Microencapsulated microbial culture using protein-carbohydrate and chitosan complex coacervate polymer

Abstract

The present invention relates to microencapsulated microbial cultures with high storage stability and method of preparation thereof. It particularly relates to probiotic microencapsulation using protein-oligosaccharide conjugate and chitosan to form a complex insoluble coacervates polymer for enhancing storage stability of microbial cultures during freeze drying and storage.

Description

[0001] MICROENCAPSULATED MICROBIAL CULTURE USING PROTEIN-CARBOHYDRATE

[0002] AND CHITOSAN COMPLEX COACERVATE POLYMER

[0003] Technical field of the invention

[0004] The present invention relates to microencapsulated microbial cultures with high storage stability and methods for producing them. In particular, the present invention relates to microbial cultures formulated in complex coacervates, wherein the complex coacervate is formed by an electrostatic interaction of a protein-carbohydrate conjugate with chitosan.

[0005] Background of the invention

[0006] In human and animal bodies, microbial cultures, such as lactic acid bacteria (LAB), are the part of normal microbiota. LAB are mostly used as starter cultures in fermented dairy foods and beverages as they can help to improve the nutritional and organoleptic characteristics, as well as extend the shelf life. Some strains of LAB have been reported to exhibit health benefits to human and animals and may thereby be referred to as probiotic strains. The typical process for the production of LAB is through fermentation followed by concentration and freezing of cell biomass. When it comes to applications of LAB, dried powder form produced using freeze drying (FD) is often desired. The dried powders are frequently kept for extended times before utilized in a final application.

[0007] Microbial cultures, such as LAB, are very sensitive to different environmental stresses applied during freezing and FD, causing the addition of cryo / lyo protectants to be necessary for protecting and retaining viability during processing. Moreover, it is well-known that storage of microbial cultures, such as LAB, at ambient (25-35°C) or higher temperature adversely affect the viability of the microbial cultures. Therefore, storage for an extended period of time necessitates expensive cooling facilitates that are not always available at the point of use.

[0008] While there are several concepts on cryo / lyo protectants to shield the LAB during freezing and FD, all of these protectants (ingredients) have limited impact on the storage stability. Therefore, there is an unmet need for methods of protecting microbial cultures during freezing and FD and which at the same time enhance the storage stability of these dried microbial cultures.

[0009] Hence, it would be advantageous to provide an improved method for preparing dried microbial cultures that maintain viability after freezing or freeze drying and over extended periods of storage even at elevated temperatures. Specifically, such methods and dry microbial cultures per se may be advantageous in the preparation of products which are exposed to conditions of increased environmental stress.

[0010] SUMMARY OF THE INVENTION

[0011] With this background it is an object of the invention to provide microencapsulated microbial cultures having first coacervate and second coacervate polymer reacted electrostatically.

[0012] In particular, the present invention discloses methods for producing microencapsulated microbial cultures that endure dry processing and exhibit enhanced storage stability upon storage at even 37°C for extended period of time.

[0013] Further object of the invention is to provide microencapsulated microbial culture comprising of; a microbial culture, a first matrix, a second matrix; wherein the first coacervate component is a protein- carbohydrate conjugate and second-coacervate component is a polysaccharide

[0014] Even further object of the invention provides microencapsulated microbial cultures using complex coacervates of Whey Protein Concentrate-Galacto-oligosaccharide conjugate (WPC-GOS conjugate) and chitosan. The method of producing these microencapsulated microbial cultures relies on complex coacervation in which sequential addition of coacervation components results in the electrostatic formation of a protective complex to shield the entrapped microbial culture. The obtained microencapsulated microbial cultures are well suited for applications in which storage at depressed temperatures is not feasible.

[0015] Thus, an object of the present invention relates to the provision of methods for preparing a microbial culture that may be utilized under conditions independent of refrigerated storage.

[0016] In particular, it is an object of the present invention to provide an improved method for production of dry microbial cultures that retain cell viability after dry processing and storage at elevated temperatures.

[0017] In a first aspect, this and further objectives are achieved with microencapsulated microbial culture, said microencapsulated microbial culture comprising; i. a microbial culture, ii. a first matrix comprising a first coacervate component, and, iii. a second matrix comprising a second coacervate component, wherein said first matrix component is a protein-carbohydrate conjugate of Whey Protein Concentrate (WPC) and Galacto-oligosaccharide (GOS) and the second matrix component is a polysaccharide like chitosan.

[0018] In yet another aspect of the present invention, the microbial culture is a bacterium or a yeast. The microbial culture may be, or comprise, a lactic acid bacteria (LAB) of a genus selected from the group consisting of Lactobacillus, Holzapfelia, Amylolactobacillus, Bifidobacterium, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquor ilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus,Apilactobacillus, Levilactobacillus, Secundilactobacillus, Lentilactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Enterococcus, Brevibacterium, and Staphylococcus. The microbial culture may be a probiotic culture.

[0019] In one preferred embodiment, the microencapsulated microbial culture is selected from the group consisting of Ligilactobacillus animalis deposited as DSM 33570 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Bifidobacterium animalis subsp. lactis deposited as DSM 15954 at DSMZ, Streptococcus thermophilus deposited as DSM 15957 at DSMZ and Lactococcus lactis subsp. \actis deposited as DSM 21404 at DSMZ.

[0020] In one embodiment of the microencapsulated microbial culture, the ratio (wt% / wt%) of WPC- GOS conjugate to Chitosan is between 50:50 to 98:2, preferably between 75:25 to 95:5.

[0021] In one embodiment of the microencapsulated microbial culture, the ratio (wt% / wt%) of the first and second matrix combined to microbial culture is between 0.25-10.

[0022] In one embodiment, the first or second matrix may further comprise an additive like antioxidant, such as trisodium citrate, glutathione, ascorbates or derivatives thereof.

[0023] Another aspect of the present invention relates to the microencapsulated microbial culture as described herein, wherein the microencapsulation may further comprise one or more additives selected from the group consisting of food-grade ingredients, pharmaceutical ingredients and excipients.

[0024] A further aspect of the present invention relates to a product comprising the microencapsulated microbial culture, wherein the product is selected from the group consisting of a feed, a plant health product, a food, a beverage and a pharmaceutical product.

[0025] An aspect of the present invention relates to a method for preparing a microencapsulated microbial culture or composition as described herein, said method comprising the steps of; i) mixing a microbial culture with a first matrix comprising a first coacervate components and optionally one or more antioxidants to form a pre-complex solution, and ii) mixing the pre-complex solution with a second matrix comprising a second coacervate component to form a microencapsulated microbial culture, wherein said first coacervate component is a WPC-GOS conjugate and second coacervate component is chitosan. Said first coacervate components are Whey protein concentrate (WPC) and Galacto-oligosaccharide (GOS) that form a first matrix using a Whey Protein Concentrate and Galacto-oligosaccharide conjugate and second coacervate component of second matrix is chitosan.

[0026] Further, the method may comprise the following steps of i) mixing microbial culture with the first matrix comprising first coacervate component for a time period in the range of 5 min to 6 hours, at a temperature in the range of 4°C to 45°C, to form the precomplex solution, and in step ii) mixing the pre-complex solution with the second matrix comprising chitosan for a time period in the range of 5 min to 6 hours, at a temperature in the range of 4°C to 45°C.

[0027] In one embodiment, the microbial culture of step i) of the method according as above is mixed with the first matrix for a time period in the range of 5 min to 6 hours, such as 2 hours at a temperature in the range of 4°C to 45°C, such as ambient temperature or 25°C, and / or wherein mixing of the precomplex solution with the second matrix in step ii) is carried out for a time period in the range of 5 min to 6 hours, such as 2 hours at a temperature in the range of 4°C to 45°C such as ambient temperature or 25°C.

[0028] In one embodiment, the pH in step ii) of the method according to the above aspects of is in the range between 3-10, 3-8, 4-8 preferably 5-7, more preferably between 5.5-7.5.

[0029] An aspect of the present invention relates to a method for preparing a first matrix comprising a first coacervate components, said method comprising the steps of: a) preparing individual solutions of first coacervate components at desired pH and hydrating them overnight, b) mixing the overnight hydrated individual solutions of first coacervate components at desired pH in desired proportion and heat incubating to form the conjugate.

[0030] In further aspect the pH in step b) of the method according to the above aspect may be in the range of 7-10. In some embodiments the pH is adjusted to 7, 8, 9 or 10, where pH 9 may be preferred.

[0031] In one embodiment, the method according to the above aspect further comprise a step iii) subsequent to step ii), wherein step iii) comprises freezing said microencapsulated microbial culture to obtain a frozen microencapsulated microbial culture. Further, a step iv) subsequent to step iii), wherein step iv) comprises sublimating water from said frozen microencapsulated microbial culture to obtain a dried microencapsulated microbial culture.

[0032] Yet another aspect of the present invention relates to a microencapsulated microbial culture or a composition obtainable by a method as described herein. Still another aspect of the present invention relates to use of a microencapsulated microbial culture, or a composition as described herein in a product selected from the group consisting of a feed, a plant health product, a food, a beverage and a pharmaceutical product.

[0033] BRIEF DESCRIPTION OF THE FIGURES

[0034] Fig. 1 Pictures of heat-treated WPC, WPC-GOS and GOS solutions.

[0035] Fig. 2 UV Spectrum of heat-treated WPC, WPC-GOS and GOS solutions.

[0036] Fig. 3 % Total protein and carbohydrate contents of WPC, WPC-GOS and GOS solutions.

[0037] Fig. 4 Turbidity curve of Conjugate-Chi tosan complex during titration while changing pH from 3 to 8, pH max 1 and pH max 2 are the insoluble complex formation events of Conjugate-Chitosan complex formed at various biopolymer ratios and different pH.

[0038] Fig. 5 Microscopic images of a) Conjugate-Chitosan complex coacervates; b) Bacteria; c) Bacteria with conjugates and d) Bacteria microencapsulated in Conjugate-Chitosan complex coacervates.

[0039] Fig. 5a shows the insoluble complex coacervates of Conjugates and chitosan without bacteria produced at optimal pH.

[0040] Fig. 5b shows the microscopic image of rod shaped LA51 (microbial cells / bacteria / probiotic) used in the present invention.

[0041] Fig. 5c shows the microbial cells uniformly distributed in the WPC-GOS conjugate.

[0042] Fig. 5d depicts the microbial cells / LA51 entrapped into the insoluble complex coacervates of Conjugates and chitosan and the aggregation observed, therefore.

[0043] Fig. 6 depicts comparison of Conjugate-Chitosan complex with WPC, GOS, Conjugate and chitosan containing sodium ascorbate on the viability of (microbial cells / 'Lactobacillus animalis LA51) in the freeze dried granulates of conjugate-chitosan complex.

[0044] Fig. 7 Comparison of viability of LA51 microencapsulated in WPC-GOS conjugate and only Cells, only WPC and only GOS after 12 weeks of storage at aw< 0.10, T=37°C.

[0045] Fig. 8 Comparison of viability of LA51 microencapsulated in Conjugate-Chitosan complex at varying proportions along with only Cells, only Chitosan, and only WPC-GOS conjugate without chitosan after 12 weeks of storage at aw< 0.10, T=37°C.

[0046] The present invention will in the following be described in more detail. DETAILED DESCRIPTION OF THE INVENTION

[0047] Definitions

[0048] Prior to outlining the present invention in more details, a set of terms and conventions is first defined:

[0049] Microbial culture

[0050] In the present context, the term “microbial culture” refers to a population of microorganisms. Microorganisms include all unicellular organisms, such as archaea and bacteria, but also many multicellular organisms, such as fungi and algae.

[0051] Probiotic culture

[0052] In the present context, the terms "probiotic" or “probiotic culture” refers to microbial cultures which, when ingested in the form of viable cells by humans or animals, confer an improved health condition, e.g. by suppressing harmful microorganisms in the gastrointestinal tract, by enhancing the immune system or by contributing to the digestion of nutrients. Probiotics may also be administered to plants. Probiotic cultures may comprise bacteria and / or fungi.

[0053] Lactic acid bacteria (LAB)

[0054] In the present context, the term “lactic acid bacteria (LAB)” refers to a group of Gram positive, catalase negative, non-motile, microaerophilic or anaerobic bacteria that ferment sugar with the production of acids including lactic acid as the predominantly produced acid, acetic acid, formic acid and propionic acid. The industrially most useful lactic acid bacteria include, but are not limited to, Lactococcus species (spp.), Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Brevibacterium spp, Enterococcus spp. and Propionibacterium spp. Additionally, lactic acid producing bacteria belonging to the group of the strict anaerobic bacteria, Bifidobacteria, i.e. Bifidobacterium spp. which are frequently used as food starter cultures alone or in combination with lactic acid bacteria, are generally included in the group of lactic acid bacteria. Even certain bacteria of the genus Staphylococcus (e.g. S. carnosus, S. equorum, S. sciuri, S. vitulinus and S. xylosus) have been referred to as LAB (Seifert & Mogensen (2002).

[0055] Viability

[0056] In the present context, the term “viability” refers to living cells in a culture. Thus, the viability of a cell culture may be determined by measuring the number of colony forming units (CFU). CFU refer to the number of individual colonies of any microbe that grow on a plate of media. This value in turn represents the number of bacteria or fungi capable of replicating as they have formed colonies on the plate. In brief, the CFU / g can be determined as follows; A known amount of sample (e.g. freeze dried) is homogenized with a specific volume of diluent (1 :100), using a stomacher, the solution is then resuspended by using a vortex mixer and is then subjected to decimal dilutions in peptone saline diluent (also referred to as ‘maximum recovery diluent (MRD)’). MRD comprises peptone, NaCl and demineralized water.

[0057] Dilutions are poured on the plates, mixed with MRS Agar (Hi -media, M641) and incubated. After incubation, colonies are counted manually.

[0058] Particularly, stability of a sample is assessed by counting the colony -forming units (CFU) per gram, using the following assay. Viable cell counts are determined in freeze-dried granulates sampled immediately after freeze-drying and at selected time points during the stability studies. A standard pour-plating method is used. The freeze-dried material is suspended in sterile peptone saline diluent (BD Difco™ Lactobacilli MRS Agar, Fisher Scientific) and homogenized by stomaching using stomacher (bioMerieux, Inc. Durham, NC). After 30 minutes of revitalization, stomaching is repeated and the cell suspension is serially diluted in peptone saline diluent. For the cfu of Bifidobacterium animalis sv .lactis deposited as DSM 15954 (BB-12™), the dilutions are plated in duplicates on MRS agar (BD Difco™ Lactobacilli MRS Agar, Fisher Scientific) supplemented with 0.5g / L of L-cysteine hydrochloride (Sigma- Aldrich, Inc.). The agar plates are incubated anaerobically for three days at 37°C. For the cfu of Ligilactobacillus animalis LA51, deposited as DSM 33570, the dilutions are plated in duplicates on MRS agar (BD Difco™ Lactobacilli MRS Agar, Fisher Scientific). The agar plates are incubated anaerobically for three days at 37°C. In case of Streptococcus thermophilus TH4 (DSM 15957) cfu, the dilutions are plated in duplicates on Ml 7 agar (BD Difco™ Lactobacilli MRS Agar, Fisher Scientific) supplemented with 0.5g / L of monosodium phosphate (Sigma-Aldrich, Inc.) and 0.5g / L of disodium phosphate (Sigma-Aldrich, Inc.). The agar plates are incubated aerobically for three days at 37°C. For the cfu of Lactococcus lactis subsp. lactis R607 (DSM 21404), the dilutions are plated in duplicates on M17 agar (BD Difco™ Lactobacilli MRS Agar, Fisher Scientific) supplemented with 0.5g / L of monosodium phosphate (Sigma-Aldrich, Inc.) and 0.5g / L of disodium phosphate (Sigma-Aldrich, Inc.). The agar plates are incubated aerobically for three days at 37°C. Plates with 30 - 300 colonies are chosen for counting of colony forming units (CFU). The result is reported as average CFU / g freeze-dried sample, calculated from the duplicates.

[0059] Microencapsulated

[0060] In the present context, the term “microencapsulated” refers to an entity, which on a micrometric scale are secluded from the surrounding environment. Thus, a microencapsulated microbial culture is a microbial culture which are compartmentalized into distinct entities separated from each other and the medium into which they are dispersed. Complex coacervate

[0061] In the present context, the term “complex coacervate” refers to an aqueous phase (or droplet) rich in the microbial culture that is formed upon complex coacervation using two or more biopolymers of opposite charge. The complex coacervate forms due to liquid-liquid phase separation and is a dense phase that exist in equilibrium with a dilute phase. Thus, the complex coacervate may be characterized as a lyophilic colloid.

[0062] The method of complex coacervation involves the mixing of an entity to be encapsulated, such as a microbial culture, with at least two biopolymers of opposite charge. Herein, the biopolymers of opposite charge are referred to as coacervate components and comprised in a first and second matrix, respectively.

[0063] Matrix

[0064] In the present context matrix refers to biopolymers of opposite charges that are interacted to form coacervates upon complexation at appropriate temperature and pH. In addition to the biopolymers, the matrix may also be comprised of food-grade ingredients such as monosaccharides or disaccharides, and antioxidants etc. Optionally other components may also be added to the matrix such as excipients or pharmaceutical ingredient. In present context, the matrix embeds the active ingredient (probiotic cells) and secludes the bacterial cells from the surrounding environment on a micrometric scale.

[0065] Chitosan

[0066] In the present context, the term “chitosan” refers to a linear polysaccharide composed of randomly distributed -(1 4)-linked D-glucosamine (deacetylated unit) and N- acetyl-D-glucosamine (acetylated unit). Chitosan is produced by deacetylation of chitin and the degree of acetylation ranges from 60% to 100%. Thus, the term chitosan as used herein includes variants with different degree of deacetylation. Moreover, the term chitosan encompasses chitosan with a molecular weight in the range of 3 kDa to 20 kDa.

[0067] Whey Protein Concentrate (WPC)

[0068] Whey protein concentrate in the concentrated dry whey protein powder as procured from standard vendor. The WPC comprises globular proteins such as alpha-lactalbumin, beta-lactoglobulin, serum albumin and immunoglobulins. Additionally, it may contain glycomacropeptide. The WPC may contain typically between 29%-89% protein by dry weight, and traces of fat and lactose. Galactose-Oligosaccharide (GOS)

[0069] Galacto-oligosaccharides are oligosaccharides composed of galactosyl residues and a terminal glucose linked by P-glycosidic bonds, such as P(1 —> 2), P(1 —> 3), P(1 —> 4), or P(1 —> 6) depending on the enzyme origin, these are used as procured from standard vendor. GOS are produced through the enzymatic conversion of lactose. Depending on the enzyme used and the degree of conversion of lactose, the degree of polymerization of GOS can vary from 2 to 8 monomeric units.

[0070] Antioxidant

[0071] In the present context, the term “antioxidant” refers to a compound that inhibit oxidation. The antioxidant may be industrial chemicals or natural compounds. As used herein, antioxidants include, but are not limited to, trisodium citrate, vitamin C, vitamin E, glutathione, ascorbates and derivatives thereof.

[0072] It is to be understood that antioxidants as used herein also include mineral salts of vitamin C, such as sodium ascorbate.

[0073] Food-grade ingredient

[0074] In the present context, the term “food-grade ingredient” refers to any compound that is non-toxic and safe for consumption and comply with the Food Chemicals Codex (FCC). Food-grade ingredients include, but are not limited to, compounds that can alter attributes such as aroma, flavour, acidity, colour, viscosity and texture, as well as preservatives, nutrients, thickeners, sweeteners, and emulsifiers.

[0075] Preferred food-grade ingredients include, but are not limited to, lactose, maltodextrin, whey protein, casein, corn starch, dietary fibres, gums and gelatine.

[0076] Pharmaceutical ingredient

[0077] In the present context, the term “pharmaceutical ingredient” refers to an ingredient in a pharmaceutical formulation that is not an active ingredient.

[0078] Pharmaceutical ingredients include, but are not limited to, calcium carbonate, sodium carboxymethyl cellulose, talc, polydimethylsiloxane, hydroxypropyl cellulose and hydroxypropyl methylcellulose.

[0079] Excipient

[0080] In the present context, the term “excipient” refers to a natural or synthetic substance formulated alongside the active ingredient or pharmaceutical ingredient (an ingredient that is not the active ingredient) of a medication, included for the purpose of stabilization, bulking, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, enhancing solubility, adjusting tonicity, mitigating injection site discomfort, depressing the freezingpoint, or enhancing stability.

[0081] Excipients include, but are not limited to, microcrystalline cellulose, titanium dioxide and aluminum silicate.

[0082] Storage stability

[0083] In the present context, the term “storage stability” refers to the ability of a microencapsulated microbial culture to maintain viability when stored at accelerated storage conditions over an extended duration of time, such as at a temperature of 37°C and a water activity Aw < 0.15 for a period of 4 weeks. A of a food is the ratio between the vapor pressure of the microencapsulated microbial culture itself, when in a completely undisturbed balance with the surrounding air media, and the vapor pressure of distilled water under identical conditions. In the present context A is measured either by a resistive electrolytic, a capacitance or a dew point hygrometer.

[0084] Storage stability can be determined by analyzing how the count of viable microbial cells develop over time. Viability of the microbial culture is measured by determining the CFU / g as described herein. Thus, a measure of the storage stability of the microencapsulated microbial culture may be determined by evaluating CFU / g of the dry granulates of microencapsulated microbial culture at time point 0 (just after drying) and after 4 weeks of storage at accelerated storage conditions.

[0085] In brief, the storage stability of FD granulates, or FD grinded powder is investigated as follows; A sample of FD granulates of microbial culture (60 mesh grinded powder) is blended in CaCO3 to achieve a sample with a water activity Aw < 0.15. The sample is placed in an aluminum bag and the bag is sealed so that no air is trapped within it. The bag is stored at 37°C for 4 weeks and the CFU / g is determined for the sample.

[0086] Microencapsulated microbial cultures, compositions comprising the same and methods for their production

[0087] Microbial cultures, such as lactic acid bacteria (LAB), play key parts in many fermented products, in which they add nutritional value to the product and improve the organoleptic and textural profile of e.g. food products. The microbial cultures are typically acquired separately as powdered compositions and mixed with additional ingredients to yield a final product. Thus, the powdered composition comprising the microbial culture need as a minimum to maintain viability from the point of becoming a dried granulate to the point at which the powdered microbial cultures is included in a final product. Ideally, the microbial cultures are kept refrigerated during transport, supplementary processing and as part of the final product. However, this is not always possible as cold transport and storage is both expensive and, in many cases, not feasible in e.g. developing countries or remote regions. Moreover, the final product may be an article that is not readily stored under refrigerated conditions. This is typically the case of animal feed.

[0088] To deliver microbial cultures of high quality, e.g. high viability, under such environmental stress conditions, it is a necessary to decrease yield loss during downstream processing and eliminate the requirement of refrigerated transport and storage. However, no methods exist that both provide adequate cryo / lyo protection and enhance storage stability at elevated temperatures.

[0089] Herein are provided different embodiments that relate to microencapsulation of microbial cultures in complex coacervates using protein-carbohydrate conjugate and chitosan based on liquid-liquid phase separation and electrostatic interaction of protein-carbohydrate conjugate and chitosan at desired pH and temperature to form a unique polymer.

[0090] The present invention relates to a microencapsulation approach for microbial cultures using complex coacervates containing Whey Protein Concentrate-Galacto-oligosaccharide conjugate (WPC-GOS conjugate) and chitosan. In particular, the present invention discloses methods for producing microencapsulated microbial cultures.

[0091] Thus, an aspect of the present invention relates to a microencapsulated microbial culture, said microencapsulated microbial culture comprising: i. a microbial culture, ii. a first matrix comprising a first coacervate component, and, iii. a second matrix comprising a second coacervate component, wherein said first matrix component is a protein-carbohydrate conjugate of Whey Protein Concentrate (WPC) and Galacto-oligosaccharide (GOS) and the second matrix component is chitosan. The first or second matrix may further comprise one or more antioxidant(s).

[0092] The first and second matrix components interact electrostatically in aqueous medium to entrap the microbial culture in the form of dense phase droplets. The inclusion of WPC-GOS conjugate and chitosan in the first and second matrices is interchangeable, meaning that either WPC-GOS conjugate or chitosan may be included in the first matrix. However, both coacervate components must be present for the complex coacervation process to unfold. An embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein said first coacervate component is WPC-GOS conjugate and said second coacervate component is chitosan.

[0093] It is the selection of coacervate component that drives the microencapsulation process. Thus, the microencapsulation technique by complex coacervation as presented herein is not limited to a specific type of microbial culture but is a general microencapsulation concept. Thus, it is contemplated that any type of microbial culture may advantageously be microencapsulated as described herein. Two types of microorganisms that are of great importance in many consumer goods are bacteria and yeast. These microorganisms are included e.g. in fermented food, feed mixes and nutritional supplements, wherein their health benefits are well-documented. Therefore, an embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the microbial culture is a bacterium or a yeast.

[0094] Another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the microbial culture is or comprises a genus selected from the group consisting of Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Enterococcus, Bifidobacterium, Propionibacterium, Brevibacterium, Staphylococcus, Bacillus and Saccharomyces. Of particular interest are lactic acid bacteria (LAB) that are an order of Gram-positive bacteria sharing common metabolic and physiological characteristics. LAB produce lactic acid as the major metabolic outcome of carbohydrate fermentation. Ever since it was discovered that acidification by food fermentation could preserve food by inhibiting growth of spoilage agents, LAB has been utilized purposefully in food fermentation. However, since efficient food fermentation requires high quality viable microorganisms, the development of fermented foods has been halted in areas that do not have advanced facilities to hand i.e. the fragile microorganisms.

[0095] Specifically, microbial cultures, such as LAB, are not easily handled in some developing countries or remote regions due to the requirement and cost of refrigerated facilities. The microencapsulated microbial cultures described herein tolerate storage at elevated temperatures and may thus open-up development of products containing microbial culture, such as LAB, to a broader ensemble of product developers.

[0096] Thus, an embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the microbial culture is a lactic acid bacteria (LAB). Another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the microbial culture is or comprises a lactic acid bacteria (LAB) of a genus selected from the group consisting of Lactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus and Lentilactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Enterococcus, Bifidobacterium, Brevibacterium, and Staphylococcus.

[0097] It will be appreciated that the Lactobacillus genus taxonomy was updated in 2020. The new taxonomy is disclosed in Zheng et al. 2020 and will be cohered to herein if nothing else is noticed. For the purpose of the present invention, table presents a list of new and old names of some Lactobacillus species relevant to the present invention.

[0098] Table: Old and New name of Lactobacillus species relevant to the present invention

[0099] Bacteria of the Lactobacillus genus, as well as the related newly updated genera, have for a long time been known to constitute a significant component of the microbiota in the human body, such as in the digestive system, urinary system and genital system. For this reason, these bacteria have been heavily utilized in in health and / or nutritional products aimed at aiding, maintaining, or restoring the natural balance of microbiota in the human body. Examples of application of Lactobacillus include treatment or amelioration of diarrhea, vaginal infections, and skin disorders such as eczema.

[0100] Thus, an embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the microbial culture is or comprises a lactic acid bacteria (LAB) of a genus selected from the group consisting of Lactobacillus, Limosilactobacillus, Lacticaseibacillus, Ligilactobacillus, Lacticaseibacillus, Lacticaseibacillus, Lactiplantibacillus, Limosilactobacillus, Ligilactobacillus, Lentilactobacillus, Latilactobacillus, Companilactobacillus, Latilactobacillus and Lactiplantibacillus. Another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the microbial culture is of a species of Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus, Ligilactobacillus salivarius, Lacticaseibacillus casei, Lacticaseibacillus paracasei subsp. paracasei, Lactiplantibacillus plantarum subsp. plantarum, Limosilactobacillus fermentum, Ligilactobacillus animalis, Lentilactobacillus buchneri, Latilactobacillus curvatus, Companilactobacillus futsaii, Latilactobacillus sakei subsp., Lactiplantibacillus pentosus, Lactobacillus acidophillus, Lactobacillus helveticus, Lactobacillus gasseri and Lactobacillus delbrueckii.

[0101] Yet another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the microbial culture is or comprises at least one selected from the group consisting of:

[0102] Bifidobacterium animalis subsp. lactis deposited as DSM 15954 at Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH (DSMZ) by Chr. Hansen A / S, Hoersholm, Denmark on 30 September 2003,

[0103] Bifidobacterium animalis subsp. lactis deposited as ATCC 27536 and publicly available through the ATCC collection (https: / / www.lgcstandards-atcc.org / en.aspx),

[0104] Bifidobacterium animalis subsp. lactis deposited as DSM 10140 and publicly available through the DSMZ collection (https: / / www.dsmz.de / ),

[0105] Lactobacillus acidophilus deposited as DSM 13241 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) by Chr. Hansen A / S, Hoersholm, Denmark on 20 January 2000,

[0106] Lactobacillus rhamnosus deposited as ATCC 53103 and publicly available through the ATCC collection (https: / / www.lgcstandards-atcc.org / en.aspx),

[0107] Lactobacillus rhamnosus deposited as ATCC 55826 and publicly available through US patent 6,479,051,

[0108] Lactobacillus reuteri deposited as ATCC 55845 and publicly available through US patent 6,479,051,

[0109] Lactobacillus paracasei subsp. paracasei deposited as ATCC 55544 and publicly available through US patent 6,033,091,

[0110] Lactobacillus paracasei deposited as LMG-17806 and publicly available through US patent 6,599,504,

[0111] Streptococcus thermophilus deposited as DSM 15957 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) by Chr. Hansen A / S, Hoersholm, Denmark on 30 September 2003,

[0112] Lactobacillus fermentum deposited as NM02 / 31074 and publicly available through European patent EP 1539927,

[0113] Lactobacillus paracasei subsp. paracasei deposited as CCTCC M204012 and publicly available through US patent 6,599,504, and

[0114] Ligilactobacillus animalis deposited as DSM 33570 at Leibniz Institute DSMZ- German Collection of Microorganisms and Cell cultures Inhoffenstr. 7B, 38124 Braunschweig Germany (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) 5 GmbHl Inhoffenstr. 7B, D-38124 Braunschweig, Germany) by Chr. Hansen A / S, Hoersholm, Denmark on 8 July 2020, Lactococcus lactis subsp. lactis deposited as DSM 21404 deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) by Chr.Hansen A / S, Hoersholm, Denmark on 23 April 2008.

[0115] The applicant requests that a sample of the deposited microorganisms stated above may only be made available to an expert, subject to available provisions governed by Industrial Property Offices of States Party to the Budapest Treaty, until the date on which the patent is granted.

[0116] For US patents, the Budapest Treaty provides that any restriction of public access to samples of deposited biological material must be irrevocably removed as of the date of grant of the relevant patent. Accordingly, the above strains are publicly available because they have been deposited under the Budapest Treaty and a US patent referring to the strain has issued.

[0117] For EP patents, the Budapest Treaty provides that if no expert solution has been requested the deposited biological material becomes available upon request to any person from the date of publication of the European patent application.

[0118] If an expert solution has been requested, restrictions concerning the furnishing of samples apply. Information as to a request for an expert solution is published on the front page of an issued patent.

[0119] The applicant has made the following deposits at a Depositary institution having acquired the status of international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Leibniz Institute DSMZ- German Collection of Microorganisms and Cell Cultures Inhoffenstr. 7B, 38124 Braunschweig, Germany.

[0120] There is no indication on the front pages of EP 1359924 or EP 1539927 (referred to above) that a request for expert solution has been made. Thus, Bifidobacterium animalis subsp. lactis deposited as ATCC 27536 referred to in EP 1359924 and Lactobacillus fermentum deposited as NM02 / 31074 referred to in EP 1539927 are publicly available.

[0121] A further embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the microbial culture is or comprises Ligilactobacillus animalis 51 (LA51) deposited as DSM 33570 at Leibniz Institute DSMZ- German Collection of Microorganisms and Cell cultures Inhoffenstr. 7B 38124 Braunschweig Germany (Deutsche Sammlung von Mikroorganismen und Zellkulture (DSMZ) 5 GmbHl Inhoffenstr. 7B, D-38124 Braunschweig, Germany) by Chr. HansenA / S, Hoersholm, Denmark on 8 July 2020.

[0122] A further embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the microbial culture is or comprises Bifidobacterium animalis subsp. lactis deposited as DSM 15954 at DSMZ.

[0123] A further embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the microbial culture is or comprises Streptococcus thermophilus deposited as DSM 15957 at DSMZ.

[0124] A further embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the microbial culture is or comprises Lactococcus lactis subsp. lactis deposited as DSM 21404 at DSMZ,

[0125] Probiotic culture are cultures of live microorganisms, which upon ingestion by a subject provide health benefits to the subject. Products comprising probiotic cultures include dairy products, animal feed and beverages. Thus, it is to be understood that the microencapsulated microbial cultures described herein may be administered not only to humans but also animals, and even plants.

[0126] Thus, an embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the microbial culture is a probiotic culture.

[0127] The process of complex coacervation is driven by the coacervate components that is WPC-GOS conjugate and chitosan. The two coacervate components may be included in varying amounts and ratios. It has been found that complex coacervates with a predominant fraction of WPC-GOS conjugate compared to chitosan result in better cryo / lyo protection and enhanced storage stability. Thus, variants of complex coacervates with high content of WPC-GOS conjugate are preferred.

[0128] Therefore, an embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the content of WPC-GOS conjugate in the composition is between 20-40 wt%, such as between 25-37 wt%, preferably between 30-35 wt%.

[0129] Another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the content of chitosan in the composition is between 1-10 wt%, such as between 2-8 wt%, preferably between 4- 6 wt%.

[0130] A further embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the ratio (wt% / wt%) of WPC-GOS conjugate to chitosan is between 75:25 to 98:2, preferably between 85: 15 to 95:5. A still further embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the ratio (wt% / wt%) of WPC-GOS conjugate to chitosan is 85: 15 or 95:5.

[0131] Oxidation is the loss of electrons of an atom or ion. In the present context, oxidation refers to oxidation of molecular oxygen and means that oxygen is metabolized to unstable free radicals, which can pry away electrons from other molecules. Oxidation may therefore lead to damaging of cell membranes and other cellular components, such as proteins, lipids and DNA. To avoid damage to the microencapsulated microbial culture, one or more antioxidants are included in the first matrix to prevent oxidation. The antioxidants may be of either natural or synthetic origin. Therefore, an embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the one or more antioxidants are selected from the group consisting of trisodium citrate, sodium ascorbate, vitamin E and combinations thereof.

[0132] Another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the one or more antioxidants is sodium ascorbate.

[0133] The content of antioxidants may be tailored to the specific microbial culture to be encapsulated and the expected application of the final powdered product. If the microencapsulated microbial culture is intended for an application for which extended storage is expected, it may for instance be preferable to increase the content of antioxidants. The content of antioxidants may also be adjusted depending on the conditions of storage, e.g. the degree of exposure to air.

[0134] Therefore, an embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the content of antioxidants in the composition is between 2-20 wt%, such as between 5-15 wt%, preferably between 8-12 wt%.

[0135] The ratio (wt% / wt%) of matrix material to microbial culture in the formulation may be customized to suit the specific application. This ratio is also referred to as the encapsulation index (El). Without being bound by theory, it is contemplated that microencapsulated microbial cultures formulated at high El will more efficiently restrict absorption of water and thereby yield increased storage stability. However, increasing the content of matrix material will dilute the content of microbial culture in the final product. Consequently, the optimal balance between matrix material and microbial culture is decided also by factors such as the time scale of storage and storage conditions, which may vary depending on the intended application.

[0136] Therefore, an embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the ratio (wt% / wt%) of the first and second matrix combined to microbial culture is between 0.25-10.

[0137] Another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the ratio (wt% / wt%) of the first and second matrix combined to microbial culture is 1.

[0138] The complex coacervation process is a result of the interactions of oppositely charged coacervate components in aqueous medium. The pKa of the carboxyl groups of most amino acids in the polypeptide sequence of the proteins such as proteins in the Whey Protein Concentrate have pKa values in the range of 2.8 to 4.1. The conjugated Whey Protein Concentrate and Galactooligosaccharide will also typically have the pKa in the acidic range (pH < 4.7). In contrast, the amino groups of chitosan have a pKa value of 6.5 and protonation is therefore significant at pH 6. By varying the pH around the pKa of the amino groups of chitosan it is possible to alter the interaction between the coacervation components. Moreover, the interaction depends on the ratio (wt% / wt%) of WPC- GOS conjugate to chitosan.

[0139] Thus, an embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the pH is in the range between 3-8, preferably between 5-7, such as pH 5, 5.5, 6, 6.5 or 7.

[0140] Another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the pH is in the range of 5 to 7, such as in the range of 5.5 to 6.8, and the ratio (wt% / wt%) of WPC-GOS conjugate to chitosan is in the range of 90: 10 to 99: 1, such as in the range of 92:8 to 98:2.

[0141] Yet another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the pH is in the range of 6.75 to 8, such as in the range of 7 to 7.5, and the ratio (wt% / wt%) of WPC-GOS conjugate to chitosan is in the range of 70:30 to 90: 10, such as in the range of 80:20 to 87: 13.

[0142] It is appreciated that the interaction between chitosan and WPC-GOS conjugate will also be affected by the extent of deacetylation of the chitosan.

[0143] Storage stability may be further increased by applying a hydrophobic coating on the exterior of the microencapsulated microbial culture. Thus, an embodiment of the present invention may relate to the microencapsulated microbial culture comprising a hydrophobic coating.

[0144] The combination of coacervate components and careful selection of process parameters has resulted in microencapsulated microbial cultures that readily withstand drying and storage at harsh conditions for extended periods of time. The count of viable microbes after storage is the most important product quality to the downstream developer or consumer. Thus, an embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the microbial culture comprises a content of viable microbes in the range from 106- 1012CFU / g after storage for 4 weeks at 37°C and Aw < 0.15. Another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the microbial culture comprises a content of viable microbes of at least 106CFU / g, such as at least 107CFU / g, such as at least 108CFU / g, preferably at least 109CFU / g, more preferably at least 1010CFU / g after storage for 4 weeks at 37°C and Aw < 0.15.

[0145] The microencapsulated microbial culture will typically be utilized as an additive in an end product comprising also other ingredients. It is therefore contemplated that the microencapsulated microbial culture for many applications will be part of a more complex composition. Thus, an aspect of the present invention relates to a composition comprising the microencapsulated microbial culture as described herein.

[0146] As the microencapsulated microbial culture is admixed with other ingredients the complex coacervates formed during the mixing of WPC-GOS conjugate and chitosan stay intact to safeguard the microbial culture from any environment potentially harmful to the microorganisms. Therefore, an embodiment of the present invention relates to the composition as described herein, wherein the composition comprises complex coacervates comprising the microbial culture, the first matrix and the second matrix.

[0147] The order of addition of coacervate components may affect the arrangement of the components within complex coacervate. Therefore, an embodiment of the present invention relates to the composition as described herein, wherein the complex coacervates are arranged with an interior part comprising the microbial culture and the first matrix, and an exterior part comprising the second matrix.

[0148] The microencapsulated microbial culture may be utilized for many different types of applications spanning from e.g. health products, nutritional supplement and pharmaceutics to animal feed. Thus, a composition encompassing the microencapsulated microbial culture may a wide range of additives. Therefore, an embodiment of the present invention relates to the composition as described herein, wherein the composition further comprises one or more additives selected from the group consisting of food-grade ingredients, pharmaceutical ingredients, and excipients.

[0149] Another embodiment of the present invention relates to the composition as described herein, wherein the food-grade ingredients are selected from the group consisting of lactose, maltodextrin, whey protein, casein, com starch, dietary fibres, gums and gelatine.

[0150] A further embodiment of the present invention relates to the composition as described herein, wherein the pharmaceutical ingredients are selected from the group consisting of calcium carbonate, sodium carboxymethyl cellulose, talc, polydimethylsiloxane, hydroxypropyl cellulose and hydroxypropyl methylcellulose.

[0151] Yet another embodiment of the present invention relates to the composition as described herein, wherein the excipients are selected from the group consisting of microcrystalline cellulose, titanium dioxide and aluminum silicate.

[0152] Many additives are provided in dry form to extent the shelf life and ease the handling of the additive. The microencapsulated microbial culture is no exception and is also provided in dry form. Therefore, an embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the microencapsulated microbial culture is in a dry form. Another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the microencapsulated microbial culture is freeze dried. Yet another embodiment of the present invention relates to the composition as described herein, wherein the composition is a freeze-dried composition. A still further embodiment of the present invention relates to the microencapsulated microbial culture or the composition as described herein, wherein the microencapsulated microbial culture or composition is in the form of a powder and / or a granulate.

[0153] Typically, the storage stability of food products can be extended by formulating the product with low water activity. By controlling the water activity (Aw), it is possible to predict and regulate the effect of moisture migration on the product. Therefore, an embodiment of the present invention relates to the microencapsulated microbial culture or the composition as described herein, wherein the water activity (Aw) of the microencapsulated microbial culture is in the range from 0.01-0.8, preferably in the range from 0.05-0.4. Microbial cultures may find diverse application across many different consumer sectors. Therefore, an aspect of the present invention relates to a product comprising the microencapsulated microbial culture or the composition as described herein, wherein the product is selected from the group consisting of a feed, a plant health product, a food, a beverage and a pharmaceutical product.

[0154] Especially the utilization of the microencapsulated microbial culture in animal feed is a preferred application. Thus, an embodiment of the present invention relates to a product comprising the microencapsulated microbial culture or the composition as described herein, wherein the product is a feed product selected from the group consisting of a feed premix, a feed blend, a pet food and a domestic animal feed.

[0155] Another preferred area of application is within products comprising LAB. Therefore, an embodiment of the present invention relates to a product comprising the microencapsulated microbial culture or the composition as described herein, wherein the product is a fermented product.

[0156] Probiotic-containing products are also a prioritized field of application. Thus, an embodiment of the present invention relates to a product comprising the microencapsulated microbial culture or the composition as described herein, wherein the product is a dairy product selected from the group consisting of yoghurt, cheese, butter, an inoculated sweet milk and a liquid fermented milk product.

[0157] Decreasing yield loss of microbial cultures during freezing / FD downstream processing is of great importance, both in terms of quality but also in terms of process efficiency.

[0158] Similarly, the ability to retain maximum viability of the microbial cultures during ambient storage conditions has commercial and technical advantages. Thus, the methods described herein has been developed with the aim of achieving both objectives. The method described herein relies on complex coacervation of a set of coacervate components that upon complexation entrap the microbial culture within dense phase droplets. In contrast to other conventional techniques in which the microbial culture is just blended with different matrices, the method described herein provides enhanced protection of the microbial culture both during drying and ambient storage.

[0159] Thus, an aspect of the present invention relates to a method for preparing a microencapsulated microbial culture or a composition as described herein, said method comprising the steps of i) mixing a microbial culture with a first matrix comprising a first coacervate component, and optionally one or more antioxidants, to form a pre-complex solution, and ii) mixing the pre-complex solution with a second matrix comprising a second coacervate component, and optionally one or more antioxidants, to form a microencapsulated microbial culture, wherein said first and second coacervate components are Whey Protein Concentrate and Galactooligosaccharide conjugate (WPC-GOS conjugate) or chitosan and wherein said first and second coacervate components are not the same.

[0160] An embodiment of the present invention relates to a method as described herein, wherein said first coacervate component is Whey Protein Concentrate and Galacto-oligosaccharide conjugate (WPC- GOS conjugate) and said second coacervate component is chitosan.

[0161] Further embodiment of the present invention relates to a method for preparing a first matrix comprising a first coacervate component, said method comprising the steps of i) preparing individual solutions of first coacervate components at desired pH and hydrating them overnight, ii) mixing the overnight hydrated individual solutions of first coacervate components at desired pH in desired proportion and heat incubating to form the conjugate.

[0162] To obtain a final dry microbial culture product with as high viability over time as possible, it is necessary to carefully select and adjust content of the starting materials. Therefore, an embodiment of the present invention relates to the method as described herein, wherein the microbial culture of step i) is a concentrated microbial culture comprising a dry matter content in the range from 5-80 wt%, preferably 15-25 wt%. Another embodiment of the present invention relates to the method as described herein, wherein the microbial culture of step i) is a concentrated microbial culture comprising a dry matter content of at least 5 wt%, such as at least 10 wt%, such as at least 15 wt%, such as at least 20 wt%.

[0163] A further embodiment of the present invention relates to the method as described herein, wherein the microbial culture of step i) is a concentrated microbial culture comprising a content of viable microbes in the range from 109- 1012CFU / g, preferably approximately 1011CFU / g. A still further embodiment of the present invention relates to the method as described herein, wherein the microbial culture of step i) is a concentrated microbial culture comprising a content of viable microbes of at least 109CFU / g, such as at least IO10CFU / g, preferably at least 1011CFU / g.

[0164] The starting materials are utilized in a complex coacervation process to yield the microencapsulated microbial cultures. Each process step is adjusted to obtain coacervate complexes suitable for the intended final application, with some overall parameters, such as the temperature and duration of mixing, being defined. Thus, an embodiment of the present invention relates to the method as described herein, wherein the microbial culture of step i) is mixed with the first matrix for a time period in the range of 5 min to 6 hours at a temperature in the range of 4°C to 45°C. Another embodiment of the present invention relates to the method as described herein, wherein the microbial culture of step i) is mixed with the first matrix for a time period in the range of 10-30 min.

[0165] Yet another embodiment of the present invention relates to the method as described herein, wherein the microbial culture of step i) is mixed with the first matrix at a temperature in the range of 10°C to 15°C.

[0166] A further embodiment of the present invention relates to the method as described herein, wherein mixing of the pre-complex solution with the second matrix in step ii) is carried out for a time period in the range of 5 min to 6 hours at a temperature in the range of 4°C to 45°C.

[0167] A still further embodiment of the present invention relates to the method as described herein, wherein mixing of the pre-complex solution with the second matrix in step ii) is carried out for a time period in the range of 5 min to 2 hours, preferably for a time period in the range of 10-20 min.

[0168] An even further embodiment of the present invention relates to the method as described herein, wherein mixing of the pre-complex solution with the second matrix in step ii) is carried out at a temperature in the range of 10°C to 15°C.

[0169] As explained herein, the pH of the solution during complex formation guides the extent of electrostatic interactions between the coacervate components. Therefore, it is necessary to control the pH throughout the complex coacervation process. Thus, an embodiment of the present invention relates to the method as described herein, wherein the pH in step ii) is adjusted using an acid or a base.

[0170] Another embodiment of the present invention relates to the method as described herein, wherein the acid is selected from the group consisting of phosphoric acid, hydrochloric acid and acetic acid, and mixtures thereof.

[0171] A further embodiment of the present invention relates to the method as described herein, wherein the base is selected from the group consisting of NaOH, KOH and liquid ammonia, and mixtures thereof.

[0172] It is to be understood that the skilled person may also select and use other acids and bases suitable for adjusting the pH during the complex coacervation process. Given the relevant pKa values of (WPC- GOS conjugate) and chitosan, only pH values suitable for facilitating electrostatic interactions between these two coacervation components are applicable. As explained herein, the pH may for instance be adjusted to vary the protonation stage of the amine groups of chitosan. Thus, an embodiment of the present invention relates to the method as described herein, wherein the pH in step ii) is in the range between 3-8, preferably between 5-7.

[0173] As an additive for inclusion in a variety of different end products, microbial cultures, such as LAB, are often supplied as dried powders. The dry form makes the microbial cultures easy to transport, store and handle prior to final processing. Therefore, an embodiment of the present invention relates to the method as described herein further comprising a step iii) subsequent to step ii), wherein step iii) comprises freezing said microencapsulated microbial culture to obtain a frozen microencapsulated microbial culture.

[0174] Another embodiment of the present invention relates to the method as described herein further comprising a step iv) subsequent to step iii), wherein step iv) comprises sublimating water from said frozen microencapsulated microbial culture to obtain a dried microencapsulated microbial culture.

[0175] A further embodiment of the present invention relates to the method as described herein, wherein step iv) is carried out by a technique selected from the group consisting of spray drying, vacuum drying, air drying, freeze drying, tray drying and vacuum tray drying.

[0176] A still further embodiment of the present invention relates to the method as described herein, wherein the technique used in step iv) is freeze drying and wherein said freeze drying is performed at 0.005 to 1 mbar at -45°C to 45°C until complete water removal.

[0177] An even further embodiment of the present invention relates to the method as described herein, wherein said freeze drying is performed at 0.1 to 0.4 mbar. Yet another embodiment of the present invention relates to the method as described herein, wherein said freeze drying is performed at atleast 0.1 mbar, such as at least 0.2 mbar, such as at least 0.3 mbar, such as at least 0.4 mbar.

[0178] Another embodiment of the present invention relates to the method as described herein, wherein said freeze drying is performed at 15°C to 35°C. An additional embodiment of the present invention relates to the method as described herein, wherein said freeze drying is performed at at-least 15°C, such as at least 20°C, such as at least 25°C, such as at least 30°C.

[0179] Depending on the application of the microencapsulated microbial culture, it may be advantageous to receive the microencapsulated microbial culture in frozen or dried form. Therefore, an embodiment of the present invention relates to the method as described herein, wherein said method further comprises: step (v) packing said frozen microencapsulated microbial culture obtained in step (iii) or the dried microencapsulated microbial culture obtained in step (iv).

[0180] The microencapsulated microbial culture is the result of the complex coacervation process as described herein. Consequently, an aspect of the present invention relates to a microencapsulated microbial culture or a composition obtainable by a method as described herein.

[0181] Preferably, the microencapsulated microbial culture is utilized as additive in the preparation of an end product. Thus, an aspect of the present invention relates to the use of a microencapsulated microbial culture or a composition as described herein in a product selected from the group consisting of a feed, a plant health product, a food, a beverage and a pharmaceutical product.

[0182] The listing or discussion of an apparently prior published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0183] Preferences, options and embodiments for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences, options and embodiments for all other aspects, features and parameters of the invention. This is especially true for the description of the microencapsulated microbial culture and all its features, which may readily be part of the final composition obtained by the method as described herein. Embodiments and features of the present invention are also outlined in the following items.

[0184] Specific embodiments

[0185] The present disclosure comprises a microencapsulated microbial culture, said microencapsulated microbial culture comprising: i. a microbial culture, ii. a first matrix comprising a first coacervate component, and iii. a second matrix comprising a second coacervate component, wherein said first matrix component is a protein-carbohydrate conjugate of Whey Protein Concentrate (WPC) and Galacto-oligosaccharide (GOS) and the second matrix component is chitosan. The first or second matrix may comprise one or more antioxidants.

[0186] The microencapsulated microbial culture may comprise a first coacervate component, which is a conjugate of Whey Protein Concentrate (WPC) and Galacto-oligosaccharide (GOS) and said second coacervate component, which is chitosan.

[0187] The microencapsulated microbial culture may comprise a microbial culture, which is a bacterium or a yeast.

[0188] The microencapsulated microbial culture may comprise a microbial culture, which is a probiotic culture.

[0189] In one embodiment, the microbial culture is or comprises a lactic acid bacteria (LAB) of a genus selected from the group consisting of Lactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus,A grilactobacillus,

[0190] Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus and Lentilactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Enterococcus, Bifidobacterium, Brevibacterium, and Staphylococcus.

[0191] In one embodiment, the microbial culture is or comprises a lactic acid bacterium (LAB) of a genus selected from the group consisting of Lactobacillus, Limosilactobacillus, Lacticaseibacillus, Ligilactobacillus, Lacticaseibacillus, Lacticaseibacillus, Lactiplantibacillus, Limosilactobacillus, Ligilactobacillus, Lentilactobacillus, Latilactobacillus, Companilactobacillus, Latilactobacillus and Lactiplantibacillus.

[0192] In one embodiment, the microbial culture is or comprises of a species of Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus, Ligilactobacillus salivarius, Lacticaseibacillus casei, Lacticaseibacillus paracasei subsp. paracasei, Lactiplantibacillus plantarum subsp. plantarum, Limosilactobacillus fermentum, Ligilactobacillus animalis, Lentilactobacillus buchneri, Latilactobacillus curvatus, Companilactobacillus futsaii, Latilactobacillus sakei subsp., Lactiplantibacillus pentosus, Lactobacillus acidophillus, Lactobacillus helveticus, Lactobacillus gasseri and Lactobacillus delbrueckii.

[0193] In one embodiment, the microbial culture is or comprises Ligilactobacillus animalis 51 (LA51) deposited as DSM 33570 at Leibniz Institute DSMZ-German Collection of Microorganisms and Cell cultures Inhoffenstr. 7B 38124 Braunschweig Germany (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) 5 GmbHl Inhoffenstr. 7B, D-38124 Braunschweig, Germany) by Chr. Hansen A / S, Hoersholm, Denmark on 8 July 2020; Bifidobacterium animalis subsp. lactis deposited as DSM 15954 at DSMZ; Streptococcus thermophilus deposited as DSM 15957 at DSMZ; or Lactococcus lactis subsp. lactis deposited as DSM 21404 at DSMZ.

[0194] The content of WPC-GOS conjugate in the microencapsulated microbial culture composition may be between 20-40 wt%, such as between 25-37 wt%, preferably between 30-35 wt%.

[0195] The content of chitosan in the microencapsulated microbial culture composition may be between 1- 10 wt%, such as between 2-8 wt%, preferably between 4-6 wt%.

[0196] The ratio (wt% / wt%) of WPC-GOS conjugate to chitosan may be between 50:50 to 98:2, preferably between 75:25 to 98:2, more preferably between 85:15 to 95:5.

[0197] The ratio (wt% / wt%) of WPC-GOS conjugate to chitosan may be 85: 15 or 95:5.

[0198] The one or more antioxidants may be selected from the group consisting of trisodium citrate, sodium ascorbate, vitamin E and combinations thereof.

[0199] In one specific embodiment, the microencapsulated microbial culture is sodium ascorbate.

[0200] The content of antioxidants in the composition may be between 2-20 wt%, such as between 5-15 wt%, preferably between 8-12 wt%.

[0201] The ratio (wt% / wt%) of the first and second matrix combined to microbial culture may be between 0.25-10.

[0202] The ratio (wt% / wt%) of the first and second matrix combined to microbial culture may be 1.

[0203] The pH may be in the range between 3-8, preferably between 5-7.

[0204] In one embodiment, the microencapsulated microbial culture may further comprising a hydrophobic coating. The hydrophobic coating may be one or more fats or waxes and mixture thereof.

[0205] In one specific embodiment, the microbial culture comprises a content of viable microbes in the range from 106- 1012CFU / g after storage for 4 weeks at 37°C and Aw < 0.15.

[0206] In one embodiment, a composition comprising the microencapsulated microbial culture according to embodiments of the invention is provided.

[0207] The composition may comprise complex coacervates comprising the microbial culture, the first matrix and the second matrix.

[0208] The complex coacervates may be arranged with an interior part comprising the microbial culture and the first matrix, and an exterior part comprising the second matrix.

[0209] The composition may further comprise one or more additives selected from the group consisting of food-grade ingredients, pharmaceutical ingredients, and excipients.

[0210] The food-grade ingredients may be selected from the group consisting of lactose, maltodextrin, whey protein, casein, com starch, dietary fibres, gums and gelatine.

[0211] The pharmaceutical ingredients may be selected from the group consisting of calcium carbonate, sodium carboxymethyl cellulose, talc, poly dimethyl siloxane, hydroxypropyl cellulose and hydroxypropyl methylcellulose.

[0212] The excipients may be selected from the group consisting of microcrystalline cellulose, titanium dioxide and aluminum silicate.

[0213] The composition may be a freeze-dried composition.

[0214] In one embodiment, the microencapsulated microbial culture according to embodiments of the invention or the composition according to embodiments of the invention is in the form of a powder and / or a granulate.

[0215] The water activity (Aw) of the microencapsulated microbial culture may be in the range from 0.01- 0.8, preferably in the range from 0.05-0.4, such as 0.1, 0.2, 0.3.

[0216] In one embodiment, a product selected from the group consisting of a feed, a plant health product, a food, a beverage and a pharmaceutical product is provided, said product comprising the microencapsulated microbial culture according to embodiments of the invention or the composition according to embodiments of the invention.

[0217] In one embodiment, a method for preparing a microencapsulated microbial culture according to embodiments of the invention or a composition according to embodiments of the invention is provided, said method comprising the steps of: i. mixing a microbial culture with a first matrix comprising a first coacervate component and optionally one or more antioxidants to form a pre-complex solution, and ii. mixing the pre-complex solution with a second matrix comprising a second coacervate component and optionally one or more antioxidants to form a microencapsulated microbial culture, wherein said first matrix component is a protein-carbohydrate conjugate of Whey Protein Concentrate (WPC) and Galacto-oligosaccharide (GOS) and the second matrix component is chitosan and wherein said first and second coacervate components are not the same.

[0218] Said first coacervate component may be Whey Protein Concentrate-Galacto-oligosaccharide (WPC- GOS) conjugate and said second coacervate component may be chitosan.

[0219] The microbial culture of step i) above may be a concentrated microbial culture comprising a dry matter content in the range from 5-80 wt%, preferably 15-25 wt%, such as 20 wt%.

[0220] The microbial culture of step i) above may be a concentrated microbial culture comprising a content of viable microbes in the range from 109- 1012CFU / g, preferably approximately 1011CFU / g.

[0221] The microbial culture of step i) above may be mixed with the first matrix for a time period in the range of 5 min to 6 hours at a temperature in the range of 4°C to 45°C.

[0222] The microbial culture of step i) above may be mixed with the first matrix for a time period in the range of 10-30 min.

[0223] The microbial culture of step i) above may be mixed with the first matrix at a temperature in the range of 10°C to 15°C.

[0224] Mixing of the pre-complex solution with the second matrix in step ii) above, may be carried out for a time period in the range of 5 min to 6 hours at a temperature in the range of 4°C to 45°C.

[0225] Mixing of the pre- complex solution with the second matrix in step ii) above, may be carried out for a time period in the range of 5 min to 2 hours, preferably for a time period in the range of 10-20 min.

[0226] Mixing of the pre- complex solution with the second matrix in step ii) above, may be carried out at a temperature in the range of 10°C to 15°C. The pH in step ii) above, may be adjusted using an acid or a base.

[0227] The acid may be selected from the group consisting of orthophosphoric acid, hydrochloric acid and acetic acid, and mixtures thereof.

[0228] The base may be selected from the group consisting of NaOH, KOH and liquid ammonia, and mixtures thereof.

[0229] The pH in step ii) above may be in the range between 3-8, preferably between 5-7.

[0230] In one embodiment, the method above further comprising a step iii) subsequent to step ii), wherein step iii) comprises freezing said microencapsulated microbial culture to obtain a frozen microencapsulated microbial culture.

[0231] In one embodiment, the method above further comprises a step iv) subsequent to step iii), wherein step iv) comprises sublimating water from said frozen microencapsulated microbial culture to obtain a dried microencapsulated microbial culture.

[0232] In one embodiment, step iv) is carried out by a technique selected from the group consisting of spray drying, vacuum drying, air drying, freeze drying, tray drying and vacuum tray drying.

[0233] In one specific embodiment, the drying is freeze drying and said freeze drying is performed at 0.005 to 1 mbar at - 45°C to 45°C until complete water removal.

[0234] In one preferred embodiment, said freeze drying is performed at 0.1 to 0.4 mbar. In one preferred embodiment, said freeze drying is performed at 15°C to 35°C. The method may further comprise:

[0235] (v) packing said frozen microencapsulated microbial culture obtained in step (iii) or the dried microencapsulated microbial culture obtained in step (iv).

[0236] In one embodiment, a microencapsulated microbial culture or a composition obtainable by a method according to embodiments of the invention is provided.

[0237] In one embodiment, use of a microencapsulated microbial culture or a composition according to embodiments of the invention, in a product selected from the group consisting of a feed, a plant health product, a food, a beverage and a pharmaceutical product, is provided.

[0238] In yet another embodiment of the present invention is described preparing a first matrix comprising a first coacervate component, said method comprising the steps of: iii) preparing individual solutions of first coacervate components at desired pH and hydrating them overnight, iv) mixing the overnight hydrated individual solutions of first coacervate components at desired pH in desired proportion and heat incubating to form the conjugate. The invention will now be described in further details in the following non-limiting examples.

[0239] EXAMPLE-1- WPC-GOS conjugate.

[0240] Described herein is an example of preparation of the WPC-GOS conjugate in a preferred embodiment. The WPC-GOS conjugates were prepared separately using heating method. The individual solutions of WPC (8%) and GOS (16%) were prepared by suspending w / w component of each in RO water. The solutions so obtained were allowed to stand overnight and were used the day next. In this method, 8% (w / w) of WPC, 16% (w / w) of GOS were used to prepare the WPC-GOS conjugate having concentration of 24% (w / w). The ratio of components in the final WPC-GOS conjugate was kept 1 :2 respectively of the WPC and GOS. These solutions of 8% (w / w) of WPC, 16% (w / w) of GOS and the 24% (w / w) WPC-GOS were prepared in RO water for further experimental use. The pH of these solutions was adjusted to 9.0 using 10% NaOH to facilitates the conjugation reaction. These solutions were subjected in thermomixer (Thermo Fisher Scientific) and allowed to incubate at 90°C for 3 hr to form the said WPC-GOS conjugates (Fig. 1).

[0241] The WPC-GOS conjugate thus obtained was cooled to room temperature and diluted for further spectrophotometer analysis.

[0242] Qualitative analysis: Spectrophotometry

[0243] The qualitative determination of WPC-GOS conjugates was done using UV-Vis spectrophotometer (Thermo Fisher Scientific) by measuring optical density (O.D.) at 600 nm. (Fig. 1). For this purpose, the 24% (w / w) conjugate solution was diluted 100 times with RO water and subjected to spectrum analysis to determine the formation of WPC-GOS conjugate.

[0244] The spectrum analysis showed that newly formed conjugate had significantly higher km ax (1.473) as compared to WPC alone ( max=1.056) (Fig. 2). The increase in km ax or absorbance could be contributed to the formed covalent complex between GOS to WPC (Conjugate) which resulted into the formation of coloured compounds as a mixture.

[0245] The WPC-GOS conjugate formed was also evaluated for its total carbohydrate and total protein content.

[0246] Quantitative analysis: Protein-Carbohydrate content analysis

[0247] The WPC-GOS conjugate thus formed were further investigated to determine the % protein and % carbohydrate content of conjugates as described below.

[0248] Quantification of Total carbohydrates using Anthrone test

[0249] The quantitative analysis of total carbohydrates was done using Anthrone method. In this method, the carbohydrate content can be measured by hydrolyzing the polysaccharides into simple sugars by acid hydrolysis and estimating the resultant monosaccharides. Detailed methodology is given in (SOP / PTL- 35). Weigh 50mg glucose in distilled water and make up the volume to 50mL. Allow it to dissolve for 5 min in sonicator water bath. Pipette out 5mL of the solution and make up the volume to 50 mL using distilled water in a volumetric flask. Take ImL of above solution in test tube and then add 4mL anthrone reagent. Incubate the content in test tube in hot water bath at 95 °C for 10 min followed by cooling at room temperature. Similarly, take 50mg of sample and make up the volume to 50mL using distilled water (dissolve well first using lOmL water using sonication for 5 mins). Filter the prepared solution to get a clear solution and then pipette 5mL of the solution and make up the volume to 50 mL using distilled water in a volumetric flask. Take ImL of above solution in test tube and then add 4mL anthrone reagent. Incubate the content in test tube in hot water bath at 95 °C for 10 min followed by cooling at room temperature. For blank, take ImL of distilled water in test tube and then add 4mL anthrone reagent. Incubate the content in test tube in hot water bath at 95 °C for 10 min followed by cooling at room temperature. Finally, measure the absorbance at 630 nm using UV-visible spectrophotometer. The Carbohydrate content of given was calculated using below given formula, Calculation: Carbohydrate content

[0250] CC = Sample Abs * Standard Cone (mg / mL) * Standard Potency Where, Standard Potency= 99%

[0251] Quantification of Protein using Bradford

[0252] The quantitative estimation of protein in conjugates and other samples was done using Bradford method. Bradford reagent was prepared by mixing 100 mg of coomassie brilliant blue dye with 50 ml of ethanol (95%) and 100 ml of ortho-phosphoric acid (85%) which is then finally made up to 1000 ml using distilled water. The solution before its use was filtered through Whatman no. 1 filter paper. Bovine serum albumin was used a standard for the assay which is prepared by mixing 100 mg of BSA with 100 ml of phosphate buffer (PBS) (pH 7.0) in standard volumetric flask. The standard curve was obtained for BSA solution concentration ranging from 0 to 100 pg / ml (R2>0.9). For sample analysis, weigh 100 mg of sample and dissolve it in PBS (pH 7) and make up the volume to 25 ml in standard flask. Reaction mixture contains 1 ml of sample with 5 ml of Bradford reagent. Allow the mixture to incubate at 25-30°C for 5 mins and finally take absorbance at 595 nm (SOP / PTL- 34).

[0253] Calculate protein content of sample:

[0254] > .

[0255] Protein content (

[0256] Calculate % protein content:n / . Y*Sample final volume*100

[0257] % protein content = - weight of the sample

[0258] It was found that the protein content of WPC was 85% whereas in conjugates, it remained 37.79% (Fig. 3). The carbohydrate content of GOS was 69% and it was measured to be 37.5% in the conjugate. This could be due to the partial grafting between WPC to GOS which has quantitively reduced both protein and carbohydrate fraction in conjugates.

[0259] A further aspect of the present invention relates to the composition and method of preparation thereof; of a second matrix the protein-oligosaccharide chitosan conjugate.

[0260] Described herein is an exemplary embodiment of the present invention that describes the composition and method of preparation thereof of the complex insoluble coacervates of conjugate and chitosan.

[0261] EXAMPLE-2 WPC-GOS chitosan conjugate.

[0262] In an exemplary preparation of complex insoluble coacervates polymer of WPC-GOS conjugate and chitosan, the WPC-GOS conjugate solution of 24% strength was diluted using RO water to get final solution with a concentration of 0.1 wt %.

[0263] Each of the solutions were weighed separately to get the desired concentration of biopolymers and mixed together in a glass beaker. Both these solutions were allowed to hydrate overnight on magnetic stirrer at 400 rpm at temperature of 25°C. Chitosan solution was prepared by dispersing 0.1 g of chitosan in 99.9 g of 0.5 wt% acetic acid solution. Before starting the complexation experiment, the pH of 0.1 wt% chitosan dispersion was adjusted to pH 5 using IM acetic acid.

[0264] Both these solutions of WPC-GOS conjugate and chitosan were allowed to hydrate overnight on magnetic stirrer at 400 rpm at temperature of 25°C to obtain the said conjugate.

[0265] EXAMPLE-3 This example describes experimental data for variations of ratios of conjugate to chitosan and demonstrates their effect on viability as seen in Fig.6.

[0266] The conjugate: chitosan complexes were obtained for a varying range of combination of different biopolymer ratio of conjugate: chitosan (50:50 to 95:5) wherein the total biopolymer concentration was kept constant to 0.1 wt%. Biopolymer solution of each of the varying strength were obtained by weighing each component (conjugate and chitosan) of the desired concentrations separately and then mixed together in a glass beaker and then allowed to stir on magnetic stirrer at 25°C. This mixing and incubation at right temperature and pH allows for the desired polymer formation by electrostatic interactions.

[0267] The electrostatic interaction between conjugates and chitosan were studied by varying pH from 3 to 8 where the pH of reaction mixture was adjusted using 1 M NaOH solution and 1 M phosphoric acid. The maximum insoluble complex coacervates (Fig. 4) formation between Conjugate-Chi tosan were found at conjugate: Chitosan at pHmax 2 (85: 15, pH 6.80) and pHmax 1 (95:5, pH 5.60). A) Qualitative analysis

[0268] 1) Spectrophotometry

[0269] For spectrophotometry analysis small amount of sample was withdrawn at predetermined pH interval of 0.25 for varying pH range and the optical density (OD) of the reaction mixture was measured at 600 nm using UV-vis spectrophotometer (Thermo Fisher Scientific).

[0270] The absorbance data from UV-vis spectrophotometer was collected to plot pH versus OD to find out the maximum complex formation between conjugates and chitosan. The maximum insoluble complex coacervate (Fig. 4) formation between Conjugate-Chi tosan were found at conjugate: Chitosan at pHmax 2 (85: 15, pH 6.80) and pHmax 1 (95:5, pH 5.60).

[0271] 2) Microscopic analysis

[0272] The same samples of prepared conjugates and insoluble complex polymer were also used to check interaction and macroscopic structure formation under the phase contrast microscope at magnification of 40X.

[0273] The microscopic examination revealed the biopolymers formed between WPC-GOS. The biopolymer complex formed between WPC-GOS and chitosan due to the net charge neutralization, and the complex coacervates of WPC-GOS and chitosan was clearly seen as in Fig. -5.

[0274] A further embodiment of the present invention describes the microencapsulation of microbial cells in the complex insoluble coacervates wherein the microbial cells comprises of but not limited to; a lactic acid bacteria (LAB) of a genus selected from the group consisting of Lactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquor ilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfur ilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus and Lentilactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Enterococcus, Bifidobacterium, Brevibacterium, and Staphylococcus.

[0275] The microencapsulation of microbial cells by complex coacervation as described in the present invention is not limited to any specific microbial culture but is a general concept emphasizing the novel complex coacervates formed. Thus, it is contemplated that any type of microbial culture may advantageously be microencapsulated as described herein. EXAMPLE- 4

[0276] An exemplary embodiment describes the microencapsulation of the microbial culture in the said insoluble complex coacervates of WPC-GOS and chitosan as depicted in FIG.- 5 (here LA51). The conjugate and chitosan are mixed with desired quantity of microbial biomass and allowed to interact at proper temperature and pH. The ratio of matrix components to microbial culture is 1 : 1 (wt% / wt%). Fig. 5 clearly depicts macro assemblies of both the biopolymers formed due to the net charge neutralization which was used for microencapsulation of microbial cells (LA51). While the electrostatic interaction is happening between conjugate and chitosan the microbial cells / probiotic (LA51) gets encapsulated or sandwiched between these two matrices. Additives like antioxidants can be added to WPC-GOS and microbial cells mixture before electrostatic interaction with chitosan to form the final desired insoluble complex polymer.

[0277] Yet another aspect of the present invention describes storage of these microencapsulated microbial cultures at ambient temperature until further use. For this purpose, the prepared composition of microbial cells encapsulated in conjugate and complex insoluble coacervate is filled in sterile pipette and the entire composition along with antioxidant is added dropwise into liquid nitrogen to pelletize forming the Pre-Freeze Dried (PFD) pellets which are then stored at -80°C prior to freeze drying. These PFDs were dried using freeze drier (Martin Christ, GmbH) using safe profile (0.3 mbar, 37°C for a time of 26 h). These freeze-dried granulates of each composition were weighed and sealed in an airtight aluminum bag. The bag was then stored at 37°C for about 12 weeks and viability count of each composition stored was determined intermittently and at the end of 12thweek.

[0278] EXAMPLE-5 Storage stability study of the Microencapsulated product.

[0279] The following example demonstrates the cryo / lyo protectant activity of the prepared conjugate and complex coacervate polymer of the present invention. Both the matrices of WPC-GOS and WPC-GOS and chitosan along with sodium ascorbate and LA51 were tested for their cryo / lyo protectant activity. The solutions of WPC-GOS and WPC-GOS and chitosan were heat sterilized before they were used in the formulations to be added along with microbial cell concentrate. The WPC, GOS, conjugate and chitosan dispersions were sterilized at 90°C for 20 mins and then cooled down to room temperature before its use. Then, both these conjugate and conjugate-chi tosan complexes were reacted for 10 min at 10°C using tube Revolver / Rotator (Thermo Fisher Scientific) to mimic the production conditions at the manufacturing plant.

[0280] 30% Sodium ascorbate solution was filtered through a 0.22-micron syringe filter for sterilizing it and then added to the compositions. Fresh biomass of LA51 of desired composition (as also depicted in the table-1 below) was added to the pre-sterilized WPC-GOS, WPC-GOS and chitosan conjugate along with sodium ascorbate.

[0281] These formulations along with microbial culture were filled in the sterile pipette and added dropwise into liquid nitrogen to pelletize (referred as PFD) and then stored at -80°C prior to freeze drying. These PFDs were dried using freeze drier (Martin Christ, GmbH) using safe profile (0.3 mbar, 32 °C for a time of 26 h). After freeze drying, freeze dried granulates (FD granulates) were tested for water activity (aw), colony forming unit (cfu / g) and FlowCyto analysis for active (%).

[0282] Untreated, non-reacted WPC, GOS, reacted conjugate and chitosan along with sodium ascorbate devoid of microbial cells were kept as a control for comparison (Table 1).

[0283] Table 1. Composition of freeze dried product containing cryoprotectants and microbial cells along with their cryo / lyo protectant activities.

[0284] The WPC-GOS and chitosan containing conjugate-chitosan complex was prepared with varying range of biopolymer ratios from 25:75 to 95:5, the complexation pH was adjusted varying from 3 to 8 for these combinations. The pH was adjusted using 1 M NaOH solution and 1 M phosphoric acid.

[0285] The WPC, GOS, Conjugate and chitosan were used as benchmark to compare the cryo / lyo protectant activity of the conjugate and conjugate complexes. It was seen that when only chitosan was present it had negative effect on the viability and showed significantly lesser protection compared to the other ingredients like WPC, GOS alone and conjugate-chi tosan complexes at higher chitosan ratio. Thus, also iterating the changed physio-chemical nature of the newly formed polymer that exhibited lesser log loss of viable cell count when the polymer had chitosan along with WPC-GOS.

[0286] EXAMPLE-6

[0287] In yet another exemplary embodiment of the present invention the said Conjugate-Chitosan complex were prepared from varying biopolymer ratios ranging from 50:50 to 95:5 with pH varying from 4.03 to 7.4. This study showed that with decrease in the concentration of chitosan in the complex there is slight increase in initial CFU count with corresponding increase in % active cell count (Fig.6).

[0288] The output of this study showed that the WPC-GOS conjugate exhibited enhanced cryo / lyo protectant activity (7.65E+11 cfu / g) compared to WPC (2.25+11 cfu / g), GOS (3.10E+11 cfu / g) and cells (no protectant) (1.28E+11 cfu / g) alone. The present invention also claims an insoluble complex coacervate polymer of the conjugate along with chitosan. This complex insoluble coacervate conjugate also showed enhanced cryo / lyo protectant activity (4.25E+11 cfu / g) compared to the cells (no protectant) (1.28E+11 cfu / g) alone. The conjugate (5.10 LoglO loss) and the insoluble complex coacervates of conjugate and chitosan (0.90 LoglO loss) provided enhanced storage stability compared to cells (no protectant) (6.26 LoglO loss) at the elevated temperature (37°C) after 12 weeks of storage.

[0289] The results of the above study with varying ratio of biopolymers in the conjugate showed that conjugates of WPC and GOS showed enhanced cryo / lyo-protectant compared to WPC, GOS or microbial cells alone (no protectants). Furthermore, the complex insoluble coacervate polymer of conjugates and chitosan with different ratios of conjugates and chitosan combined with different pH exhibited higher lyo / cryo-protectant activity compared to the microbial cells only (no protectant) (table 1 & Fig. 6).

[0290] A further embodiment of the present invention describes the use of antioxidant or a combination thereof, selected from the group consisting of sodium ascorbate, salts of citric acid such as sodium citrate; glutathione etc; in the composition along with WPC, GOS, chitosan and microbial biomass.

[0291] Stability Study- Storage stability of conjugates and insoluble coacervates of conjugates and chitosan along with antioxidant.

[0292] The freeze dried granulates of each composition as in table 1 were weighed and placed in an aluminum bag and the bag was sealed such that there was no air inside it. The bag was then stored at 37°C for about 12 weeks and the CFU / g count of each composition stored was determined at the end of 12th week. Samples were also withdrawn at predetermined time intervals and analyzed for CFU / g to give the viability of microbial cultures under storage for different time duration in these 12 weeks of span.

[0293] EXAMPLE-7 The following exemplary description describes the results of the Viability study.

[0294] Viable cell counts were determined in freeze-dried granulates sampled immediately after freeze-drying and at selected time points during the stability studies. A standard pour-plating method was used. For this study freeze-dried material was suspended in sterile peptone saline diluent and homogenized by stomaching. After 30 minutes of revitalization, stomaching was repeated, and the cell suspension was serially diluted in peptone saline diluent. The dilutions were plated in duplicates on MRS agar (BD Difco™ Lactobacilli MRS Agar, Fisher Scientific). The agar plates were incubated anaerobically for three days at 37°C. Plates with 30 - 300 colonies were chosen for counting of colony forming units (CFU). The result presented herein is average CFU / g freeze-dried sample, calculated from the duplicates.

[0295] The viability study of the present invention showed that different type of ingredient of the composition had different impact on the storage stability of microbial culture (LA51). The use of single ingredients such as WPC, GOS, Conjugate and Chitosan, did not impart the desired protection to the microbial culture during storage at aw< 0.10, T=37°C. Use of single ingredient in the composition showed more than 4 LogioLoss (CFU / g). Further, all the compositions which were prepared using the advanced polymer of conjugates-chitosan complex showed better cryo / lyo protection in terms of less than 3 Logio Loss after 12 weeks of storage at accelerated conditions (aw< 0.10, T=37°C) compared to their single ingredient form. (Table-1)

[0296] The conjugate-chitosan complex polymer prepared at pH 6.8 with biopolymer ratio of 85: 15 showed significantly higher protection at accelerated conditions among all the other compositions. (Fig. -6) This composition showed only 0.90 Log 10 Loss after 12 weeks of storage, which proved that the optimal biopolymer ratio and pH are important parameters to form best complex coacervation pair that will extend shelf life of microbial biomass at accelerated storage conditions.

[0297] Fig. 7 depicts survival rate of LA51 at condition of (aw= 0.05, T=37°C) for 12 weeks. Cells with only WPC showed higher rate of log loss, followed by only cells with no protectant, followed by GOS. WPC-COS conjugate showed better protectant activity in the given conditions.

[0298] Fig. 8 depicts survival rate of LA51 at condition of (aw= 0.05, T=37°C) for 12 weeks. Only Cells showed higher rate of log loss, followed by conjugate and chitosan when used alone with cells. Conjugate- chitosan complex coacervate polymer with ratio of 85:15 and 95:5 showed least LoglO Loss after 12 weeks of storage.

[0299] The covalent conjugated product of GOS to WPC prepared in the present invention is based on the principles of Maillard reaction. The Maillard reaction is a nonenzymatic covalent conjugation reaction, which is seen mostly in food processing during heat treatment for example in bread and other baked goods, milk powder, meat products, and fried foods. In the initial steps of the Maillard reaction there is a formation of Schiff-base conjugates via covalent bonding of a reducing sugar and an amino compound present in proteins. In the present invention, the reactions were planned in such a manner that only mild conditions prevail for the formation of the conjugates and advanced Maillard reaction products such as Melanoidins (the browning pigments) were avoided.

[0300] The WPC used in the present conjugate was from commercially available from Aria foods, Denmark. Similarly, the GOS was commercially obtained from Zytex Biotech Private Limited, Mumbai, India. The present invention harbors the technique of complexation for entrapping desired product in the formed polymer wherein the microbial cells get encapsulated / entrapped between two different biopolymers. Conventional in the art techniques involved simple blending of microbial cells with different matrices which did not provide the complete protection to microbial cells due to deficient encapsulation. The conjugate-chi tosan complex coacervate polymer of the present invention is such that it has allowed to combine the functional properties of WPC, GOS and chitosan together resulting in the novel functional composition that has not only provided cryo / lyo protection but also provided a better stability to microbial cells under accelerated storage conditions compared to benchmark.

[0301] The complex polymer of the present invention is prepared using thermal processing. The method of the present invention using novel combination of matrices has technological advancement to the present invention over existing methods in the prior art. The prior art majorly describes matrices with negatively charged polymers that are food-grade. The present invention however makes use of two matrices to form a single complex coacervate polymer wherein the two matrices are of contrast charges. Furthermore, the two matrices used in the present invention are such that when interacted electrostatically at desired pH give a product that is completely different to its parent compounds in its physical and chemical properties.

[0302] The use of the composition of the present invention for providing storage stability can also be extended to other genera of microorganisms including bacteria and yeast (probiotic and lactic acid bacteria) such as Bifidobacterium, Leuconostoc, Pediococcus, Enterococcus, Propionibacterium, Streptococcus, Bacillus and Saccharomyces. In addition to probiotics, the complex coacervation formulations can also be used for microencapsulation of natural colors and other bio-actives such as omega fatty acids, antioxidant extracts etc.

[0303] The present invention thus provides for an advanced solution for obtaining stable polymers for encapsulation techniques. The present invention describes the composition and method to obtain a complex polymer with opposite charge molecules that electrostatically interact with each other giving a thermostable conjugate that can encapsulate a wide range of products and provide thermostability to products like microbial culture when encapsulated in them and increase shelf storage life.

Claims

CLAIMS1. A microencapsulated microbial culture, said microencapsulated microbial culture comprising: i) a microbial culture, ii) a first matrix comprising a first coacervate component, and iii) a second matrix comprising a second coacervate component, wherein said first coacervate component is a protein-carbohydrate conjugate and second- coacervate component is a polysaccharide.

2. The microencapsulated microbial culture according to claim 1, wherein the protein- carbohydrate conjugate is a Whey Protein Concentrate (WPC) and Galactooligosaccharide (GOS) conjugate, and the polysaccharide is chitosan.

3. The microencapsulated microbial culture according to claim 1, wherein the microbial culture comprises a bacterium and / or a yeast.

4. The microencapsulated microbial culture according to any one of the preceding claims, wherein the microbial culture is a probiotic culture.

5. The microencapsulated microbial culture according to any one of the preceding claims, wherein the microbial culture is or comprises a lactic acid bacteria (LAB) of a genus selected from the group consisting of Lactobacillus, Holzapfelia, Amylolactobacillus, Bifidobacterium, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfur ilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, Lentilactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Enterococcus, Bifidobacterium, Brevibacterium, and Staphylococcus.

6. The microencapsulated microbial culture according to any one of the preceding claims,wherein the ratio (wt% / wt%) of WPC-GOS to chitosan is between 50:50 to 95:5.

7. The microencapsulated microbial culture according to any one of the preceding claims, wherein the ratio (wt% / wt%) of the first and second matrix to microbial culture is between 0.25-10.

8. A composition comprising the microencapsulated microbial culture according to any one of the preceding claims, wherein the composition further comprises one or more additives selected from the group consisting of food-grade ingredients, pharmaceutical ingredients and excipients.

9. A product comprising the microencapsulated microbial culture according to any one of the preceding claims, wherein the product is selected from the group consisting of a feed, a plant health product, a food, a beverage and a pharmaceutical product.

10. A method for preparing a microencapsulated microbial culture according to any one of the preceding claims or a composition according to claim 8, said method comprising the steps of: i) mixing a microbial culture with a first matrix comprising first coacervate component and optionally one or more antioxidant to form a pre-complex solution, and ii) mixing the pre-complex solution with a second matrix comprising a second coacervate component to form a microencapsulated microbial culture, wherein said first coacervate component is a WPC-GOS conjugate and second coacervate component is chitosan.

11. The method according to claim 10, wherein in step i) the microbial culture is mixed with the first matrix comprising first coacervate component for a time period in the range of 5 min to 6 hours, at a temperature in the range of 4°C to 45°C, to form the pre-complex solution, and in step ii) mixing the pre-complex solution with the second matrix comprising chitosan for a time period in the range of 5 min to 6 hours, at a temperature in the range of 4°C to 45°C.

12. The method according to claims 10 or 11, wherein the method for preparing the first matrix comprising WPC and GOS prior to step i), comprises the steps of: a) preparing individual solutions of WPC and GOS at desired pH and hydratingthem overnight, b) mixing the overnight hydrated individual solutions of WPC and GOS of step a) at desired pH in desired proportion and heat incubating to form the WPC-GOS conjugate.

13. The method according to claim 12, wherein in step a) the WPC and GOS solutions are individually prepared at the pH range of 7-10 and hydrated overnight at room temperature, and in step b) the overnight hydrated individual solutions of WPC and GOS of step a) are mixed at the pH in the range of 3-10, preferably at pH 9, and incubated at 65-95°C for 2-4 hours to form the conjugate.

14. A microencapsulated microbial culture or a composition obtainable by a method according to any one of claims 12-14.

15. Use of a microencapsulated microbial culture according to claims 1-7 or a composition according claim 8 in a product selected from the group consisting of a feed, a plant health product, a food, a beverage and a pharmaceutical product.

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