined based on measurements to reveal the average of the actual distance separating the starch granules. The average diameter of the starch channel may be determined using optical and/or electron microscope at different grain moisture, followed by measuring representative channel diameters and determining the average. Th ewater distribution on the structure of the grain and the calculation the average diameter of the starch channel grains can also be produced by low - field NMR (e.g. using an NMI20 NMR Imager and Analyser (NIUMAG, China)). Fluctuation of the average diameter of the channel between the starch granules was 0.009 - 2 μιτι, depending on moisture.

Using the methods of photon correlation spectrometry and dynamic light scattering, the hydraulic diameter of enzymes was calculated (B), i.e., the diameter of the enzyme molecule in aqueous solution under defined conditions. Optimally, the diameter should be determined in authentic systems, i.e. at conditions in which the cereal grains are contacted with the aqueous solution comprising enzyme(s). More specifically, the hydraulic diameter was determined by Dynamic Light Scattering in accordance with the ISO 22412 standard using a Nanotrac Wave (Microtrac, United States) as well as by photon correlation spectrometry by the 180° DLS-method using a NANO-flex® (ParticleMetrix, Germany). Fluctuation of the average hydraulic diameter of the enzymes is typically between 0.001 - 0.1 μιτι.

Figure 2 shows the results of the measurement of the hydraulic radii of different enzyme molecules, in particular presenting data for the enzyme preparations Diazyme® (amyloglucosidase), Termamyl® (a-amylase) and laminex 3G (a complex enzyme preparation comprising β-glucanase and xylanase). By varying certain physical factors (such as moisture level and optionally pH of the water), a particular value of B/A was achieved, such that B/A< 0.9. At this ratio, conditions are created which are believed to improve mass transport of the enzyme from the aqueous solution to the grain.

Example 2

DIFFERENCE BETWEEN MALT OBTAINED BY THE METHOD OF THE INVENTION AND MALT OBTAINED BY THE STANDARD METHOD.

The experiment utilized barley grains of a non-husked variety. The temperature of the water for soaking of the grain was 14° C, the pH of the water was 7.4, and the conductivity was 120 β.

The barley grains were steeped (step of conditioning) for 2.5 hours, followed by incubation for 4 hours for germination (airbag pause). Then exogenous enzymes were added and the grains were incubated for 1.5 hours. The grains were kiln dried for 8 hours.

The initial moisture content of the barley grains was 1 1.1 %. Moisture content was adjusted to 24% during steeping and enzymes were observed to enter the grains at a moisture content of 28%.

Specimens of fast malt (malting time 3 days), classical malt (malting time 6 days) and eco-malt obtained according to the present invention (malting time as indicated above) were micro-malted under standard regimes. The enzyme preparation used in the process of producing the eco-malt was a combination of Diazyme® (Dupont), Laminex® 3G ( Dupont) and Termamyl (Novozymes, Denmark) (D+3G+T). The extract content (in % of dry matter) was calculated by the Analytica-EBC (method 4.5.1 ).

Table 1

Table 1 shows the difference in extract content between the studied types of malt. The findings indicate that the extract content of eco-malt, obtained with a short malting time, is comparable to the extract content of classical malt, obtained by the standard method of malting with a malting time of 6 days. Accordingly, the method of malting according to the present invention generates malt in a quality comparable to classical malt, but in a substantially shorter time. At the same time, fast malt is characterized by the lowest value of extract content. The malt obtained according to the invention is also characterized by a friability of 74% determined according to Analytica-EBC method 4.15. No DMS precursors could be detected in the malt.

Example 3

VERIFICATION OF THE MASS TRANSPORT COEFFICIENT OF ENZYME. To assess the significance of the mass transport of enzyme from the aqueous solution to the grain volume, which in accordance with the proposed invention is not less than 80% when calculated as explained in this example, barley specimens were analyzed as follows. The eco-malt was produced essentially as described in Example 2. The enzyme preparation used in the process of production of the eco-malt was a combination of Diazyme® (2 g/kg) and Laminex® 3G (1 g/kg) (D+3G) or Diazyme® (2 g/kg), Laminex® 3G (1 g/kg), enzyme NZ26062 (1 g/kg)(Novozymes, Denmark) and Termamyl SCDS (0.5 g/kg) (D+3G+P+T). The amount of enzyme is provided per kg barley grains. The extract content (in % dry matter) was calculated by the Analytica-EBC (method 4.5.1). The control was specimens of dry barley.

Table 2

* Enzymes added to barley grains during germination according to the methods of the invention. ** Enzymes added to solution during mashing.

The eco-malt was obtained from the same barley variety that was used as the control. To verify the transport coefficients, the same weight of enzyme was used for preparation of eco-malt (indicated in the table column "Enzymes for malting") and also for preparation of the wort from dry barley (indicated in table column "Aqueous solution of enzymes for ground barley"). The ratio of the mean values of their extract content shows the coefficient of mass transport of the enzyme. To calculate the ratio, the mean value of the extract content from two repetitions, indicated in the last column of table 2, was used.

Thus, for example for the system D+3G the coefficient of mass transport is equal to

and for the system D+3G+P+T the coefficient of mass transport is equal to

Thus, the results presented in table 2 show that, based on the extract content (converting to % of dry matter), the value of the coefficient of mass transport for the eco-malt was 98%.

Thus, the method of production of malt presented in the present invention can significantly shorten the malting time (down to one day), and increase the savings of electrical energy. Consequently, the entire production process of malt is notably reduced. Specifically, up to 90% energy savings have been observed when compared to the standard malting methods. Furthermore, the method features no stringent requirements on the temperature and salt composition of the water used for the malting. Also, the method may be ecologically superior, because frequently there are no waste products such as sprouts, rinse water with high level of COD and BOD), which are obtained using conventional methods. This may in particular be the case, when only enough water to obtain the desired moisture content is employed, resulting in no waste water. The malt product (eco-malt) obtained by carrying out the biological regime for malting of cereals with or without husks is also characterized by improved quality, in particular, a rather high extract content and, moreover, its subsequent use in the brewing industry makes it possible to improve the organoleptic properties of the beer, since no DMS-p is formed during the malting.

Example 4

Wort having a wort extract content of 11 ° Plato and 78% fermentable sugars was prepared. Said 78% indicates the amount of fermentable sugars as a percentage of the total dry matter.

For the production of such a wort from 100% eco-malt prepared according to the methods of the invention, the following enzyme quantities were employed (in g per kg barley grains):

Diazyme ® (2 g / kg),

Laminex® 3G (1 g / kg),

26062 (1 g / kg) and

Termamyl® (1 g / kg). Alternatively, 100% unmalted barley grains were used for production of wort, the following quantities of enzyme were added during mashing to arrive at wort having the properties outlined above. The quantites were (g per kg barley grains).

Diazyme® (2,4 g / kg),

Laminex® 3G (1 ,8 g / kg),

26 062 (1 ,4 g / kg) and Termamyl® (2,2 g / kg).

It was tested whether the quantity of enzyme could be further reduced. Accordingly, an additional wort was prepared according to the methods of the invention. The following enzyme quantity of enzyme was employed (g per kg barley grains):

Diazyme ® (1.5 g / kg),

Laminex® 3G (1 g / kg), and

Termamyl® (0.5 g / kg).

This wort also had satisfying extract content and fermentable sugars.