阅读说明：本技术 生产谷物提取物的方法以及将这种提取物加工成饮料的方法 (Method for producing cereal extract and method for processing the extract into beverage ) 是由 芬恩·洛克 K·克鲁塞维奇 L·马里 伯吉特·斯卡德豪奇 索伦·克努森 T·文特 奥利· 于 2018-12-21 设计创作，主要内容包括：本发明涉及制备基于谷物的饮料的方法。例如,本发明提供了在连续通气下浸泡和使谷物谷粒发芽的方法。特别地,本发明提供了加热发芽的谷物谷粒,然后湿磨发芽的谷物谷粒,以及将发芽的谷粒在不经干燥的情况下直接转移到啤酒厂进行进一步加工的方法。与目前的方法相比,本发明的方法显著减少了水消耗、能量消耗和运输需求。(The present invention relates to a method of preparing a cereal-based beverage. For example, the present invention provides a method of steeping and germinating cereal grains under continuous aeration. In particular, the present invention provides a method of heating a malted cereal grain, then wet milling the malted cereal grain, and transferring the malted grain directly to a brewery for further processing without drying. The process of the present invention significantly reduces water consumption, energy consumption and transportation requirements compared to current processes.)

1. A method of producing an aqueous extract of cereal grains, the method comprising the steps of:

a) providing a grain of a cereal;

c) subjecting a cereal grain to a germination step, thereby obtaining a germinated grain;

d) subjecting the germinated grain to a heat treatment step at a temperature in the range of 35 to 55 ℃;

e) subdividing said germinated kernels to obtain ground germinated kernels, while said germinated kernels have a moisture content of at least 20%, with the proviso that the moisture content of said cereal kernels is not less than 20% at any time between steps c) and e); and

f) preparing an aqueous extract of the milled germinated grain,

thereby producing an aqueous extract of the grain.

2. The method of claim 1, wherein the germinating step includes incubating the grain in an aqueous solution until the grain has a moisture content of at least 30%, wherein at least 2L O is allowed to pass per hour₂Per kg dry weight cereal grain is passed through the aqueous solution.

3. The method according to any one of the preceding claims, wherein the germinating step comprises:

i. incubating the grain in an aqueous solution for 16 to 40 hours, wherein at least 2L O is allowed per hour₂(ii) per kg dry weight of cereal grains passing through the aqueous solution and wherein the grains are submerged in the aqueous solution during the incubation;

removing excess aqueous solution; incubating the moist grain in air at a temperature in the range of 15 to 30 ℃ for 20 to 50 hours.

4. A method of producing an aqueous extract of cereal grains, the method comprising the steps of:

a) providing a grain of a grain, wherein the grain is a shelled grain;

b) treating the cereal grain to remove hulls, wherein the treatment results in at least a 2% total weight loss of the cereal grain;

c) subjecting a cereal grain to a germination step, thereby obtaining a germinated grain, wherein the germination step comprises:

i. incubating the grain in an aqueous solution for 16 to 40 hours, wherein at least 2L O is allowed per hour₂(ii) per kg dry weight of cereal grains passing through the aqueous solution and wherein the grains are submerged in the aqueous solution during the incubation;

removing excess aqueous solution; and

incubating the moist grain in air at a temperature in the range of 15 to 30 ℃ for 18 to 50 hours;

d) subdividing the germinated grain to obtain ground germinated grain, while the germinated grain has a moisture content of at least 20%; with the proviso that at any time between steps c) and d) the moisture content of the cereal grain is not less than 20%; and

e) preparing an aqueous extract of the milled malted grain, thereby producing an aqueous extract of cereal.

5. A method according to any one of the preceding claims, wherein the method comprises a further step after the germination step and before the subdividing step, the further step comprising subjecting the germinated grain to a heat treatment step at a temperature in the range of 35 to 55 ℃.

6. A method according to any one of the preceding claims wherein the step of incubating the moist cereal grain in air is carried out at a flow rate of 85 to 95L/h atmospheric air/kg dry cereal grain.

7. A method according to any preceding claim wherein the step of incubating the moist grain in air is at 17 to 21L/h O₂At a flow rate per kg of dry cereal grains.

8. A method according to any one of the preceding claims wherein the duration of the step of incubating the moist grain in air is in the range 20 to 30 hours.

9. The method according to any of the preceding claims, wherein the duration of the heat treatment is in the range of 1 to 5 hours.

10. The method according to any of the preceding claims, wherein the entire germination step (step c.) is carried out for 44 to 72 hours.

11. The method according to any one of the preceding claims, wherein the germination step is carried out at a temperature in the range of 15 to 30 ℃.

12. The method according to any preceding claim, wherein removing the hulls results in a loss of total weight of cereal grain in the range of 2.5% to 7.5%, such as a loss in the range of 3% to 6%.

13. The method according to any one of the preceding claims, wherein the moisture content of the germinated cereal grain is not less than 25%, even preferably not less than 30%, and still more preferably not less than 35% at any time between the completion of the germination step and the time of subdividing the cereal grain.

14. The method according to any one of the preceding claims, wherein the cereal is barley.

15. A method of producing a beverage, the method comprising the steps of:

i. preparing an aqueous extract according to the method of any one of the preceding claims;

processing said extract into a beverage.

16. A method according to any of the preceding claims, wherein the method does not comprise the step of kiln drying the germinated grain.

17. The method according to any one of the preceding claims, wherein the method does not comprise a step of removing the rootlets.

18. A method according to any one of the preceding claims, wherein the germinated grain contains at most 4g rootlets (dry matter)/100 g germinated cereal grain (dry matter).

19. A method according to any one of the preceding claims, wherein the germinated grain contains at most 2g rootlets (dry matter)/100 g germinated cereal grain (dry matter).

Technical Field

The present invention generally relates to the germination of cereals and the preparation of aqueous extracts of cereals (e.g. by mashing), including processes for the production of beer. Thus, the present invention provides a low input, rapid and continuous process for the preparation of beer from barley grain. The method may be performed in a single facility. The invention is also applicable to the germination of other cereal grains, including rice, sorghum, corn, millet and wheat, the preparation of aqueous extracts of the grains, and brewing processes containing auxiliary materials.

Background

In commercial malting processes, barley grains are germinated or malted under controlled conditions that allow the starch and protein reserves of the starchy endosperm to be partially mobilized over a period of 4-6 days (mobilisation). The malting process is typically initiated by steeping dry barley grains in water. This process, known as steeping, is not only aimed at cleaning the grain but also to increase its moisture content to about 40-45% (w/w) in order for the subsequent endosperm mobilization step to occur more quickly. During the steeping process, the water is drained once to re-aerate the grains. This step is called "air resting" and is considered necessary, mainly because the submerged grains become anoxic after about 16 hours. After approximately 8 hours of "air rest", the grains are re-steeped in water to complete the steeping process for an additional 8 hours period — or in a series of re-steeping steps. A total of about 32 hours is required for a two-step steeping process to increase the moisture content of the dried grain to 40% or more. In some malting plants, spray soaking techniques are used.

The soaked grains are spread for germination, during which the enzymes secreted by the aleurone and scutellum epithelial cells-together with some pre-existing enzymes in the starchy endosperm cells-degrade the cell wall, starch and proteins. Under normal germination conditions, the plant hormone Gibberellic Acid (GA) is thought to be synthesized in the nodal region (nodal region) or elsewhere in the embryo, from where it diffuses along a water gradient (Fincher, 2011).

Malting manufacturers generally aim to rapidly induce the synthesis of as many starch degrading enzymes in the grain as possible. In many commercial malting programs GA is added to accelerate the process of enzyme secretion from the aleurone layer. Starch degrading enzymes-which include alpha-and beta-amylases, starch debranching enzymes and alpha-glucosidases-depolymerize the starch reserve portion of the grain into mono-, oligo-and glucose (Smith et al, 2005; concerning the beta-amylase it is noteworthy that these are deposited in the starch endosperm during grain development). The depolymerized products of the starch are then used as a carbon source by the yeast cells and fermented to beer ethanol. Mashing power (diastatic power) is a malting quality parameter that refers to the level of activity of this group of starch degrading enzymes, with high values required for brewing.

Other major components of barley grain include storage proteins, which are also present in dead starch endosperm cells, and include hordeins as well as water and salt soluble proteins. Depolymerization of these proteins also starts naturally in the malting process, but the brewer can manage the degree of degradation of these proteins, releasing enough peptides and amino acids to support yeast growth during the subsequent germination step of the brewery. However, if the degradation of the storage protein is excessive, the released protein may cause difficulties in the brewing process. In particular, high levels of released soluble protein can precipitate and form undesirable haze (haze) in the final beer product or increase the likelihood of Strecker aldehyde formation during beer storage. In the specification of malt production quality, yeast growth during fermentation requires a sufficient level of Free Amino Nitrogen (FAN). The Kolbach index is the soluble protein: a measure of the total protein ratio, generally preferred is that malt produce a sufficient Kolbach index. Thus, the extent of protein degradation is an ongoing challenge for malt makers. In addition to the beer sedimentation problem that may be associated with over-extracted proteins, very high levels of FAN can also cause difficulties by possible off-taste formation.

Malting manufacturers have also attempted to induce high levels of enzymes that degrade cell wall polysaccharides in barley grains, particularly (1, 3; 1,4) - β -glucan and arabinoxylan. Incompletely degraded (1, 3; 1,4) -ss-glucans are particularly troublesome for beer manufacturers, since they can be extracted from malt in soluble form, forming highly viscous aqueous solutions, thus slowing down the filtration process in breweries and leading to undesirable cloudiness in the final beer. Thus, low levels of soluble (1, 3; 1,4) - β -glucanase represent an important malt production quality parameter, while high levels of (1, 3; 1,4) - β -glucanase remain an important measure of malt quality.

As mentioned above, the germination process usually takes about 4-5 days. After the controlled germination step, the wet malt is dried from a moisture content of about 40% to a moisture content of 4-5%. This drying process, known as kilning, is very energy consuming and represents a major cost to the industry.

Kiln drying is considered an important part of beer production for a number of reasons. One important reason is the formation of rootlets (also known as "culms") during germination. Rootlets have a bitter taste, which affects the aftertaste of beer, and, in addition, may add undesirable color to beer (see beer brewing Technology (1999):183 and US9,326,542, published by Shokuhin Sangyo Shimbun). Once the green malt has been dried by the kiln, the rootlets can be easily removed, for example, using a rooter (deculmer). Then, according to the general textbook by d.e. briggs on "Maltsand Malting", the culms "have to be removed [. so ] because they are extremely hygroscopic, rich in soluble nitrogenous substances, substances with bad flavour and bitterness, and can be rich in sulphur dioxide and/or nitrosamines. After the malt has been stripped from the kiln to help it cool, and before the rootlets absorb moisture from the air to become loose and flexible (less brittle) and thus more difficult to break and separate, rootage should be carried out as soon as possible (D.E. Briggs, Malts and Malting; p695, 1 st edition, 1998, published by Blackie & Profesials, London, ISBN 041229800).

Kiln dried malt typically has a moisture content of 4.5-5.0%. The kiln dried malt is then transported from the malt manufacturing plant to the brewery by road, rail or sea transport. This involves the following facts: the malting and brewing processes are traditionally performed at different locations and often by different corporate entities.

In a brewery, kiln dried malt is milled to break open the grains and the resulting contents are extracted with hot water in a process known as mash. The extracted material comprises partially degraded starch, proteins and cell wall molecules as described above, and these substances are further degraded by endogenous granzymes extracted from malt. At this stage, some brewers add additional and often cheaper carbon sources (adjuncts) to support the subsequent yeast fermentation process and offset the higher malt costs. The adjunct may be barley, rice, wheat or other cereal flour from unmalted grain, but their addition may necessitate the concomitant addition of hydrolytic enzymes, as endogenous enzymes in the malt are not sufficient to degrade the components of the adjunct. The added enzymes are typically from unpurified and relatively inexpensive extracts of fungal and/or bacterial cultures. In some countries, especially in countries where beer must be produced in a strictly regulated environment, the addition of exogenous enzymes is not legal.

Further degradation of starch and other endosperm components extracted in hot water occurs in a process known as saccharification. After the mash, the extract is filtered, usually in a filter tank, and cooled. The extract can be boiled in the presence of hops or hops extract and, after cooling, yeast culture is added for fermentation of the released sugars to ethanol. The beer so produced is typically matured and filtered before bottling. The beer can also be carbonated before bottling.

Disclosure of Invention

The present invention provides methods for rapid germination and for preparing aqueous extracts of cereals. The method significantly speeds up the process of preparing a wort for the production of a cereal-based beverage, while maintaining the possibility of preparing said wort with high levels of fermentable sugars and preferably with low levels of beta-glucans and xylans. In particular, beverages prepared from the wort may be characterized by low levels of astringency.

The present invention demonstrates that a continuous and integrated brewing process from dry barley grain to beer can be performed at a single location. In this regard, the present invention provides a method of steeping and germinating a cereal grain combination to a moisture content, for example > 30%, preferably > 35%, by constant aeration. Thus, the present invention can significantly save water by eliminating the drying of the malt and energy, for example, by omitting the kiln drying step.

In one aspect of the invention, the grain is submerged in an aqueous solution (typically water) and incubated, wherein the liquid is optionally supplied with O₂. Typically, the O is₂The supply is continuous throughout the cultivation, which may for example last from 20 to 72 hours, and the grains are generally allowed not only to reach a suitable moisture content but also to germinate in a controlled manner. The germination step may also include one or more air rests, typically after the step of incubating in aqueous solution under aeration.

This controlled germination process can be shortened by adding one or more compounds capable of accelerating germination. For example, the plant hormone Gibberellic Acid (GA) may be added to the aqueous liquid-from the beginning or during incubation. GA 'activates' gene expression in its target cells (i.e. aleurone and scutellum epithelium of barley grains), including genes encoding starch, storage proteins and endogenous enzymes necessary for cell wall polysaccharide hydrolysis. Thus, using the present invention, the total time required for steeping and germination can be reduced from more than 5 days in conventional brewing processes to-2 days or less. However, in some embodiments, no exogenous GA is added during germination.

After the germination step, the present invention may comprise a step of heat treating the germinated cereal grain. The temperature is preferably controlled at a predetermined level throughout the germination step, which is typically below 35 ℃. In one aspect of the invention, the germination step is followed by a step of heat treatment at a temperature in the range of, for example, 35 to 55 ℃. The present invention has shown that the heat treatment improves the beneficial properties of the germinated grain, such as increased alpha-amylase levels, as well as limiting dextrinase in the early stages of germination.

In another aspect of the invention, the grain is a shelled grain that is dehulled prior to germination to remove at least a portion of the hulls. These peeling and/or heat treatment steps have proven to be very beneficial in order to increase the level of hydrolytic enzymes in the malted grain to produce a wort with a high level of fermentable sugars.

After the germination and/or heat treatment step, the method may comprise a step of subdividing the germinated and/or heat treated cereal grain. A particularly interesting aspect of the present invention is that the method of the invention allows for the subdivision of malted cereal grains immediately after malting and/or heat treatment. Thus, the method typically does not include a step of drying the malted cereal grain. In particular, the method does not include the step of kiln drying the malted cereal grain. Kiln drying is a very energy-consuming step, and therefore the omission of kiln drying can lead to significant energy savings. As mentioned above, an important aspect of kiln drying is to allow easy removal of the rootlets. Removal of the rootlets prior to drying is difficult to achieve. However, the malted cereal grain produced according to the present method has significantly reduced rootlets (typically less than 4g/100g malted barley (dry weight)), and as shown in the present invention, the cereal malted according to the present method does not require a kiln drying step.

The malted cereal grain may be subdivided, for example by wet milling the malted cereal grain, followed by a step of preparing an aqueous extract, for example by mashing at a predetermined temperature for any suitable time as described below in the "preparing an aqueous extract" section. Conversion of released sugars (e.g. polysaccharides) and proteins can be facilitated by the addition of a mixture of exogenous enzymes that catalyze the degradation of starch, storage proteins and cell wall polysaccharides during the pulping process. The enzymes may be partially purified from the barley itself, malt or other source parts-or from fungal and/or bacterial enzyme mixtures that may be purchased from commercial sources.

In addition to the benefits described above, the present invention eliminates the need not only for kiln drying of the malt, but also for transporting the dried grain from the malting plant to the brewery. The invention through this combined soaking and germination can save energy costs by as much as 50%, which can greatly reduce the carbon emissions of the industry. This is very important because most countries have ever-increasing legislative and tax pressures on a global scale in reducing carbon emissions from the malting and brewing industries.

Furthermore, in the context of sustainability, the present invention allows the entire production of beer to be carried out in already existing brewery facilities, requiring little additional capital expenditure.

Thus, in one aspect, there is provided a method of producing an aqueous extract of cereal grain, the method comprising the steps of:

a) providing a grain of a cereal;

c) subjecting a cereal grain to a germination step, thereby obtaining a germinated grain;

d) subjecting the germinated grain to a heat treatment step at a temperature in the range of 35 to 55 ℃;

e) subdividing said germinated kernels to obtain ground germinated kernels, while the moisture content of said germinated kernels is at least 20%, with the proviso that at any time between steps c) and e) the moisture content of said cereal kernels is not less than 20%; and

f) preparing an aqueous extract of the milled germinated grain,

thereby producing an aqueous extract of the grain.

It is to be understood that the term "no moisture content of the cereal grain is lower than 20% at any time between steps c) and e)" includes, for example, that the cereal grain may have a moisture content higher than 20% at any time during steps c), d) and e), at any time from the completion of step c) to the completion of step e) or at any time from the completion of step c) to the start of step e).

In another aspect, there is provided a method of producing an aqueous extract of cereal grain, the method comprising the steps of:

a) providing a grain of a grain, wherein the grain is a shelled grain;

b) treating the cereal grain to remove hulls, wherein the treatment results in at least a 2% total weight loss of the cereal grain;

c) subjecting a cereal grain to a germination step, thereby obtaining a germinated grain, wherein the germination step comprises:

i. incubating the grain in an aqueous solution for 16 to 40h, wherein at least 2L O is allowed per h₂(ii) per kg dry weight of cereal grains passing through the aqueous solution and wherein the grains are submerged in the aqueous solution during the incubation;

removing excess aqueous solution; and

incubating moist cereal grain in air at a temperature in the range of 15 to 30 ℃ for 18 to 50 h;

d) subdividing the germinated grain to obtain ground germinated grain, while the water content of the germinated grain is at least 20%; with the proviso that at any time between steps c) and d), the moisture content of the cereal grain is not less than 20%; and

e) preparing an aqueous extract of the milled germinated grain,

thereby producing an aqueous extract of the grain.

In another aspect, there is provided a method of producing a beverage, the method comprising the steps of:

i. preparing an aqueous extract by the methods disclosed herein;

processing said extract into a beverage.

The invention is further defined in the appended claims.

Drawings

Figure 1 shows an example of apparatus which may be used to carry out the process of the invention in which grain may be immersed in an aqueous solution and aerated continuously. The apparatus comprises a cereal grain inlet (1); a tank, such as a soaking tank (2); gas inlets, such as calculus (3); a pump, such as an air pump (4); a cereal grain outlet (5); a grain pump (6); a device for subdividing cereal grains, such as a mill (7); an inlet (8); a container, such as a container for mash (9), and an outlet (10).

Figure 2 shows an example of apparatus which may be used to perform the steps of the invention in which grain may be immersed in an aqueous solution and aerated continuously. The apparatus comprises: a cereal grain inlet (1); a tank (2); a grid or mesh, such as a metal mesh (3); gas inlets, such as gallstones (4); and a pump, such as an air pump (5).

Figure 3 shows hulless (hull-less) barley grains after incubation in water at 15 ℃ or 25 ℃ for 24 hours or 48 hours under the indicated air flow. A shows a collection of grains, while B shows representative individual grains. It is noted that after 24 hours at 15℃ the grain has already started to germinate (visible buds (chi) are visible even with a 30L/h air flow only, after 48 hours a few small rootlets are visible. after 24 hours at 25℃ the grain has started to germinate and contains visible buds even with a 30L/h air flow only, after 48 hours a few small rootlets are visible.

Figure 4 shows the hulled barley grain after incubation in water at 15 ℃ or 25 ℃ for 24 hours or 48 hours under the indicated air flow. A shows a collection of grains, while B shows representative individual grains.

FIG. 5 shows the enzymatic activity of A) alpha-amylase, B) beta-amylase and C) free limiting dextrinase after incubation of hulled barley in Water (WA) at 25 ℃ for 24 hours under aeration (90l/h/kg), followed by incubation in air (A) at 25 ℃ for 18 hours under aeration (90l/h/kg), followed by air standing in atmospheric air at different temperatures (25-40-50-60 ℃) for 6 hours (grey) or 24 hours (black).

FIG. 6 shows the enzymatic activity of A) alpha-amylase, B) beta-amylase and C) free limiting dextrinase after incubation of hulled barley in Water (WA) for 24 hours at 25 ℃ under aeration (90l/h/kg), followed by incubation in air (A) for 24 hours at 25 ℃ and then aeration for another 2 hours (grey) or 4 hours (black) at different temperatures (40, 50, 60 ℃).

FIG. 7 shows the effect of combining different individual treatments to optimize the production of hydrolases from barley grain incubated in tanks under aeration (treatment 1, 48H WA soak (grey), at 25 ℃ under aeration with 90l/H/kg air at 0.01% H₂O₂1000nM GA3 and 0.01% antifoam in water for 48h (WA); treatment 2, peeling _24h WA +24h A soak, black: under the condition of aeration by 90l/H/kg of air, the mixture is added with 0.01 percent of H₂O₂1000nM GA3 and 0.01% antifoam in tap water for 24h, followed by incubation in air at 25 ℃ for 24h with 90l/h/kg air aeration). The numbers 1 to 10 represent results from different barley varieties (cultivars).

Figure 8 shows the% weight loss after removal of rootlets in both hulled and hulless barley malted by incubation in aqueous solution for 48h (48hWA) under aeration and barley malted by standard steeping conditions for 96h (96 h for malting).

FIG. 9 shows the NDMA content in unmalted barley, barley germinated by the method of the invention (malt 1a) and 3 different conventional malts (malt 1b, malt 2 and malt 3).

Fig. 10 shows an example of a pulping process using steeped, germinated and wet milled grain.

FIG. 11 shows a filterability graph of wort prepared from grains germinated under different aeration conditions in the presence or absence of the brewing enzyme mixture Ultraflo Max (UFM).

FIG. 12 shows a graph of fermentable sugar levels in wort prepared from grains that germinated continuously for 48h at 25 ℃ under different aeration conditions. Samples supplemented with brewing enzyme mixture Ultraflo Max were labelled with UFM.

Definition of

The term "about" when used herein with respect to numerical values preferably means ± 10%, more preferably ± 5%, still more preferably ± 1%.

The term "adjunct" as used herein refers to a source of carbon-rich raw material added during the preparation of beer. The adjunct may be unmalted cereal grain, which may be milled with germinated grain prepared according to the present invention. The adjuvants can also be syrup, sugar, etc.

The term "bud" as used herein refers to an embryogenic, growing bud that is visible during the germination stage of a cereal grain.

The term "moisture content" of a grain as used herein refers to the% w/w of water in the grain.

The term "malted grain" as used herein refers to a grain that has developed visible shoots, preferably shoots of at least 1mm, for example at least 2 mm.

The term "onset of germination" as used herein refers to the point in time at which barley grain having a moisture content of less than 15% is contacted with sufficient water to initiate germination.

The term "beta-glucan" as used herein refers to the cereal cell wall polymer "(1, 3; 1,4) -beta-glucan" unless otherwise indicated.

Similarly, the term "xylan" as used herein refers to the cereal cell wall polymer "arabinoxylan", unless otherwise indicated.

The terms "kiln dried malt" and "kilnedmalt" as used herein refer to malted cereal grain that has been dried by kiln drying. The moisture content of kiln dried malt is typically about 4-5%.

The term "germinated grain" as used herein refers to a grain that has developed visible buds and visible stems.

The term "steeping" as used herein refers to a method of increasing the moisture content of cereal grain (cereal kernel).

The term "beta-glucanase" as used herein refers to an enzyme having the potential to depolymerise cereal beta-glucans. Thus, unless otherwise indicated, the term "β -glucanase" refers to an endonuclease or exonuclease or mixture thereof characterized by (1, 3; 1,4) - β -and/or (1,4) - β -glucanase activity.

The term "xylanase" as used herein refers to an enzyme having the potential to degrade the backbone and side chains of xylans and arabinoxylans. Thus, unless otherwise indicated, the term "xylanase" refers to an enzyme or mixture of enzymes characterized by an enzymatic activity derived from one or more of the following enzymes: endo-1, 4-xylanase; exo-1, 4-xylanase; arabinofuranosidase; feruloyl esterase.

As used herein, enzymatic activity of a cereal grain refers to activity measured in flour prepared from a specified grain type. For example, an alpha-amylase activity of 10U/g per gram of cereal grain refers to the alpha-amylase activity (10U) measured in an aqueous extract of 1g flour (dry matter) of the cereal. Alpha-amylase activity was determined by K-CERA 01/12 (protocol and kit from Megazyme, Ireland). BETA-amylase activity was determined by K-BETA3 (protocol and kit from Megazyme, Ireland). Limited dextrinase activity was determined by T-LDZ1000 (protocol and kit from Megazyme, Ireland).

The volume of gas as described herein refers to the volume of the gas at 1atm and 20 ℃.

O as described herein₂Volume means O at 1atm and 20 ℃₂The volume of (a). In which O is₂In the embodiment of the invention contained in the gas mixture, the total volume of the gas mixture, O, can be determined₂Can be calculated as O₂Percentage of the total volume constituted. For example, atmospheric air contains 21% O₂. Thus, it is possible to provideAtmospheric air O as used herein₂The volume of (b) is 21% of the total volume of atmospheric air.

Detailed Description

Cereal grains

The method of the invention is a method of producing an aqueous extract of cereal or a method of producing a beverage from an aqueous extract of said cereal. The method of the invention comprises a germination step comprising incubating a cereal grain in an aqueous solution. It should be noted that the mixture of aqueous solution and cereal grain may be considered a suspension.

The cereal grain may be the grain of any cereal, for example a cereal selected from barley, rice, sorghum, maize, millet, triticale, rye, oats and wheat. In a preferred embodiment of the invention, the cereal grain is barley grain. The grain may be of any barley variety, for example any barley variety described below in the "barley" section.

The cereal grain may have a relatively low water content prior to incubation in the aqueous solution. For example, the cereal grain may have a moisture content of at most 30%, preferably at most 20%, such as at most 15%, for example in the range of 5 to 15%.

Prior to incubation in the aqueous solution, the cereal grain may be subjected to one or more antimicrobial treatment steps. The antimicrobial treatment may be any useful antimicrobial treatment that does not impair the germination potential of the grain. The antimicrobial treatment may be, for example, treatment with one or more antimicrobial agents. The antimicrobial agent can be any antimicrobial agent that is non-toxic to the cereal grain at the concentrations used. For example, the antimicrobial agent may be a chlorine-containing compound, such as hypochlorite. The antimicrobial agent may also be a peroxide, such as hydrogen peroxide and/or peracetic acid. Non-limiting examples of useful commercial antimicrobial agents includeOr P3-oxonia activeThe cereal grain may be treated with hypochloren at a concentration in the range 0.1-10%, for example 0.5-5%, for example about 1%, for example 1%. Cereal grains may be treated with said hypochlororan for 15 minutes to 10 hours, such as 1 to 5 hours, such as 2 to 4 hours. After treatment, the cereal grain may be washed one or more times.

In some embodiments of the invention, the antimicrobial treatment is performed by incubating the cereal grain in an aqueous solution comprising an antimicrobial agent. The germination step may be started immediately after said incubation, e.g. by starting aeration. Thus, in such embodiments, no change to the aqueous solution is required, and the same aqueous solution may be used for the antimicrobial treatment and at least during the start of the subsequent germination step. This may be particularly the case when the antimicrobial agent is a peroxide, such as hydrogen peroxide.

It may be preferred that the cereal grain has not undergone germination prior to incubation in aqueous solution according to the invention. Thus, it may be preferred that the cereal grain is not subjected to a pre-germination step.

As noted above, the cereal grain may be any cereal grain. Some cereal grains include hulls, while other cereal grains are hulless. The shelled cereal grain may be treated to remove at least a portion of the shell prior to the germination step. Typically, if a hulless cereal grain is used, no dehulling treatment is required. Hulless grains include, for example, hulless barley varieties and wheat.

The shelled cereal grain may be treated to remove the hulls by physically treating the cereal grain to remove the hulls. The physical treatment may for example be selected from polishing, sanding, peeling and smoothing. Preferably, the physical treatment results in a loss of the shell. The shell loss can be determined as the total weight loss. Thus, the physical treatment preferably results in a loss of at least 2%, for example at least 3% of the total weight of the cereal grain. For example, the physical treatment may result in a total weight loss of the cereal grain in the range of 2-7%, for example a total weight loss of the cereal grain in the range of 36%.

Germination

The method of the invention comprises the step of germinating the cereal grain. The germination step may comprise the step of incubating the cereal grain in an aqueous solution, typically under aeration. The germinating step includes the step of incubating the cereal grain in an aqueous solution, optionally under aeration. In some embodiments, the germinating step includes the step of incubating the cereal grain in an aqueous solution without aeration. In other embodiments, the step of incubating the cereal grain in an aqueous solution is performed under aeration.

Incubation in aqueous solution under aeration

The cereal grain may be any cereal grain described above in the "cereal grain" section and the aqueous solution may be any aqueous solution described below in the "aqueous solution" section.

It may be preferred that during said incubation in aqueous solution, the cereal grain is completely covered by said aqueous solution throughout the incubation. Thus, the cereal grain may for example be incubated in an aqueous solution of at least 1L, preferably at least 1.5L, more preferably at least 2L, for example in the range of 1 to 10L, such as in the range of 1 to 5L, for example in the range of 1.5 to 3L per kg cereal grain (dry weight).

Thus, in some embodiments, the grain is submerged in the aqueous solution throughout the incubation in the aqueous solution.

In other embodiments, the cereal grain absorbs the aqueous solution in a manner such that at the end of incubation in the aqueous solution, all of the aqueous solution is absorbed by the cereal grain.

In other embodiments, the aqueous solution remaining after incubation under aeration may be drained from the cereal grain.

In some embodiments, it is preferred that after incubating the cereal grain in the aqueous solution, a majority, e.g., at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, e.g., substantially all of the cereal grain contains visible shoots of at least 1 mm.

In some embodiments, a majority, e.g., at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., at least 95%, e.g., substantially all of the cereal grain, after incubating the cereal grain in the aqueous solution, contains one or more visible rootlets.

In some embodiments, a majority, e.g., at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., at least 95%, e.g., substantially all of the cereal grain, after incubating the cereal grain in the aqueous solution, contains one or more visible rootlets and visible stems.

In some embodiments, the cereal grain has a moisture content of at least 30%, preferably at least 35%, more preferably at least 37%, for example in the range of 35-60%, for example in the range of 35-50%, for example in the range of 37-60%, for example in the range of 37-50%, after incubation of the cereal grain in the aqueous solution. In some embodiments, the cereal grain has a water content of at least 30%, such as at least 31%, such as at least 32%, such as at least 33%, such as at least 34%, such as at least 35%, such as at least 36%, such as at least 37%, such as at least 38%, such as at least 39%, such as at least 40%, such as at least 45%, such as at least 46%, such as at least 47%, such as at least 48%, such as at least 49%, such as at least 50%, such as at least 51%, such as at least 52%, such as at least 53%, such as at least 54%, such as at least 55%, such as at least 56%, such as at least 57%, such as at least 58%, such as at least 59%, such as 60%, after incubating the cereal grain in an.

The moisture content of a cereal grain may be determined by measuring the weight of the cereal grain, then drying the cereal grain, and measuring the weight of the dry cereal grain. The weight difference between wet and dry cereal grains is considered to be water, and the moisture content is given by the weight of water divided by the total weight of the cereal grains (wet cereal grains). The water content, expressed as% is thus w/w%.

The cereal grain may be incubated in the aqueous solution for a sufficient time to allow a majority of the cereal grain to germinate as described above. The cereal grain may also be incubated in the aqueous solution for a sufficient time to achieve the moisture content described above. Typically, the grain kernel is incubated in the aqueous solution for at least 20 hours, for example at least 24 hours. Typically, the cereal is incubated in the aqueous solution for up to 72 hours, such as up to 60 hours, such as up to 48 hours.

In some embodiments, the cereal grain is incubated in aqueous solution under aeration for 20 to 72 hours, preferably 40 to 55 hours, more preferably 45 to 50 hours. This is especially the case in embodiments of the invention wherein the germination does not comprise a step of air standing.

In some embodiments of the invention, the germinating step includes incubation in an aqueous solution followed by air resting. In such embodiments, the incubation in the aqueous solution is performed for a sufficient time to allow sufficient enzyme activity in the germinated cereal grain after air standing. The enzymatic activity is preferably as described below in the section "malted cereal grain".

In such embodiments, the cereal grain may be incubated, for example, in aqueous solution for at least 16 hours, such as at least 20 hours. Typically, the grain is incubated in the aqueous solution for a period of up to 72 hours, such as up to 40 hours, such as up to 35 hours, such as up to 30 hours. Thus, in some embodiments, the cereal grain is incubated in the aqueous solution for 20 to 72 hours, such as 20 to 60 hours, such as 20 to 48 hours, such as 20 to 30 hours, such as 22 to 26 hours.

In some embodiments, the cereal grain is incubated in aqueous solution under aeration for 16 to 40 hours, preferably 20 to 35 hours, such as 20 to 30 hours, more preferably 22 to 26 hours. This is especially true in embodiments of the invention wherein the germination further comprises an air resting step.

The cereal grain may be incubated at any useful temperature, but it may be preferred to incubate at a temperature sufficiently high to allow for a rapid increase in moisture content. As shown in example 3 below, an increase in temperature can significantly increase the water content. Thus, it may be preferred to incubate the cereal grain in the aqueous solution at a temperature of at least 15 ℃, such as at least 20 ℃, such as at least 25 ℃. In particular, the cereal grain may be incubated at a temperature in the range of 10 to 35 ℃, preferably in the range of 15 to 30 ℃, for example in the range of 20 to 30 ℃, for example in the range of 25 to 30 ℃, for example at about 25 ℃.

In particular, in embodiments of the invention wherein the cereal grain is incubated at a temperature in the range of 20 to 30 ℃, then the cereal grain may be incubated for 20 to 48 hours, for example 16 to 30 hours.

As described above, O is then often made₂Incubating the cereal grain in an aqueous solution while passing through the aqueous solution. This is also known as incubating the cereal grain in an aqueous solution under aeration. Preferably, O is allowed to stand for the entire incubation period₂Continuously through the aqueous solution. Said O is₂The aqueous solution may be passed through in any useful manner, but often the O-containing is introduced at the bottom and/or lower portion of a vessel containing the aqueous solution and the cereal grain₂The gas of (2). Typically, the gas will diffuse through the aqueous solution/cereal grain mixture and exit the aqueous solution/cereal grain mixture from the top of the aqueous solution. In particular, the incubation can be performed in a device as described below in the "device" section. Heavy gases, in particular CO, can also be withdrawn from the bottom of the vessel₂So that fresh air/O can be supplied from the upper part of the container₂。

Said O is₂Can be used as pure O₂Adding to the aqueous solution. However, often, the O is₂Is contained in the gas mixture. In one embodiment, the O is₂Contained in atmospheric air. Thus, the method of the invention may comprise passing atmospheric air through the aqueous solution.

Typically, at least 2L, preferably at least 3L, more preferably at least 4L, still more preferably at least 5L, even more preferably at least 6L O per kilogram cereal grain per hour₂Passing through the aqueous solution. The weight of the cereal grain is dry weight. For example, O per kg cereal grain (dry weight) per hour is in the range of 2 to 100L, such as in the range of 2 to 75L, such as in the range of 2 to 50L, such as in the range of 4 to 100L, such as in the range of 4 to 75L, such as in the range of 4 to 50L, such as in the range of 6 to 100L, such as in the range of 6 to 75L, such as in the range of 6 to 50L₂By said aqueous solution/cereal grain mixtureA compound (I) is provided.

In one embodiment, preferably at least 20g O per kilogram of cereal grain per hour₂More preferably at least 30g O per kg of cereal grain₂More preferably at least 40g O per kg of cereal grain₂E.g. in the range of 40 to 100g O per kg cereal grain₂E.g. in the range of 40 to 80g O per kg cereal grain₂E.g. in the range of 60g O per kg cereal grain₂Passing the aqueous solution/cereal grain mixture. The weight of the cereal grain is provided in dry weight. During incubation, the cereal grain typically absorbs at least some of the aqueous solution, thus O in the aqueous solution₂The concentration of (b) is typically varied during the incubation. Typically, O is supplied per liter of aqueous solution per hour₂The amount of (B) is in the range of 40 to 200g, preferably in the range of 50 to 150 g.

As noted above, atmospheric air is often passed through the aqueous solution. Thus, the method may comprise passing at least 10L, preferably at least 15L, more preferably at least 20L, still more preferably at least 25L, even more preferably at least 30L of atmospheric air through the aqueous solution per kg of cereal grain per hour. The weight of the cereal grain is dry weight. For example, atmospheric air in the range of 10 to 500L, such as in the range of 10 to 375L, such as in the range of 20 to 500L, such as in the range of 20 to 375L, such as in the range of 20 to 250L, such as in the range of 30 to 500L, such as in the range of 30 to 375L, such as in the range of 30 to 250L per kilogram of cereal grain (dry weight) per hour is passed through the aqueous solution. In one embodiment, in the range of 50 to 110L, preferably 80 to 100L, of atmospheric air is passed through the aqueous solution per kilogram of grain (dry weight) per hour.

Air standing

In addition to the incubation in aqueous solution under aeration, the cereal grain may also be incubated in air (e.g., in the absence of aqueous solution). The step of incubation in air may also be referred to as "air resting". Thus, after incubation in aqueous solution under aeration, the remaining aqueous solution can be drained and the cereal grain can be incubated in air. Or, atAfter incubation in aqueous solution under aeration, all of the aqueous solution has been absorbed by the cereal grain, which can then be incubated in air. The incubation in air is preferably carried out under aeration, e.g. O₂May be passed through a container containing the cereal grain. Preferably, O is allowed to stand for the entire air₂Through the vessel. O through a container comprising cereal grains₂The amount of (b) may be in contact with O passing through the aqueous solution as described above₂The same amount. O is₂May be provided in the form of a gas mixture, for example atmospheric air.

The air resting may be performed for any suitable amount of time, for example in the range of 18 to 50 h. In a preferred embodiment, the air resting is carried out for at least 20h, preferably at least 22h, more preferably in the range of 20 to 38h, such as in the range of 20 to 35h, preferably in the range of 20 to 30h, such as in the range of 22 to 26 h.

The air resting may be carried out at ambient temperature, for example at a temperature in the range of 20 to 30 ℃, for example at a temperature in the range of 23 to 27 ℃, preferably at about 25 ℃. In particular, the air resting may be carried out at a temperature in the range of 20 to 30 ℃ for at least 20h, for example in the range of 20 to 30 h.

In some embodiments of the invention, the air flow rate is from 85 to 95L/h, e.g., from 87 to 93L/h, e.g., about 90L/h, of atmospheric air or about 20% O/kg of dry cereal grain₂Air standing was performed in the case of the gas mixture of (1). In other embodiments of the invention, the O at an air flow rate of 17 to 21L/h, e.g., 18 to 20L/h, e.g., about 19L/h, per kilogram of dry cereal grain₂Air standing was performed in the case of (1).

During air resting, additional water or aqueous solution may be added to the cereal grain, for example by irrigation or spraying. However, during air standing, the cereal grain should not be submerged in the aqueous solution.

Germination method

In one embodiment, the cereal grain may be germinated using standard methods known in the art to obtain germinated grain. The duration of the germination may preferably be at most 96 hours, more preferably at most 72 hours. Germination may also comprise incubation in aqueous solution under aeration, and optionally followed by air resting as defined above.

Germination may also comprise several steps of incubation in aqueous solution and/or several steps of air-standing. Typically, the first step is a step of incubating the cereal grain in an aqueous solution under aeration as described above. Thus, germination may comprise or consist of the following steps:

incubating cereal grains in aqueous solution under aeration as described above in the section "incubation in aqueous solution under aeration" section

Incubating the cereal grain in air as described above in the "air resting" section

In this embodiment, incubating the cereal grain in the aqueous solution under aeration may for example be carried out for 16 to 30 hours, such as 20 to 30 hours, such as 22 to 26 hours, while the air resting may be carried out for 20 to 38 hours, such as 22 to 35 hours, such as 20 to 30 hours, such as 22 to 26 hours.

Germination may also comprise or consist of the following steps:

incubating cereal grains in aqueous solution under aeration as described above in the section "incubation in aqueous solution under aeration" section

Incubating the cereal grain in air as described above in the "air resting" section

Incubating cereal grains in aqueous solution under aeration as described above in the section "incubation in aqueous solution under aeration" section

Germination may also comprise or consist of the following steps:

incubating cereal grains in aqueous solution under aeration as described above in the section "incubation in aqueous solution under aeration" section

Incubating the cereal grain in air as described above in the "air resting" section

Incubating cereal grains in aqueous solution under aeration as described above in the section "incubation in aqueous solution under aeration" section

Incubating the cereal grain in air as described above in the "air resting" section

Germination may also comprise or consist of the following steps:

incubating cereal grains in aqueous solution under aeration as described above in the section "incubation in aqueous solution under aeration" section

Incubating the cereal grain in air as described above in the "air resting" section

Incubating cereal grains in aqueous solution under aeration as described above in the section "incubation in aqueous solution under aeration" section

Incubating the cereal grain in air as described above in the "air resting" section

Incubating cereal grains in aqueous solution under aeration as described above in the section "incubation in aqueous solution under aeration" section

The time of each incubation may vary, but typically the total germination step, i.e. the total time of all incubations in aqueous solution and all air stills, does not exceed 72 hours, more preferably does not exceed 60 hours, even more preferably does not exceed 54 hours. Thus, it may be preferred that the entire germination step is carried out for 20 to 72 hours, such as 24 to 60 hours, such as 24 to 48 hours. Thus, if the germination comprises several steps of incubation in aqueous solution and/or air standing, each incubation step is typically shorter.

In one embodiment, the entire germination step is carried out for at least 44 hours, such as at least 46 hours, such as at least 48 hours. Thus, the entire germination step may be carried out for 44 to 72 hours, such as 46 to 72 hours, such as 48 to 72 hours, such as 46 to 60 hours. The germination time may be determined, for example, from the start of germination until the start of heat treatment as described below. In embodiments of the invention lacking a heat treatment step, the time of germination may be determined, for example, from the start of germination until the start of subdivision of the cereal grain.

In some embodiments, one or more exogenous enzymes may be added. For example, one or more enzymes may be added during the germination step, e.g. as described in WO 2016/071463.

In one embodiment, the germination is carried out during germination without acidifying the grains. Acidification is the application of acidic substances to the grain during germination. The acidic substance may, for example, be a strong to moderately strong organic or inorganic acid, such as sulfuric acid, nitric acid, hydrochloric acid, perchloric acid, phosphoric acid or a monocarboxylic acid or an acid salt thereof. In this regard, atmospheric air components and gibberellic acids are not considered acidic species.

Thermal treatment

In one embodiment of the invention, the malted cereal grain is heat treated after the malting step or as a final part of the malting step. The heat treatment increases the level of hydrolytic enzymes in the malted cereal grain.

During the steps of germination and heat treatment, the temperature is preferably controlled. The temperature may be controlled by any conventional means. During germination in the incubation step in an aqueous solution, the temperature may be controlled by controlling the temperature of the aqueous solution. During germination in the air incubation step, the temperature may be controlled by controlling the temperature of the air, for example by using a temperature controlled air flow. The germination step itself may be hot and therefore may require temperature control at a desired level by cooling.

During the heat treatment step, the temperature may be controlled in various ways. For example, the temperature may be controlled by air flow or by incubation in a temperature controlled aqueous solution. As described above, the germination itself may generate heat, and thus the temperature can be controlled by cooling even during the heat treatment step. Cooling may be performed with air or water.

It is an aspect of the present invention that the air resting at a temperature of at most 30 c, preferably at about 25 c, as described above, is carried out for at least 20 hours, such as at least 22 hours, preferably at least 24 hours, before the heat treatment.

In one embodiment, the duration of the heat treatment step is carried out for at least 1 hour, such as at least 2 hours, such as in the range of 1 to 12 hours, preferably in the range of 1 to 5 hours, such as in the range of 2 to 4 hours. The heat treatment may be carried out at a temperature in the range of 35 to 55 ℃, for example in the range of 40 to 50 ℃.

Preferably, all steps of germination and/or heat treatment are performed in the same container. The container may in particular be a tank, such as any of the tanks described below in the section "plant".

The heat treatment step, if present, is generally carried out in such a way that: reducing the moisture content of the malted cereal grain subjected to the heat treatment to less than 20% at any time between initiation of malting and heat treatment. In other words, the heat treatment step is not a kiln drying step. In some embodiments, the moisture content of the malted cereal grain is not less than 25%, even more preferably not less than 30%, still more preferably not less than 35% at any time during the heat treatment step.

Preferably the cereal grain is subdivided substantially immediately after germination and/or heat treatment. Thus, the process of the invention preferably does not comprise a drying step between the germination and/or heat treatment step and the step of subdividing the cereal grain. Accordingly, it is an aspect of the invention that the moisture content of the malted cereal grain, when subdividing the cereal grain, is at least 25%, preferably at least 30%, even more preferably at least 35%. Another aspect of the invention is that the moisture content of the malted cereal grain is not less than 25%, preferably not less than 30%, even more preferably not less than 35% at any time between the time the malting step is completed and the step of subdividing the cereal grain.

The process of the present invention preferably does not include the step of actively cooling the heat-treated cereal grain prior to subdividing the cereal grain. It is therefore an aspect of the invention that the temperature of the germinated grain is above 35 ℃ at any point between the heat treatment step and the step of subdividing the germinated grain.

Sprouted cereal grain

The present invention relates to a method comprising the step of producing a malted cereal grain and optionally the step of heat treating the malted grain.

The germinated and/or heat treated cereal grain preferably comprises one or more hydrolase activities provided, for example, by alpha-amylase, beta-amylase, starch debranching enzyme (e.g., limit dextrinase), alpha-glucosidase, and protease.

Often, the onset of hydrolase activity can occur in a timely coordinated manner, and thus the activity of some hydrolases can be used as a marker for other hydrolase activities.

Thus, it is preferred that the malted and/or heat-treated cereal grain has a sufficient level of measurable alpha-amylase activity. Preferably, the malted cereal grain has a measurable alpha-amylase activity of at least 40U/g, such as at least 50U/g cereal grain (dry weight). In some embodiments of the invention, the cereal grain may have an alpha-amylase activity of at least 50U/g, for example at least 60U/g cereal grain (dry weight).

The alpha-amylase activity is preferably determined according to standard methods, e.g., by using the Ceralpha kit (K-CERA) from Megazyme in Ireland. In particular, alpha-amylase activity can be determined as described in example 2 below.

It may also be preferred that the malted cereal grain has a sufficient level of measurable β -amylase activity. Preferably, the malted cereal grain has a measurable β -amylase activity of at least 5U/g cereal grain (dry weight). Thus, preferably, the malted cereal grain may have a measurable β -amylase activity of at least 10U/g, such as at least 15U/g cereal grain (dry weight).

Preferably, the BETA-amylase activity is determined according to standard methods, for example, by using the Betamyl kit (K-BETA3) from Megazyme, Ireland. In particular, β -amylase activity can be determined as described in example 2 below. It is also preferred that the malted and/or heat treated cereal grain has a sufficient level of limiting dextrinase activity. Preferably, the malted cereal grain has a limiting dextrinase activity of at least 5mU/g cereal grain (dry weight). Thus, preferably, the malted cereal grain may have a limiting dextrinase activity of at least 9mU/g cereal grain (dry weight).

Preferably, the limiting dextrinase activity is determined according to standard methods, e.g.by using the Limit Dextrizyme kit T-LDZ1000 from Megazyme, Ireland. In particular, the limiting dextrinase activity may be determined as described in example 2 below.

Interestingly, the malted and/or heat-treated cereal grain according to the invention has significantly reduced rootlets compared to conventional green malt. Thus, the malted cereal grain according to the invention preferably comprises at most 4g of rootlets per 100g of malted barley, preferably at most 3g of rootlets per 100g of malted barley, even more preferably at most 2g of rootlets per 100g of malted barley, e.g. at most 1.1g of rootlets per 100g of malted barley, wherein the mass of the rootlets and the mass of the malted barley are both provided in dry weight. The quality of the rootlets is preferably determined as described in example 6 below. Thus, in one embodiment, the method of the invention does not comprise the step of removing the rootlets.

Nitrosamines (NDMA) are of the chemical structure R¹N(–R²) -N ═ O, i.e. nitroso groups bonded to amines. Most nitrosamines are carcinogenic. Despite the low content of nitrosamines in modern malt, the malted barley grain according to the invention still has a significantly reduced NDMA content compared to conventional malt. In one embodiment, the malted barley grain according to the invention comprises at most 0.15 μ g/kg NDMA, preferably at most 0.12 μ g/kg NDMA, for example at most 0.10 μ g/kg NDMA.

Aqueous solution

The aqueous solution may be any aqueous solution. The aqueous solution may be considered a solution, even though the mixture of aqueous solution and cereal grain may be considered a suspension. Often, the aqueous solution is water, such as tap water. One or more additional reagents may be added to the water, and thus the aqueous solution may be water comprising one or more additional reagents, such as tap water. The additional reagents may be included in the aqueous solution from the beginning or they may be added during the incubation.

The additional agent may be, for example, a compound capable of accelerating germination of a cereal grain. Thus, the aqueous solution may comprise Gibberellic Acid (GA), e.g. the aqueous solution mayComprises GA at a concentration of at least 100nM, e.g., at a concentration of at least 1000nM, e.g., in the range of 100 to 100,000nM, e.g., in the range of 500 to 2000 nM. The GA may be present in the aqueous solution from the start of incubation, or it may be added during incubation. The GA can be any GA, e.g., GA₃Or GA₇. In one embodiment, the GA is a GA₃。

The additional agent may also be an antifoaming agent. The anti-foaming agent may be, for example, any food grade anti-foaming agent, such as Foamzol FCD511(AB Vickers, UK).

Device for measuring the position of a moving object

The method of the present invention may be performed using one or more devices adapted to perform the method.

For example, the step of incubating the cereal grain in an aqueous solution may be performed in a vessel equipped with one or more air pumps. The container may be any container in which the cereal grain may be incubated in an aqueous solution. In some embodiments, the container may be a tank, such as a tank as described below.

One example of an apparatus for incubating cereal grains is provided in fig. 2. The apparatus includes a tank (2) having sufficient volume to contain the cereal grain and the aqueous solution. The can shown in fig. 2 is cylindrical, but the can for use with the invention may have any suitable shape, for example it may be a cylindrical can, for example a cylindrical can with a conical bottom. The can may be made of any suitable material, such as plastic (e.g., Plexiglas) or metal (e.g., stainless steel or copper).

The tank comprises at least one cereal grain inlet (1) which can be used to add cereal grains to the tank. The inlet may also be used to add other compounds to the tank, for example, the inlet may be used to add an aqueous solution, such as water. The inlet may be positioned at any useful location in the tank, and in some embodiments, the inlet is located in an upper portion of the tank, e.g., the top of the tank. The inlet should be of sufficient size to allow addition of the cereal grain. The tank may include additional, if not necessary, inlets in addition to the cereal grain inlet.

The tank may optionally comprise a grid or mesh (3) positioned substantially horizontally in the tank. If present, the grid or mesh, for example, is typically located at the lower 1/3 of the tank, such as at the lower 1/5 of the tank. The grid or mesh preferably contains only openings which are smaller than the cereal grain. The grid or mesh may be made of any suitable material, such as plastic or metal, and may be, for example, a metal mesh. Thus, a grid or mesh may be used to separate the cereal grain from the bottom of the tank. However, tanks often do not include a mesh. In particular, if the tank includes a cereal grain outlet at or near the bottom, the tank typically does not include a grid or mesh.

Furthermore, the tank comprises one or more gas inlets (4). The inlet may be any inlet through which O is contained₂Can enter the tank. The gas inlet may have a shape that allows the gas to enter the aqueous solution at a high velocity, thereby ensuring diffusion of the gas through the aqueous solution. Thus, for example, the gas inlet may be a jet (nozzle), a nozzle (jet), a diffusing stone or a sintered stone. In one embodiment, the gas inlet is a calculus. The gas inlet is typically connected to a pump (5), through which the pump (5) pumps gas into the tank. The pump may be any pump capable of pumping gas, such as air, through the gas inlet. Preferably, the tank comprises a plurality of gas inlets, such as at least 2, such as at least 3, such as 3 to 20. The gas inlets may be positioned anywhere in the tank, but typically they are located at the bottom 1/3 of the tank, for example at the bottom 1/5 of the tank. This allows gas to enter the aqueous solution from the bottom and diffuse upward through the aqueous solution. Excess gas may leave the tank through any opening in the tank, such as through an inlet to add grain. In one embodiment it may be preferred that the inlet (3) is positioned directly on the side wall of the tank (2), preferably in the lower part of the side wall, e.g. as shown in fig. 1.

An example of an apparatus for performing several steps of the method of the present invention is shown in fig. 1. The apparatus comprises a cereal grain inlet (1), a tank (2), a gas inlet (3) and a pump (4), which may be any of the cereal grain inlets, tanks, gas inlets and pumps described above in relation to fig. 2. The tank (2) may comprise a cereal grain outlet (5) located at the lower 1/3 of the tank, for example at the lower 1/5 of the tank, for example at the bottom of the tank. The outlet may be used to remove both cereal grains and other components remaining in the tank, such as an aqueous solution. The outlet may be connected to a grain pump (6), for example by a pipe. The grain pump (6) may be any pump capable of pumping grain from the tank (2) to the device (7) for subdividing cereal grains and optionally further to the container (9) for mashing.

The apparatus comprises a device (7) for subdividing cereal grains. The apparatus may be any apparatus capable of subdividing cereal grains having a moisture content of greater than 20%, for example greater than 35%. The apparatus may be, in particular, a grinder (grinder) or mill (mill), for example a wet mill. The device (7) may be connected to the tank (2) and the container (9) by means of pipes. The movement of the grains from the tank (2) to the device (7) and further to the container (9) can be ensured by a pump (6).

The device may further comprise a container (9). The container (9) may be any container which is capable of containing the aqueous extract and which is capable of withstanding the temperatures used for the mashing, e.g. temperatures up to 90 ℃, e.g. up to 85 ℃, e.g. up to 80 ℃. Thus, the container may be made of any material that is resistant to such temperatures, such as a metal (e.g., stainless steel or copper). The container may have any useful shape, for example, it may be substantially cylindrical. The container may be associated with a device for temperature control. The container may be used to prepare an aqueous extract of the subdivided cereal grain by a method involving incubation at one or more predetermined temperatures as described herein in the "preparation of an aqueous extract" section. The temperature-controlled apparatus is capable of controlling the temperature of the liquid within the container, including being capable of heating the liquid within the container to a predetermined temperature, such as any of the temperatures described herein in the section "preparing an aqueous extract". The container (9) may also comprise means for stirring or rotating any liquid contained in said container. In particular, the container (9) may be a container for mash. The container for mash is well known in the art and the container (9) may be any conventional container for mash.

The container (9) typically contains an inlet (8) through which the subdivided malted cereal grain can enter the container. The inlet (8) is typically located in the upper half of the vessel, e.g. at the upper 1/3 of the vessel, e.g. at the upper 1/5 of the vessel, e.g. at the top of the vessel. The subdivided cereal grains may pass from the apparatus (7) for subdividing cereal grains through a conduit to an inlet (8) of a container (9).

Typically, the container (9) also contains an outlet (10) through which the aqueous extract can leave the container after preparation of the aqueous extract (see below for details on the aqueous extract in the sections "aqueous extract" and "preparation of aqueous extract"). The outlet is typically located in the lower half of the vessel, e.g., at the lower 1/3 of the vessel, e.g., at the lower 1/5 of the vessel, e.g., at the bottom of the vessel.

Finely divided germinated cereal grains

The method of the present invention comprises the step of subdividing a cereal grain that has been peeled and/or germinated and/or heated according to the methods defined herein.

When the cereal grains are subdivided they preferably still have a high moisture content, preferably the cereal grains have a moisture content of at least 20%, more preferably at least 25%, even more preferably at least 30%, still more preferably at least 35%. For example, the malted cereal grain may be transferred directly from the malting and/or heat treatment to the apparatus for subdividing the cereal grain. Thus, the moisture content of the germinated cereal grain when subdivided may be the same as the moisture content of the cereal grain immediately after germination and/or heat treatment of the cereal grain, e.g., the moisture contents described above in the "germinated" section. In particular, the method generally does not include a step of drying the malted cereal grain. Thus, the method preferably does not comprise the step of kiln drying the malted cereal grain. As mentioned above, kiln drying results in a reduction of the moisture content to a level of about 4.5-5%. Preferably, the moisture content of the germinated cereal grain is not less than 20%, preferably not less than 25%, even more preferably not less than 30%, yet more preferably not less than 35% at any time after germination and/or heat treatment and before subdividing the cereal grain.

In some embodiments of the invention, the malted cereal grain is heat treated after the malting step. The process of the present invention preferably does not include the step of actively cooling the heat-treated cereal grain prior to subdividing the cereal grain. Thus, it is an aspect of the invention that the temperature of the malted grain is above 35 ℃ at any point between the heat treatment step and the step of subdividing the malted cereal grain.

The malted cereal grain may be subdivided using any equipment suitable for subdividing cereal grains having a moisture content of greater than 20%, such as greater than 25%, such as greater than 30%, such as greater than 35%. For example, the malted cereal grain may be subjected to milling, e.g. wet milling. Useful mills for milling the germinated cereal grain include mills available from Millstar, USA. The sprouted cereal grain may also be subjected to grinding.

The cereal grain is typically subdivided to an extent such that an aqueous extract of fermentable sugars of the cereal grain can be prepared. Thus, the cereal grain is split sufficiently so that 1kg of a 7-L aqueous extract of said subdivided cereal grain has a specific gravity of at least 8 ° Plato.

In an embodiment of the invention, wherein the aqueous extract is made from malted cereal grains and one or more auxiliary materials, which may also be subdivided. This may be the case in particular when the adjunct comprises unmalted cereal grains. The auxiliary material may be subdivided, for example in a separate procedure, for example milling. However, the invention also includes subdividing the adjunct with the malted cereal grain. Similarly, if the aqueous extract is made from malted cereal grain and kiln-dried malt, the kiln-dried malt may be subdivided in a separate procedure, e.g. milling. However, the invention also comprises the subdivision of kiln dried malt together with malted cereal grains.

Preparation of aqueous extract

The method of the invention further comprises the step of preparing an aqueous extract of the finely divided malted cereal grain. The step may for example be a pulping step.

Such aqueous extracts can generally be prepared by incubating finely divided cereal grains in water or in an aqueous solution. The aqueous solution used to prepare the aqueous extract is typically a different aqueous solution than the aqueous solution used to incubate the cereal grain during germination.

For the sake of distinction, the aqueous solution used to prepare the aqueous extract may also be referred to as a "slurrying solution". The pulping solution may be any aqueous solution, but it typically consists of water (e.g., tap water) to which one or more additional reagents may be added. These additional agents may be referred to as "additional mash agents" in order to distinguish them from other agents added during germination. Thus, the mash solution may consist of water (e.g. tap water) to which one or more further mash agents are added. The mash agents may be present in the mash solution from the beginning or they may be added during the process of preparing the aqueous extract.

The additional mash agent may be an enzyme. Thus, the pulping solution may comprise one or more enzymes. The enzyme may be added to the mash solution initially or subsequently during the process.

The enzyme may be, for example, one or more hydrolases. Suitable enzymes include lipases, starch degrading enzymes (e.g.amylases), glucanases [ preferably (1-4) -and/or (1, 3; 1,4) -beta-glucanases ], and/or xylanases (e.g.arabinoxylanases), and/or proteases, or enzyme mixtures comprising one or more of the above enzymes, e.g.Cereflo, Ultraflo or Ondea Pro (Novozymes). For example, the pulping solution may comprise one or more hydrolases, wherein at least one hydrolase is selected from the group consisting of-amylase, beta-amylase, dextrinase, pullulanase, beta-glucanase, xylanase, glucoamylase, and protease.

In one embodiment of the invention, the mash solution comprises one or more of the following enzymes:

-beta-glucanases, such as endo- (1, 3; 1,4) -beta-glucanases or endo-1, 4-beta-glucanases.

Xylanases, e.g. endo-or exo-1, 4-xylanases, arabinofuranosidases or feruloyl esterases

-alpha-amylase

Pullulanase or dextrinase

-a glucoamylase.

The decision whether to add an enzyme to the mash solution, and which enzyme to add, may depend on the cereal grain used. Thus, in embodiments of the invention wherein the cereal is a barley plant with low levels of β -glucans (e.g., as described below in the "barley" section), little or no β -glucanase may be added to the mash solution.

In one embodiment, it is preferred that no exogenous protease is added during the mashing. The addition of proteases may be less preferred as proteases may affect the enzymatic activity. In one embodiment, preferably no exogenous lipase is added during the mashing.

In one embodiment, up to 700U, preferably up to 350U of exogenous glucoamylase per g of malted cereal grain (dry matter) is preferably used during the preparation of the aqueous extract.

In one embodiment, preferably up to 400AGU, preferably up to 200AGU of exogenous glucoamylase per kg of malted cereal grain (dry matter) is used during the preparation of the aqueous extract. The determination of AGU can be performed as described in US 7060468.

In another embodiment, preferably no more than 700U, preferably no more than 350U per g of malted cereal grain (dry matter) of the exogenous glucoamylase and alpha-amylase combined is used during the preparation of the aqueous extract. The combined glucoamylase and alpha-amylase activity can be determined, for example, using K-CERA 01/12 (protocol and kit available from Megazyme, Ireland).

In one embodiment, preferably up to 20U of exogenous pullulanase or limiting dextrinase per kg of malted cereal grain (dry matter) is used during the preparation of the aqueous extract.

In one embodiment, preferably at most 100PUN of pullulanase per kg of malted cereal grain (dry matter) is used during the preparation of the aqueous extract. The determination of the PUN can be performed as described in US 7060468.

The additional mash may also be an adjuvant, such as unmalted cereal grain, syrup or sugar. If auxiliary materials are added, these may also have been finely divided, for example by milling or grinding. If the adjunct is a cereal grain, for example an unmalted cereal grain, it may typically be subdivided or milled. If the excipients are syrups, sugars, etc., they are usually not milled. Adjuvants such as sugar or syrup can be added to the mash solution at any time during the process; however, these adjuvants may also be added to the aqueous extract or later during the process of preparing the beverage as described below. Typically, the adjunct is added in a smaller amount than the malted cereal grain. Thus, at least 50%, preferably at least 70%, e.g. at least 90% of the carbohydrates of the aqueous extract are derived from the malted cereal grain, whereas the adjunct preferably only comprises a minor part of the carbohydrates. If the adjunct is an unmalted cereal grain, preferably the germinated cereal grain constitutes at least 50% (w/w), preferably at least 70% (w/w), more preferably at least 90% (w/w) of the total amount of cereal grain as determined on a dry weight basis.

The additional mash agent may also be kiln dried malt. If kiln dried malt is added, it may also have been subdivided, for example by milling or grinding. Typically, kiln dried malt is added in smaller amounts than malted cereal grains. Thus, the malted cereal grain constitutes at least 80% (w/w), preferably at least 90% (w/w), more preferably at least 95% (w/w) of the total amount of cereal grain and malt as determined on a dry weight basis. In a preferred embodiment, no kiln dried malt is added.

The additional mash, preferably of food grade quality, may also be a salt, such as CaCl₂。

The additional mash agent may also be an acid, preferably a food grade acid, such as H₃PO₄。

The aqueous extract is typically prepared by incubating the subdivided malted cereal grain in a mash solution at one or more predetermined temperatures. The predetermined temperature may also be referred to herein as the "mash temperature". The temperature of the mash may for example be the conventional temperature used for mash.

The temperature of the bud slurry is generally maintained constant (isothermal bud slurry) or is increased gradually, e.g., in a sequential manner. In either case, soluble material from the subdivided sprouted cereal grain is released into the mash solution, thereby forming an aqueous extract.

The pulping temperature is typically a temperature in the range of 30 to 90 ℃, such as a temperature in the range of 40 to 85 ℃, such as a temperature in the range of 50 to 85 ℃. The temperature of the mash may be selected depending on the type of grain used. Thus, in embodiments of the invention wherein the cereal grain is barley (see below for details in the "barley" section) with low or no level of Lipoxygenase (LOX) activity and/or Methyl Methionine Transferase (MMT) activity, the temperature of the mashing may be lower, for example in the range of 35 to 69 ℃.

Incubation in the slurrying solution may be performed for any suitable amount of time. The time of incubation in the slurrying solution in the slurrying container may be, for example, 60 to 300 minutes, for example 60 to 240 minutes, for example 90 to 300 minutes, for example 90 to 240 minutes, for example 90 to 270 minutes. For example, the time of incubation in the slurrying solution may be any time used in conventional slurrying. One non-limiting example of a suitable mash is:

(1) the slurry is slurried (Mashing-in) at a temperature in the range of 50-60 deg.C, for example about 55 deg.C, for a period of 10-30 minutes, for example about 15 minutes.

(2) To a temperature in the range of 60 to 70 c, preferably in the range of 60 to 65 c, for example about 62 c, for 30 to 90 minutes, for example about 60 minutes.

(3) To a temperature in the range of 70 to 75 c, for example about 72 c, for 5-30 minutes, for example about 15 minutes.

(4) To a temperature in the range of 75 to 80 c, preferably in the range of 75 to 78 c, for example about 78 c, for 5 to 15 minutes, for example about 10 minutes.

Following incubation in the mash solution in the mash vessel, the finely divided malted cereal grains in the mash solution may be transferred to another vessel, such as a filter tank, and incubated at an elevated temperature for an additional time, such as for 30 to 120 minutes at a temperature in the range of 70 to 78 ℃.

Therefore, in addition to the above steps, the incubation in the slurried solution may further include the following step (5):

(5) to a temperature in the range of 70 to 78 c, preferably in the range of 75 to 78 c, for example about 78 c, for 30 to 120 minutes, for example about 60 minutes.

One non-limiting example of useful slurrying temperatures and times is shown in FIG. 10 herein. The first approximately 120 minutes of incubation may be performed, for example, in a container for mash, while the remaining incubation may be performed, for example, in another container. Other non-limiting examples can be found in the brewing literature, for example, in Briggs et al (supra) and Hough et al (supra).

After incubation in the mash solution, the aqueous extract can generally be separated, for example, by filtration into an aqueous extract and residual undissolved solid particles, the latter also referred to as "spent grain". The filtration can be carried out, for example, in a filter tank. Alternatively, the filtration may be performed by a filter for mash filtration. The aqueous extract thus obtained may also be referred to as "first wort".

Additional liquid (e.g., water) may be added to the spent grain in a process known as sparging (sparging). After bubbling and filtration, a "second wort" can be obtained. Further wort may be prepared by repeating the procedure.

Thus, the aqueous extract may be a wort, such as a first wort, a second wort, a further wort or a combination thereof.

Aqueous extract

The aqueous extract prepared by the method of the present invention may have a number of useful properties, including, but not limited to, the properties described in this section.

As mentioned above, the aqueous extract may be subjected to a filtration step. Therefore, it may be preferred that the wort has good filterability. For example, filtering highly viscous liquids can be technically challenging, which is one reason that aqueous extracts may be preferred to have low viscosity.

Filterability can be determined in a number of ways. In one embodiment, filterability is determined as the amount of liquid obtained after 1 hour of filtration through a filter funnel fitted with filter paper. Preferably, the aqueous extract has a filterability of at least 250mL when 400mL of a mash solution comprising 100g of finely divided cereal grains is added to the filter funnel. Filterability may also be determined as the percentage of the liquid volume obtained after 60 minutes of filtration as described above compared to the liquid volume of the aqueous extract added to the funnel. Thus, the filterability may be at least 50%, for example at least 60% (v/v). In particular, filterability can be determined as described in example 8 below.

Filterability may often depend on the level of beta-glucan. Therefore, it may be preferred that the level of β -glucan is not too high. For example, the aqueous extract may comprise up to 200mg/L, preferably up to 150mg/L, of beta-glucan.

It is also preferred that the aqueous extract contains a sufficient level of fermentable sugars. In particular, it may be preferred that the aqueous extract comprises at least 10g, such as at least 15g maltose per L. For example, it may be preferred that the aqueous extract comprises at least 1g/L/° Plato maltose. It may also be preferred that the aqueous extract comprises at least 1g, such as at least 2g, of glucose per L.

It is generally desirable that the wort contain high levels of Free Amino Nitrogen (FAN) sufficient to obtain good yeast viability, while very high levels may be undesirable. Thus, it may be preferred that the aqueous extract comprises from 150 to 400mg/L, such as from 150 to 300mg/L, such as from 150 to 250mg/L of FAN.

It is generally desirable for the wort to contain a high level of the amino acid valine, as this reduces the likelihood of undesirable diacetyl formation. Thus, it may be preferred that the aqueous extract comprises at least 55mg/L, such as at least 60mg/L, valine. In one embodiment, the aqueous extract comprises at least 65mg/L valine.

The sugar, FAN and amino acid levels mentioned above are preferably levels in the aqueous extract prior to any fermentation.

Method for producing aqueous extract of shelled cereal grains

In one embodiment, the present invention relates to a method of producing an aqueous extract of cereal grains, the method comprising the steps of:

a) providing a grain of a grain, wherein the grain is a shelled grain;

b) treating the cereal grain to remove hulls, wherein the treatment results in at least a 2% loss in total weight of the cereal grain, for example, results in a 2-7% loss in total weight of the cereal grain. Cereals, for example resulting in a total weight loss of 3-6% of the cereal grain;

c) subjecting a cereal grain to a germination step, thereby obtaining a germinated grain, wherein the germination step may comprise

i. Incubating the grain in aqueous solution for 16 to 40 hours, for example 20 to 30 hours, wherein at least 2L O is allowed per hour₂(ii) per kg dry weight of cereal grains passing through the aqueous solution and wherein the grains are submerged in the aqueous solution during the incubation;

removing excess aqueous solution; and

at least 2L O₂Incubating moist cereal grains in air at a flow rate per kg dry weight cereal grains per hour at a temperature in the range of 15 to 30 ℃ for 20 to 50 hours, for example 20 to 30 hours;

d) subdividing the germinated grain while the germinated grain has a moisture content of at least 20%; with the proviso that at any time between steps c) and d) the moisture content of the cereal grain is not less than 20% thereby obtaining milled germinated grain; and

e) preparing an aqueous extract of the milled germinated grain,

thereby producing an aqueous extract of the grain.

It is to be understood that the term "no moisture content of the cereal grain is less than 20% at any time between steps c) and d)" includes that the moisture content of the cereal grain may be greater than 20% at any time during steps c) and d), at any time from the completion of step c) to the completion of step d), or at any time from the completion of step c) to the start of step d), for example.

When the cereal grains are subdivided they preferably still have a high moisture content, preferably the cereal grains have a moisture content of at least 20%, more preferably at least 25%, even more preferably at least 30%, still more preferably at least 35%. For example, the malted cereal grain may be transferred directly from the malting and/or heat treatment to the apparatus for subdividing the cereal grain. Thus, the moisture content of the germinated cereal grain when subdivided may be the same as the moisture content of the cereal grain immediately after germination and/or heat treatment of the cereal grain, e.g., the moisture contents described above in the "germinated" section. In particular, the method generally does not include a step of drying the malted cereal grain. Thus, the method preferably does not comprise the step of kiln drying the malted cereal grain. As mentioned above, kiln drying results in a reduction of the moisture content to a level of about 4.5-5%. Preferably, the moisture content of the germinated cereal grain is not less than 20%, preferably not less than 25%, even more preferably not less than 30%, yet more preferably not less than 35% at any time after germination and/or heat treatment and before subdividing the cereal grain.

The method may include a heat treatment step as described above in the "heat treatment" section.

O passing through cereal grains in aqueous solution or air₂The amount of (C) may be O as described above in the "Germination" section₂Any amount of (a). In one embodiment, during step i, 85 to 95L of atmospheric air per kg dry weight of cereal grain per hour is passed through the aqueous solution. In one embodiment, the incubation in air of step iii is carried out at a flow rate of 85 to 95 litres of atmospheric air per kg of dry weight of cereal grain.

The step of subdividing the malted grain may be as described above in the section "subdividing malted cereal grains".

The steps for preparing the aqueous cereal extract may be as described in the "aqueous extract" section above.

Preparation of beverages

In some embodiments, the methods of the present invention further comprise the step of processing the aqueous extract prepared by the methods of the present invention into a beverage. Any of the above-described methods described herein can be used to obtain an aqueous extract of the grain, which is then further processed into a beverage.

The aqueous extract may be boiled with or without hops, after which it may be referred to as boiled wort.

The first, second and further wort may be mixed and then heated or boiled. The aqueous extract may be heated or boiled for any suitable amount of time, for example, 60 minutes to 120 minutes. During heating or boiling, the volume of the aqueous extract may decrease due to evaporation. It may be preferred that the aqueous extract has a volume reduction of less than 8%, preferably less than 5%. This can significantly reduce energy consumption.

The beverage may be prepared by fermenting an aqueous extract, for example by fermenting wort. Thus, the beverage may be prepared by fermenting the aqueous extract with yeast.

In one embodiment, the beverage may be an alcoholic beverage, such as beer. In other embodiments, the beverage may be a non-alcoholic beverage based on malted cereal grains. The non-alcoholic beverage may for example be a non-alcoholic beer or other kind of non-alcoholic beverage, such as maltina.

In a preferred embodiment, the beverage is a beer, for example the beer may be a lager beer (ale) or an ale beer (ale). Thus, the beer may, for example, be selected from Altbier, Amber Eler, barley wine (Barleywine), Berlin white beer (Berlinier weisse), Bi e de Garde, Bitter, Blonde Ale, Bock, Brown Eler (Brown Ale), California Common, Cream Ale, Dortminder Export, Doppelbock, Dunkel, Eisbock, Fruit Rabike beer (Fruit lamb), Goden Ale, Gose, Guize, Hefeweizen, Helles, Indian pale Elder (India pale Ale),Lambic, Light ale, Maibock, Malt liquor (Malt liquor), Mill,Old ale (Old ale), Oud bruin, light ale (Pale ale), Pilsener, Porter, Red ale (Red), Roggenbier, Saison, Scotch ale, Steam beer (Steam beer), Stout, Schwarzbier, Lagger, Witbier, Weissbier, and Weizenbock. The aqueous extract according to the invention is prepared from malted cereal grains that have not been kiln dried. The malted cereal grains that have not been kiln dried generally have a lighter colour and the process of the present invention is therefore particularly suitable for the production of lighter beer, in particular for the production of lager beer. Deeper beers can also be made by the process of the present invention, e.g., adding one or more kiln dried malts during the mashing process, as described in the "making aqueous extract" section.

The invention therefore also relates to a method of producing a beverage comprising the steps of:

-preparing an aqueous extract by the method according to the invention.

-processing said extract into a beverage.

Alcoholic beverages, such as beer, may be produced from malted cereal grains according to the method of the invention. In addition to hops and yeast, the malted cereal grains contribute to the flavor and color of beer.

Once the aqueous extract is prepared, it can be processed into beer by any method, including conventional brewing methods. A non-limiting description of examples of suitable methods for brewing can be found in publications such as Briggs et al (1981) and Hough et al (1982). Many regularly updated barley and beer product analysis methods are available, such as, but not limited to, American Association of Central Chemists (1995), American Society of Brewing Chemists (1992), European Brewery Convention (1998), and Institute of Brewing (1997). It is recognized that many specific procedures are employed for a given brewery, with the most significant variations being related to local consumer preferences. Any such method of producing beer may be used in the present invention.

The first step in the production of beer from the aqueous extract preferably comprises heating the aqueous extract as described above, followed by a subsequent cooling and optional vortexing standing (whirlpool rest) stage. One or more additional compounds may be added to the aqueous extract, for example, one or more additional compounds described below in the "additional compounds" section. After cooling, the aqueous extract may be transferred to a fermentor containing yeast, e.g. brewer's yeast such as saccharomyces pastorianus (s.pastorianus) or saccharomyces cerevisiae (s.cerevisiae). The aqueous extract may be fermented for any suitable period of time, typically in the range of 1 to 20 days, for example 1 to 10 days. The fermentation is carried out at any useful temperature, for example at a temperature in the range of from 10 to 20 ℃. The method may further comprise adding one or more enzymes, for example, one or more enzymes may be added to the wort before or during fermentation. In particular, the enzyme may be a proline specific endoprotease. A non-limiting example of a proline specific endoprotease is "Brewer's Clarex" available from DSM. In other embodiments, no exogenous enzyme is added during the process.

During the fermentation process over several days, the sugars are converted into alcohol and CO₂With some flavor development. The fermentation can be terminated at any desired time, for example, once no further reduction in% P is observed.

Subsequently, the beer can be further processed, for example iced (chilled). It can also be filtered and/or long-term cellared, a process that produces a pleasant aroma and less yeast-like flavor. Additives may also be added. In addition, CO may be added₂. Finally, the beer may be pasteurized and/or filtered before it is packaged (e.g., transferred to a keg, bottled or canned). Beer can also be pasteurized by standard methods.

The beer produced by the process of the present invention generally has a pleasant taste and has no or only little astringency. For example, taste can be analyzed by professional beer taste panels.

Barley

In a preferred embodiment of the invention, the cereal grain for use in the process of the invention is barley grain.

The grain may be grain of any barley plant. However, in some embodiments, the barley plant may comprise one or more specific characteristics, for example, one or more characteristics as described below. Although various features are discussed separately below, the barley plants of the present invention may have a combination of these features.

In one embodiment of the invention, the barley may be the hulless barley variety (var.). The invention also includes that the barley is a barley variety with natural thin hulls, such as the variety Admiral. For example, the hull (husks) may comprise less than 7% of the total weight of the grain and hull.

As mentioned above, it is preferred that the aqueous extract obtained during the mashing has a sufficiently low viscosity to give a good filterability of the mash mixture. As also described in detail above, soluble β -glucan may contribute to the high viscosity of the aqueous extract. Thus, in some embodiments of the invention, it may be preferred to use cereal plants-and in particular barley plants with low β -glucan levels (e.g. without β -glucan, e.g. below the detected level). Such barley plants are known in the art and include, for example, barley plants carrying a mutation in the gene encoding β -glucan synthase. The gene may be a gene encoding SEQ ID NO: 2. For example, the barley plant may be a plant comprising SEQ ID NO: 1 or SEQ ID NO: 18, or a β -glucan deficient gene. The barley plant may also be a plant containing the silenced CslF6 gene, resulting in barley grains with very low (1, 3; 1,4) - β -glucan levels (as described by Taketa et al, 2011).

The barley plant may also be a barley plant with low levels of LOX activity. Such barley plants are known in the art and include, for example, barley plants carrying a mutation in the gene encoding LOX-1. For example, the barley plant may be one carrying any mutation in the LOX-1 gene described in WO02/053721, WO 2005/087934 and WO 2004/085652.

The barley plant may also be a barley plant carrying a mutation in the gene encoding lipoxygenase 1(LOX-1) and/or in the gene encoding LOX-2. For example, the barley plant may be a barley plant carrying any mutation in the LOX-1 and LOX-2 genes described in WO 2010/075860.

The barley plant may also be a barley plant with low levels of MMT activity. Such barley plants are known in the art and include, for example, barley plants carrying a mutation in the gene encoding MMT. In particular, the barley plant may be a barley plant carrying any mutation in the MMT gene described in WO 2010/063288. The barley plant may also be any of the barley plants described in WO 2011/150933.

The barley plant may also be a barley plant characterized as having increased GA signaling. In particular, the barley plant may be a barley plant carrying a mutation in the Slender1 gene encoding a del protein. For example, the barley plant may be one carrying the mutations described in Chandler et al, Journal of Experimental botanic, Vol.64, No.6, pp.1603-1613,2013, doi:10.1093/jxb/ert022, for example in Table 1 therein. For example, a barley plant may carry a mutation in the Slender1 gene, resulting in a mutant Slender1 gene encoding a mutant DELLA protein, wherein the mutant DELLA protein carries a mutation of one or more of amino acid numbers 46, 490, 280, 268, 271, 277, 231, 481, 282, 277, 227, 485 or 237, e.g. a mutation selected from G46E, S490F, R268H, G271D, a277T, V231M, R481H, V282F, a277T, G227E, S485F and C237Y. Amino acid numbering is provided for the order of DELLA proteins obtained under Genbank accession numbers AK372064 or AF035820 (versions up to 2013, month 2 and day 4).

Beverage and its preparing process

Beverages prepared by processing an aqueous extract according to the present invention into beverages may have many useful properties, including, but not limited to, the properties described in this section.

It is generally desirable that the beverage according to the invention contains as little diacetyl as possible. Thus, it may be preferred that the beverage comprises a diacetyl level below the threshold value that is considered to be off-flavour in lager beer. Preferably, the beverage comprises at most 30ppb of diacetyl, more preferably at most 25ppb of diacetyl, even more preferably at most 20ppb of diacetyl. This is especially the case if the beverage is a beer, such as lager.

The beverage according to the invention may be an aqueous extract, such as described herein, which has optionally been fermented. Thus, the beverage may comprise or consist of said aqueous extract or fermented aqueous extract and optionally one or more additional compounds. The additional compound may for example be any other compound described in the "additional compound" section below.

Additional compounds

The method of the invention may comprise the step of adding one or more additional compounds. The further compound may for example be a flavouring compound, a preservative, a functional ingredient, a pigment, a sweetener, a pH adjuster or a salt. The pH adjusting agent may for example be a buffer or an acid, such as phosphoric acid.

The functional ingredient may be any ingredient added to achieve a given function. Preferably, the functional ingredient makes the beverage healthier. Non-limiting examples of functional ingredients include vitamins or minerals.

The preservative may be any food grade preservative, for example it may be benzoic acid, sorbic acid, a sorbate salt (e.g. potassium sorbate), a sulfite salt and/or a salt thereof.

The further compound may also be CO₂. In particular, CO may be added₂To obtain a carbonated beverage.

The flavour compounds used in the present invention may be any useful flavour compound. The flavour compounds may for example be selected from the group consisting of aromas, plant extracts, plant concentrates, plant parts and herbal infusions. In particular, the flavour compound may be hops.

Item

The invention may also be described by the following items:

1. a method of producing an aqueous extract of cereal grains, the method comprising the steps of:

a) providing a grain of a cereal;

b) subjecting a cereal grain to a germination step, thereby obtaining a germinated grain;

c) subjecting the germinated grain to a heat treatment step at a temperature in the range of 35 to 55 ℃;

d) subdividing said germinated kernels, with said germinated kernels having a moisture content of at least 20%, with the proviso that at any time between steps b) and d) the moisture content of said cereal kernels is not less than 20%; and

e) preparing an aqueous extract of the milled germinated grain,

thereby producing an aqueous extract of the grain.

2. A method of producing an aqueous extract of cereal grains, the method comprising the steps of:

a) providing a grain of a cereal;

c) subjecting a cereal grain to a germination step, thereby obtaining a germinated grain;

d) subjecting the germinated grain to a heat treatment step at a temperature in the range of 35 to 55 ℃;

e) subdividing said germinated kernels to obtain ground germinated kernels, while said germinated kernels have a moisture content of at least 20%, with the proviso that at any time between steps c) and e) the moisture content of said cereal kernels is not less than 20%; and

f) preparing an aqueous extract of the milled germinated grain,

thereby producing an aqueous extract of the grain.

3. The method of clause 1, wherein step b) is performed for up to 72 hours.

4. The method according to any one of the preceding items, wherein the germinating step includes incubating the grain in an aqueous solution until the grain has a moisture content of at least 30%, wherein at least 2L O is allowed per hour₂Per kg dry weight cereal grain is passed through the aqueous solution.

5. The method of any of clauses 1 to 4, wherein the germinating step comprises:

i. incubating the grain in an aqueous solution for 16 to 40 hours, wherein at least 2L O is allowed per hour₂(ii) per kg dry weight of cereal grains passing through the aqueous solution and wherein the grains are submerged in the aqueous solution during the incubation;

removing excess aqueous solution; and

incubating the moist grain in air at a temperature in the range of 15 to 30 ℃ for 20 to 50 hours.

6. The method of any of items 1-5, wherein the grain is a shelled grain, and wherein the method comprises an initial step of removing at least a portion of the hulls.

7. A method according to any one of the preceding items, wherein the method does not comprise the step of kiln drying the germinated grain.

8. A method of producing an aqueous extract of cereal grains, the method comprising the steps of:

a) providing a grain of a grain, wherein the grain is a shelled grain;

b) treating the cereal grain to remove hulls, wherein the treatment results in at least a 2% total weight loss of the cereal grain;

c) subjecting a cereal grain to a germination step, thereby obtaining a germinated grain, wherein the germination step comprises:

i. incubating the grain in an aqueous solution for 16 to 40 hours, wherein at least 2L O is allowed per hour₂(ii) per kg dry weight of cereal grains passing through the aqueous solution and wherein the grains are submerged in the aqueous solution during the incubation;

removing excess aqueous solution; and

incubating the moist grain in air at a temperature in the range of 15 to 30 ℃ for 18 to 50 hours;

d) subdividing the germinated grain to obtain ground germinated grain, while the germinated grain has a moisture content of at least 20%; with the proviso that at any time between steps c) and d) the moisture content of the cereal grain is not less than 20%; and

e) preparing an aqueous extract of the milled germinated grain,

thereby producing an aqueous extract of the grain.

9. A method according to item 8, wherein the method comprises a further step after the germination step and before the subdividing step, the further step comprising the step of heat treating the germinated grain at a temperature in the range of 35 to 55 ℃.

10. The method of any of items 5 to 9, wherein the step of incubating the moist grain in air is performed at a flow rate of 85 to 95L/h atmospheric air/kg dry cereal grain.

11. A method according to any of items 5 to 9 wherein the step of incubating the moist grain in air is at 17 to 21L/h O₂At a flow rate per kg of dry cereal grains.

12. The method according to any one of the preceding items, wherein the heat treatment is performed at a temperature in the range of 40 to 50 ℃.

13. The method of any one of the preceding items, wherein the duration of the heat treatment is in the range of 1 to 5 hours.

14. The method of any one of the preceding items, wherein the temperature is controlled, for example by cooling.

15. The method according to any one of the preceding items, wherein step d) is optional.

16. A method according to any of items 4 to 15, wherein the grain is incubated in the aqueous solution for 20 to 72 hours.

17. The method according to any of clauses 5 to 1616, wherein step i) · comprises incubating the grain in the aqueous solution for 16 to 40 hours, such as 20 to 35 hours, preferably 20 to 30 hours.

18. The method according to any of clauses 55 to 17, wherein the grain of the cereal is submerged in the aqueous solution during the entire step i) of germination.

19. The method according to any of items 4, 6 and 12 to 16, wherein the grain is submerged in the aqueous solution throughout the germination step.

20. The method of any of clauses 4 to 19, wherein the grains of the grain are submerged in the aqueous solution with at least 2L O per hour₂Per kg dry weight of cereal grain is passed through the aqueous solution for 20 to 72 hours.

21. The method of any of clauses 4 to 20, wherein the grains of the grain are submerged in the aqueous solution with at least 2L O per hour₂Per kg dry weight of cereal grain is passed through the aqueous solution for 16 to 40 hours, such as 20 to 35 hours, preferably 20 to 30 hours.

22. The method of any of clauses 5 to 21, wherein the step of incubating the moist cereal grain in air is performed under aeration.

23. The method according to any of clauses 5 to 22, wherein the duration of the step of incubating the moist cereal grain in air is in the range of 20 to 50 hours, more preferably in the range of 20 to 35 hours, for example in the range of 20 to 30 hours.

24. The method of any of clauses 5 to 23, wherein the step of incubating the moist cereal grain in air has a temperature in the range of 20 to 30 ℃.

25. The method according to any of the preceding items, wherein the entire germination step does not exceed 72 hours, more preferably does not exceed 60 hours, even more preferably does not exceed 54 hours.

26. The method according to any of the preceding items, wherein the entire germination step is carried out for at least 44 hours, such as at least 46 hours.

27. The method according to any one of the preceding items, wherein the entire germination step is carried out for 44 to 72 hours.

28. The method according to any one of the preceding items, wherein the entire germination step is performed at a temperature in the range of 15 to 30 ℃, such as at a temperature in the range of 20 to 30 ℃.

29. The method of any of clauses 4 to 24, wherein the aqueous solution is water.

30. The method of any of clauses 4-26, wherein the method further comprises adding Gibberellic Acid (GA) to the aqueous solution.

31. The method of clause 30, wherein the GA is added to the aqueous solution at a concentration of at least 100nM, e.g., at least 1000 nM.

32. The method of any of clauses 4 to 31, wherein the aqueous solution further comprises an antifoaming agent.

33. The method of any of clauses 4 to 32, wherein at least 3L, more preferably at least 4L, still more preferably at least 5L, even more preferably at least 6L O, are allowed to stand per hour₂Per kg dry weight cereal grain is passed through the aqueous solution.

34. The method of any of clauses 4 to 33, wherein at least 20g O is consumed per hour₂Per kg cereal grain, more preferably at least 30g O₂Per kg cereal grain, still more preferably at least 40g O₂Per kg of cereal grain, e.g. 40-100g O₂Per kg of cereal grain, e.g. 40-80g O₂Per kg of cereal grain, e.g. 60g O₂/kg cereal grain (dry matter) was passed through the aqueous solution/cereal grain mixture.

35. The method of any of clauses 4 to 34, wherein the O is₂Is contained in the gas mixture.

36. The method of clause 35, wherein the gas mixture is atmospheric air.

37. The method of any of clauses 4 to 36, wherein at least 10L, preferably at least 15L, more preferably at least 20L, still more preferably at least 25L, even more preferably at least 30L, of atmospheric air per kg dry weight of cereal grain is passed through the aqueous solution per hour.

38. The method of any of clauses 4-37, wherein incubating in aqueous solution is performed at a temperature in the range of 15 to 30 ℃, preferably at about 25 ℃.

39. A method according to any of items 4 to 38, wherein in the germination and heat treatment steps the grain is kept in the same container.

40. A method according to any of items 4 to 39, wherein the cereal grains are incubated in the aqueous solution until they have a moisture content of at least 35%, for example at least 37%.

41. A method according to any one of the preceding items, wherein the grain provided in step a.

42. The method of clause 40, wherein the antimicrobial agent is a peroxide, such as hydrogen peroxide.

43. The method of any of items 4 to 42, wherein the method does not comprise a rootlet removal step.

44. A method according to any preceding item, wherein removing the hulls results in a total weight loss of cereal grain in the range 2.5% to 7.5%, for example in the range 3% to 6%.

45. The method of any one of the preceding items, wherein the moisture content of the germinated cereal grain is at least 25%, even more preferably at least 30%, and still more preferably at least 35% when subdividing the cereal grain.

46. The method of any one of the preceding items, wherein the moisture content of the germinated cereal grain is not less than 25%, even more preferably not less than 30%, and still more preferably not less than 35% at any time between the completion of the step of germinating and the time of subdividing the cereal grain.

47. The method of any one of the preceding items, wherein the temperature of the germinated grain is greater than 35 ℃ at any time between completion of the heat treatment step and when the cereal grain is subdivided.

48. The method according to any of the preceding items, wherein the cereal is barley.

49. The method of item 48, wherein said barley is a hulless barley or a barley variety with thin hulls.

50. The method of any of clauses 1-48, wherein the cereal is a hulless cereal, such as wheat or hulless barley.

51. The method of any of clauses 1-48, wherein the grain is a hulled grain, such as hulled barley.

52. The method of any of clauses 1-48, wherein the grain is barley characterized by one or more of the following:

B. carrying mutations in the gene encoding beta-glucan synthase

C. Carrying a mutation in the gene encoding LOX-1

D. Carrying a mutation in the gene encoding LOX-2

E. Carry a mutation in the gene encoding MMT; and/or

F. Carrying a mutation in the gene encoding DELLA.

53. The method according to any one of the preceding items, wherein the germinated grain has an alpha-amylase activity of at least 40U/g, such as at least 50U/g, preferably at least 60U/g cereal grain on a dry weight basis.

54. Method according to any of the preceding items, wherein the germinated grain has a limiting dextrinase activity of at least 5mU/g, preferably at least 9U/g grain on a dry weight basis.

55. The method according to any of the preceding items, wherein the germinated grain contains at most 4g rootlets (dry matter)/100 g germinated cereal grain (dry matter).

56. The method according to any of the preceding items, wherein the germinated grain contains at most 2g rootlets (dry matter)/100 g germinated cereal grain (dry matter).

57. The method according to any one of the preceding items, wherein the germinated grain has a nitrosamine content of at most 0.15 μ g/kg, preferably at most 0.12 μ g/kg, such as at most 0.10 μ g/kg of cereal grain (dry matter) immediately prior to the step of subdividing the germinated grain.

58. The method of any one of the preceding items, wherein step c.

59. The method of clause 58, wherein the pulping is conducted in the presence of one or more added hydrolytic enzymes.

60. The method of clause 59, wherein the at least one hydrolase is selected from the group consisting of cell-wall degrading enzymes and starch degrading enzymes, including but not limited to alpha-amylase, beta-amylase, dextrinase, pullulanase, beta-glucanase, xylanase, glucoamylase, and protease.

61. The method of any of clauses 58 to 60, wherein the pulping is conducted in the presence of at least one β -glucanase and at least one xylanase.

62. The method of any of clauses 58 to 61, wherein during the mashing up to 700U, preferably up to 350U, of exogenous glucoamylase and/or alpha-amylase are added per g of malted cereal grain (dry weight).

63. The method of any of clauses 58 to 62, wherein during the mashing, up to 100PUN of exogenous pullulanase is added per g of malted cereal grain (dry weight).

64. The method of any of clauses 58 to 59 and 62 to 63, wherein the grain is characterized by low levels of β -glucans in the grain, and wherein no β -glucanase is added during the mashing.

65. The method of any one of the preceding items, wherein the method further comprises the step of filtering the aqueous extract.

66. The method according to any one of the preceding items, wherein the aqueous extract has a filterability of at least 50%, such as at least 60%.

67. The method according to any one of the preceding items, wherein the aqueous extract comprises at most 200mg/L of β -glucan.

68. The method according to any one of the preceding items, wherein the aqueous extract comprises at least 10g, such as at least 15g maltose per litre.

69. The method of any one of the preceding items, wherein the aqueous extract comprises 150 to 400 mg/LFAN.

70. The method according to any one of the preceding items, wherein the aqueous extract comprises at least 60mg/L, preferably at least 65mg/L, valine.

71. The method of any of the preceding items, wherein the method does not include a kiln drying step.

72. A method according to any one of the preceding items, wherein the method does not comprise the step of kiln drying the germinated grain.

73. The method of any one of the preceding items, wherein the method does not comprise a step of removing the rootlet.

74. A method according to any preceding item, wherein the germinated grain contains at most 4g rootlets (dry matter)/100 g germinated cereal grain (dry matter).

75. A method according to any preceding item, wherein the germinated grain contains at most 2g rootlets (dry matter)/100 g germinated cereal grain (dry matter).

76. A method of producing a beverage, the method comprising the steps of:

i. preparing an aqueous extract by the method of any one of the preceding items;

processing said extract into a beverage.

77. The method of item 72, wherein step ii.

a) Optionally heating the aqueous extract in the presence of hops or hops extract;

b) cooling the aqueous extract;

c) fermenting the aqueous extract with yeast, thereby producing a fermented beverage.

78. The method of clause 77, wherein the method further comprises a settling step performed after step a.

79. The method according to any one of the preceding claims, wherein the entire method is carried out at one site.

80. The method of any of clauses 72 to 79, wherein the beverage comprises at most 25ppb, e.g., at most 20ppb, diacetyl.

81. A beverage produced by the method of any of items 76 to 80.

82. An aqueous cereal extract produced by the method of any of clauses 1 to 75.

83. A method of producing an aqueous extract of cereal grains, the method comprising the steps of:

a) providing a grain of a cereal;

c) subjecting a cereal grain to a germination step, thereby obtaining a germinated grain;

d) subjecting the germinated grain to a heat treatment step at a temperature in the range of 35 to 55 ℃;

e) subdividing said germinated kernels, with said germinated kernels having a moisture content of at least 20%, with the proviso that the moisture content of said cereal kernels is not less than 20% at any time between steps b) and d); and

f) preparing an aqueous extract of the milled germinated grain,

thereby producing an aqueous extract of the grain.

78. A method of producing an aqueous extract of cereal grains, the method comprising the steps of:

a) a) providing a grain of a cereal, wherein the cereal is a shelled cereal;

b) treating the cereal grain to remove hulls, wherein the treatment results in at least a 2% total weight loss of the cereal grain;

c) subjecting a cereal grain to a germination step, thereby obtaining a germinated grain, wherein the germination step comprises

i. Incubating the grain in an aqueous solution for 16 to 40 hours, wherein at least 2L O is allowed per hour₂(ii) per kg dry weight of cereal grains passing through the aqueous solution and wherein the grains are submerged in the aqueous solution during the incubation;

removing excess aqueous solution; and

incubating the moist grain in air at a temperature in the range of 15 to 30 ℃ for 18 to 50 hours;

e) subdividing the germinated grain while the germinated grain has a moisture content of at least 20%; with the proviso that at any time between steps c) and d) the moisture content of the cereal grain is not less than 20%; and

f) preparing an aqueous extract of the milled germinated grain,

thereby producing an aqueous extract of the grain.

Examples

The invention is further illustrated by the following examples. However, these should not be construed as limitations of the present invention. The barley samples used in the following examples were all analyzed as follows:

germination test

All barley samples used in the examples were evaluated for parameters germination index, germination energy and water sensitivity. The data are based on sample sizes of 100 barley grains subjected to a 4-mL germination test and 100 barley grains subjected to an 8-mL germination test according to Analytica-EBC method 3.6.2 barley germination energy (BRF method).

Characterization of the barley samples

One thousand kernel weights were determined by automated counting using a Data Count JR instrument, while size grading was adjusted to 4 different particle sizes (X) using pfeffer Sortimat K3: x is greater than 2.8 mm; 2.8< X >2.5 mm; 2.5< X >2.2 mm; x <2.2 mm. Size grading data was calculated based on 100-g grain samples.

The protein, water and starch content of the barley samples were determined using a Foss 1241NIT instrument using a barley calibration (FOSS BY 213271; supplied BY Foss, DK). Prior to incubation in aqueous solution (e.g., 24 hours), the moisture content of 100-g grain samples was re-determined using a Foss 1241NIT instrument using a barley calibrated Foss BY303300(Foss, Denmark).

The moisture content of the grain is determined by first measuring the weight of the respective barley sample, then drying the sample and determining the weight of the dried sample. The weight difference between the wet and dry samples is considered water, and the water content is equal to the weight of water divided by the total weight of the sample (wet sample).

Analysis of germinated grains

The germinated grain samples were tested for the following parameters (w/w): moisture content, protein content, soluble protein and extract of malt samples. Values were determined using a Foss 1241NIT instrument calibrated against data provided by Foss (DK; calibration MA 000010).