FIELD OF THE INVENTION

The present invention concerns a process for treating brewers' spent grains (BSG) obtained from the brewing process such that the growth of microbes in said grains and subsequent production of microbial toxins are kept below levels herewith specified (microbial stabilization). This invention covers a method for acidification of BSG with one or a combination of acids, or organic acids, or food-grade organic acids to achieve microbial stability of BSG. The present invention further concerns microbiologically stable BSG (MS-BSG) obtained from said process. The invention further concerns the stability of BSG for longer time periods than currently possible, the consequential expansion in the range of applications for MS-BSG and the necessary increase in value of MS-BSG originating from said applications. The invention further concerns the use of microbiologically stable brewers' spent grain (MS-BSG) for animal feed and/or for the production of human food and/or food-grade ingredient and/or for the production of a food-grade beverage ingredient. BACKGROUND TO THE INVENTION

Brewers' spent grain (BSG) is the most abundant co-product generated in the beer-brewing process, making up to 85% of total waste products. This material consists of the barley grain husks obtained as solid portion after wort filtration. Since BSG is rich in carbohydrates and proteins, the main use to date for the utilization of this product has been as animal feed. Beyond the conventional use as cattle feed, several other applications have been proposed for BSG (reviewed by Mussatto et al., 2006 ¹ and Xiros and Christakopoulos, 2012 ² ): energy production in the form of direct combustion, reduction to BSG charcoal or for biogas production; as a raw material for brick building and paper making; in biotechnology applications such as enzyme production, and propagation of microorganisms and; fractionation and enrichment of high-value components, such as proteins or phenolic compounds.

However, the nutritional value of BSG also makes it an interesting candidate for human foodstuff or ingredient. Several applications of BSG as food or food ingredient have been described (reviewed by Mussatto et. Al, 2006 ¹ and Lynch et al., 2016 ³ ). Brewers' spent grain has been used as an ingredient for baking goods such as bread, cookies, bread sticks, baked snacks and also sausages and as a beverage additive. A number of health benefits are associated with including BSG in a diet, including reduction of postprandial blood glucose levels, lowering of cholesterol, prebiotic, immunomodulatory and antioxidant activities.

Microbial stability is a major concern in the use of BSG for either animal or human consumption. The availability of water, sugars and proteins make BSG an attractive substrate for microbes, which can quickly colonize it and compromise its integrity for its subsequent use as food. There are problems associated with microbial spoilage of BSG: first, the possibility of growth of pathogenic microbes, next, the depletion of nutrients from BSG by spoilage microorganisms and, last, the production of toxic compounds by fungi. The latter is of concern, particularly the production of stable mycotoxins.

The very short stability of wet BSG has been identified as an obstacle for its use as animal feed, human food or human food ingredient. Without an effective preservation method, BSG becomes quickly infected by microbes. This compromises the nutritional integrity and general food safety of BSG.

Current methods for preventing spoilage of BSG include (reviewed by Mussato et al., 2006):

• Drying in a rotary-drum drier or in an oven. The former consumes large amounts of energy while the latter can introduce unpleasant aromas and flavors to BSG derived from chemical reactions at high drying temperatures. An alternative experimental method of drying involves the use of superheated steam ⁵ . • Freezing. Storage of large amounts of frozen BSG is not practical or economical. Additionally, thawed BSG can have a lower arabinose content than fresh BSG, possibly due to microbial growth during thawing ⁶ .

• Pressing and vacuum packing. Good stability of BSG was achieved by El-Shafey et al. (2004) ⁷ membrane filter press coupled with vacuum drying. The resulting BSG had 20% moisture which was reduced to 10% after storing in open air. No microbial growth was observed up to 6 months after treatment ¹ .

Preservation of BSG using organic acids was investigated by Al Hadithi et al. (1985 quoted by Mussato et al., 2006). They found that organic acids prevented spoilage and preserved the nutritional value of BSG for long periods of time.

Acidification is a traditional means of preserving food. Food can be acidified either by direct addition of acid (e.g. pickling with vinegar), by microbial fermentation of food by lactic or acetic acid bacteria species (e.g. sauerkraut, kimchi) or a combination of both. The low pH (<4.1) and presence of organic acids inhibits the growth of most harmful bacteria and fungi. Moreover, lactic and acetic acid are generally perceived as pleasant in food at the right concentrations and are considered harmless to human health.

Brewing manufacturing process are in place to keep the final product, beer, but not its co-products, microbiologically stable. Brewers' spent grain is not microbiologically compromised out of the filter or lauter tun. The temperature conditions during mashing and filtering are up to 75°C. Out of the filter, BSG has relatively low counts of microbes, with the exception of thermophilic bacteria, and can be considered microbiologically stable ⁴ . However, as the grains cool down, and because the grains are treated as a waste product without any food safety concerns, mesophilic bacteria and fungi are able to grow on them. After one day of storage at 25°C, the colony count of bacteria (aerobic and anaerobic) and fungi increases from less than 100 colony forming units (CFU) per gram of BSG to 10 ^s and 10 ^s CFU/gram, respectively. After two days, all microbes are found in the 10 ^s CFU/gram order of magnitude.

Mycotoxins are a type of compound produced by fungi which grow on cereal crops. They can be toxic and deadly for humans and animals if consumed in high doses. Because of this, they are of great concern to the cereal food industry, and to the brewing industry, and limits have been set for the maximum levels allowed in food. Fungal contamination of BSG can result in the production of mycotoxins, which is an irreversible process, i.e. once the level of mycotoxins in BSG is above a set limit, it is considered unsafe for consumption.

Considering the time frames on which microbial growth and spoilage occurs after BSG has left the filter/lauter tun, the opportunities for its use as animal or human food ingredient are limited. Within hours, BSG microbial and/or mycotoxin levels can be above the recommended levels for human consumption.

The use of BSG as human food or food ingredient requires that BSG be available at its freshest state, before any significant microbial growth and/or mycotoxin production compromises its integrity. For any food production process, this poses an incredibly difficult operational barrier. Therefore, there remains a need for keeping the integrity of BSG for use as animal feed and/or as human food or food ingredient.

SUMMARY OF THE INVENTION

The present invention concerns A process for stabilizing fresh brewer's spent grains (BSG) microbiologically, the process comprising the steps of:

Producing a mash comprising barley malt;

Separating the mash from BSG;

Collecting the BSG;

acidifying the BSG to a pH of 4 or lower, characterized in that the BSG:

is acidified prior to reaching mycotoxin levels higher than 3μg/kg Ochratoxin A

(OTA), higher than 750 μg/kg deoxynivalenol (DON), higher than 20 μg/kg nivalenol

(NIV), and higher than 75 μg/kg zearalenone (ZEA) and/or

having a colony count of not higher not higher than 10 ³ CFU/g MS-BSG total aerobic bacteria and; not higher than 10 ³ CFU/g MS-BSG fungi and; not higher than 10 ³ CFU/g MS-BSG yeast and; not higher than 10 ³ CFU/g MS-BSG mesophilic aerobic bacteria and; not higher than 10 ³ CFU/g MS-BSG total anaerobic bacteria, after one week of storage at 25°C.

More specifically, the use of food-grade organic acids makes the resulting BSG microbiologically stable and safe for human consumption. Even more specifically, the use of a combination of 0.4% lactic acid and 0.4% acetic acid results in a microbiologically stable product, safe for human consumption with acceptable organoleptic characteristics.

An objective of this invention is to render BSG microbiologically stable over longer periods of time than currently possible. As a result, the range of applications of MS-BSG is much broader than that of untreated BSG, rendering MS-BSG into a much more valuable raw material than untreated BSG.

Thus, a further aspect of this invention concerns MS-BSG and uses thereof. One application of MS- BSG is as part of animal feed diet, with fewer time or distance restrictions between the brewhouse and the animals. A more specific application is the use of food-grade MS-BSG as part of human diet, for example as, but not limited to, an ingredient in flours, a baking-product additive or a food fortification ingredient. A further application of food-grade MS-BSG is as the starting material for a beverage resulting from the bacterial fermentation of previously milled and deacidified MS-BSG. DEFINITIONS

BSG consists of the seed coat-pericarp-husk layers that covered the original barley grain. The starch content is usually low, and the composition of BSG mainly contains fibers , which are non-starch polysaccharides (NSP, ~38%; hemicellulose in the form of arabinoxylans (AX) and cellulose) and significant quantities of proteins (~19%), lignin (~15%), bound phenolics (10%), lipids (~10%) and ash (5%) ⁴ . Therefore, BSG is basically a lignocellulosic material. This high fiber and protein content makes BSG an interesting raw material for food applications.

As it is released from the wort filter or lauter tun, BSG can contain anywhere from 70 to 85% water. The high level of water and presence of nutrients make BSG a good substrate for bacterial and fungal growth. In fact, fresh BSG is quickly colonized by different types of bacterial and fungal species if no measures are taken against this. After two days of incubation of BSG at 25 ^Q C, there is an increase of total bacteria, mesophilic bacteria, anaerobic bacteria and fungi to 10 ⁷ -10 ⁸ CFU/g of BSG. Storage of BSG at 25 and 35°C is also associated with a decrease in the nutritional value of BSG: there is a decrease in total protein, soluble sugars and total dry mass of the BSG ⁹ . Wang et al. (2014) ⁹ and the authors here have observed a significant increase in both yeast and mold in BSG after 2 days storage at 25 ^Q C. In both cases, mold colony counts are as high as 10 ^s after 1 day and 10 ^s after 2 days ⁹ ( and Lynch, unpublished). There is evidence that portion of the mold community in stored BSG is composed of mycotoxin-producing molds, such as Penicillum and Fusarium species ¹⁰ . Mycotoxins are compounds produced by fungi that infect food crops. They are particularly prevalent in cereal crops, such as in barley or wheat used for brewing. Different types of mycotoxins have various effects on the health of animals that are fed the contaminated crop. Mycotoxins are very stable molecules, resisting cold or heat treatments, and even animal digestion, which means they can enter human food chain through contaminated animals. The main fungi affecting barley are those of Fusarium genus ^11,12 . Fusarium species produce a variety of toxins, including zearalenone (ZEA) and the trichocethenes nivalenol (NIV) and deoxynivalenol

(DON). Table 1 shows the maximum levels allowed in Europe for these mycotoxins and for ochratoxin A (OTA) (full dataset is found in ^13"16 ). Table 1 Maximum levels of mycotoxins allowed in foodstuff and animal feed in Europe.

Reducing the pH of BSG using an acid compound to no more than 4 pH units significantly inhibits the growth of total and aerobic mesophilic bacteria in BSG stored at 25°C. We find 10 ^{2 7} and 10 ² CFU total and mesophilic bacteria, respectively, per gram acidified BSG after 1 week storage at 25°C. More specifically, we find 10 ³ and 10 ^{2 1} CFU total and mesophilic bacteria, respectively, per gram of acidified BSG after 2 weeks storage at 25°C

Reducing the pH of BSG using an acid compound to no more than 4 pH units significantly inhibits the growth of mold in BSG stored at 25°C. We find 10 ² -10 ^{2 4} CFU/g acidified BSG after 1 week storage at 25°C, and more specifically, less than 10 ² CFU/g acidified BSG after two weeks storage at 25°C. The levels of mycotoxins monitored in acidified BSG after one week storage at 25°C are correspondingly low: DON, not detected (detection threshold (DT) = 20 μg/kg); NIV, not detected (DT = 20 μg/kg); ZEA, not detected (DT = 30 μg/kg) and; OTA, 0.6 μg/kg (DT = 0.5 μg/kg). Therefore, one object of this invention is to reduce the content of mycotoxins in brewers' spent grain (BSG) for animal or human consumption by minimizing the proliferation of mycotoxin- producing fungi by means of acidification of BSG.

In this invention, acidification of BSG results in a microbiologically stable BSG (MS-BSG) with the same protein, soluble fiber and insoluble fiber values as fresh BSG, with bacterial and fungal counts not higher than 10 ³ CFU/g after two weeks storage at 25°C, and with low or undetectable levels of mycotoxins after one week of storage at 25°C.

An additional object of this invention is to produce microbially stable BSG (MS-BSG) with organoleptical characteristics that are agreeable to consumers. In this invention, a mixture of acetic acid and lactic acid to a final concentration of 0.4% each are used as acidifying agents. Acetic acid is more volatile (vapor pressure = 15.8 mm Hg at 20°C) than lactic acid (0.0813 mm Hg at 20°C). Acetic acid has a lower detection threshold (200 mg/L in beer) than lactic acid (400 mg/L in beer). At high concentrations, acetic acid has a pungent, vinegar-like aroma. We examined the impact of acetic acid concentration in MS-BSG on consumer acceptance of downstream beverage product made with MS-BSG. MS-BSG acidified with various acetic acid concentrations was used to produce a base for a fermented beverage. MS-BSG was milled, its pH brought up to 6.1 pH units, treated with saccharification enzymes and fermented with lactic acid bacteria. We found that levels of acetic acid higher than 0.4% in the starting MS-BSG resulted in consumer rejection of the fermented MS-BSG beverage and that a beverage made with MS-BSG stabilized with 0.4% lactic acid and no more than 0.4% acetic acid was accepted by a consumer panel.

DETAILED DESCRIPTION OF THE INVENTION

Fresh brewers' spent grain (BSG) with a moisture content of 70% is retrieved from the wort filter or lauter tun. Fresh BSG is preferably processed no later than 8 hrs from release from the filter/lauter tun and are preferably collected by transferring the BSG from a mash separation unit to a collection tank by or through a BSG transfer line, wherein the acidification of the BSG is done during transfer of the BSG to the collection tank. Preferably, BSG is processed 'in-line' as it is conveyed from the filter/lauter tun to storage or transport vessels. The present invention concerns the process of chemical acidification of BSG by addition of one or a combination of acid compounds to reduce the pH of BSG to a level not higher than 4.1 pH units, more specifically 3.85-3.95 pH units. Specifically, the process makes use of one or a combination of organic acids such as, but not limited to lactic acid, acetic acid, citric acid, benzoic acid, malic acid, formic acid or ascorbic acid to reduce the pH of BSG to a level not higher than 4.1 pH units, more specifically 3.85-3.95 pH units. Even more specifically, the process makes use of 0.4% food-grade acetic and 0.4% food-grade lactic acid to reduce the pH of BSG to a level not higher than 4.1 pH units, more specifically 3.85-3.95 pH units and to obtain a sensorially agreeable product.

It is the object of this invention to provide microbiologically stable BSG (MS-BSG) which is characterized by: · a pH level not higher than 4.1 pH units, more specifically 3.85-3.95 pH units

the same nutritional value as fresh BSG

• after one week storage at 25°C, a colony count not higher than 10 ³ CFU/g MS-BSG total aerobic bacteria and; not higher than 10 ³ CFU/g MS-BSG fungi and; not higher than 10 ³

CFU/g MS-BSG yeast and; not higher than 10 ³ CFU/g MS-BSG mesophilic aerobic bacteria and; not higher than 10 ³ CFU/g MS-BSG total anaerobic bacteria

• after one week storage at 25°C, mycotoxin levels not higher than 3μg/kg Ochratoxin A

(OTA), preferably not higher than ^g/kg OTA, even more preferably undetectable levels of OTA and; not higher than 750 μg/kg deoxynivalenol (DON), preferably not higher than

20 μg/kg DON, even more preferably undetectable levels of DON and; 20 μg/kg nivalenol (NIV), more preferably undetectable levels of nivalenol and; not higher than 75 μ§/1¾ zearalenone (ZEA), preferably not higher than 30 μg/kg ZEA, and more preferably undetectable levels of ZEA

In one embodiment of this invention, fresh BSG is mixed with stock solutions of acetic acid and lactic acid, to a final concentration of 0.4% each, in storage vessels no later than 8 hrs after release from filters/lauter tun.

In the preferred embodiment of this invention, fresh BSG is mixed with stock solutions of acetic and lactic acid, to a final concentration of 0.4% each, 'in-line' as it is conveyed from filter/lauter tun to storage. This embodiment represents the most efficient application of the method here described.

The above described microbiologically stable brewers' spent grain (MS-BSG) can be used in the following applications:

• Animal feed. MS-BSG can be used as feed or feed complement for animals. More specifically, it can be used as feed or feed complement for ruminant cattle, such as dairy cows.

• As human food or food ingredient. MS-BSG can be used as an ingredient in the manufacture of foods such as breads, cookies, cereal products, baked snacks, extrusion cooked snacks or pasta products; and/or in the manufacture of food ingredients such as flours; and/or in the manufacture of dietary supplements such as fiber supplements.

In a further embodiment of this invention, MS-BSG is used as the starting material to produce a bacterially fermented beverage. MS-BSG is de-acidifed to an pH level optimal for the bacteria to grow and ferment. MS-BSG is de-acidified using food-grade calcium hydroxide to bring the pH up to 6.1-6.2 pH units. De-acidified MS-BSG is then used as a starting material for a beverage made by enzymatic digestion of MS-BSG fiber and bacterial fermentation of MS-BSG. The process for making such bacterially fermented beverage preferably comprises:

^■ Providing the microbiologically stabilized brewer's spent grain;

^■ De-acidification of the MS-BSG, preferably by addition of food-grade calcium hydroxide to achieve a pH of 6.1-6.2 pH units;

^■ Performing a saccharification by enzymatic treatment of the brewer's spent grain and a fermentation of the saccahrified brewer's spent grain with lactic acid bacteria and/or acetic acid bacteria and/or probiotics to obtain a fermented broth; and

^■ filtering the fermented broth and collecting the permeate to obtain the beverage or beverage component; or

^■ 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) that is subsequently stabilized by a method in accordance with the present invention.

For the saccharification the MS-BSG is mixed with water and exposed to saccharification and fermentation, preferably to a simultaneous process of saccharification and fermentation (SSF). Commercial enzymatic products used for the 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.

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 3

Example 4

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 S. cerevisiae).

Examples of lactic acid bacteria include:

L. fermentum AB15 Heterofermentative Sourdough

L. fermentum AB31 Heterofermentative Sourdough

L. fermentum F23 Heterofermentative Sourdough

L. gallinarum AB13 Homofermentative Sourdough

L. plantarum F6 Heterofermentative Sourdough

L. plantarum FIO Heterofermentative Brewery

L. plantarum F21 Heterofermentative Sourdough

L. plantarum ll Heterofermentative Cheese

L. plantarum R13 Heterofermentative Cheese

L. reuteri AB38 Heterofermentative Sourdough

L. reuteri DSM20016 Heterofermentative Human 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 lactis Ell Heterofermentative Sourdough

Leuc.

DSM20240 Heterofermentative Root beer

Mesenteroides

Weissella cibaria MG1 Heterofermentative Sourdough

Examples of acetic acid bacteria include 6. oxydans and K. xylinus.

Preferably, the strains L. planetarum FIO 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), 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 can follow two different subsequent processes, leading to two different types of beverages or beverage components:

1. Fermented broth can be filtered to produce a filtered beverage with the following nutritional claims: low energy, fat free, sugar free, high in protein, very low salt content.

■ The fermented base is swirled to re-suspend settled particles.

^■ Solid (insoluble) particles are allowed to settle, preferably by centrifugation.

^■ The resulting supernatant is filtered, preferably through mash filters. Further filtration steps are possible to reduce the size of particles in the final beverage.

2. Fermented broth can be 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.

^■ 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 filtering the fermented broth, a beverage or beverage component (type 1) can be obtained that is a low in energy (<20 kcal/100 g) and/or fat free (<0.5%) and/or sugar free (<0.5%) and/or high in protein (>12%, preferably >20% of the energy provided by proteins) and/or very low in salt content (<0.4%).

By homogenizing a beverage or beverage component (type 2) the fermented broth, a beverage or beverage 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 high in protein (>12%, preferably >20% of the energy provided by proteins) and/or very low in salt content (<0.4%).

Since no dairy product is used in the described process, the beverage or beverage component obtained 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 final beverage or beverage component obtained by the above process 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.

In a further embodiment of this invention, MS-BSG is used as the starting material for recovery of proteinaceous and/or fibrous material therefrom.

After fermentation, the pH of the fermentation broth is preferably adjusted to a pH in a range of 2.5 to 3.5, preferably to a pH of 2. 7 - preferably by additions of acids such as phosphoric acid and even more preferably by addition of strong acids such as sulfuric acid - allowing hydrolising the proteins in the fermentation broth by enzymatic treatment with eg. FP2 (Falcipain-2, a papain family cysteine protease).

Subsequently, proteinaceous material can be recovered (extracted, purified and/or separated) from the fermentation broth by for example an adsorption process. Such process may typically include three subsequent process steps. A first step in the protein recovery process is the separation of the solid particles. Typically, disc stack centrifuges, scroll decanters or hydrocyclones can be used for this purpose. A secondary solid removal step may be included to ensure that minimal quantities of particles are introduced to the equipment involved in subsequent protein purification steps. A failure to achieve this might imply a serious reduction in process outputs. Typical equipment used for secondary filtration might include filter bags or filter cartridges with a maximum pore diameter of 5, suitably 4, 3, 2 or 1 μηη. The insoluble solids containing stream from protein recovery steps 1 and 2 above can be dried. For the recovery of proteinaceous material, the purified liquid stream comprising the hydrolyzed protein components is fed to a primary protein concentration process that can be achieved by a chromatography step. Types of chromatography that can be utilised include adsorption matrices with properties such as ion exchange (I EX), size exclusion, affinity or any other appropriate type used in liquid chromatography systems. After the primary protein concentration step, a further step might be necessary in order to increase concentration and the purity of a particular protein or proteins of interest. For this step an additional chromatographic step may be included. An ultrafiltration/diafiltration or evaporation step can be used to concentrate the protein mixture further after the chromatography steps. The type of filter for ultrafiltration/diafiltration will depend on the physical and chemical properties of the desired protein or proteins. A suitable filter material will then have for example, hydrophilic or hydrophobic properties and a nominal molecular weight cut off between 3-1000 kDa. A final step in the overall process includes further concentration, specifically removal of water. Typical moisture content of protein powders are less than 20%. For this purpose, driers might be used that might include: cross- circulation and through-circulation driers, tray driers, tunnel driers, rotary driers, drum driers, spray driers and/or freeze drier. The protein depleted waste stream and optionally streams resulting from equilibration and regeneration of the adsorption matrix during used in the first or subsequent chromatography steps can enter waste water treatment systems. These streams are particularly suited to anaerobic digestion systems.

Examples of chromatography resins applicable in the first and further chromatography steps include, but are not limited to: Capto S (GE Healthcare) and Food-Grade Zeolite.

Elution of the proteinaceous material from the resins can be achieved by various eluents well known to persons skilled in the art and comprise, for example: NaCI solutions, NaHC03 solutions, Na2C03, NaOH, ...

The eluted proteinaceous material obtained by a method according to the present invention, is believed to have desired organoleptic properties and functionalities differing from proteinaceous material recovered from BSG without a step of fermenting the BSG. The proteinaceous material obtained by a method according to the present invention is believed to be particularly well suited for use as supplement (ingredient) for food and/or as foaming agent, emulsifying agent, egg or animal protein substitute in food recipes, diary protein substitute, baking ingredient, ...

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