Microencapsulated microbial culture formulations with high storage stability

Abstract

The present invention relates to microencapsulated microbial cultures with high storage stability and methods of producing the same. In particular, the present invention relates to microbial cultures formulated by encapsulating a matrix material to a core material in a high ratio.

Description

Technical Field

[0001] The present invention relates to a microencapsulated microbial culture having high storage stability and a method for producing the same. In particular, the present invention relates to a microbial culture prepared with a high ratio of encapsulation matrix material to core material. Background Art

[0002] In the human body and animals, microbial cultures such as lactic acid bacteria (LAB) are part of the normal microbial flora. Lactic acid bacteria are mainly used as starter cultures for fermented milk foods and beverages because they help improve nutritional and sensory properties and extend shelf life. It is reported that some LAB strains show health benefits to humans and animals and can therefore be called probiotic strains. The typical process for producing LAB is fermentation, followed by concentration and freezing of cell biomass. When applying LAB, it is usually necessary to use freeze drying (FD) to produce a dry powder form. Before being used in final applications, dry powders are usually stored for a long period of time.

[0003] Microbial cultures (e.g., LAB) are very sensitive to environmental stresses imposed during freezing and FD, so cryoprotectants / lyophilization protectants must be added during processing to protect and maintain viability. In addition, it is well known that storage of microbial cultures (e.g., LAB) at ambient temperature (25-35° C.) or higher can adversely affect the viability of the microbial cultures. Therefore, storage for long periods of time requires expensive cooling equipment that is not always available when used.

[0004] Although there are some cryoprotectant / lyoprotectant concepts for protecting LAB during freezing and FD, all of these protectants (ingredients) have limited effects on storage stability. Therefore, there is an unmet need for methods to protect microbial cultures during freezing and FD while enhancing the storage stability of these dry microbial cultures.

[0005] Therefore, it would be advantageous to provide an improved method for preparing dried microbial cultures which, after freezing or freeze drying, retain viability even when stored at elevated temperatures for extended periods of time. In particular, such methods and the dried microbial cultures themselves may be advantageous in the preparation of products exposed to conditions of increased environmental stress. SUMMARY OF THE INVENTION

[0007] The present invention relates to a microencapsulation method, wherein a microbial culture is formulated with a high ratio of encapsulation matrix material to core dry matter, and an encapsulation matrix is ​​selected that effectively protects the encapsulated microbial culture, forming agglomerates. In particular, the present invention discloses a method for producing microencapsulated microbial cultures that can withstand dry processing and exhibit enhanced storage stability even when stored for long periods of time at a temperature of 25°C. The obtained microencapsulated microbial cultures are very suitable for applications where storage at low temperatures is not feasible.

[0008] It is therefore an object of the present invention to provide a method for preparing a microbial culture which can be used independently of refrigerated storage.

[0009] In particular, it is an object of the present invention to provide improved methods for producing dry microbial cultures that retain cell viability after dry processing and high temperature storage.

[0010] Thus, one aspect of the present invention relates to a microencapsulated microbial culture, wherein the microbial culture is encapsulated in an agglomerate comprising i) and ii):

[0011] i) core material comprising microbial cultures, and

[0012] ii) an encapsulating matrix comprising one or more matrix components,

[0013] wherein the ratio of encapsulating matrix to core material (wt% / wt%) is at least 2.

[0014] Another aspect of the present invention relates to a composition comprising the microencapsulated microbial culture described herein.

[0015] Another aspect of the present invention relates to a product comprising the microencapsulated microbial culture or composition described herein, wherein the product is selected from the group consisting of feed, plant health products, food, beverages and pharmaceuticals.

[0016] Another aspect of the present invention relates to a method for preparing the microencapsulated microbial culture or composition described herein, the method comprising the steps of:

[0017] i) mixing a microbial culture with a first matrix comprising one or more first matrix components to form a first mixture, and

[0018] ii) mixing the first mixture with a second matrix comprising one or more second matrix components to form a microencapsulated microbial culture,

[0019] The first matrix component and the second matrix component comprise:

[0020] a) maltodextrin, trehalose dihydrate and sodium ascorbate;

[0021] b) trehalose dihydrate, sodium ascorbate, sodium caseinate and high methoxyl pectin;

[0022] c) trehalose dihydrate, sodium ascorbate, sodium caseinate and guar gum;

[0023] d) trehalose dihydrate, sodium ascorbate, sodium caseinate and sodium carboxymethylcellulose;

[0024] e) trehalose dihydrate, sodium ascorbate, high methoxyl pectin and rice protein; or

[0025] f) Trehalose dihydrate, sodium ascorbate, amidated pectin and hydroxypropyl methylcellulose thickener.

[0026] Yet another aspect of the present invention relates to a microencapsulated microbial culture or composition obtainable by the method described herein.

[0027] Yet another aspect of the present invention relates to the use of the microencapsulated microbial culture or composition described herein in a product selected from the group consisting of feed, plant health products, food, beverages and pharmaceuticals.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Initial CFU / g of homogeneous and heterogeneous preparations diluted in CaC03 at an EI of 1 or 4 are shown, respectively.

[0030] Figure 2 The Log of homogeneous and heterogeneous formulations at EI of 1 or 4 after storage at 37°C and accelerated conditions of 0.4Aw for 2 weeks are shown. 10 CFU / g reduction.

[0031] Figure 3 Shown are the absolute CFU / g counts of homogeneous and heterogeneous formulations at EI 4 after storage at accelerated conditions of 37°C and 0.4 Aw for up to 4 weeks, respectively.

[0032] Figure 4 The log of the matrix preparation is shown when EI=1. 10 Graph of loss (CFU / g).

[0033] Figure 5 The log of animal combined with lactobacillus of matrix preparation at EI = 4 is shown 10 Graph of loss (CFU / g).

[0034] Figure 6 The logs show the comparison of animal combined lactobacillus at EI 1 and 4 after 12 weeks. 10Graph of loss (CFU / g).

[0035] Figure 7 The Lactococcus lactis subsp. Lactis Log of the matrix preparation at EI = 1 10 Graph of loss (CFU / g).

[0036] Figure 8 The log of Lactococcus lactis subsp. lactis in the matrix preparation at EI=4 is shown 10 Graph of loss (CFU / g).

[0037] Fig. 9 The log of the comparison of Lactococcus lactis subsp. lactis at EI of 1 and 4 after 12 weeks 10 Graph of loss (CFU / g).

[0038] Fig.10 The Streptococcus thermophilus log of the matrix preparation at EI = 1 is shown 10 Graph of loss (CFU / g).

[0039] Fig.11 Shown is the log of Streptococcus thermophilus in matrix preparation at EI=4 10 Graph of loss (CFU / g).

[0040] Fig.12 The logs show the comparison of Streptococcus thermophilus at EI 1 and EI 4 after 12 weeks. 10 Graph of loss (CFU / g).

[0041] Fig.13 The Logarithm of Bifidobacterium animalis subsp. lactis of the matrix preparation when EI=1 is shown. 10 Graph of loss (CFU / g).

[0042] Fig.14 is a graph showing the Log10 loss (CFU / g) of Bifidobacterium animalis subsp. lactis of a matrix formulation at EI=4.

[0043] Fig.15 Shows the comparison of Bifidobacterium animalis subsp. lactis at EI 1 and 4 after 12 weeks 10 Graph of loss (CFU / g).

[0044] Fig.16is a microscopic image of Lactobacillus animalis (DSM 33570) encapsulated in MF-01.

[0045] Fig.17 is a microscopic image of Lactobacillus animalis (DSM 33570) encapsulated in MF-02.

[0046] Fig.18 is a microscopic image of Lactobacillus animalis (DSM 33570) encapsulated in MF-03.

[0047] Fig.19 is a microscopic image of Lactobacillus animalis (DSM 33570) encapsulated in MF-04.

[0048] Fig. 20 is a microscopic image of Lactobacillus animalis (DSM 33570) encapsulated in MF-05.

[0049] Fig.21 is a microscopic image of Lactobacillus animalis (DSM 33570) encapsulated in MF-06.

[0050] The present invention will be described in more detail below. DETAILED DESCRIPTION OF THE INVENTION

[0052] definition

[0053] Before outlining the present invention in more detail, a number of terms and conventions should first be defined:

[0054] Core Materials

[0055] In the present context, the term "core material" refers to a preparation comprising a microbial culture. The microbial culture is preferably provided in the form of a liquid cell concentrate.

[0056] Microbial cultures

[0057] In this context, the term "microbial culture" refers to a population of microorganisms. Microorganisms include all single-celled organisms, such as archaea and bacteria, and also many multicellular organisms, such as fungi and algae. Microbial cultures as referred to herein do not include harmful microorganisms that contribute to spoilage or disease risks.

[0058] Probiotic cultures

[0059] In this context, the term "probiotic" or "probiotic culture" refers to a culture of microorganisms that, when ingested by humans or animals in the form of viable cells, improves health, for example by inhibiting harmful microorganisms in the gastrointestinal tract, enhancing the immune system, or promoting nutrient digestion. Probiotics can also be applied to plants. Probiotic cultures can contain bacteria and / or fungi.

[0060] Lactic acid bacteria (LAB)

[0061] In this context, the term "lactic acid bacteria (LAB)" refers to a group of Gram-positive, catalase-negative, non-motile, microaerophilic or anaerobic bacteria that ferment sugars while producing acids (including lactic acid, acetic acid, formic acid and propionic acid as the main acids produced). The most useful industrial lactic acid bacteria include, but are not limited to, Lactococcus spp., Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Brevibacterium spp., Enterococcus spp. and Propionibacterium spp. In addition, lactic acid-producing bifidobacteria belonging to the group of strictly anaerobic bacteria, i.e., Bifidobacterium spp., are often used alone or in combination with lactic acid bacteria as food starter cultures and 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) are also referred to as LAB (Seifert & Mogensen (2002)).

[0062] Encapsulation matrices and matrix components

[0063] In the present context, the term "encapsulation matrix" refers to a coating or layer that encloses a microbial culture. The coating or layer comprises one or more matrix components.

[0064] Preferably, all matrix components of the encapsulation matrix are food grade, e.g., conforming to the Food Chemicals Codex (FCC) and / or generally recognized as safe (GRAS) components. Matrix components include, but are not limited to, antioxidants, cryoprotectants, carbohydrates, proteins, alone or in combination with each other.

[0065] Microencapsulation

[0066] In the present context, the term "microencapsulated" refers to an entity isolated from the surrounding environment on a micrometer scale. Thus, a microencapsulated microbial culture is a microbial culture that is partitioned into different entities that are separated from each other and dispersed in a culture medium.

[0067] Aggregates

[0068] In the present context, the term "agglomerates" refers to an aqueous phase (or droplets) rich in a microbial culture. In the present context, agglomerates are formed when a heterogeneous (phase-separated) encapsulation matrix is ​​applied. In turn, agglomerates are formed due to liquid-liquid phase separation providing a dense phase (or droplets) and a dilute phase in thermodynamic equilibrium with each other. This process is also known as separate phase separation.

[0069] Therefore, the coacervation formulation described herein refers to a formulation comprising aggregates of a microbial culture.

[0070] As used herein, aggregates are distinguished from complex aggregates, which are limited to unique forms of aggregates formed by electrostatic interactions of oppositely charged biopolymers. This process is also known as associative phase separation.

[0071] vitality

[0072] In this context, the term "viability" refers to living cells in a culture. Thus, the viability of a cell culture can be determined by measuring the number of colony forming units (CFU). CFU refers to the number of individual colonies of any microorganism that grow on a plate of culture medium. This value in turn represents the number of bacteria or fungi that are able to replicate because they have formed colonies on the plate.

[0073] Samples were taken immediately after lyophilization and at selected time points during the stability study to determine the viable cell count. The standard pour-plating method was used. In brief, a known amount of sample was homogenized with a specific volume of diluent (1:100) using a stomacher, and the solution was resuspended with a vortex mixer and then decimal dilutions were made in peptone saline diluent (also known as "maximum recovery diluent (MRD)"). MRD contains peptone, NaCl and deionized water. The diluent was poured into a plate, mixed with MRS Agar (Hi-media, M641), and incubated anaerobically at 37°C for three days. After incubation, colonies were counted manually. Results were reported as average CFU / g lyophilized sample calculated in duplicate.

[0074] Antioxidants

[0075] In this context, the term "antioxidant" refers to a compound that inhibits oxidation. Antioxidants can be industrial chemicals or natural compounds. As used herein, antioxidants include, but are not limited to, trisodium citrate, vitamin C, vitamin E, glutathione, ascorbic acid, ascorbyl palmitate, quercetin, gallic acid, and tocotriene.

[0076] It should be understood that antioxidants as used herein include mineral salts of vitamin C, such as sodium ascorbate. Vitamin E should also be understood to include all variants (alpha, beta, gamma, delta) of tocopherol and tocotrienol.

[0077] Hydrophobic coating

[0078] In this context, the term "hydrophobic coating" refers to a hydrophobic layer or shell located on the surface of the aggregate. Such a hydrophobic layer or shell may comprise one or more hydrophobic compounds or molecules containing hydrophobic moieties that render the outer surface of the aggregate hydrophobic.

[0079] The hydrophobic coating may include, but is not limited to, fats and waxes such as carnauba wax, beeswax, cocoa butter, hydrogenated palm oil, palm stearin, shea butter, mango butter, soybean oil, olive oil, coconut oil, rice bran oil, sunflower oil, candelilla wax, rice bran wax, and laurel wax.

[0080] Food grade ingredients

[0081] In this context, the term "food-grade ingredient" refers to any compound that is non-toxic, safe to eat, and complies with the Food Chemicals Codex (FCC) and / or Generally Recognized as Safe (GRAS). Food-grade ingredients include, but are not limited to, compounds that can change properties such as aroma, flavor, acidity, color, viscosity, and texture, as well as preservatives, nutrients, thickeners, sweeteners, and emulsifiers.

[0082] Drug integrity

[0083] In the present context, the term "drug ingredient" refers to an ingredient in a pharmaceutical preparation that is not the active ingredient.

[0084] Pharmaceutical ingredients include, but are not limited to, calcium carbonate, sodium carboxymethylcellulose, talc, polydimethylsiloxane, hydroxypropylcellulose, and hydroxypropylmethylcellulose.

[0085] excipient

[0086] In this context, the term "excipient" refers to a natural or synthetic substance formulated with a pharmaceutically active ingredient or a pharmaceutical ingredient (an ingredient that is not an active ingredient) and includes substances used to stabilize, bulk or impart therapeutic enhancement to the active ingredient in the final dosage form, such as promoting drug absorption, reducing viscosity, increasing solubility, adjusting tonicity, alleviating discomfort at the injection site, lowering the freezing point or increasing stability.

[0087] Examples of excipients include, but are not limited to, microcrystalline cellulose, titanium dioxide, and aluminum silicate.

[0088] feed

[0089] In this context, the term "feed" refers to food given to livestock. Livestock include, but are not limited to, pets such as dogs, cats, rabbits, hamsters, etc.; livestock such as cattle, sheep, pigs, goats, etc.; and beasts of burden such as horses, camels, donkeys, etc.

[0090] Feed can be blended from various raw materials and additives and specially formulated according to the requirements of the recipient animal. Feed can be provided in the form of, for example, powdered feed, crumbled feed or pelleted feed.

[0091] The term "feed" also includes premixes, which consist of ingredients such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products and combinations thereof. Premixes are usually added to the feed given to livestock as a nutritional supplement.

[0092] Water activity

[0093] In this context, the term "water activity" refers to the partial vapor pressure of water in a substance divided by the partial vapor pressure of water in its standard state. Water activity is denoted by Aw. Specifically, the Aw of a food is the ratio of the vapor pressure of the microencapsulated microbial culture itself when it is in equilibrium and completely undisturbed by the surrounding air medium to the vapor pressure of distilled water under the same conditions.

[0094] Generally speaking, water will migrate from areas of high Aw to areas of low Aw. By formulating a product with a low Aw, the storage stability of a food can often be extended.

[0095] The water activities mentioned in this article were measured using a Rotronics water activity analyzer with an HC2-AW probe. WA-1 / Pt-100, 1 / 3DIN B-class humidity sensor, and humidity calculation by dew point or frost point method.

[0096] In short, the sample is placed in the sample cup, filling it to within 3mm of the rim, while ensuring that there is as little air as possible in the container to ensure faster equilibration time. Next, the measuring head is placed on the sample holder, ensuring a seal. Water activity is measured using the predictive model of the Rotronic water activity analyzer.

[0097] Water activity is calculated using the following formula: Aw = p / p s and %ERH = 100 X Aw, where p = partial pressure of water vapor at the product surface; p s = saturated vapor pressure of water vapor above pure water at product temperature / partial pressure of water vapor; ERH = equilibrium relative humidity.

[0098] Storage stability

[0099] In this context, the term "storage stability" refers to the ability of a microencapsulated microbial culture to maintain viability when stored for extended periods of time under accelerated storage conditions, such as 2 weeks at 37°C and a water activity (Aw) ≤ 0.4.

[0100] Storage stability can be determined by analyzing how the count of viable microbial cells develops over time. As described herein, the viability of a microbial culture is measured by determining CFU / g. Thus, a measure of storage stability of a microencapsulated microbial culture can be determined by evaluating the CFU / g of the microencapsulated microbial culture particles at time 0 (just after drying) and after 2 weeks (or 4 weeks) of storage under accelerated storage conditions.

[0101] Briefly, the storage stability of FD ground powder was studied as follows: FD granule samples of microbial culture were ground for 30 seconds in a coffee blender and then passed through a 60 mesh (250 μ) sieve, followed by dilution 100 times in CaCO3 to obtain a sample with a water activity (Aw) of 0.4. The sample was placed in an aluminum bag, which was then sealed so that no air was trapped inside. The bag was stored at 37°C for 2 weeks (or 4 weeks), and then the CFU / g of the sample was determined.

[0102] Microencapsulated microbial cultures, compositions comprising microencapsulated microbial cultures, and methods for producing the same

[0103] Microbial cultures, such as lactic acid bacteria (LAB), play a key role in many fermented products, where they increase the nutritional value of the product and improve, for example, the sensory and textural characteristics of food. In order to obtain the final product, the microbial culture is usually obtained separately in the form of a powdered composition and mixed with other ingredients. Therefore, the powdered composition containing the microbial culture needs to remain active at least from the time it becomes dry granules to the time when the powdered microbial culture is included in the final product. Ideally, the microbial culture should be kept refrigerated during transportation, supplementary processing and as part of the final product. However, this is not always possible, because refrigerated transportation and storage are both expensive and, in many cases, such as in developing countries or remote areas, are not feasible. In addition, the final product may be an item that is not easy to store under refrigerated conditions. Animal feed is usually in this case.

[0104] In order to deliver high quality (e.g., high viability) microbial cultures under such environmental stress conditions, it is necessary to reduce yield losses during downstream processing and eliminate the requirement for refrigerated transportation and storage. However, there is no method that provides both sufficient freezing / lyophilization protection and enhanced storage stability at high temperatures.

[0105] Provided herein is a method for microencapsulating a microbial culture in an encapsulation matrix that isolates the microbial culture from the surrounding environment. The microbial culture is encapsulated at a high ratio of encapsulation matrix to core material, thereby maintaining the viability of the microencapsulated microbial culture during long-term storage at high temperatures.

[0106] Thus, aspects of the present invention relate to microencapsulated microbial cultures, wherein the microbial cultures are encapsulated in agglomerates comprising i) and ii):

[0107] i) core material comprising microbial cultures, and

[0108] ii) an encapsulating matrix comprising one or more matrix components,

[0109] wherein the ratio of encapsulating matrix to core material (wt% / wt%) is at least 2.

[0110] The ratio of encapsulating matrix material to core material in a formulation (wt% / wt%) is also referred to as the encapsulation index (EI). Without being bound by theory, it is believed that microencapsulated microbial cultures formulated with a high EI will more effectively limit water absorption, thereby improving storage stability.

[0111] Thus, embodiments of the present invention relate to microencapsulated microbial cultures as described herein, wherein the ratio of encapsulation matrix to core material (wt% / wt%) is in the range of 2-10, such as in the range of 2-8, such as in the range of 4-8.

[0112] Another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the ratio of encapsulation matrix to core material (wt% / wt%) is at least 3, preferably at least 4.

[0113] In some variants, the core material can be composed entirely of microbial cultures. Therefore, microencapsulated microbial cultures can also be defined according to the ratio of encapsulation matrix to microbial culture (wt% / wt%). Therefore, embodiments of the present invention relate to microencapsulated microbial cultures as described herein, wherein the ratio of encapsulation matrix to microbial culture (wt% / wt%) is at least 2, such as at least 3, preferably at least 4.

[0114] The encapsulation matrix preferably comprises more than one component. Combinations of encapsulation matrix components having different chemical properties allow for tailoring of microencapsulation according to a specific microbial culture or a specific application. Thus, embodiments of the present invention relate to microencapsulated microbial cultures as described herein, wherein the encapsulation matrix comprises at least two matrix components, such as at least three matrix components, such as at least four matrix components.

[0115] The encapsulation matrix and its components are selected so that aqueous droplets (agglomerates) are formed after mixing with the microbial culture. These agglomerates are enriched with the microbial culture. Preferably, a heterogeneous encapsulation matrix is ​​used to form agglomerates. A heterogeneous matrix refers to a matrix containing components that do not mix uniformly when mixed but are separated in discrete phases. Therefore, embodiments of the present invention relate to microencapsulated microbial cultures as described herein, wherein the encapsulation matrix is ​​a heterogeneous matrix.

[0116] In addition to driving the formation of agglomerated formulations, the matrix components can also act as cryoprotectants and / or antioxidants to further enhance the tolerance of the microbial culture to processing and storage. Thus, embodiments of the present invention relate to microencapsulated microbial cultures as described herein, wherein one or more matrix components are selected from carbohydrates, proteins, antioxidants, and combinations thereof.

[0117] Cryoprotectants are used to improve the ability of microbial culture concentrates to survive the deleterious effects of freezing, frozen storage, and freeze drying. Preferably, these cryoprotectants should not be metabolized by the microbial strain to produce acid, as it may lead to loss of viability due to impairment of ATPase membrane-bound enzymes, β-galactosidase, and cell membrane fluidity. Generally, cryoprotectants that do not produce acid are more effective in improving the survival rate of freeze-dried microbial cultures. One preferred class of cryoprotectants is carbohydrates and their related subgroups.

[0118] Thus, embodiments of the present invention relate to the microencapsulated microbial cultures described herein, wherein the carbohydrate is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, polysaccharides, and combinations thereof.

[0119] Another embodiment of the present invention relates to the microencapsulated microbial culture described herein, wherein the monosaccharide is selected from the group consisting of glucose, fructose, galactose, fucose, xylose, erythrose, and combinations thereof.

[0120] Yet another embodiment of the present invention relates to the microencapsulated microbial culture described herein, wherein the disaccharide is selected from trehalose, lactose, sucrose, maltose, cellobiose and combinations thereof. A preferred embodiment of the present invention relates to the microencapsulated microbial culture described herein, wherein the disaccharide is trehalose.

[0121] Another embodiment of the present invention relates to the microencapsulated microbial culture described herein, wherein the oligosaccharide is selected from the group consisting of fructo-oligosaccharide (FOS), galactooligosaccharide (GOS), mannan oligosaccharide (MOS) and combinations thereof.

[0122] Yet another embodiment of the present invention relates to the microencapsulated microbial culture described herein, wherein the polysaccharide is selected from the group consisting of pectin, cellodextrin, gum, alginate, starch, glycogen, cellulose, chitin, inulin, dextran, carrageenan, chitosan and combinations thereof. A preferred embodiment of the present invention relates to the microencapsulated microbial culture described herein, wherein the polysaccharide is pectin.

[0123] Yet another embodiment of the present invention relates to the microencapsulated microbial culture described herein, wherein the gum is selected from the group consisting of acacia, agar, alginate, cassia, dammar, beta-glucan, glucomannan, mastic, chicle, psyllium, spruce, gellan, guar, locust bean, xanthan gum, and combinations thereof.

[0124] Therefore, embodiments of the present invention relate to microencapsulated microbial cultures as described herein, wherein the protein is selected from caseinate, whey protein, gelatin, plant protein (e.g., pea protein, potato protein, and rice protein), and combinations thereof. Preferred embodiments of the present invention relate to microencapsulated microbial cultures as described herein, wherein the protein is sodium caseinate.

[0125] Oxidation is the loss of electrons from an atom or ion. In this context, oxidation refers to the oxidation of molecular oxygen, meaning that the oxygen is metabolized into unstable free radicals that can pry electrons from other molecules. As a result, oxidation can cause damage to cell membranes and other cellular components such as proteins, lipids, and DNA. To avoid damage to the microencapsulated microbial culture, one or more antioxidants can be included in the encapsulation matrix to prevent oxidation. Antioxidants can be of natural or synthetic origin.

[0126] Thus, embodiments of the present invention relate to the microencapsulated microbial cultures described herein, wherein the antioxidant is selected from the group consisting of citrate, ascorbate, tocopherol, ascorbyl palmitate, quercetin, gallic acid, tocotriene, tocotrienols, glutathione, and combinations thereof.

[0127] Another embodiment of the present invention relates to the microencapsulated microbial culture described herein, wherein the antioxidant is selected from vitamin C and / or vitamin E. It should be understood that the antioxidants used herein include mineral salts of vitamin C, such as sodium ascorbate. In addition, vitamin E should also be understood to include all variants (α, β, γ, δ) of tocopherol and tocotrienol.

[0128] Yet another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the antioxidant is selected from trisodium citrate and / or sodium ascorbate. A preferred embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the antioxidant is trisodium citrate.

[0129] An encapsulation matrix surprisingly effectively enhances the storage stability of microbial cultures when stored under ambient storage conditions, the encapsulation matrix comprising four encapsulation matrix components (see Example 2). Thus, a preferred embodiment of the present invention relates to the microencapsulated microbial cultures described herein, wherein the encapsulation matrix comprises sodium caseinate, pectin, trehalose and trisodium citrate.

[0130] Example 3 (Table 2) shows a more specific and more advantageous encapsulation matrix. Therefore, a preferred embodiment of the present invention relates to a microencapsulated microbial culture as described herein, wherein the encapsulation matrix comprises maltodextrin, trehalose dihydrate and sodium ascorbate. A preferred embodiment of the present invention relates to a microencapsulated microbial culture as described herein, wherein the encapsulation matrix comprises trehalose dihydrate, sodium ascorbate, sodium caseinate and high methoxy pectin. A preferred embodiment of the present invention relates to a microencapsulated microbial culture as described herein, wherein the encapsulation matrix comprises trehalose dihydrate, sodium ascorbate, sodium caseinate and guar gum. A preferred embodiment of the present invention relates to a microencapsulated microbial culture as described herein, wherein the encapsulation matrix comprises trehalose dihydrate, sodium ascorbate, sodium caseinate and sodium carboxymethylcellulose. A preferred embodiment of the present invention relates to a microencapsulated microbial culture as described herein, wherein the encapsulation matrix comprises trehalose dihydrate, sodium ascorbate, high methoxy pectin and rice protein. A preferred embodiment of the present invention relates to the microencapsulated microbial culture described herein, wherein the encapsulation matrix comprises trehalose dihydrate, sodium ascorbate, amidated pectin and a hydroxypropylmethylcellulose thickener (e.g., Methocel TM K4).

[0131] The content of encapsulation matrix components can be customized according to the expected application of the specific microbial culture to be packaged and the final powdered product. For example, if the microencapsulated microbial culture is used for the need for long-term storage, the content of antioxidant is preferably increased. The content of antioxidant also can be adjusted according to storage conditions (for example, the degree of exposure to air). Equally, the combination and content of cryoprotectants can also be adjusted to be suitable for specific freezing processing selection. It should be understood that the content of each encapsulation matrix component is provided with wt% relative to the gross weight of the encapsulation matrix.

[0132] Thus, an embodiment of the present invention relates to a microencapsulated microbial culture as described herein, wherein the content of sodium caseinate in the encapsulation matrix is ​​1-6 wt%, such as 2-5 wt%, preferably 3-4 wt%.

[0133] Another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the content of pectin in the encapsulation matrix is ​​0.5-3 wt%, such as 0.75-2.5 wt%, preferably 1-2 wt%.

[0134] Another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the content of trehalose in the encapsulation matrix is ​​10-40 wt%, such as 15-35 wt%, preferably 20-30 wt%.

[0135] Yet another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the content of trisodium citrate in the encapsulation matrix is ​​1-10 wt%, such as 2-8 wt%, preferably 4-6 wt%.

[0136] Yet another embodiment of the present invention relates to the microencapsulated microbial culture described herein, wherein the encapsulation matrix comprises 1-6 wt% sodium caseinate, 0.5-3 wt% pectin, 10-40 wt% trehalose and 1-10 wt% trisodium citrate.

[0137] It is the selection of a large number of encapsulation matrices and encapsulation matrix components that drives the microencapsulation process via agglomeration and enhances storage stability under ambient storage conditions. Therefore, the microencapsulation technology presented herein is not limited to a specific type of microbial culture, but rather a general microencapsulation concept. Therefore, it is contemplated that any type of microbial culture can be advantageously microencapsulated as described herein.

[0138] Bacteria and yeasts are two very important groups of microorganisms that are found in many consumer products. For example, they are included in fermented foods, feed mixes and nutritional supplements, and their health benefits are well documented.

[0139] Thus, embodiments of the present invention relate to the microencapsulated microbial cultures described herein, wherein the microbial culture is a bacterium or a yeast.

[0140] Another embodiment of the present invention relates to the microencapsulated microbial culture 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.

[0141] Of particular interest are lactic acid bacteria (LAB), a class of Gram-positive bacteria with common metabolic and physiological characteristics. Lactic acid produced by LAB is the primary metabolic outcome of carbohydrate fermentation. Since the discovery that acidification of food fermentation can preserve food by inhibiting the growth of rancidity factors, LAB has been purposefully used in food fermentation. However, since efficient food fermentation requires high-quality live microorganisms, the development of fermented foods has been halted in areas where there are no advanced facilities to handle fragile microorganisms. Specifically, microbial cultures, such as LAB, are not easy to handle in some developing countries or remote areas due to the requirements and costs of cold storage facilities. The microencapsulated microbial cultures described herein can tolerate storage at high temperatures, and therefore can open up the development of products containing microbial cultures such as LAB to a wider group of product developers.

[0142] Thus, an embodiment of the present invention relates to a microencapsulated microbial culture as described herein, wherein the microbial culture is a lactic acid bacterium (LAB). Another embodiment of the present invention relates to a microencapsulated microbial culture as described herein, wherein the microbial culture is or comprises a lactic acid bacterium (LAB) selected from the genera Lactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacillus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquilactobacillus. quorilactobacillus), Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, and Lentilactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Enterococcus, Bifidobacterium, Brevibacterium, and Staphylococcus.

[0143] It should be recognized that the Lactobacillus taxonomy has been updated in 2020. The new taxonomy is disclosed in Zheng et al. 2020, and this article will be consistent with it unless otherwise noted. For the purposes of the present invention, Table 1 lists the old and new names of some lactobacilli related to the present invention.

[0144] Table 1. New and old names of some lactobacilli related to the present invention

[0145]

[0146] It has long been known that bacteria of the genus Lactobacillus and related more recently updated genera constitute an important component of the human microbiome, for example in the digestive, urinary and reproductive systems. For this reason, these bacteria have been used extensively in health products and / or nutritional products intended to assist, maintain or restore the natural balance of the microbiome in the human body. Examples of the use of Lactobacillus include the treatment or improvement of diarrhea, vaginal infections and skin diseases, such as eczema.

[0147] Thus, embodiments of the present invention relate to microencapsulated microbial cultures as described herein, wherein the microbial culture is or comprises lactic acid bacteria (LAB) selected from the genera Lactobacillus, Lactobacillus mucilaginosus, Lactobacillus casei, Lactobacillus associating, Lactobacillus casei, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus mucilaginosus, Lactobacillus associating, Lactobacillus lentus, Lactobacillus spp., Lactobacillus associating, Lactobacillus spp. and Lactobacillus plantarum. Another embodiment of the present invention relates to a microencapsulated microbial culture as described herein, wherein the microbial culture is or comprises the following species: Lactobacillus mucilaginosus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus casei, Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum subsp. plantarum, Lactobacillus fermentum mucilaginosus, Lactobacillus animalis, Lactobacillus lentus, Lactobacillus curvifolius, Lactobacillus fuchsii, Lactobacillus sakei subsp. sakei, Lactobacillus pentosus, Lactobacillus acidophillus, Lactobacillus helveticus, Lactobacillus gasseri, and Lactobacillus delbrueckii.

[0148] Yet another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the microbial culture is or comprises a strain selected from the group consisting of:

[0149] Animal combined lactobacillus, which was purchased from Chr. Hansen A / S of Denmark on July 8, 2020, Deposited as DSM33570 at the Leibniz DSMZ Institute - German Collection of Microorganisms and Cell Cultures, 5 GmbH 1 Inhoffenstr. 7B, D-38124 Braunschweig, Germany;

[0150] Bifidobacterium animalis subsp. lactis, which was purchased from Chr. Hansen A / S of Denmark on September 30, 2023, Deposited as DSM 15954 at the Leibniz DSMZ Institute - German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B 38124 Braunschweig, Germany (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) 5 GmbH1 Inhoffenstr. 7B, D-38124 Braunschweig, Germany);

[0151] Streptococcus thermophilus, which was acquired by Chr. Hansen A / S of Denmark on September 30, 2023, Deposited as DSM15957 at the Leibniz DSMZ Institute - German Collection of Microorganisms and Cell Cultures, 5 GmbH 1 Inhoffenstr. 7B, D-38124 Braunschweig, Germany; and

[0152] Lactococcus lactis subsp. lactis, which was purchased from Chr. Hansen A / S, Denmark on April 23, 2008. Deposited as DSM 21404 at the Leibniz DSMZ-German Collection of Microorganisms and Cell Cultures, Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) 5 GmbH 1 Inhoffenstr. 7B, D-38124 Braunschweig, Germany.

[0153] The applicant requests that the above-mentioned deposited microbial samples be made available only to experts, subject to the effective regulations of the industrial property offices of the contracting states of the Budapest Treaty until the date of patent grant.

[0154] Probiotic cultures are live microbial cultures that provide health benefits to individuals after being ingested by individuals. Probiotic microorganisms can be LABs. Products containing probiotic cultures include dairy products, animal feeds, and beverages. Therefore, it should be understood that the microencapsulated microbial cultures described herein can be administered not only to humans, but also to animals and even plants.

[0155] Thus, embodiments of the present invention relate to the microencapsulated microbial cultures described herein, wherein the microbial culture is a probiotic culture.

[0156] Another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the genus of the probiotic culture is selected from the genus Lactobacillus or Bifidobacterium.

[0157] Yet another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the probiotic culture is selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus animalis and Bifidobacterium animalis subsp. lactis.

[0158] Another embodiment of the present invention relates to the microencapsulated microbial culture described herein, wherein the microencapsulated microbial culture is a dry preparation.

[0159] Another embodiment of the present invention relates to the microencapsulated microbial culture described herein, wherein the dry preparation is selected from the group consisting of a freeze-dried preparation, a spray-dried preparation, a vacuum-dried preparation, and an air-dried preparation.

[0160] Another embodiment of the present invention relates to the microencapsulated microbial culture described herein, wherein the dry preparation is freeze-dried.

[0161] During the microencapsulation process, environmental conditions can be controlled to provide agglomerates of high stability. Therefore, an embodiment of the present invention relates to a microencapsulated microbial culture as described herein, wherein the pH of the microencapsulated microbial culture is 6-8, preferably 6.25-7.5. Another embodiment of the present invention relates to a microencapsulated microbial culture as described herein, wherein the pH of the microencapsulated microbial culture is at least 6, such as at least 6.25, such as at least 6.5.

[0162] Storage stability can be further improved by applying a hydrophobic coating on the outside of the microencapsulated microbial culture. Therefore, an embodiment of the present invention relates to a microencapsulated microbial culture as described herein that also contains a hydrophobic coating. Another embodiment of the present invention relates to a microencapsulated microbial culture as described herein, wherein the hydrophobic coating comprises one or more fats or waxes or mixtures thereof. Another embodiment of the present invention relates to a microencapsulated microbial culture as described herein, wherein the hydrophobic coating comprises one or more ingredients selected from carnauba wax, beeswax, cocoa butter, hydrogenated palm oil, palm stearin, shea butter, mango butter, soybean oil, olive oil, coconut oil, rice bran oil, sunflower seed oil, candelilla wax, rice bran wax and laurel wax and combinations thereof.

[0163] In order to bring into play the beneficial effects of the core material, it is preferred that the core material is high quality and comprises a large amount of live microbial cultures. These microorganisms will contribute to the function of the natural population regulation of live microorganisms in the consumer (e.g., intestinal). Therefore, embodiments of the present invention relate to microencapsulated microbial cultures as herein described, wherein the core material comprises 1.0E+07 to 5.0E+11 CFU / g of microbial culture, preferably 1.0E+09 to 1.0E+11 CFU / g of microbial culture, more preferably 1.0E+10 to 5.0E+10 CFU / g of microbial culture.

[0164] Corresponding to certain applications, for example in the feed industry, the microencapsulated microbial culture can be diluted with CaCO. Thus, an embodiment of the present invention relates to a microencapsulated microbial culture as described herein, wherein the microencapsulated microbial culture is diluted with CaCO by at least 10 times, such as by at least 20 times, such as by at least 50 times, such as by at least 100 times, such as by at least 1000 times. Another embodiment of the present invention relates to a microencapsulated microbial culture as described herein, wherein it is a freeze-dried preparation of a diluted microencapsulated microbial culture.

[0165] The combination of encapsulation matrix ingredients and carefully selected process parameters make the microencapsulated microbial culture easily withstand drying and long-term storage under harsh conditions. Therefore, the compositions and products described herein are particularly suitable for distribution to areas where cooling is not possible. For downstream developers or consumers, the viable microbial count after storage is the most important product quality. Therefore, an embodiment of the present invention relates to a microencapsulated microbial culture as described herein, wherein the microbial culture contains 1.0E+06 to 1.0E+11 CFU / g of viable microorganisms after storage for 2 weeks at 37°C and Aw≤0.4. Another embodiment of the present invention relates to a microencapsulated microbial culture as described herein, wherein after storage for 4 weeks at 37°C and Aw≤0.15, the microbial culture contains a content of viable microorganisms of at least 1.0E+05 CFU / g, such as at least 1.0E+06 CFU / g, such as at least 1.0E+07 CFU / g, such as at least 1.0E+08 CFU / g, preferably at least 1.0E+09 CFU / g, more preferably at least 1.0E+10 CFU / g.

[0166] Yet another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein after storage for 2 weeks at 37°C and Aw≤0.4, the loss of viability of the microbial culture is less than 3 log units, preferably less than 2.5 log units, when measured in CFU / g.

[0167] Microencapsulated microbial cultures are often used as additives in final products that also include other ingredients. Therefore, it is expected that for many applications, microencapsulated microbial cultures will become part of a more complex composition. Therefore, aspects of the present invention relate to compositions comprising microencapsulated microbial cultures as described herein.

[0168] When the microencapsulated microbial culture is mixed with other ingredients, the agglomerates formed during the mixing of the encapsulation matrix and the core material remain intact to protect the microbial culture from any environmental influences that may be harmful to the microorganisms. Therefore, embodiments of the present invention relate to compositions as described herein, wherein the composition comprises agglomerates comprising a microbial culture and an encapsulation matrix.

[0169] Microencapsulated microbial cultures can be used in a variety of different types of applications, such as from health products, nutritional supplements, pharmaceuticals to animal feed. Therefore, compositions comprising microencapsulated microbial cultures can include a variety of additives. Therefore, embodiments of the present invention relate to compositions as described herein, wherein the composition further comprises one or more additives selected from food grade ingredients, feed grade ingredients, pharmaceutical ingredients and excipients.

[0170] Food-grade ingredients are compounds that are non-toxic, safe for consumption, and comply with the Food Chemical Codex (FCC). Food-grade ingredients include, but are not limited to, compounds that alter properties such as aroma, flavor, acidity, color, viscosity, and texture, as well as preservatives, nutrients, thickeners, sweeteners, and emulsifiers.

[0171] Embodiments of the present invention relate to the compositions, products or dairy products described herein, wherein the one or more food-grade ingredients are selected from compounds that can modify properties such as aroma, flavor, acidity, color, viscosity and texture, as well as preservatives, nutrients, thickeners, sweeteners, emulsifiers and combinations thereof.

[0172] Another embodiment of the present invention relates to the composition as described herein, wherein the food grade ingredient is selected from the group consisting of lactose, maltodextrin, whey protein, casein, corn starch, dietary fiber, gums and gelatin.

[0173] Another embodiment of the present invention is directed to the composition as described herein, wherein the pharmaceutical ingredient is selected from the group consisting of calcium carbonate, sodium carboxymethylcellulose, talc, polydimethylsiloxane, hydroxypropylcellulose and hydroxypropylmethylcellulose.

[0174] Yet another embodiment of the present invention relates to the composition as described herein, wherein the excipient is selected from the group consisting of microcrystalline cellulose, titanium dioxide and aluminum silicate.

[0175] Many additives are provided in dry powder form to extend shelf life and facilitate the handling of the additive. Microencapsulated microbial cultures are no exception and can preferably be provided in dry form. Therefore, embodiments of the present invention relate to compositions as described herein, wherein the composition is a dry preparation.

[0176] The dry powder formulation comprising the microbial culture can be obtained by any suitable method that does not significantly reduce the viability of the microbial culture. Therefore, an embodiment of the present invention relates to the composition described herein, wherein the composition is a freeze-dried composition.

[0177] Yet another embodiment of the present invention relates to the microencapsulated microbial culture or composition as described herein, wherein the microencapsulated microbial culture or composition is in the form of powder and / or granules.

[0178] Keeping the water activity of the dry formulation low will help the dry formulation not to spoil over time. By controlling the water activity (Aw), the effects of moisture migration on the product can be predicted and adjusted. Generally, a water activity of less than 0.6 should avoid any negative microbial growth. Therefore, embodiments of the present invention relate to microencapsulated microbial cultures or compositions as described herein, wherein the water activity (Aw) of the microencapsulated microbial culture is 0.01-0.8, preferably 0.05-0.6, and most preferably 0.1-0.4.

[0179] Microbial cultures can find different applications in many different consumer sectors. Thus, aspects of the present invention relate to products comprising a microencapsulated microbial culture or composition as described herein, wherein the product is selected from feed, plant health products, food, beverages and pharmaceuticals.

[0180] In particular, the use of microencapsulated microbial cultures in animal feed is a preferred application. Therefore, embodiments of the present invention relate to products comprising the microencapsulated microbial cultures or compositions described herein, wherein the product is a feed product selected from feed premixes, feed blends, pet food and livestock feed.

[0181] It should be understood that the term "food" also includes "extruded food" and "bar", and that the "food" or "feed" may be in the form of a premix, intended to be further mixed with other ingredients to obtain the final product. Therefore, an embodiment of the present invention relates to a product or composition as described herein, wherein the product or composition is a premix. Another embodiment of the present invention relates to a product or composition as described herein, wherein the product or composition is an extruded food or a bar. Bars are expanded products made by extrusion, in which the different food ingredients are held together with an edible binder (such as, but not limited to, sugar).

[0182] Any such product may also contain other excipients suitable for any particular type of product. Such excipients include, but are not limited to, lactose, rice husks, and microcrystalline cellulose (MCC).

[0183] Another preferred area of ​​application is products comprising LAB.Accordingly, embodiments of the present invention relate to products comprising a microencapsulated microbial culture or composition as described herein, wherein the product is a fermentation product.

[0184] Products containing probiotics are also a preferred application area. Therefore, aspects of the present invention relate to dairy products comprising the microencapsulated microbial culture or composition described herein. Another embodiment of the present invention relates to a dairy product as described herein, wherein the dairy product is selected from yogurt, cheese, butter, inoculated sweet milk and liquid fermented dairy products.

[0185] Dairy-based products such as yogurt are well-characterized vehicles suitable for protecting and delivering microbial cultures such as probiotics in the intestine. One reason is that yogurt is rich in nutrients, proteins, fatty acids, carbohydrates, vitamins, minerals and calcium, which improves the ability of probiotic strains to bind to epithelial cells. Another reason is that consumers consider yogurt to be a nutritious, healthy and natural vehicle for live bacteria, and therefore prefer to drink it every day.

[0186] Thus, embodiments of the present invention relate to the dairy product described herein, wherein the dairy product is yogurt.

[0187] Reducing yield losses of microbial cultures during downstream processing of frozen / FD is very important not only for quality but also for processing efficiency. Similarly, the ability to maintain maximum viability of microbial cultures during ambient storage conditions also has commercial and technical advantages. Therefore, the methods described herein were developed to achieve both of these goals. The methods described herein rely on encapsulating microbial cultures in dense phase droplets (agglomerates) formed by mixing a large amount of a first and a second matrix. Compared to other traditional techniques that simply mix microbial cultures with different matrices, the methods described herein provide enhanced protection of microbial cultures during drying and ambient storage.

[0188] Thus, aspects of the present invention relate to methods of preparing the microencapsulated microbial cultures or compositions described herein, the methods comprising the steps of:

[0189] i) mixing a microbial culture with a first matrix comprising one or more first matrix components to form a first mixture, and

[0190] ii) mixing the first mixture with a second matrix comprising one or more second matrix components to form a microencapsulated microbial culture,

[0191] wherein the one or more first matrix components are different from the one or more second matrix components.

[0192] In a variation of the method, the microbial culture may be mixed simultaneously with the first and second matrices to form a microencapsulated culture. Thus, an embodiment of the present invention relates to the method described herein, wherein steps i) and ii) are performed simultaneously.

[0193] Another embodiment of the present invention is directed to a method of preparing a microencapsulated microbial culture or composition as described herein, comprising the steps of simultaneously mixing a microbial culture with i) and ii) to form a microencapsulated microbial culture:

[0194] i) a first matrix comprising one or more first matrix components, and

[0195] ii) a second matrix comprising one or more second matrix components,

[0196] wherein the one or more first matrix components and the one or more second matrix components are different.

[0197] The first and second matrix components are selected to form agglomerates in which the microbial culture is encapsulated after mixing. Thus, the first and second matrix components together form a phase-separated encapsulation matrix. This can be achieved by using first and second matrix components with different physicochemical properties (Example 1).

[0198] Thus, embodiments of the present invention relate to the methods described herein, wherein the one or more first matrix components are selected from the group consisting of carbohydrates, proteins, antioxidants, and combinations thereof.

[0199] Another embodiment of the present invention relates to the methods described herein, wherein the one or more first matrix components comprise one or more carbohydrates selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, polysaccharides, and combinations thereof.

[0200] It has been found that in particular disaccharides, such as trehalose, are suitable for inclusion as the first matrix component. Thus, embodiments of the present invention relate to the methods described herein, wherein the one or more first matrix components comprise one or more disaccharides selected from the group consisting of trehalose, lactose, sucrose, maltose, cellobiose and combinations thereof, preferably trehalose.

[0201] Likewise, it has also been found that the first matrix may advantageously comprise one or more antioxidants and certain proteins. Thus, embodiments of the present invention relate to methods as described herein, wherein the one or more first matrix ingredients comprise one or more proteins selected from caseinate, whey protein, gelatin, vegetable protein (e.g., pea protein, potato protein, and rice protein) and combinations thereof, preferably sodium caseinate.

[0202] Another embodiment of the present invention relates to the method described herein, wherein the one or more first matrix components comprise one or more antioxidants selected from the group consisting of citrate, ascorbate, tocopherol, ascorbyl palmitate, quercetin, gallic acid, tocotriene, tocotrienols, glutathione, and combinations thereof, preferably trisodium citrate.

[0203] The second matrix may comprise one or more second matrix components. Embodiments of the invention relate to the methods described herein, wherein the one or more second matrix components comprise one or more carbohydrates selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, polysaccharides, and combinations thereof.

[0204] The inventors have found that polysaccharides such as pectin are suitable second matrix components. Therefore, embodiments of the present invention relate to the methods described herein, wherein the one or more second matrix components comprise one or more polysaccharides selected from pectin, gum, maltodextrin, cellodextrin, alginate, starch, glycogen, cellulose, chitin, inulin, dextran, carrageenan, chitosan and combinations thereof, preferably pectin.

[0205] In some variants of the microencapsulated microbial culture, gums are preferred second matrix components. Gums are polysaccharides that can substantially increase the viscosity of a solution even at relatively low concentrations. Gums can be of plant or marine origin and can be in charged and uncharged variants. Embodiments of the present invention relate to methods described herein, wherein the one or more second matrix components comprise one or more gums selected from the group consisting of gum arabic, agar, alginate, cassia gum, dammar gum, beta-glucan, glucomannan, mastic, chicle, psyllium gum, spruce gum, gellan gum, guar gum, locust bean gum, xanthan gum, and combinations thereof.

[0206] For some combinations of first and second matrix, using only a single component in the second matrix is ​​sufficient to obtain agglomerates with a protective effect. Thus, embodiments of the present invention relate to methods described herein, wherein the second matrix comprises only a single second matrix component.

[0207] Many combinations of the first and second matrix components can be expected, and the combinations shown herein should not be construed as limitations of the present invention. However, some selections of matrix components are preferred. Therefore, embodiments of the present invention relate to methods described herein, wherein the first matrix comprises sodium caseinate, trehalose, and trisodium citrate.

[0208] Another embodiment of the present invention relates to the methods described herein, wherein said second matrix comprises pectin.

[0209] A preferred embodiment of the present invention relates to the method described herein, wherein the first matrix comprises sodium caseinate, trehalose and trisodium citrate, and the second matrix comprises pectin.

[0210] The first and second matrices should be provided as sterile matrices to ensure a high quality product and to avoid contamination. Thus, embodiments of the present invention relate to the methods described herein, wherein prior to step (i) the first and second matrices are heat sterilized and subsequently cooled to ambient temperature.

[0211] The microbial culture in step i) can be provided in the form of a liquid cell culture concentrate. In order to obtain a final dry microbial culture that has as high a viability as possible over time, it is necessary to carefully select and adjust the content of the starting material. Therefore, an embodiment of the present invention relates to the method described herein, wherein the microbial culture of step i) is a concentrated microbial culture having a dry matter content of 5-80 wt%, preferably 15-25 wt%. Another embodiment of the present invention relates to the method described herein, wherein the microbial culture of step i) is a concentrated microbial culture having 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%.

[0212] Another embodiment of the present invention relates to the method described herein, wherein the microbial culture of step i) is a concentrated microbial culture having a live microbial content of 1.0E+09 to 1.0E12 CFU / g, preferably about 1.0E11 CFU / g. Yet another embodiment of the present invention relates to the method described herein, wherein the microbial culture of step i) is a concentrated microbial culture having a live microbial content of at least 1.0E09 CFU / g, such as at least 1.0E10 CFU / g, preferably at least 1.0E11 CFU / g.

[0213] The starting material is used in an agglomeration process to produce a microencapsulated microbial culture. Each process step is adjusted to obtain an agglomerated formulation suitable for the intended final application, while determining some overall parameters, such as mixing temperature and duration. Therefore, an embodiment of the present invention relates to the method described herein, wherein the microbial culture of step i) is mixed with the first matrix at a temperature of 4°C to 20°C for a period of time of 15 min to 2 hours.

[0214] 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 substrate for a period of 15 min to 2 hours, such as 30 min to 2 hours, such as 1-2 hours.

[0215] 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 substrate at a temperature of 4 to 20°C, such as 4 to 15°C, for example 4 to 10°C.

[0216] Another embodiment of the present invention relates to the process as described herein, wherein in step ii), the mixing of the first mixture with the second matrix is ​​carried out at a temperature of 4°C to 20°C for 15 min to 2 hours.

[0217] Yet another embodiment of the present invention relates to the method as described herein, wherein in step ii), the mixing of the first mixture with the second matrix is ​​performed for 15 min to 2 hours, such as 30 min to 2 hours, such as 1-2 hours.

[0218] Yet another embodiment of the present invention relates to the method as described herein, wherein in step ii), the mixing of the pre-complex solution with the second matrix is ​​carried out at a temperature of 4°C to 20°C, such as 4°C to 15°C, such as 4°C to 10°C.

[0219] As additives included in a variety of different final products, microbial cultures such as LAB are usually provided in the form of dry powders. The dry powder form makes the microbial culture easy to transport, store and handle before final processing. Therefore, an embodiment of the present invention relates to the method described herein further comprising a step iii) subsequent to step ii), wherein step iii) comprises freezing the microencapsulated microbial culture to obtain a frozen microencapsulated microbial culture.

[0220] Another embodiment of the present invention relates to the method described herein further comprising a step iv) subsequent to step iii), wherein step iv) comprises sublimating the water in said frozen microencapsulated microorganism culture to obtain a dry microencapsulated microorganism culture.

[0221] Another embodiment of the present invention relates to the process as described herein, wherein step iv) is performed by a process selected from the group consisting of spray drying, vacuum drying, air drying, freeze drying, tray drying and vacuum tray drying.

[0222] Yet another embodiment of the present invention relates to the process as described herein, wherein the technique used in step iv) is freeze drying and wherein said freeze drying is carried out at a pressure of 0.005-1 mbar (millibar) and a temperature of -45°C to 75°C until complete removal of water.

[0223] Yet another embodiment of the present invention relates to the method as described herein, wherein the freeze drying is carried out at a pressure of 0.1-0.4 mbar. Yet another embodiment of the present invention relates to the method as described herein, wherein the freeze drying is carried out at a pressure of at least 0.1 mbar, such as at least 0.2 mbar, such as at least 0.3 mbar, such as at least 0.4 mbar.

[0224] Another embodiment of the present invention relates to the method as described herein, wherein said freeze drying is carried out at a temperature of 15° C. to 35° C. Another embodiment of the present invention relates to the method as described herein, wherein said freeze drying is carried out at a temperature of at least 15° C., such as at least 20° C., such as at least 25° C., such as at least 30° C.

[0225] 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 described herein, wherein the method further comprises:

[0226] (v) packaging the frozen microencapsulated microbial culture obtained in step (iii) or the dried microencapsulated microbial culture obtained in step (iv).

[0227] The microencapsulated microbial culture is the result of the agglomeration process described herein. Thus, aspects of the present invention relate to a microencapsulated microbial culture or composition obtainable by the methods described herein.

[0228] Preferably, the microencapsulated microbial culture is used as an additive for the preparation of a final product.Thus, aspects of the invention relate to the use of a microencapsulated microbial culture or composition as described herein in a product selected from feed, plant health products, food, beverages and pharmaceuticals.

[0229] Microencapsulated microbial cultures are particularly suitable for use in dairy products where probiotics are often delivered to consumers as a health supplement. Thus, embodiments of the present invention relate to the use of a microencapsulated microbial culture or composition as described herein, wherein the product is a dairy product.

[0230] Another embodiment of the present invention relates to the use of the microencapsulated microbial culture or composition described herein, wherein the dairy product is selected from the group consisting of yogurt, cheese, butter, inoculated sweet milk and liquid fermented dairy products.

[0231] The listing or discussion of an apparently prior-published document in this specification is not necessarily taken as an admission that the document is part of the state of the art or is common general knowledge in the art.

[0232] Unless the context indicates otherwise, preferences, options and embodiments for a given aspect, feature or parameter of the invention should be considered to have been disclosed in conjunction with any and all preferences, options and embodiments for all other aspects, features and parameters of the invention. This is particularly true for the description of microencapsulated microbial cultures and all their features, which are readily part of the final composition obtained by the methods described herein. Embodiments and features of the invention are also summarized in the following items.

[0233] project

[0234] X1. A microencapsulated microbial culture, wherein the microbial culture is encapsulated in an aggregate comprising i) and ii):

[0235] i) core material comprising microbial cultures, and

[0236] ii) an encapsulating matrix comprising one or more matrix components,

[0237] wherein the ratio of encapsulating matrix to core material (wt% / wt%) is at least 2.

[0238] X2. A microencapsulated microbial culture according to item X1, wherein the ratio of encapsulation matrix to core material (wt% / wt%) is in the range of 2-10, such as in the range of 2-8, such as in the range of 4-8.

[0239] X3. A microencapsulated microbial culture according to any one of items X1 or X2, wherein the ratio of encapsulation matrix to core material (wt% / wt%) is at least 3, preferably at least 4.

[0240] X4. The microencapsulated microbial culture according to any of the preceding items, wherein the encapsulation matrix comprises at least two matrix components, such as at least three matrix components, such as at least four matrix components.

[0241] X5. The microencapsulated microbial culture according to any of the preceding items, wherein the encapsulation matrix is ​​a heterogeneous matrix.

[0242] X6. A microencapsulated microbial culture according to any of the preceding items, wherein one or more matrix components are selected from carbohydrates, proteins, antioxidants and combinations thereof.

[0243] X7. A microencapsulated microbial culture according to item X6, wherein the carbohydrate is selected from monosaccharides, disaccharides, oligosaccharides, polysaccharides and combinations thereof.

[0244] X8. A microencapsulated microbial culture according to item X7, wherein the monosaccharide is selected from glucose, fructose, galactose, fucose, xylose, erythrose and combinations thereof.

[0245] X9. A microencapsulated microbial culture according to any one of items X7 or X8, wherein the disaccharide is selected from trehalose, lactose, sucrose, maltose, cellobiose and combinations thereof.

[0246] X10. A microencapsulated microbial culture according to any one of items X7-X9, wherein the disaccharide is trehalose.

[0247] X11. A microencapsulated microbial culture according to any one of items X7-X10, wherein the oligosaccharides are selected from fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), manno-oligosaccharides (MOS) and combinations thereof.

[0248] X12. A microencapsulated microbial culture according to any one of items X7-X11, wherein the polysaccharide is selected from pectin, cellodextrin, gum, alginate, starch, glycogen, cellulose, chitin, inulin, dextran, carrageenan, chitosan and combinations thereof.

[0249] X13. A microencapsulated microbial culture according to any one of items X7 to X12, wherein the polysaccharide is pectin.

[0250] X14. A microencapsulated microbial culture according to any one of items X12 or X13, wherein the gum is selected from gum arabic, agar, alginate, cassia gum, dammar gum, β-glucan, glucomannan, frankincense, chicle, psyllium gum, spruce gum, gellan gum, guar gum, locust bean gum, xanthan gum and combinations thereof.

[0251] X15. A microencapsulated microbial culture according to any one of items X6 to X14, wherein the protein is selected from caseinate, whey protein, gelatin, plant protein (such as pea protein, potato protein and rice protein) and combinations thereof.

[0252] X16. A microencapsulated microbial culture according to any one of items X6-X15, wherein the protein is sodium caseinate.

[0253] X17. A microencapsulated microbial culture according to any one of items X6-X16, wherein the antioxidant is selected from citrate, ascorbate, tocopherol, ascorbyl palmitate, quercetin, gallic acid, tocotriene, tocotrienols, glutathione and combinations thereof.

[0254] X18. A microencapsulated microbial culture according to any one of items X6-X17, wherein the antioxidant is trisodium citrate.

[0255] X19. A microencapsulated microbial culture according to any of the preceding items, wherein the encapsulation matrix comprises sodium caseinate, pectin, trehalose and trisodium citrate.

[0256] X20. A microencapsulated microbial culture according to any one of items X16 to X19, wherein the content of sodium caseinate in the encapsulation matrix is ​​1-6wt%, such as 2-5wt%, preferably 3-4wt%.

[0257] X21. A microencapsulated microbial culture according to any one of items X12 to X20, wherein the pectin content in the encapsulation matrix is ​​0.5-3wt%, such as 0.75-2.5wt%, preferably 1-2wt%.

[0258] X22. A microencapsulated microbial culture according to any one of items X9 to X21, wherein the content of trehalose in the encapsulation matrix is ​​10-40wt%, such as 15-35wt%, preferably 20-30wt%.

[0259] X23. A microencapsulated microbial culture according to any one of items X18 to X22, wherein the content of trisodium citrate in the encapsulation matrix is ​​1-10wt%, such as 2-8wt%, preferably 4-6wt%.

[0260] X24. A microencapsulated microbial culture according to any of the preceding items, wherein the encapsulation matrix comprises 1-6 wt% sodium caseinate, 0.5-3 wt% pectin, 10-40 wt% trehalose and 1-10 wt% trisodium citrate.

[0261] X25. The microencapsulated microbial culture according to any of the preceding items, wherein the microbial culture is bacteria or yeast.

[0262] X26. A microencapsulated microbial culture according to any of the preceding items, wherein the microbial culture is or comprises lactic acid bacteria (LAB) selected from the following genera: Lactobacillus, Holzapfel, Lactobacillus amyloliquefaciens, Lactobacillus bee, Lactobacillus associatus, Lactobacillus stonewall, Lactobacillus agriculturae, Lactobacillus schleifer, Lactobacillus putrefaciens, Lactobacillus casei, Lactobacillus broad, Draglio, Lactobacillus liquid, Lactobacillus united, Lactobacillus plantarum, Lactobacillus furfur, Lactobacillus oligovorus, Lactobacillus mucus, Lactobacillus fruiti, Lactobacillus aceticus, Lactobacillus bee, Lactobacillus promotility, Lactobacillus subtilis and Lactobacillus lentus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Enterococcus, Bifidobacterium, Brevibacterium and Staphylococcus.

[0263] X27. A microencapsulated microbial culture according to any of the preceding items, wherein the microbial culture is or comprises lactic acid bacteria (LAB) selected from the following genera: Lactobacillus, Lactobacillus mucilaginosus, Lactobacillus casei, Lactobacillus united, Lactobacillus casei, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus mucilaginosus, Lactobacillus united, Lactobacillus lentus, Lactobacillus spp., Lactobacillus symbioticus, Lactobacillus spp. and Lactobacillus plantarum.

[0264] X28. A microencapsulated microbial culture according to any of the preceding items, wherein the microbial culture is or comprises the following species: Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus casei, Lactobacillus casei subsp. casei, Lactobacillus plantarum subsp. plantarum, Lactobacillus fermentum, Lactobacillus animalis, Lactobacillus lentus, Lactobacillus curvatum, Lactobacillus fuchsii, Lactobacillus sakei subsp. sake, Lactobacillus pentosus, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus gona and Lactobacillus delbrueckii.

[0265] X29. A microencapsulated microbial culture according to any of the preceding items, wherein the microbial culture is or comprises Lactobacillus animalis, which was purchased from Chr. Hansen A / S of Denmark on July 8, 2020. Deposited as DSM 33570 at the Leibniz DSMZ-German Collection of Microorganisms and Cell Cultures, Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) 5 GmbH 1 Inhoffenstr. 7B, D-38124 Braunschweig, Germany.

[0266] X30. The microencapsulated microbial culture according to any of the preceding items, wherein the microbial culture is a probiotic culture.

[0267] X31. The microencapsulated microbial culture according to any of the preceding items, wherein the microencapsulated microbial culture is a dry preparation.

[0268] X32. A microencapsulated microbial culture according to item X31, wherein the dry preparation is selected from freeze-dried preparations, spray-dried preparations, vacuum-dried preparations and air-dried preparations.

[0269] X33. A microencapsulated microbial culture according to any one of items X31 or X32, wherein the dry preparation is freeze-dried.

[0270] X34. The microencapsulated microbial culture according to any of the preceding items, wherein the pH of the microencapsulated microbial culture is 6-8, preferably 6.25-7.5.

[0271] X35. The microencapsulated microbial culture according to any of the preceding items, wherein the pH of the microencapsulated microbial culture is at least 6, such as at least 6.25, such as at least 6.5.

[0272] X36. The microencapsulated microbial culture according to any of the preceding items, further comprising a hydrophobic coating.

[0273] X37. A microencapsulated microbial culture according to item X36, wherein the hydrophobic coating comprises one or more fats or waxes, or a mixture thereof.

[0274] X38. A microencapsulated microbial culture according to any one of items X36 or X37, wherein the hydrophobic coating comprises one or more ingredients selected from carnauba wax, beeswax, cocoa butter, hydrogenated palm oil, palm stearin, shea butter, mango butter, soybean oil, olive oil, coconut oil, rice bran oil, sunflower seed oil, candelilla wax, rice bran wax and laurel wax and combinations thereof.

[0275] X39. A microencapsulated microbial culture according to any of the preceding items, wherein the core material comprises 1.0E+07 to 5.0E+11 CFU / g of microbial culture, preferably 1.0E+09 to 1.0E+11 CFU / g, and more preferably 1.0E+10 to 5.0E+10 CFU / g.

[0276] X40. The microencapsulated microbial culture according to any of the preceding items, wherein after storage for 2 weeks at 37°C and Aw≤0.4, the content of live microorganisms contained in the microbial culture is 1.0E+06 to 1.0E+11 CFU / g.

[0277] X41. A microencapsulated microbial culture according to any of the preceding items, wherein after storage for 2 weeks at 37°C and Aw≤0.4, the loss of viability of the microbial culture when measured in CFU / g is less than 3 log units, preferably less than 2.5 log units after storage for 2 weeks at 37°C and Aw≤0.4.

[0278] X42. A composition comprising the microencapsulated microbial culture described in any of the preceding items.

[0279] X43. A composition according to item X42, wherein the composition further comprises one or more additives selected from food-grade ingredients, feed-grade ingredients, pharmaceutical ingredients and excipients.

[0280] X44. A composition according to item X43, wherein the food-grade ingredient is selected from lactose, maltodextrin, whey protein, casein, corn starch, dietary fiber, gum and gelatin.

[0281] X45. A composition according to any one of items X43 or X44, wherein the drug ingredient is selected from calcium carbonate, sodium carboxymethylcellulose, talc, polydimethylsiloxane, hydroxypropyl cellulose and hydroxypropyl methylcellulose.

[0282] X46. A composition according to any one of items X43-X45, wherein the excipient is selected from microcrystalline cellulose, titanium dioxide and aluminum silicate.

[0283] X47. A composition according to any one of items X42-X46, wherein the composition is a freeze-dried composition.

[0284] X48. A microencapsulated microbial culture according to any one of items X1 to X41 or a composition according to any one of items X42 to X47, wherein the microencapsulated microbial culture or the composition is in the form of a powder and / or granules.

[0285] X49. A microencapsulated microbial culture according to any one of items X1 to X41 or a composition according to any one of items X42 to X47, wherein the water activity (Aw) of the microencapsulated microbial culture is in the range of 0.01-0.8, preferably in the range of 0.05-0.6, and most preferably in the range of 0.1-0.4.

[0286] X50. A product comprising a microencapsulated microbial culture as described in any one of items X1-X41 or X48-X49 or a composition as described in any one of items X42-X49, wherein the product is selected from feed, plant health products, food, beverages and pharmaceuticals.

[0287] X51. A dairy product comprising a microencapsulated microbial culture as described in any one of items X1-X41 or X48-X49 or a composition as described in any one of items X42-X49.

[0288] X52. A dairy product according to item X51, wherein the dairy product is selected from yogurt, cheese, butter, inoculated sweet milk and liquid fermented dairy products.

[0289] Y1. A method for preparing a microencapsulated microbial culture as described in any one of items X1-X41 or X48-X49 or a composition as described in any one of items X42-X49, the method comprising the following steps:

[0290] i) mixing a microbial culture with a first matrix comprising one or more first matrix components to form a first mixture, and

[0291] ii) mixing the first mixture with a second matrix comprising one or more second matrix components to form a microencapsulated microbial culture,

[0292] wherein the one or more first matrix components and the one or more second matrix components are different.

[0293] Y2. A method according to item Y1, wherein the one or more first matrix components are selected from carbohydrates, proteins, antioxidants and combinations thereof.

[0294] Y3. A method according to any one of items Y1 or Y2, wherein the one or more first matrix components comprise one or more carbohydrates selected from monosaccharides, disaccharides, oligosaccharides, polysaccharides and combinations thereof.

[0295] Y4. A method according to any one of items Y1-Y3, wherein the one or more first matrix components comprise one or more disaccharides selected from trehalose, lactose, sucrose, maltose, cellobiose and combinations thereof, preferably trehalose.

[0296] Y5. A method according to any one of items Y1-Y4, wherein the one or more first matrix ingredients comprise one or more proteins selected from caseinate, whey protein, gelatin, plant protein (such as pea protein, potato protein and rice protein) and combinations thereof, preferably sodium caseinate.

[0297] Y6. A method according to any one of items Y1-Y5, wherein the one or more first matrix components comprise one or more antioxidants selected from citrate, ascorbate, tocopherol, ascorbyl palmitate, quercetin, gallic acid, tocotrienols, tocotrienols, glutathione and combinations thereof, preferably trisodium citrate.

[0298] Y7. A method according to any one of items Y1-Y6, wherein the one or more second matrix components comprise one or more carbohydrates selected from monosaccharides, disaccharides, oligosaccharides, polysaccharides and combinations thereof.

[0299] Y8. A method according to any one of items Y1-Y7, wherein the one or more second matrix components comprise one or more polysaccharides selected from pectin, gum, maltodextrin, cellodextrin, alginate, starch, glycogen, cellulose, chitin, inulin, dextran, carrageenan, chitosan and combinations thereof, preferably pectin.

[0300] Y9. A method according to any one of items Y1-Y8, wherein the one or more second matrix components comprise one or more gums selected from gum arabic, agar, alginate, cassia gum, dammar gum, β-glucan, glucomannan, frankincense, chicle, psyllium gum, spruce gum, gellan gum, guar gum, locust bean gum, xanthan gum and combinations thereof.

[0301] Y10. A method according to any one of items Y1-Y9, wherein the second matrix comprises only a single second matrix component.

[0302] Y11. A method according to any one of items Y1-Y10, wherein the first matrix comprises sodium caseinate, trehalose and trisodium citrate.

[0303] Y12. A method according to any one of items Y1-Y11, wherein the second matrix comprises pectin.

[0304] Y13. A method according to any one of items Y1-Y12, wherein the first matrix comprises sodium caseinate, trehalose and trisodium citrate, and the second matrix comprises pectin.

[0305] Y14. A method according to any one of items Y1 to Y13, wherein the first and second matrices are heat sterilized prior to step (i) and subsequently cooled to ambient temperature.

[0306] Y15. A method according to any one of items Y1 to Y14, wherein the microbial culture of step i) is a concentrated microbial culture having a dry matter content of 5-80 wt%, preferably 15-25 wt%.

[0307] Y16. A method according to any one of items Y1 to Y15, wherein the microbial culture of step i) is a concentrated microbial culture containing live microorganisms in a content of 1.0E+09 to 1.0E12 CFU / g, preferably about 1.0E11 CFU / g.

[0308] Y17. A method according to any one of items Y1 to Y16, wherein the microbial culture of step i) is mixed with the first substrate at a temperature of 4°C to 20°C for a period of 15 min to 2 hours.

[0309] Y18. A method according to any one of items Y1 to Y17, wherein the microbial culture of step i) is mixed with the first substrate for 15 min to 2 hours, such as 30 min to 2 hours, such as 1-2 hours.

[0310] Y19. A method according to any one of items Y1-Y18, wherein the microbial culture of step i) is mixed with the first substrate at a temperature of 4°C to 20°C, such as 4°C to 15°C, such as 4°C to 10°C.

[0311] Y20. A method according to any one of items Y1-Y19, wherein in step ii), the mixing of the first mixture with the second matrix is ​​carried out at a temperature of 4°C to 20°C for a period of 15 min to 2 hours.

[0312] Y21. A method according to any one of items Y1-Y20, wherein in step ii), the mixing of the first mixture with the second matrix is ​​carried out for a period of 15 min to 2 hours, such as 30 min to 2 hours, such as 1-2 hours.

[0313] Y22. A method according to any one of items Y1-Y21, wherein in step ii), the mixing of the pre-complex solution with the second matrix is ​​carried out at a temperature of 4°C to 20°C, such as 4°C to 15°C, such as 4°C to 10°C.

[0314] Y23. The method according to any one of items Y1-Y22, further comprising a step iii) following step ii), wherein step iii) comprises freezing the microencapsulated microbial culture to obtain a frozen microencapsulated microbial culture.

[0315] Y24. The method according to item Y23, further comprising a step iv) following step iii), wherein step iv) comprises sublimating the water in the frozen microencapsulated microbial culture to obtain a dry microencapsulated microbial culture.

[0316] Y25. A method according to item Y24, wherein step iv) is carried out by a technique selected from spray drying, vacuum drying, air drying, freeze drying, tray drying and vacuum tray drying.

[0317] Y26. A method according to any one of items Y24 or Y25, wherein the technology used in step iv) is freeze drying, which is carried out at a pressure of 0.005-1 mbar and a temperature of -45°C to 75°C until the water is completely removed.

[0318] Y27. A method according to item Y26, wherein the freeze drying is carried out at a pressure of 0.1-0.4 mbar.

[0319] Y28. A method according to any one of items Y26 or Y27, wherein the freeze-drying is carried out at a temperature of 15°C to 35°C.

[0320] Y29. A method according to any one of items Y24 to Y28, wherein the method further comprises:

[0321] (v) packaging the frozen microencapsulated microbial culture obtained in step (iii) or the dried microencapsulated microbial culture obtained in step (iv).

[0322] Z1. A microencapsulated microbial culture or composition obtainable by the method described in any one of items Y1-Y29.

[0323] Q1. Use of the microencapsulated microbial culture or composition described in any one of items X1-X49 or Z1 in a product selected from feed, plant health products, food, beverages and medicines.

[0324] Q2. Use of the microencapsulated microbial culture or composition according to item Q1, wherein the product is a dairy product.

[0325] Q3. Use of the microencapsulated microbial culture or composition according to claim Q2, wherein the dairy product is selected from yogurt, cheese, butter, inoculated sweet milk and liquid fermented dairy products.

[0326] The present invention will be described in further detail in the following non-limiting examples. Example

[0327] Example 1: Preparation of microencapsulated microbial cultures

[0328] Microencapsulation of microbial cultures was prepared as either agglomerated (heterogeneous) or homogenous preparations to evaluate the effect of the encapsulation matrix on the subsequent storage stability of the microbial cultures.

[0329] method

[0330] The agglomerated formulation consisted of Lactobacillus animalis DSM 33570 incorporated into a heterogeneous encapsulation matrix consisting of sodium caseinate (3.6%), pectin (1.4%), trehalose (23%), trisodium citrate (5%) and water (67%).

[0331] The matrix solutions were prepared as a first (sodium caseinate, trehalose, trisodium citrate and water) matrix solution and a second (pectin) matrix solution, respectively. The first and second matrix solutions were heat sterilized and subsequently cooled to RT (room temperature). The animal-associated Lactobacillus bacterial culture was mixed with the first matrix solution at 10° C. for 10-15 min and then with the second matrix solution at 10° C. for 10-15 min. The final composition was frozen in liquid nitrogen to form granules, which were then freeze-dried to obtain freeze-dried granules (FD).

[0332] The homogenized formulation consisted of Lactobacillus animalis incorporated into a reference encapsulation matrix consisting of maltodextrin, trehalose dihydrate, trisodium citrate dihydrate, and water.

[0333] A freezing solution containing maltodextrin and trehalose dihydrate was prepared in water. The freezing solution was autoclaved and then cooled to RT and stored in a refrigerated container overnight. Filter-sterilized trisodium citrate dihydrate was added to the freezing solution, and the animal combined lactobacillus culture was subsequently mixed with the freezing solution at below 10°C for 2h. The final mixture was frozen in liquid nitrogen to obtain pre-freeze dried particles (PFD). These PFDs were then freeze dried using a safety curve (32°C, 0.3mbar) to obtain freeze dried particles (FD).

[0334] Agglomerated and homogenous formulations were prepared with an encapsulating matrix to core material ratio of 1 or 4 (wt% / wt%).

[0335] Samples for exploring storage stability under ambient conditions were prepared. Freeze-dried particles of agglomerated or homogenized formulations were ground in a coffee blender for 30 seconds and then passed through a 60 mesh (250 μm) sieve. The ground material was diluted 100 times in calcium carbonate (CaCO3) powder to a final water activity of 0.4 Aw.

[0336] result

[0337] After dilution with calcium carbonate at 1:100, the viable bacterial concentrations of agglomerated and homogenized preparations containing Lactobacillus animalis were comparable and acceptable (approximately 2E+8 to 5E+8 CFU / g) (see Figure 1 ).

[0338] in conclusion

[0339] This example demonstrates that agglomerated formulations comprising a heterogeneous encapsulation matrix are suitable for formulating microbial cultures, as the number of viable bacteria after preparation is comparable to a homogeneous formulation based on a reference homogeneous encapsulation matrix.

[0340] Example 2: Storage stability of microencapsulated microbial cultures

[0341] Microencapsulated microbial cultures were studied to evaluate the effects of formulation strategies (encapsulation matrix and EI) on culture viability over time when exposed to environmental stress conditions.

[0342] The blend of FD powder and CaCO3 was filled into Alu bags, which were then heat-sealed and stored for a long time.

[0343] method

[0344] The blend of FD powder and CaCO3 of Example 1 was packed into aluminum bags, which were then heat sealed and subjected to storage stability studies under accelerated conditions (temperature = 37°C, Aw = 0.4). Samples were retrieved after 2 and 4 weeks and analyzed for CFU / g and Aw. Storage stability was determined according to the definition of storage stability as described herein.

[0345] result

[0346] The results clearly show that at EI = 1, neither the heterogeneous agglomerated nor the homogeneous formulations provided any stability under accelerated storage conditions, with a reduction in CFU / g of more than 5 log units after 2 weeks. In contrast, when the EI was increased to 4, the CFU / g of the homogeneous formulation decreased by 3 log units, while the CFU / g of the heterogeneous agglomerated formulation decreased by only 2.3 log units, which greatly enhanced the protection compared to EI 1 (see Figure 2 ).

[0347] The protective effect of the non-homogeneous agglomerated formulation was even more pronounced when absolute CFU / g counts were compared after 4 weeks under accelerated storage conditions. After 4 weeks, the homogenous formulation did not provide any further protective effect and the viable bacterial counts were below the detection limit. However, the agglomerated formulation provided extended storage stability and a considerable number of viable bacteria were still present after 4 weeks of storage (see Figure 3 ).

[0348] in conclusion

[0349] This experiment demonstrated that the combination of a heterogeneous encapsulation matrix and a high EI produced protective microcapsules that improved the stability of microbial cultures stored under accelerated storage conditions.

[0350] Example 3: Further preparation of microencapsulated microbial cultures

[0351] To further illustrate various aspects of the present invention, other microcapsules of microbial cultures were prepared.

[0352] In order to study the influence of matrix composition and its encapsulation index (EI) on the storage stability of microbial culture under environmental humid conditions, animal combined lactobacillus (DSM 33570), animal bifidobacterium lactis subspecies (DSM 15954), thermophilic streptococcus (DSM 15957) and lactococcus lactis subspecies (DSM 21404) strains were selected. According to the method given below, the cell concentrates of all strains were produced. Animal bifidobacterium lactis subspecies was inoculated into De Man, Rogosa and Sharpe (MRS) liquid culture medium (BD DifcoTM Lactobacilli MRS Agar, Fisher Scientific) supplemented with 0.5g / L of L-cysteine ​​hydrochloride (Sigma-Aldrich, Inc.), and cultured anaerobically at 37°C for 24 hours. After growing for 24 hours, centrifugation was used to concentrate the cells in the culture medium by 25 times. These concentrated cells were used for the microencapsulation experiment described in detail below. Similarly, Lactobacillus animalis, Streptococcus thermophilus and Lactococcus lactis were grown anaerobically in De Man, Rogosa and Sharpe (MRS) liquid medium at 37°C for 24 hours. These concentrated cells were used for microencapsulation experiments detailed below. Matrix formulations including homogeneous and heterogeneous (agglomeration-based phase separation) formulations were designed, as shown in Table 2. In the case of heterogeneous matrix formulations, protein-polysaccharide, polysaccharide-polysaccharide combinations were used to obtain phase separation matrices to protect cells under ambient humid conditions. The encapsulation index studied for the generation of examples of all strains was 1 and 4. The method of encapsulating core cells in homogeneous and heterogeneous phase separation matrix formulations was the same as that given in the provisional application. After the matrix formulation and cell mixture were granulated in liquid nitrogen, the pre-freeze dried (PFD) particles were stored at -80°C until freeze-dried. These PFDs were freeze-dried using a safety curve at 32°C and 0.3mbar.

[0353] Table 1. Composition of various matrix preparations

[0354]

[0355] The freeze-dried granules of all formulations with EI of 1 and 4 were ground in a coffee blender for 30 seconds and then passed through a 60 mesh (250 μ) sieve. The ground material was diluted 25 times in calcium carbonate (CaCO3) powder to obtain the desired water activity (a) of 0.35-0.4. w ). Under ambient humid conditions (T = 25°C, a w =0.35-0.4), the blends with CaCO3 were subjected to storage stability studies in sealed Alu bags. Figures 4 to 15The Log values ​​for all strains encapsulated in various matrix formulations (EI = 1 and 4) under given storage conditions for up to 12 weeks are given. 10 Loss (CFU / g).

[0356] Cell viability (CFU / g) was determined as follows: a known amount of sample (e.g., freeze-dried or calcium carbonate blend) was homogenized with a specific volume of diluent (1:100) using a homogenizer, then resuspended using a vortex mixer, and then decimal dilutions were performed in peptone saline diluent (also known as "maximum recovery diluent (MRD)"). MRD contains peptone, NaCl, and deionized water. The dilution was poured onto a plate, mixed with MRS agar (Hi-media, M641) and incubated. After incubation, colonies were counted manually. In particular, the stability of the sample was evaluated using the following assay by counting colony forming units (CFU) per gram. Samples were taken immediately after freeze drying and at selected time points during the stability study to determine the viable cell count of the dried freeze-dried particles or calcium carbonate blends. A standard pour-plating method was used. The freeze-dried material or calcium carbonate blend is suspended in sterile peptone saline diluent (BD DifcoTM Lactobacilli MRS Agar, Fisher Scientific) and homogenized by patting (stomaching) using a homogenizer (bioMérieux, Inc.Durham, NC). After activation for 30 minutes, the pat is repeated, and the cell suspension is continuously diluted in a peptone saline diluent. For the CFU of animal Bifidobacterium lactis subspecies, the dilution is plated on MRS agar (BD DifcoTM Lactobacilli MRS Agar, Fisher Scientific) supplemented with 0.5g / L L-cysteine ​​hydrochloride (Sigma-Aldrich, Inc.), in duplicate. Agar plates are incubated anaerobically at 37°C for three days. For the CFU of animal combined lactobacillus, the dilution is plated on MRS agar (BD DifcoTM Lactobacilli MRS Agar, Fisher Scientific), in duplicate. The agar plates were incubated anaerobically at 37°C for three days. In the case of CFU of S. thermophilus, the dilutions were plated on M17 agar (BDDifcoTM Lactobacilli MRS Agar, Fisher Scientific) supplemented with 0.5 g / L monosodium phosphate (Sigma-Aldrich, Inc.) and 0.5 g / L disodium phosphate (Sigma-Aldrich, Inc.) in duplicate. The agar plates were incubated aerobically at 37°C for three days.For the CFU of Lactococcus lactis, the dilution was plated on M17 agar (BD DifcoTM Lactobacilli MRS Agar, Fisher Scientific) supplemented with 0.5 g / L monosodium phosphate (Sigma-Aldrich, Inc.) and 0.5 g / L disodium phosphate (Sigma-Aldrich, Inc.) in duplicate. The agar plates were incubated aerobically at 37°C for three days. Plates with 30-300 colonies were selected for colony forming unit (CFU) counting. The results were reported as average CFU / g dried freeze-dried samples or calcium carbonate blend samples by duplicate calculation.

[0357] Storage stability results of animal combined lactobacillus

[0358] Figure 4 and Figure 5 The storage stability results clearly show that the control cells without any matrix formulation had a Log10 loss of 5 after 12 weeks of storage under the given conditions. This suggests that it is necessary to embed or encapsulate the cells in a matrix formulation to protect them under storage conditions. Matrix formulation 01 (MF-01) is a homogeneous formulation, whereas MF-02 to MF-06 are all heterogeneous (agglomeration-based) phase separated matrix formulations with encapsulation indices of 1 and 4 for protecting the cells. Figure 4 and Figure 5 The storage stability results of animal combined with lactobacillus at EI = 1 and 4 are shown respectively. At EI = 1, MF-05 has the highest protective effect on cells. After 12 weeks, the Log 10 The loss was 1.93, and MF-06 was the preparation with the lowest protection among all preparations. After 12 weeks under the given storage conditions, its Log 10 However, at EI = 4, MF-02 provided significantly higher protection to cells after 12 W, Log 10 The loss was only 0.67, which is almost a 4.3 log advantage compared to control cells. Figure 6 It is clearly shown that for all matrix formulations, the loss of viability of Lactobacillus animalis was greater at EI = 1 compared to EI = 4, indicating that the higher the encapsulation index, the higher the storage stability of the cells. 10 The loss was 1.32, whereas the Log10 loss for the heterogeneous MF-02 formulation was only 0.67. The significant protection of cells under ambient humidification conditions is apparently due to the combined effects of the higher encapsulation index and the composition of the matrix formulation.

[0359] Microscopic images of aggregates MF-01 to MF-06 (see Table 2. Composition of various matrix preparations) including animal-associated Lactobacillus were collected and are shown in Table 2. Figures 16 to 21 .

[0360] Storage stability results of Lactococcus lactis subsp.

[0361] Lactococcus lactis subsp. lactis cells are easily affected by storage conditions and suffer significant loss of viability during storage. Figure 7 and Figure 8 Storage stability results under ambient humid conditions at encapsulation indices of 1 and 4 are shown, respectively. At EI = 1, all formulations had a log reduction greater than 3 after 12W of storage, except for MF-04, which is a heterogeneous phase separation matrix formulation, which had a log loss of 2.07. The protective effect on cells under ambient humid conditions was greater when the study was performed at EI = 4. However, the most obvious protective effect was observed in the case of the MF-02 matrix formulation, which had a log reduction of only 1.29, which is almost a 3.7 log advantage compared to the control cells with a log reduction of 5 after 12W. From Fig. 9 The effect of increasing encapsulation index on the enhanced protection was clearly observed. The log loss at EI = 4 for the homogeneous MF-01 formulation was 1.88, whereas in the case of the heterogeneous phase separated MF-02 matrix formulation the log loss was only 1.29.

[0362] Storage stability results of Streptococcus thermophilus

[0363] Like L. lactis subsp. lactis, S. thermophilus cells are also very sensitive to storage conditions and lose viability when exposed to ambient moisture. Fig.10 and Fig.11 Stability results at T = 25 °C, aw = 0.35-0.4 are shown for EI of 1 and 4, respectively. At EI = 1, the homogeneous and heterogeneous phase separation matrix formulations provided very little protection to the cells. However, when these formulations were studied at a higher EI of 4, a significant increase in protection was observed, with log losses ranging from 0.8-1.65. Fig.12 Comparative stability results after 12 W storage are shown for all investigated formulations with EI of 1 and 4. At EI = 4, the homogeneous formulation MF-01 showed a log reduction of 1.65, while the heterogeneous MF-03 matrix formulation showed a log reduction of only 0.81.

[0364] Storage stability results of Bifidobacterium animalis

[0365] When B. animalis cells were studied alone, they were susceptible to storage conditions, with a log reduction of greater than 5 after 12 weeks of storage under given conditions. Fig.13 and14 Storage stability results at EI of 1 and 4 under ambient humid conditions are shown, respectively. Animal B. animalis cells were more robust when studied at EI = 1 compared to the other studied microbial cultures. When tested at EI = 1, the log reduction of the MF-02 matrix formulation after 12W storage was only 1.22. However, at EI = 4, the protective effect of all formulations on cells was enhanced compared to EI = 1 ( Fig.15 ). At T = 25 °C, a w = 0.35-0.4 after 12 W, the log reduction of the heterogeneous MF-04 matrix formulation was only 0.85, compared with 1.44 for the homogeneous MF-01 matrix formulation.

[0366] in conclusion

[0367] In general, heterogeneous matrix formulations provided better protection for different types of microbial cultures under ambient humid conditions compared to homogeneous formulations. The higher the encapsulation index, the higher the storage stability of all studied microbial cultures. The apparent increase in cell protection was due to the combined effects of the composition of the matrix formulation and the higher encapsulation index. Matrix formulations are specific to individual cultures and the same formulation is not suitable for all microbial cultures.

[0368] References

[0369] ·Seifert&Mogensen(2002), Bulletin of the IDF, 377, 10-19

[0370] ·Zheng et al. (2020), Int.J.Syst.Evol.Microbiol., 70, 2782-2858

Claims

1. A microencapsulated microbial culture, wherein the microbial culture is encapsulated in an agglomerate comprising i) and ii): i) core material comprising microbial cultures, and ii) an encapsulation matrix comprising one or more matrix components, wherein the ratio of encapsulating matrix to core material (wt% / wt%) is at least 2, and wherein the encapsulating matrix comprises: a) maltodextrin, trehalose dihydrate and sodium ascorbate; b) trehalose dihydrate, sodium ascorbate, sodium caseinate and high methoxyl pectin; c) trehalose dihydrate, sodium ascorbate, sodium caseinate and guar gum; d) trehalose dihydrate, sodium ascorbate, sodium caseinate and sodium carboxymethylcellulose; e) trehalose dihydrate, sodium ascorbate, high methoxyl pectin and rice protein; or f) Trehalose dihydrate, sodium ascorbate, amidated pectin and hydroxypropyl methylcellulose thickener.

2. The microencapsulated microbial culture according to claim 1, wherein the ratio of the encapsulation matrix to the core material (wt% / wt%) is 2 to 10.

3. The microencapsulated microbial culture according to any one of the preceding claims, wherein the microbial culture is bacteria or 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 Ligilactobacillus animalis deposited as DSM 33570, Bifidobacterium animalis subsp. Lactis deposited as DSM 15954, Streptococcus thermophilus deposited as DSM 15957 or Lactococcus lactis subsp. Lactis deposited as DSM 21404.

6. The microencapsulated microbial culture according to any one of the preceding claims, wherein after storage for 12 weeks at 25°C and Aw≤0.4, the microbial culture loses less than 3 log units of viability when measured in CFU / g, preferably after storage for 12 weeks at 25°C and Aw≤0.4, the microbial culture loses less than 1.0 log unit of viability when measured in CFU / g.

7. A composition comprising the microencapsulated microbial culture according to any one of the preceding claims.

8. The composition according to claim 7, wherein the composition further comprises one or more additives selected from the group consisting of food grade ingredients, feed grade ingredients, pharmaceutical ingredients and excipients.

9. A product comprising the microencapsulated microbial culture according to any one of claims 1 to 6 or the composition according to any one of claims 7 or 8, wherein the product is selected from the group consisting of feed, plant health products, food, beverages and pharmaceuticals.

10. A method for preparing a microencapsulated microbial culture according to any one of claims 1 to 6 or a composition according to any one of claims 7 or 8, the method The following steps are involved: i) mixing a microbial culture with a first matrix comprising one or more first matrix components to form a first mixture, and ii) mixing the first mixture with a second matrix comprising one or more second matrix components to form a microencapsulated microbial culture, The first matrix component or the second matrix component comprises: a) maltodextrin, trehalose dihydrate and sodium ascorbate; b) trehalose dihydrate, sodium ascorbate, sodium caseinate and high methoxyl pectin; c) trehalose dihydrate, sodium ascorbate, sodium caseinate and guar gum; d) trehalose dihydrate, sodium ascorbate, sodium caseinate and sodium carboxymethylcellulose; e) trehalose dihydrate, sodium ascorbate, high methoxyl pectin and rice protein; or f) Trehalose dihydrate, sodium ascorbate, amidated pectin and hydroxypropyl methylcellulose thickener.

11. A microencapsulated microbial culture or composition obtainable by the method according to claim 10.

12. Use of the microencapsulated microbial culture or composition according to any one of claims 1-8 or 11 in a product selected from the group consisting of feed, plant health products, food, beverages and pharmaceuticals.

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