The present invention concerns a beverage or beverage component obtained by the fermentation of brewer's spent grain and a process of preparing such beverage, as well as the use of a component obtained by the fermentation of brewer's spent grain for preparing a beverage and/or for prepa ring other foodstuffs. In a further aspect the present invention provides compositions of a beverage obtained through the fermentation of brewer's spent grains, in particular food compositions that comprise nutritional claims such as high protein/source of protein, high fiber/source of fiber, particularly soluble and insoluble ara binoxylans, a nd optionally prebiotics such as Beta- glucans and probiotics such us Lactobacillus. Background to the Invention

Brewers' spent grain (BSG) is the most abundant co-product generated in the beer- brewing process. This material consists of the barley grain husks obtained as solid portion after the wort production. Since BSG is rich in sugars and proteins, the main use to date for the utilization of this product has been as animal feed. However, for exactly these same reasons, because it is high in dietary fiber and proteins, BSG is of interest for application in different areas particularly when considering its valuable component composition as a potentia l source of bioactive, health-promoting compounds.

BSG consists of the seed coat-pericarp-husk layers that covered the original barley grain. The starch content is usua lly low, and the composition of BSG mainly contains fibers, which are non-starch polysaccharides (NSP; hemicellulose in the form of arabinoxylans (AX) and cellulose) and significant quantities of proteins and lignin, with arabinoxylans (AX) typically constituting the most abundant component. Therefore, BSG is basically a lignocellulosic material. Fiber constitutes about half of the BSG composition on a dry weight basis, while proteins can constitute up to 30% of the dry weight basis. This high fiber and protein content makes BSG an interesting raw material for food applications.

As would be expected, cellulose ( -(l,4)-linked glucose residues) is another abundant polysaccharide in BSG. Certain levels of (l-3,l-4)- -D-glucan may also be present. The most abundant monosaccharides in BSG are xylose, glucose, and arabinose, while traces of traces of rhamnose and galactose have also been found.

Arabinoxylans (AX) constitute up to 25% of dry weight in BSG. Most of these are associated with other fibre components (cellulose or lignin) or with protein and are not bioavailable (water-unextractable arabinoxylans, WUAX). A small fraction of WUAX can be me made soluble (water-extractable arabinoxylans, WEAX) via enzymatic treatment. Consumption of WEAX has been shown to have positive health effects, including prebiotic effects, regulation of postprandial blood glucose levels, lowering cholesterol levels, tumor suppression and immunomodulating effects. It is, therefore, desirable to increase the proportion of WEAX in BSG preparations for human consumption. The protein content of BSG typically is present at levels of approximately 30% per dry weight basis. The most abundant are hordeins, glutelins, globulins and albumins. Essential amino acids represent approximately 30% of the total protein content, with lysine being the most abundant, while non-essential amino acids in BSG constitute up to 70% of the total protein content. This is significant because lysine is often deficient in cereal foods. In addition, BSG also contains a variety of minerals elements, among which silicon, phosphorus, calcium and magnesium are the most abundant.

The present invention is directed to a particular BSG utilization for beverage production, allowing obtaining a beverage with beneficial effect on the organization of the intestinal microbial community, and comprising nutritional claims referring the high protein content or the beverage serving as source of protein and an increased level of health- promoting WEAX. This invention further covers the method for preparing such a beverage. Therefore, the present invention does not only address new uses of brewer's spent grain, but specifically addresses a higher valorization of the brewer's spent grain than currently possible. Summary of the Invention

The present invention achieves a high valorization of brewer's spent grain by use of this material for preparing healthy and/or functional beverages with specific nutritional characteristics such as high protein content or source of protein, desired by sportsmen and craftsmen to recover from intense physical exercise. Additionally, the mentioned beverage contains high fiber, a proportion of which is comprised by healt-promoting water-extractable arabinoxylans (WEAX). Furthermore, the beverage preferably contains prebiotics such as Beta-glucans and/or probiotics such as Lactobacillus.

In particular, the present invention concerns a process for preparing a beverage or beverage component comprising the steps of:

^■ Providing brewer's spent grain;

^■ Performing saccharification and fibre solubilization by enzymatic treatment of the brewer's spent grain;

^■ Fermenting the saccharified brewer's spent grain with lactic acid bacteria and/or acetic acid bacteria and/or probiotics to obtain a fermented broth; and

^■ Homogenizing the fermented broth to obtain the beverage, beverage component or food component.

The present invention also concerns a beverage, beverage component or food component obtained by fermentation of brewer's spent grain, wherein the beverage, beverage component or food component comprises proteins in a level sufficiently high such that at least 12% and preferably at least 20% of the total caloric value of the beverage, beverage component or food component originates from proteins therein; and wherein the total arabinoxylan content of said beverage, beverage component or food component is at least 5% (w/v) and the soluble arabinoxylan (WEAX) content of said beverage, beverage component or food component is at least 1% (w/v)

The present invention further concerns the use of a beverage component as defined supra for obtaining a final beverage by mixing with another beverage component. The present invention finally concerns the use of lactic acid bacteria (LAB) for fermenting brewer's spent grain in the preparation of a beverage or beverage component.

Detailed Summary of the Invention

The enzyme treatment of the brewer's spent grain preferably includes the addition of one or more enzymes with following enzymatic activity to the brewer's spent grain: alpha- amylase, gluco-amylase, cellulase, xylanase, protease, Beta-glucanase and/or admixtures thereof. Treatment with said enzymes results in an increase of the levels of health- promoting soluble arabinoxylans (WEAX). Preferably, the fermentation of the fermentable broth is achieved by lactic acid bacteria, preferably lactic acid bacteria of the species Lactobacillus plantarum and/or Lactobacillus rhamnosus, more preferably the strain Lactobacillus plantarum F10 and/or Lactobacillus rhamnosus GG (LGG ^® ). According to a preferred embodiment of the invention, the beverage or beverage component is supplemented by a probiotic microorganism after pasteurization, preferably a lactic acid bacteria, more preferably Lactobacillus rhamnosus, and more preferably the strain Lactobacillus rhamnosus GG (LGG ^® ). The beverage or beverage component can be: a low energy beverage having a caloric value of less than 20 kcal/100 g; and/or have a fat content of less than 1.5w%, preferably less than 0.5w% and/or have a sugar content of less than 2.5w%, preferably less than 0.5w%; and/or have a fiber content of at least 1.5g per 100 kcal of beverage or beverage component. The beverage or beverage component may comprise prebiotics and/or probiotics, for example by supplementing the beverage by a probiotic microorganism after pasteurization, preferably a lactic acid bacteria, more preferably Lactobacillus rhamnosus, and more preferably the strain Lactobacillus rhamnosus GG (LGG ^® ). The beverage or beverage component is preferably lactose free. Definitions

Barley is the main raw material used for the production of beer. However, other cereals such as corn or rice are typically used together with malted barley. During the brewing process the starchy endosperm of these cereals is subjected to enzymatic degradation, resulting in the liberation of fermentable (maltose and maltotriose, and a minor percentage of glucose) and non-fermentable carbohydrates (dextrins), proteins, polypeptides and amino acids. The thus produced medium (which will be fermented into beer by the action of yeast) is known as wort. The insoluble grain components (comprising mainly the grain coverings) is the brewers' spent grain (BSG). In traditional brewing employing a lauter tun, the BSG components play an important role as they form the bed through which the mash is filtered to produce wort. Therefore, the initial milling of the malt must be such that the grain coverings remain intact so as to form an adequate filter. Today, while many small or craft breweries still use this method of mash filtration, many larger breweries employ a mash filter which relies less on the filtration function of the BSG and thus malt can be milled more extensively.

The brewer's spent grain contains all the solids that have been separated from the wort by filtration; it includes what is left of the barley malt and the adjuncts. The spent grain consists mainly of the pericarp and hull portions of the barley and of non-starchy parts of corn, provided corn grits were used as an adjunct. Brewer's spent grain is a lignocellulosic material typically comprising lipids, lignin, proteins, cellulose, hemicellulose and some ash.. For the description and claims of this invention the wording "brewer's spent grain" (BSG) will be used in accordance with the definition here above.

Product water refers to water used in the brewing process, that has suffered a defined and standard process for making it suitable for consumption.

Nutritional definitions as defined by the European Commission (http://ec.europa.eu/food/safety/labelling_nutrition/claims/ nutrition_claims/index_en. htm), see Table below: Nutritional claim Definition .· ^■

Low energy <20 kCal per 100 g

Fat free <0.5% fat content

Low fat <1.5% fat content

Very low salt <0.4% salt content

Source of fiber >3% fiber content OR >1.5 g fiber per 100 kCal

Low sugar <2.5% sugar content

High in fiber >6% fiber content OR >3 g fiber per 100 kCal

Source of protein >12% of the energy provided by protein

High in protein >20% of the energy provided by protein

Digestion of AX either enzymatically or otherwise results in an increase of the soluble fraction of arabinoxylans (WEAX). This fraction is responsible for most of the health- promoting effects of arabinoxylans. Among the many positive effects WEAX have on health we find:

1. reduction of postprandial glucose levels in individuals with compromised glucose metabolism (Lu et al., 2004, Garcia et al., 2006)

2. tumor suppressing activity (Li et al., 2011)

3. reduction of obesity, cholesterol levels and restoration of beneficial gut bacteria in high fat diets (Neyrinck et al., 2011)

4. immune-enhancing effects (Zhou et al., 2010)

5. prebiotic effects, including promoting healthy gut bacteria and short chain fatty acid in distal colon (Cloetens et al., 2010, Sanchez et al., 2009)

Additionally, there is evidence that preparations of arabinoxylans from brewer's spent grains (BSG-AX) can exert the same prebiotic effects as the better-studied wheat-derived arabinoxylans, namely:

6. BSG-AX are not absorbed in the small intestine and reach the colon (Texeira et al., 2017); BSG-AX promote proliferation of gut bacteria, particularly beneficial species like, for example, those of the Bifidobacteria genus, and BSG-AX promote the production of short chain fatty acids by said bacteria (Reis et al., 2014)

The documented effects listed above were elicited by the following dosages:

(1) 0.12 g/kg body weight/day, (2) 0.4 g/kg body weight/day, (3)10% of diet , (4) 0.1 g/kg day , (5) 0.14 g/kg weight/day and 0.6% (w/v), (6) 0.6 g/kg body weight/day

Additionally, a patent concerning the use of soluble arabinoxylans extracted from wheat (Ekhart et al., 2016), recommends that a daily dosage of 0.08 g/kg day would be adequate to obtained the claimed health effects, namely prebiotic effect and decrease of symtoms associated with high-fat diets.

European Food Safety Authority has concluded that there is a cause effect relationship between the consumption of wheat arabinoxylan and the reduction of postprandial glucose levels (EFSA, 2011) . Based on the provided evidence EFSA suggests that to obrain the claimed effect, 4.8 % w/w of consumed carbohydrate should be soluble arabinoxylans. For a healthy 70kg adult with an average 2200 kcal daily intake (EFSA, 2013), of which 45% are carbohydrates (EFSA, 2010), this corresponds to 0.17 g/kg body weight/day.

It is therefore considered that no less than 0.1 g/kg body weight/day, is a sufficient dose of WEAX to have positive health effects.

The fibre-solubilization and saccharification enzyme process described here results in a beverage, beverage ingredient or food ingredient with no less than 1.4 % (w/v) soluble arabinoxylans.

Finally, lactose free refers to a product that contains no trace of this compound. The present invention refers to a beverage produced through the fermentation of BSGs, therefore containing no dairy product and thus lactose free.

Detailed Description of a Preferred Embodiment

The process according to the present invention generally comprises the steps of:

■ Providing brewer's spent grain; ^■ Performing saccharification and fibre solubilization by enzymatic treatment of the brewer's spent grain;

^■ Fermenting the saccharified brewer's spent grain with lactic acid bacteria and/or acetic acid bacteria and/or probiotics to obtain a fermented broth; and ■ homogenizing the fermented broth to obtain the beverage or beverage component.

The brewer's spent grain is preferably obtained from a regular beer production process, wherein malt and potentially some adjuncts such as corn, rice, sorghum, wheat, barley, rye, oat or combinations thereof are mixed with water to form a mash wherein enzymes - either originating from the barley malt or added separately to the mash - are allowed to break down starch into fermentable sugars, typically a mixture of glucose, maltose and maltotriose. At the end of the mashing, the mash is filtered to obtain a fermentable wort that is further processed in to beer. The retentate of the mash filtering is the brewer's spent grain (BSG).

BSG comprises the seed coat-pericarp-husk layers that covered the original barley grain. BSG's composition mainly comprises fibers, which are non-starch polysaccharides (NSP; hemicellulose in the form of arabinoxylans (AX) and cellulose) and significant quantities of proteins and lignin, with arabinoxylans (AX) typically constituting the most abundant component. Therefore, BSG is basically a lignocellulosic material. Fiber constitutes about half of the BSG composition on a dry weight basis, while proteins can constitute up to 30% of the dry weight basis. This high fiber and protein content makes BSG an interesting raw material for food applications.

As would be expected, cellulose ( -(l,4)-linked glucose residues) is another abundant polysaccharide in BSG. Certain levels of (l-3,l-4)- -D-glucan may also be present. The most abundant monosaccharides in BSG are xylose, glucose, and arabinose, while traces of traces of rhamnose and galactose have also been found.

The protein content of BSG typically is present at levels of approximately 30% per dry weight basis. The most abundant are hordeins, glutelins, globulins and albumins. Essential amino acids represent approximately 30% of the total protein content, with lysine being the most abundant, while non-essential amino acids in BSG constitute up to 70% of the total protein content. This is significant because lysine is often deficient in cereal foods. In addition, BSG also contains a variety of minerals elements, among which silicon, phosphorus, calcium and magnesium are the most abundant. The BSG obtained from a lager beer production process typically comprises hemicellulose (20-25w% on dry matter); cellulose (12-25w% on dry matter); protein (19- 30w% on dry matter); lignin (12-28w% on dry matter); lipid (ca. 10w% on dry matter); ash (2-5w% on dry matter); and low amounts of fructose, lactose, glucose and maltose.

The BSG is highly nutritious and very sensitive for spoilage by micro-organisms, hence heat treating of the BSG is desired to increase the shelf life. In this sense, the high water content of BSGs in the moment of their production (wort filtration), which is in the range of 75% (25% total solids), increases the instability of the material. For this reasons preferably fresh spent grains are used in the process of the present invention, and/or BSGs are stabilized or treated for sterilization, preferably by boiling. In a process according to the present invention, BSGs, preferably as produced during the brewing process (in the range of 25% total solid content), and more preferably collected just after their production, are mixed with distilled water, or preferably hot product water, to a final dry matter content of between 6 and 10%, more preferably between 8 and 9%. The solids in this suspension are ground, preferably using corundum stone grinding technology, to an average particle size no bigger than 80 μιη and an absolute particle size no bigger than 300 μιη. The ground suspension is subsequently treated for stabilization, for example by heat treatment such as by boiling for 60 minutes.

Subsequently, the mixture of BSGs and water is exposed to fibre solubilization, saccharification and fermentation, preferably to a simultaneous process of saccharification and fermentation (SSF). Commercial enzymatic products used for the fibre solubilization and saccharification of the BSG in the present invention will have at least one of following activities: xylanase (including endo-xylanase); cellulase; glucanase (including beta-glucanase); glucoamylase, protease, and or admixtures thereof. Preferably, the enzymatic mixture use will contain starch, dextrin, protein and fiber degrading activities. More preferably, these activities will comprise gluco-amylase, pullulanase, alpha-amylase, beta-glucanase, xylanase and protease. Enzyme treatment with xylanase and protease solubilizes WUAX and increases the levels of health- promoting WEAX. As examples of such enzyme treatment, experiments were done by adding to a mixture of BSGs and water the following commercial products:

Example 1

Example 2

Example 5

After hydrolysis, a fermentable broth is obtained that is subsequently fermented with lactic acid bacteria and/or acetic acid bacteria and/or probiotics. Preferably, such microorganisms are added during the hydrolysis, thus performing a simultaneous saccharification and fermentation process (SSF). The lactic acid bacteria can be used either alone or in combination with yeast (eg 5. cerevisiae).

Examples of lactic acid bacteria include:

L. gallinarum AB13 Homofermentative Sourdough

L. plantarum F6 Hete rofe rmentative Sourdough

L. plantarum F10 Heterofermentative Brewery

L. plantarum F21 Heterofermentative Sourdough

L. plantarum Rll Heterofermentative Cheese

L. plantarum R13 Heterofermentative Cheese

L. reuteri AB38 Heterofermentative Sourdough

Human

L. reuteri DSM20016 Heterofermentative

intestine

L. reuteri Ff2 Heterofermentative Porcine

L. reuteri hhlP Heterofermentative Porcine

L. reuteri R12 Heterofermentative Cheese

L. rhamnosus C7 Homofermentative Cheese

L. rhamnosus C8 Homofermentative Cheese

L. rhamnosus C9 Homofermentative Cheese

L. rhamnosus GG Homofermentative Human gut

L. sakei AB3a Heterofermentative Sourdough

L vaginalis AB11 Heterofermentative Sourdough

Leuconostoc

TR116 Heterofermentative Sourdough citreum

L. holzapfelii AB4 Heterofermentative Sourdough

Leuconostoc

Ell Heterofermentative Sourdough lactis

Leuc.

DSM20240 Heterofermentative Root beer

Mesenteroides

Weissella cibaria MG1 Heterofermentative Sourdough

Examples of acetic acid bacteria include G. oxydans and K. xylinus. Preferably, the strains L planetarum F10 and L. rhamnosus LGG are preferred as selected to provide desirable organoleptic properties. Possibly, a probiotic strain is added at the end of the process of production of the beverage defined in the present invention. Hydrolysis of the BSG is performed for at least 12 hours, preferably 24 hours at a temperature in function of the enzyme(s) used (typically about 55°C), to ensure solubilization of arabinoxylans and increase in the level of WEAX to health-promoting levels of at least 1.4% (w/v). Hydrolysis is followed by a 8 to 24 hours of fermentation at about 25 to 37°C, preferably at 30°C. Preferably, the hydrolysis and fermentation steps are combined in one step (SSF) and performed during between 15 and 24h at a temperature between 25 and 37°C, more preferably during 20h at a temperature of 30°C. Aerobic and static conditions are used during the fermentation or SSF process.

The fermentation or SSF is followed by critical parameters such us pH, extract, total acidity (TTA) and concentration of reducing sugars. The process is considered to be finished when, for example, total acidity (TTA) doubles its value, preferably from 4.0 to 8.0 mL/10 mL of broth, and more preferably together with a drop of between 0.2 and 0.4 pH units and increased extract of 0.5-1.0% (extract measured by Anton-Paar and defined as gram of soluble solid per 100 g of broth). Alcohol concentration in the fermented broth is also measured. Aerobic and static conditions are used to ensure a low alcohol concentration, below 0.20%, preferably below 0.15%, and more preferable below 0.10% in the fermented broth.

The above described fermented broth is subsequently homogenized to produce a beverage with the following nutritional claims: low fat content, low sugar content, high in fiber, high in protein, very low salt (see definitions). ■ The fermented base is swirled to re-suspend settled particles.

^■ The mixture is then blended, preferably by an industrial blender, until a homogenous mixture is obtained.

By homogenizing a beverage or beverage component (type 2) the fermented broth, a beverage, beverage component or food component (type 2) can be obtained that is low in fat content (<1.5%) and/or low in sugar content (<2.5%) and/or high in fiber content (>1.5 g fiber/ 100 kcal, preferably > 3 g fiber/ 100 kcal) and/or sufficient levels of health- promoting soluble arabinoxylans (no less than 1.4% w/v, preferably no less than 3%) and/or high in protein (>12%, preferably >20% of the energy provided by proteins) and/or very low in salt content (<0.4%). A 500mL serving of said beverage would provide 70g of soluble arabinoxylans, or 0.1 g/kg body weight for a 70kg adult person.

Since no dairy product is used in the described process, the beverage or beverage component obtained by a process according to the present invention is consequently lactose free. The beverage can be consumed as such or can be used as a beverage component and mixed with one or more other components prior to consumption. Such components can be beverages as for example a fruit juice. The beverage can be used as a food component or food additive for foodstuffs such as: pasta products, breads and sourdoughs, cerals and cereal products, baked goods and cookies. The final beverage, beverage component or food component obtained by the process described in this invention can be exposed to stabilization treatments, preferably pasteurization, preferably at 70 C during 12 min. Additionally, the final beverage or beverage component can be supplemented by the addition of probiotic microorganisms, preferably lactic acid bacteria.

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