The production of postbiotics from fermented pot ale and brewers spent grain

Abstract

A method of producing a postbiotic comprising: (a) obtaining liquid pot ale from whisky production in the absence of enzymes; (b) concentrating the pot ale by reverse osmosis with 0.0002 micron or les

Description

Field of the Invention The invention relates to methods of producing postbiotics from waste materials resulting from the production of whisky and to the postbiotics produced by these processes. Background to the Invention The microbial habitat within the animal / human gastrointestinal tract is the site of a complex and dynamic mutualistic relationship between the gut microbiota and the host. The human body provides a stable, nutrient-rich environment for those microorganisms, and in return, receives several benefits. These benefits include stimulation of the immune system, improved digestion and absorption of food, reduced growth of pathogenic flora, and maintenance of intestinal barrier integrity. The members of the gut microbiota also produce a wide range of compounds that can be used by both the host and by other microorganisms. This dynamic interaction between the host and its microbiota is essential for achieving and maintaining host homoeostasis and health. The composition and function of the gastrointestinal microbiota can be modulated in numerous ways. Prebiotics, probiotics and postbiotics can modulate the structure and metabolic activities of the microbial community. Prebiotics are defined as “a substrate that is selectively utilized by host microorganisms conferring a health benefit”. The following criteria are used to classify a compound as a prebiotic: (i) it should be resistant to acidic pH of stomach, cannot be hydrolyzed by mammalian enzymes, and also should not be absorbed in the gastrointestinal tract, (ii) it can be fermented by intestinal microbiota, and (iii) the growth and / or activity of the intestinal bacteria can be selectively stimulated by this compound and this process improves host’s health. Prebiotics have an established role in the promotion of human health due to their beneficial effects with a range of diseases including cardiovascular disease, gastrointestinal disorders, diabetes, colorectal cancer, obesity and allergies. The potential of prebiotics as dietary supplements in animals has been explored with regard to their ability to enhance growth performance and limit intestinal colonisation by animal and foodborne pathogens via their promotion of beneficial bacterial genera and their metabolic products in the GIT microbiota. Probiotics are “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”. Probiotics have numerous advantageous functions in humans and animals, with the most notable being their ability to prevent pathogens from adhering to the intestinal surface through the production of antimicrobial substances and competitive exclusion, as well as maintaining the epithelial barrier, and modulating the immune system. Health benefits have mainly been demonstrated for specific probiotic strains of the following genera: Lactobacillus, Bifidobacterium, Saccharomyces, Enterococcus, Streptococcus, Pediococcus, Bacillus and Escherichia coli. While probiotics are by definition alive and require an efficacious number of viable bacteria at the time of administration to the host, most probiotic preparations will include a large proportion of inanimate microbes, particularly towards the end of shelf life. Maintaining stability of live bacteria in foods is a challenge as many probiotic organisms are sensitive to certain food characteristics (acidity, water activity, specific chemical compounds) or storage conditions (moisture, temperature, package permeability to oxygen, time). Cell viability has long been regarded as the most important parameter for the efficacy and quality of probiotics to confer a health benefit. However, recently there has been increased interest in inanimate microorganisms, their cell components and metabolites and their potential health benefits. Regarding the use of these inanimate microorganisms and their metabolites, several different terminologies have been used over the years such as non-viable probiotics, heat-killed probiotics, cell lysates, paraprobiotics, inactivated probiotics and postbiotics. As of 2021, a definition of postbiotics was proposed by the International Scientific Association of Probiotics and Prebiotics (ISAPP), stating that a postbiotic is a “preparation of inanimate microorganisms and / or their components that confers a health benefit on the host”. In simple terms, postbiotics can be described as bioactive compounds produced during a fermentation process (including inactive microbial cells, cell constituents, and metabolites) that provide a health benefit. A microbe can only be regarded as a postbiotic if it is properly characterized, deliberately prepared with a reproducible method for inactivation, and shown to confer a health benefit. Thus, a probiotic microorganism that gradually loses cell viability in food does not gradually become a postbiotic at the end of its shelf life. The use of postbiotics is gaining increased attention because the administration of dead or inactivated microorganisms exerts many benefits over the use of live microorganisms. While probiotics have a range of health benefits, probiotic bacteria isolated from several commercially available dietary supplements have been shown to harbor resistance toward a range of antibiotics. There is also concern that these live bacteria may translocate from the gut lumen to the blood, thereby increasing susceptibility to bacteremia, especially in immunocompromised and vulnerable individuals. To address such issues, postbiotic components derived from probiotics may be a favourable and promising alternative. Postbiotics are often more stable and convenient to include in consumer products compared to probiotics. As postbiotics contain inanimate microorganisms, cell viability is not an issue. Postbiotics are generally stable at room temperature, and they can be applied to many product forms, offering formulators greater flexibility. This may also be of particular interest for consumers in geographical regions that do not have reliable cold storage or whose temperate climates causes problems for storage of live bacteria. Pot ale and brewers spent grain are nutrient rich co-products of whisky production which to date, have yet to be considered for use in the development of postbiotics. However, the use of these low-cost waste material as a fermentation media could offer great opportunity. The Scotch whisky definition prohibits the use of added enzymes. If enzymes are prohibited in whisky production, pot ale from such whisky production has a higher residual carbohydrate concentration compared to pot ale from other distilleries which use enzymes in manufacture. Fermenting pot ale in the presence of a culture of Lactic acid bacteria, for example, will provide a fermented product not only containing lactic acid bacteria, but also fermentation-derived metabolites that exert an array of health benefits for humans and animals. The production of single malt-based spirits, such as Scotch whisky is an important sector of the economy. The production of whisky varies depending on the type and proportion of the cereals used as raw materials and the country it originates. A characteristic feature of Scotch single malt whisky is that the only cereal used in its manufacture is malted barley. Scotch whisky production generates a significant amount of co-products. The main co-products from malt whisky are draff (brewers spent grain), the residual starch-depleted grains remaining after the mashing step, pot ale, the liquid residue from the first distillation step, and spent lees from subsequent distillations. An overview of the malt whisky production process including generation of co-products is illustrated in Figure 1. The co-products of the manufacture of malt whisky therefore comprise draff, pot ale and spent lees. The production of 1 L of alcohol yields approximately 2.5 kg of draff (brewers spent grain), 8 L of pot ale, and 10 L of spent lees (and washings). A large proportion of these co-products are used as fertiliser for animal feed, whilst the remainder requires disposal which incurs cost to the distillery along with the negative impact on the carbon footprint due to transportation. Brewers spent grain (BSG) is a rich source of fibre, protein and phenolic compounds, however due to its complex composition, high moisture content and fast deterioration, it is often underutilized as animal feed. Pot ale consists of yeast, yeast and barley residues, soluble protein and carbohydrate, and copper. A proportion of pot ale is fed to animals, mainly pigs, whilst the remainder is used as feedstock for anaerobic digestion or consensually disposed of on the land / sea which comes at a cost to the distillery. Large volumes of BSG and pot ale are generated by the brewing process all year round and at a low cost. These properties, together with its high nutritive value, have increased the exploration of alternative uses of BSG and pot ale which may encompass economic benefits. Pot ale and BSG are nutrient rich co-products of whisky production which to date, have yet to be considered for use in the development of postbiotics. However, the use of these low-cost waste materials as a fermentation media could offer great opportunity. Single strain and multi-strain postbiotics will be developed from these microorganisms which will have a range of applications in human and animals as outlined in Table 1. Raw pot ale is collected from distilleries and can be pre-treated with Carrageenan and the resulting material is used as a fermentation medium. During the fermentation, salts, surfactants and nitrogen sources are added and pH, temperature, inoculum and fermentation time is optimised to achieve high viable cell counts and growth. A complex mixture of metabolic products are secreted by bacteria during the fermentation such as enzymes, secreted proteins, short chain fatty acids, vitamins, secreted bacteriocins, amino acids, peptides, organic acids, etc. These microbial metabolites have a range of health promoting effects including anti-inflammatory, antibacterial, immunomodulator and antioxidant properties that enhance both the immune system and intestinal barrier functions by acting directly on specific tissues of the intestinal epithelium, but also on various organs / tissues. Table 1. Suitable microorganisms for use in the process and their applications Microorganisms Process Application Bacteria Lactic acid bacteria (Approximately 14 strains) Addition of salts, surfactants and nitrogen sources from products within the company to improve viable cell counts. Aswell as optimising pH, temperature, inoculum, fermentation time. -Silage additives -Postbiotic liquid product -Inoculum for BSG solid state fermentation -Mycotoxin binder -Methane mitigation Propionic bacteria cc -Silage additives -Postbiotic liquid product Corynebacterium glutamicum cc -Glutamate (umami) -Postbiotic liquid product -Other amino acids Fungus Aspergillus oryzae (KOJI) cc -Postbiotics -Enzymes (amylase, proteases etc.,) -Food applications Yeast Saccharomyces sp. cc - Postbiotic liquid product - Mycotoxin binder - Growth Ingredients Object of the Invention The object of the invention is to develop a process for the growth of microorganisms using pot ale from the whisky industry, particularly for whisky made without the use of enzymes. These microorganisms include approximately 14 EFSA registered lactic acid bacteria strains (available from Marigot Ltd), propionic bacteria, Corynebacterium glutamicum (C. glutamicum), Aspergillus oryzae (KOJI) and Saccharomyces sp. It is an object to develop single strain and multi-strain postbiotics these microorganisms which will have a range of applications in human and animals. A further object of the invention is to utilise waste products or by-products of other industries to produce value added products. A still further object of the invention is to provide novel postbiotics with a variety of advantageous properties such as antiinflammatory and immunomodulatory properties. Summary of the Invention According to the present invention there is provided a method of producing a postbiotic comprising the steps: (a) obtaining a liquid pot ale from the production of whisky in the absence of enzymes, (b) concentrating the pot ale by reverse osmosis using a membrane with a pore size of about 0.0002 micron or less to produce a fermentation medium, (c) inoculating the raw, concentrated pot ale from step (b) with one or more probiotic bacterial and / or yeast and / or fungal strains, (d) incubating the inoculated pot ale at a temperature which is appropriate to promote growth of the particular bacterial, yeast or fungal strains used in step (c), for between 15 and 30 hours, (e) harvesting the bacterial biomass from the product of step (d) and subjecting the biomass to heat treatment at temperatures of 80 - 90 °C for 90 - 120 minutes to kill the bacterial, yeast or fungal strains. Suitably the reverse osmosis membrane has a pore size of about 0.0001 micron or less. The liquid pot ale may be clarified before concentration in step (b) by mixing it with an aqueous solution of carrageenan. The aqueous solution of carrageenan ensures removal of any insoluble particles such as yeast cells and suspended solids from the pot ale. Alternatively the pot ale may be clarified by centrifugation. Preferably the heat-killed bacterial biomass is resuspended in cell-free fermented pot ale, which is then is sprayed onto mechanically or chemically processed seaweed to achieve the final product which contains a desired cell count of approximately 108CFU / g. The bacterial strains may be selected from homofermentative and heterofermentative lactic acid bacterial group Lactobacillus species, Paediococcus species. Aspergillus species, Saccharomyces species, Propionicbacterium species, Corynebacterium species and the Lactic acid bacterial strains (EFSA approved):-Lactobacillus case, Lactobacillus rhamnosus, Lactobacillus brevis - 2 strains, Paediococcus pentosaceus - 3 strains, Paediococcus acidilactici, Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus plantarum - 3 strains, Lactobacillus paracasei, Paediococcus parvulus, Lactobacillus buchneri - 2 strains, and Enetrococcus faecium. In some embodiments the postbiotic will be produced using one microbial strain and in others a mixture or cocktail of organisms will be used. Suitably the carrageenan powder is made up in a cold-water slurry just prior to adding to the hot pot ale. The pot ale may be introduced into a collection vessel, and the carrageenan may then be added. The action of filling the collection vessel by pump agitates and mixes the carrageenan together with the pot ale. The pot ale may then pumped into a whirlpool tank. Whirlpool tanks are specialized cylindrical vessels with flat bottoms where liquids are precisely swirled to precipitate solids and accumulate them at the base of the tank. The addition of carrageenan assists in removing protein fractions in the pot ale, by binding to them in solution to form aggregates that are precipitated most quickly by whirlpooling. The carrageenan treated pot ale is left in the tank until the material has fully precipitated and cooled. The pot ale is then subjected to reverse osmosis to concentrate the whirlpool material for use as a fermentation medium. Suitably the reverse osmosis step concentrates the material by a factor of 5 - 6. Preferably, one or more of magnesium (about 0.2 g / L) and manganese (about 0.04 g / L) salts, tween surfactants (about 1 g / L) and nitrogen sources, such as protein hydrolysates (about 1-5 g / L), are added to the fermentation medium to promote lactic acid bacterial growth. Also preferably the pH, temperature and fermentation time are preferably all optimised depending on the microbial species being grown, as is well known to one of skill in the art. In one particular embodiment seaweed fdter cake is added to the fermentation medium. Seaweed filter cake is one of the waste materials generated from the manufacture of bio stimulants such as seaweed extracts derived from the marine algae Ascophyllum nodusum. Supplementation with washed and dried SFC improves the cell titre in the fermentation step. Since the seaweed filter cake is a waste stream which typically contains polyphenols, the use of seaweed filter cake in the process allows the introduction of health promoting polyphenols into the final postbiotic product. The fermentation mixtures may be further supplemented with protein hydrolysates (1-5% w / v). Suitable protein hydrolysates used for supplementing the fermentation medium may be selected from chickpea, fava bean and lentil protein concentrates. In some embodiments the step (d) is performed as a primary or submerged fermentation. In other embodiments a secondary, solid-state fermentation is carried out on brewers spent grains, before the killing step (d). A liquid postbiotic product only requires primary / submerged fermentation whereas a solid postbiotic product will require both a primary / submerged and secondary / solid-state fermentation step. Preferably after the heat killing step, the heat killed cells may be mixed with additional products such as Aquamin (available from Marigot Ltd), or milled seaweed, or a human foodstuff, an animal feed or a pet food product. The invention also provides a postbiotic product whenever produced by a method as described above. Such postbiotic products have immunomodulatory activity, dampen inflammatory responses and induce innate immune training or memory. In particular the postbiotic products have immunomodulatory properties in that they can bring about an increase in IL10 levels and a decrease in TNFa levels. Brief Description of the Drawings Fig- 1- Malt whisky production and co-product generation. Fig- 2. Growth of L. rhamnosus on Pot ale containing media. Fig. 3 Effect of addition of SFC in pot ale concentrates on growth of L. rhamnosus. Fig. 4 Growth of different lactic acid bacterial strains on Pot ale medium Fig. 5. Growth of yeast on potale with / without additional nitrogen sources Fig. 6. Fermentation of whirlpool material (supplemented with Atura protein concentrates) by L. casei Fig- 7. Fermentation of pot ale concentrates (supplemented with protein hydrolysates and seaweed filter cake) by L casei Fig. 8. Growth of L rhamnosus on spent grain supplemented with salts / citric acid / skimmed milk powder Fig. 9. Growth of L.casei on spent grain supplemented with salts / citric acid / skimmed milk powder Fig. 10. Growth of L brevis on spent grain supplemented with salts / citric acid / skimmed milk powder Fig. 11 Growth of P. acidilactici on spent grain supplemented with salts / citric acid / skimmed milk powder Fig. 12. Fermentation of spent grain (supplemented with carbohydrate fractions (5%) from Atura proteins) by L casei Fig. 13. Fermentation of spent grain (supplemented with Atura protein (1%) concentrates) by L. casei Fig. 14. Fermentation of spent grain (supplemented with Atura protein (5%) concentrates) by L. casei Detailed Description of the Drawings Fermentation of pot ale Experiment 1: Use of pot ale concentrates as a nutrient source for Lactobacillus rhamnosus (L. rhamnosus) The aim of the experiment was to test pot ale concentrates as a sole nutrient source in L. rhamnosus fermentation, and to investigate the effect of addition of nitrogen and / or salts on pot ale concentrates in L. rhamnosus fermentation. In addition supplementation with another industrial by-product (wastes) was studied for its effect in L. Rhamnosus fermentation. Material and methods: The pot ale was sourced from Lochranza distillery and was treated with Carrageenan in an (intermediate bulk container) IBC tank. Intermediate bulk containers are industrialgrade containers engineered for the mass handling, transport, and storage of liquids, semi-solids, pastes, or solids. The Carrageenan treated pot ale was run through a whirlpool to precipitate insoluble particles. The supernatant (whirlpool material) was then passed through reverse osmosis to concentrate up to the desired 5X concentrate. Cultures were grown using shake flask methodology in 100 mL Scott Duran bottles containing 50 ml medium (pH 5-8) and incubated at 25 - 40°C for 20-30 h with a slow agitation (40-60 rpm). Four shake flask experiments were conducted to test pot ale concentrates as a sole nutrient source in lactic acid fermentation and to investigate the effect of addition of nitrogen and / or salts on pot ale concentrates in lactic acid fermentation. The four shake flask experiments were as follows: 1. Pot Ale concentrates (control) 2. Pot Ale concentrates + Magnesium and Manganese salts + Tween 80 3. Pot Ale concentrates + Basal salt media (salts, citric acid, buffers) 4. Pot Ale concentrates + protein hydrolysates Results: Bacterial growth was better when pot ale was combined with nitrogen sources. The addition of essential salts and nitrogen source had a positive impact on cell growth / count (Fig. 2). Experiment 2: Effect of addition of seaweed filter cake (SFC) in pot ale concentrates and its effect on the growth of L. rhamnosus The aim was to supplement pot ale medium with another industrial by-product (wastes) and study their effect in L. rhamnosus fermentation. Material and methods: The methodology was the same as in Experiment 1 but the four shake flask experiments were as follows: 1. Pot ale concentrates containing medium - control 2. 1% dried SFC in Pot Ale concentrates 3. 2% dried SFC in Pot Ale concentrates 4. 3% dried SFC in Pot Ale concentrates 5. 4% dried SFC in Pot Ale concentrates Results: Supplementing the pot ale medium with SFC had a positive effect on cell growth / count (Fig- 3) Experiment 3: Use of pot ale concentrates as a nutrient source for lactic acid bacteria The objective of this study was to determine whether distillery concentrates could be used to grow lactic acid bacterium. Materials and methods: The methodology was the generally the same as in Experiment 1 but the pot ale medium was maintained at pH 8.0. Inoculum developed on MRS broth and was added at 10% (v / v). Total viable counts (TVC); a serial dilution of fermented pot ale was carried out using 0.85% saline and 100 pl of culture / sample was spread on MRS agar plate. The bacterial cultures used were as follows: ■ Lactobacillus casei ■ Lactobacillus rhamnosus ■ Lactobacillus brevis ■ Paediococcus acidilactici Results: All LAB strains grew well on pot ale concentrates. The highest TVC was obtained with L. casei followed by L. rhamnosus, L. brevis and P. acidilactici (Fig. 4). Experiment 4: Use of pot ale concentrates as a nutrient source for yeast fermentation The objective of this study was to determine whether pot ale could be used for yeast fermentation. Material and methods: Cultures (Saccharomyces cerevisiae) were grown in Scott Duran bottle (100 ml) containing 50 ml pot ale medium (pH adjusted to 5.50) and incubated at 30°C for 24 h with a slow agitation (80 rpm). The inoculum was developed on malt extract broth and was added at 10% (v / v). Results: The highest TVC was obtained with the addition of yeast extract (3%), protein hydrolysates (3%) and yeast extract (1.5%) + protein hydrolysates (1.5%) as shown in Fig. 5. Experiment 5: Fermentation of pot ale (whirlpool material) supplemented with protein concentrates (from Marigot Ltd. - Atura proteins) by L. casei Material and Methods: The pot ale was sourced from Lagg distillery and was treated with Carrageenan in an IBC tank. The Carrageenan treated pot ale was run through a whirlpool to precipitate insoluble particles. The whirlpool fermentation was conducted with supplementation by chickpea and fava bean protein fractions (1.5 / 3%). Cultures were grown in Scott Duran bottle (100 ml) containing 50 ml medium (pH 8.0) supplemented with / without protein fractions and incubated at 37oC for 20-24 h with a slow agitation (100 rpm). The following shake flasks experiments were conducted: ■ Whirlpool material ■ Whirlpool material + Chickpea protein concentrates (1.5%) ■ Whirlpool material + Chickpea protein concentrates (3%) ■ Whirlpool material + Fava protein concentrates (1.5%) ■ Whirlpool material + Fava protein concentrates (3%) Total viable counts (TVC) were measured after 24 h. For TVC, a serial dilution was carried out using 0.85% saline and 100 pl of culture / sample was spread on MRS agar plate. Results: There was a significant increase (5- 6-fold) in the cell counts when whirlpool material was fermented with protein supplements (Fig. 6) as compared to control. There was not much difference in the cell counts yield between 1.5% or 3% protein concentrates supplemented cultures. Experiment 6: Use of pot ale concentrates as a growth medium for Aspergillus oryzae and enzyme production The purpose of this experiment is to see whether pot ale can be used as a medium to grow a filamentous fungus (Aspergillus oryzae) and produce industrially important enzymes. Material and methods: Aspergillus oryzae IM I 283862 maintained on potato dextrose agar (PDA) was used in this study. There were two stages involved in fungal fermentation. 1) Inoculum development A piece of (-1.5 cm) of fungal cultures from PDA plate was transferred into 50 ml of pot ale (concentrated (5X) using reverse osmosis) and the pH was adjusted to 5.5. Flasks were incubated at 30°C with an agitation (150 rpm) for 24 h. 2) Enzyme cocktails production Pot ale was supplemented with different inducers (chickpea, lentil, fava bean protein fractions) at 2% concentration and the pH was adjusted to 5.5. 10% (v / v) of inoculum developed on pot ale was used. Flasks were incubated at 30°C with an agitation (150 rpm) for 120 h. Fermented broth was centrifuged at 4000 rpm for 30 min and the supernatant was used for enzyme assays / concentration. Enzyme cocktails were then concentrated using 10 kDa MWCO PES membrane. The following parameters were used. Feed pump: 50 rpm Retentate pump: 40 rpm Retentate P2: 1.4 bar Initial volume: 550 ml Final volume: 58 ml Processing time: 12 h Concentration: - 9.5-fold Results: Excellent fungal growth was observed in all pot ale materials. It is concluded that any type of pot ale materials can be used to grow Aspergillus oryzae. Significant amounts of different protease activities were measured in concentrated enzyme cocktail (Table 2) Protease activity {U / ml} Enzyme U / mi pH 6.5(0.1 MMES) SD Commercial Neutrase (U / ml) Aspergillus cocktail neat (U / ml) Aspergillus cocktail concentrated (U / ml) Aspergillus cockteii permeate (U / mi) Freeze dried Eioprotease (U / mg) Commercial Alcalase {U / ml} Aspergillus cocktail neat {U / mf) Aspergillus cocktail concentrated (U / ml) Aspergillus cocktail permeate <U / mi ; freeze dried Bioprotease(U / mg) Commercial Savinase (U / mi) Aspergillus cocktail -teat (U / ml) Aspergillus cocktail concentrated (U / mi) Aspergillus cocktail permeate (U / mi) freeze dried BioprotessefU / mg) pH 10 26.18 2 15 9.55 189 16.26 0.82 9.36 2.05 0.0092 0.0016 pH 7.5(0.1 M Tris HCL) 27.31 2 31 10.08 1.7'i 17.47 0 44 0.0178 0.003 0.1 M sodium carbonate / sodium bicarbonate 40.36 0.87 ...... ...... 16.54 0 77 9.65 1.25 0.0318 0.0051 Endo Xylanase activity (U / ml) Xylanase standard control {2.4U / mlJ Aspergillus cocktail neat Aspergillus cocktail concentrated Aspergillus cocktail permeate pH4.5 {0.1M sodium acetate buffer) U / ml SD 2.32 0.29 0.13: 0.03 0.77 0.27 0.005 0.007 pH6 (0.1M sodium phosphate buffer) U / ml SO 2.34 0.26 0.14 0.036 0.97 0.41 0,005 0.006 Endo cellulase activity (U / ml) Cellulase standard control (3U / ml) Aspergillus cocktail neat Aspergillus cocktail concentrated Aspergillus cocktail permeate pH4.5 (0.1M sodium acetate buffer) U / mfi SO 2.81 0.26 0.0083 0.01 0.012 0.01 0.011 0.01 pH6 (0.1 M sodium phosphate buffer; 2.53 0.28 0.837 0.03 0.003 0.002 0.002 0.002 Betaglucanase activity pH 4.6 (0.04M sodium acetate and 0.04M sodium phosphate) U / mi SO Malt extract faetaglucanass standard n n Aspergillus cocktail neat Aspergillus cocktail concentrated Aspergillus cocktail permeate 0.002 0.0002 0.003 0.0006 0.001 0.0007 Table 2. Determination of enzyme activities in fungal enzyme cocktails Experiment 7: Use of pot ale concentrates as a nutrient source for Corynebacterium glutamicum The purpose of this experiment is to see whether we can use pot ale as a medium to grow Corynebacterium glutamicum NCIMB 10025 which will be used to produce glutamate (umami flavour) Material and methods: The pot ale was sourced from Lochranza distillery and was treated with Carrageenan in an IBC tank. The Carrageenan treated pot ale was run through a whirlpool to precipitate insoluble particles. The supernatant (whirlpool material) was then passed through reverse osmosis to concentrate up to the desired 5X concentrate. Cultures were grown in Scott Duran bottle (100 ml) containing 50 ml Pot Ale medium (pH adjusted to 5.50) and incubated at 30°C for 24 h with a slow agitation (80 rpm). The inoculum was developed on tryptic soy broth and was added at 10% (v / v). Results: Initial studies conducted with C. glutamicum NCIMB 10025 showed that this bacterium grows on Pot ale concentrates and cell counts were slightly better than standard TSB / NB media (Table 3). Nutrient Broth (Control) 24x10s mH 18 x 10« mH Tryptic soy broth (Control} 53 ± 5 x 10f- ml; 55 ± 2 x 10« mH NS - Loch Pot Ale TSB - Loch Pot Ale 143±4x1QsmH 160 ± 2 x 10s mH 64 ± 5 x 10^ mi- Table 3. Growth of C. glutamicum on Pot ale concentrates Experiment 8: Use of pot ale concentrates as a nutrient source for Propionibacterium acidipropionici The purpose of this experiment to see the growth of propionic acid producing bacteria on pot ale concentrates. Propionibacterium acidipropionici NCIMB 8070 was purchased from NCIMB. Bacterial cultures were revived using Krebs’ yeast lactate broth. KREBS’ YEAST LACTATE MEDIUM (KYL) Yeast extract 10.0 g KH2PO4 1.0 g Na2HPO4.2H2O 3.0 g Sodium lactate (70%) 40.0 ml Distilled water 960.0 ml Preparation: **Adjust pH to 7.0. Autoclave at 121°C for 15 minutes. After 48 h, broth cultures were streaked on KYL agar plates and incubated at 30°C in a sealed Anaerocult bags. Cultures were further confirmed for their purity. Growth of P. acidipropionici NCIMB 8070 on pot ale concentrates The pot ale was sourced from Lochranza distillery and was treated with Carrageenan in an IBC tank. The Carrageenan treated pot ale was run through a whirlpool to precipitate insoluble particles. The supernatant (whirlpool material) was then passed through reverse osmosis to concentrate up to the desired 5X concentrate. The pot ale concentrates (5X) was used a sole nutrient medium for growing Propionibacterium. The pH of pot ale was adjusted to 7.0 and then autoclaved. Experiments were conducted in a sterile airtight plastic container (150 ml). Inoculum developed on KYL broth (~36 h old) was used at 10% (v / v). Fermentation was stopped after 4 and 6 days of incubation. TVC and medium pH Total viable cell counts of 19 x 107 per ml was obtained after 6 days. The medium pH was dropped from 7 to 5.9. Volatile fatty acid (VFA) analysis Cell biomass was separated by centrifugation and the supernatant was acidified using ortho phosphoric acid. Samples were analyzed for VFA using GC- FID. Sample 1: Fermentation was stopped after 6 days Sample 2: Fermentation was stopped after 4 days Total VFA (volatile fatty acids) was quantified as ~16 g / L. Propionic acid and acetic acids were quantified as 5-5 - 5.9 g / L and 7- 8 g / L, respectively. In addition to major acids, other VFA’s were also quantified at low level (Table 4). 4 Days 6 Days Test Result Unit Result Unit Ascetic 7,01 g / iitre 8,81 R / itue hopswk g / liW Jsobvtytk aoi g / ihre «0.01 g / iitte Butyric 3,05 g / lftre 6,81 g / istrs <0.01 g / ihre 0,82 g / SUe Wette g / litre 0,84 8 / We 197 g / liW g / litre Ca prate iW 0 13 g / iitte <6.01 g / litte VWtiie Fatty Wife 16.3 0,10 g / iitre 166 gyiittc Table 4. VFA produced by Propionibacterium acidipropionici fermentation of pot ale after 4 and 6 days Experiment 9: Effect of addition of protein hydrolysates and seaweed filter cake in pot ale concentrates and its effects on the growth of L. casei Material and methods: The pot ale was sourced from Lochranza distillery and was treated with Carrageenan in an IBC tank. The Carrageenan treated pot ale was run through a whirlpool to precipitate insoluble particles. The supernatant (whirlpool material) was then passed through reverse osmosis to concentrate up to the desired 5X concentrate. The pot ale concentrate fermentation was conducted with supplemented protein hydrolysates and seaweed filter cake derived from dried seaweed. Cultures were grown in Scott Duran bottle (100 ml) containing 50 ml medium (pH 8.0) supplemented with protein hydrolysates and seaweed filter cake derived from dried seaweed and incubated at 37°C for 20-24 h with a slow agitation (100 rpm). Results: Supplementing the pot ale concentrate fermentations with protein hydrolysates and seaweed filter cake derived from dried seaweed increased the TVC. The highest TVC was obtained with the addition of protein hydrolysates + seaweed filter cake derived from dried seaweed, followed by the addition of protein hydrolysates alone (Fig 7). Fermentation of brewers spent grain Experiment 10: Fermentation of brewers spent grain (BSG) using Lactobacillus casei Material and methods: Distillers spent grain was received from Laag Arran Distilleries. The bacterium used in this study was Lactobacillus casei. There were two stages involved in BSG fermentation. 1. Primary / Submerged fermentation Pot Ale concentrates were used a sole nutrient medium. L. casei cultures were grown in Scott Duran bottle (100 ml) containing 50 ml of Pot ale concentrates with an initial pH 8.0 (adjusted) and fermentation was conducted at 37°C for 24 h with a slow agitation (100 rpm). 2. Secondary / solid state fermentation Secondary solid fermentation was conducted using sterilized fresh spent grain in a closed plastic container. Lactic acid bacterial cultures grown on Pot Ale concentrates (primary fermentation) was used as an inoculum at 10% (v / w) and fermentation was conducted at 37°C for 24 h. Results: Total viable cell counts were measured after 24 h in the fermented spent grain and the TVC counts were in the range of 5-10 x 107 g BSG. Fermented BSG was dried in an oven at 100°C for ~36 h. In terms of taste, all fermented BSG are palatable. Experiment 11: Fermentation of BSG using different lactic acid bacterial strains Bacterial cultures used: ■ Lactobacillus casei ■ Lactobacillus rhamnosus ■ Lactobacillus brevis ■ Paediococcus acidilactici Material and methods: There are two stages involved in spent grain fermentation. 1. Primary / Submerged fermentation Pot Ale concentrates (5X, RO) was used a sole nutrient medium. Lactic acid bacterial cultures were grown in Scott Duran bottle (100 ml) containing 50 ml of Pot ale concentrates with an initial pH 8.0 / 6.5 (adjusted) and fermentation was carried out at 37oC for 24 h with a slow agitation (80 rpm). 2. Secondary / solid state fermentation Secondary solid fermentation was carried out using sterilized fresh spent grain. Lactic acid bacterial cultures grown on Pot Ale concentrates (primary fermentation) was used as an inoculum at 10% (v / w) and fermentation was carried out at 37oC for 24 or 48 h. To see the effect of addition of salts / citric acid / skimmed milk powder on cell titre and flavour enhancement, the following experiments were conducted. ■ Spent grain ■ Spent grain + Salts ■ Spent grain + Citric acid (1%) ■ Spent grain + Citric acid (1%) + Salts ■ Spent grain + Skimmed milk powder (1%) ■ Spent grain + Skimmed milk powder (1%) + Salts Total viable cell counts were measured after 24 h in the fermented spent grain and the results are discussed individually below for each strain. Results: Spent grain fermented by L. Casei L. casei showed an excellent growth on spent grain. Highest TVC (97 ± 2 x 107 L1) was obtained on spent grain supplemented with skimmed milk powder with salts (Fig. 8). Cell counts in spent grain fermented in the presence of salts were slightly higher than control. Cell counts were lower in citric acid added samples (7 - 25 x 105 g'1). Spent grain fermented by L. rhamnosus L. rhamnosus grew well on spent grain. TVC trend was quite similar to L. casei, TVC count was high in fermented spent grain with skimmed milk (60 ± 9 x 107 g’1). However, TVC titre in citric acid added fermented sample was higher than L. casei. There was no significant difference in TVC between control and salts added samples (Fig. 9). Spent grain fermented by L. brevis Growth performance of L. brevis on spent grain was completely different from other LAB strains. TVC counts were higher in spent grain supplemented with only salts (37 ± 2 x 107 g-1) followed by skimmed milk powder (25 ± 2 x 107 g-1). Interestingly, significant counts were observed on spent grain with citric acid added sample also (Fig. 10). Spent grain fermented by Pediococcus acidilactici P. acidilactici showed good growth on spent grain. TVC counts (15 ± 3 x 107 g4) were slightly higher in spent grain supplemented with skimmed milk powder than control (Fig. 11). TVC counts were lower in spent grain fermented with citric acid. This is in confirmatory with other LAB strains. Experiment 12: Fermentation of BSG supplemented with carbohydrate fractions (from Marigot Ltd - ATURA proteins) by L.casei Material and methods: Spent grain fermentation was carried out with supplementation of carbohydrate fractions (1%). The following experiments were carried out and TVC counts was measured after 24 h. ■ Spent grain + Salts ■ Spent grain + Lentil carbohydrate fraction (1%) ■ Spent grain + Fava carbohydrate fraction (1%) ■ Spent grain + Chickpea carbohydrate fraction (1%) Results: There was a significant increase in the cell counts in fermented BSG samples supplemented with 1% lentil, fava and chickpea carbohydrate fractions (Fig. 12) as compared to BSG with salts. Experiment 13: Fermentation of BSG supplemented with 1% protein concentrates (from Marigot Ltd - ATURA proteins) by L. casei Material and methods: Spent grain fermentation was carried out with supplementation of protein concentrates (1%). The following experiments were carried out and TVC counts was measured after 24 h. ■ Spent grain ■ Spent grain + Salts ■ Spent grain + Chickpea protein (1%) ■ Spent grain + Fava protein (1%) ■ Spent grain + Lentil protein (1%) ■ Spent grain + Umami (1%) ■ Spent grain + Chickpea + Fava + Lentil + Umami (0.25% each) Results: There was a significant increase in the cell counts in fermented BSG samples supplemented with 1% lentil, fava, chickpea and umami protein concentrates (Fig. 13) as compared to control. Experiment 14: Fermentation of BSG supplemented with 5% protein concentrates (from Marigot Ltd - ATURA proteins) by L. Casei Material and methods: Spent grain fermentation was carried out with supplementation of protein concentrates (5%). The following experiments were carried out and TVC counts was measured after 24 h. ■ Spent grain ■ Spent grain + Salts ■ Spent grain + Chickpea protein concentrates (5%) ■ Spent grain + Fava protein concentrates (5%) ■ Spent grain + Lentil protein concentrates (5%) ■ Spent grain + Umami isolate (5%) Results: There was a significant increase in the cell counts in fermented BSG samples supplemented with 5% chickpea protein concentrates (Fig. 14) as compared to control. Experiment 15: The effect of postbiotics on immune modulation (in vitro study); Materials and Methods The immunomodulatory and anti-inflamatory properties of the following postbiotics were determined :- Raw pot ale (Yeast postbiotic); Pot ale fermented with Lactobacillus casei (Lactic acid bacteria postbiotic), and Pot ale fermented with Aspergillus oryzae (Fungus postbiotic). Cell toxicity studies were carried out using standard methods. Raw blue mouse line wasused as a reporter cell line for Quanti-blue assay to measure TFN-alpha and IL10. Results: These postbiotics showed the ability to increase levels of IL 10 and decrease levels of TFNa in vitro. The words “comprises / comprising” and the words “having / including” when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. 31 07 23

Claims

1. A method of producing a postbiotic comprising the steps:(a) obtaining a liquid pot ale from the production of whisky in the absence of enzymes,(b) concentrating the pot ale by reverse osmosis using a membrane with a pore size of about 0.0002 micron or less to produce a fermentation medium,(c) inoculating the raw, concentrated pot ale from step (b) with one or more probiotic bacterial and / or yeast and / or fungal strains,(d) incubating the inoculated pot ale at a temperature which is appropriate to promote growth of the particular bacterial, yeast or fungal strains used in step (c), (e) raising the temperature of the incubated pot ale of step (d) to a temperature sufficient to kill the bacterial, yeast or fungal strains.

2. A method as claimed in claim 1 wherein the reverse osmosis membrane has a pore size of about 0.0001 micron or less.

3. A method as claimed in claim 1 or 2 wherein the liquid pot ale is clarified before concentration in step (b) by mixing it with an aqueous solution of carrageenan.

4. A method as claimed in claim 3 wherein the carrageenan powder is made up in a cold-water slurry just prior to adding to hot pot ale.

5. A method as claimed in claim 1 or 2 wherein the pot ale is clarified by centrifugation.

6. A method as claimed in any preceding claim wherein the bacterial, yeast or fungal strains are selected from homofermentative and heterofermentative lactic acid bacterial group Lactobacillus species, Paediococcus species. Aspergillus species, Saccharomyces species, Propionicbacterium species, Corynebacterium species and the Lactic acid bacterial strains (EFSA approved):-Lactobacillus case, Lactobacillus rhamnosus, Lactobacillus brevis - 2 strains, Paediococcuspentosaceus - 3 strains, Paediococcus acidilactici, Lactobacillus rhamnosus, Lactobacillus fermentum,31 07 23Lactobacillus plantarum - 3 strains, Lactobacillus paracasei, Paediococcus parvulus, Lactobacillus buchneri - 2 strains, and Enetrococcus faecium.

7. A method as claimed in any preceding claim wherein the mixture is subjected to whirlpooling to clarify the pot ale.

8. A method as claimed in claim 7 wherein the whirlpooled material is then subjected to reverse osmosis to concentrate for use as a fermentation medium.

9. A method as claimed in any preceding claim wherein one or more of salts, surfactants and nitrogen sources are added to the fermentation medium.

10. A method as claimed in any preceding claim wherein seaweed filter cake is added to the fermentation medium.

11. A method as claimed in any preceding claim wherein the fermentation mixture is further supplemented with yeast extract, and / or protein hydrolysates.

12. A method as claimed in claim 10 wherein the protein hydrolysate is selected from chickpea, fava bean and lentil protein concentrates.

13. A method as claimed in any preceding claim wherein the step (d) is performed as a primary or submerged fermentation.

14. A method as claimed in any preceding claim wherein further comprising a secondary, solid-state fermentation carried out on brewers spent grains, before the killing step (e).

15. A method as claimed in any preceding claim wherein after the heat killing step, the heat killed cells are mixed with additional products such as Aquamin, milled seaweed, a foodstuff, an animal feed or a pet food product.

16. A postbiotic product whenever produced by a method as claimed in any preceding claim.

17. A postbiotic product as claimed in claim 16 having immunomodulatory properties such that they ring about an increase in IL 10 levels and a decrease in TNFa levels31 07 23

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