Particles comprising bioactive agent

Abstract

The present invention generally relates to particles comprising a bioactive agent embedded in a biofilm, wherein the biofilm is embedded in a matrix. More specifically, but not exclusively, the present invention relates to particles comprising biofilm-forming microorganisms embedded in a biofilm wherein the biofilm is embedded in a matrix. Food or beverage products, and methods of increasing the stability of bioactive agents, are also provided.

Description

Technical Field

[0001] The present invention generally relates to particles comprising a bioactive agent embedded in a biofilm, wherein the biofilm is embedded in a matrix. More specifically, but not exclusively, the present invention relates to particles comprising biofilm-forming microorganisms. Food or beverage products, personal care products, cleaning products and methods of increasing the stability of bioactive agents are also provided.

[0002] The particles described herein can be used to increase the shelf life of bioactive agents such as probiotic microorganisms, particularly in high water activity environments at ambient temperature. These particles are particularly useful for environmentally stable high water activity food and beverage products, such as UHT processed products, which are stored for extended periods of time without refrigeration. Background Art

[0003] The consumption of products containing bioactive agents is associated with a range of health benefits.Such bioactive agents may include various microorganisms, such as probiotics.

[0004] However, bioactive agents may have limited stability and / or shelf life. For example, when the bioactive agent is a microorganism, a well-known problem in the art is the stability of the microorganism. The microorganism will tend to become inactive over time, especially when incorporated into a product with a high water activity. This makes it difficult to provide a product with a high level of active microorganisms.

[0005] Furthermore, if microorganisms in a food or beverage product grow unchecked, this can lead to changes in the product's characteristics, or even spoilage.

[0006] Known methods for improving the stability and / or shelf life of bioactive agents generally include storage at low temperatures (e.g., by refrigeration), or storage in an environment with very low water activity (e.g., dry powder). It should be understood that these methods are not universally applicable because they cannot be used to incorporate bioactive agents into food or beverage products with high water activity, or products stored at room temperature.

[0007] Therefore, there is a need for methods and compositions for increasing the stability and / or shelf life of bioactive agents. This is particularly desirable when the bioactive agents are microorganisms such as probiotics; when they are incorporated into food or beverage products with high water activity; and when the food or beverage products will be stored at room temperature for an extended period of time.

[0008] It is an object of the present invention to address one or more of these needs in some way, or at least to provide the public with a useful choice. Summary of the invention

[0009] In a first aspect, the present invention provides a particle comprising a bioactive agent embedded in a biomembrane, wherein the biomembrane is embedded in a matrix, wherein the matrix has a water vapor transmission rate (WVTR) of 0.1 to 500.

[0010] In a second aspect, the invention provides a particle comprising a bioactive agent embedded in a biological membrane, wherein the biological membrane is embedded in a matrix comprising at least 6 wt% of one or more lipids.

[0011] In a third aspect, the invention provides a food or beverage product comprising the particle of the first or second aspect.

[0012] In a fourth aspect, the present invention provides a personal care product comprising the particles of the first or second aspect.

[0013] In a fifth aspect, the present invention provides a cleaning product comprising a particle of the first or second aspect.

[0014] In a sixth aspect, the present invention provides a method for increasing the stability of a bioactive agent, the method comprising the steps of:

[0015] a. Encapsulation of bioactive agents in biofilms,

[0016] b. embedding the biofilm in a matrix having a water vapor transmission rate (WVTR) of 0.1 to 500, and

[0017] c. Granulate the matrix.

[0018] In a seventh aspect, the present invention provides a method for increasing the stability of a bioactive agent, the method comprising the steps of:

[0019] a. Encapsulation of bioactive agents in biofilms,

[0020] b. embedding the biofilm in a matrix comprising at least 6 wt % of one or more lipids, and

[0021] c. Granulate the matrix.

[0022] In an eighth aspect, the present invention provides a method for increasing the stability of a bioactive agent, the method comprising the steps of:

[0023] a. embedding the bioactive agent in a matrix having a water vapor transmission rate (WVTR) of 0.1 to 500,

[0024] b. Inducing bioactive agents to produce biofilm, and

[0025] c. forming the matrix into particles,

[0026] Steps b) and c) can be performed in any order.

[0027] In some embodiments, step (b) comprises inducing the bioactive agent embedded in the matrix to produce a biofilm.

[0028] In a ninth aspect, the present invention provides a method for increasing the stability of a bioactive agent, the method comprising the steps of:

[0029] a. embedding the bioactive agent in a matrix comprising at least 6wt% of one or more lipids,

[0030] b. Inducing bioactive agents to produce biofilm, and

[0031] c. forming the matrix into particles,

[0032] Steps b) and c) can be performed in any order.

[0033] In some embodiments, step (b) comprises inducing the bioactive agent embedded in the matrix to produce a biofilm.

[0034] In some embodiments, the method of the sixth, seventh, eighth or ninth aspect further comprises the following steps:

[0035] d. combining the granules of step c with a medium having a water activity of at least 0.5, preferably UHT treated yogurt, and

[0036] e. Incubate the culture medium for 3 to 30 days.

[0037] In another aspect, the present invention provides a method for producing a food or beverage product supplemented with a bioactive agent, the method comprising the method of the above embodiment, and further comprising the following steps:

[0038] f. harvesting particles from the culture medium of step e, and

[0039] g. Combining the harvested particles with a food or beverage product to obtain a bioactive agent-supplemented food or beverage product.

[0040] On the other hand, the present invention provides a method for producing a food or beverage product supplemented with a bioactive agent, which method includes the method of the sixth, seventh, eighth or ninth aspect, and also includes the step of combining the particles with the food or beverage product to obtain a food or beverage product supplemented with a bioactive agent.

[0041] The following embodiments may be applied to any of the above aspects.

[0042] In one embodiment, the matrix comprises at least 6 wt% of one or more lipids.

[0043] In one embodiment, the substrate has a density of 0.1 to 500 g / m 2 / 24h, for example 0.1 to 400, 0.1 to 300, 0.1 to 200, 0.1 to 150, 0.1 to 100, 0.1 to 90, 0.1 to 80, 0.1 to 70, 0.1 to 60, 0.1 to 50, 0.1 to 40, 0.1 to 30, 0.1 to 20, 0.1 to 10, 0.1 to 9, 0.1 to 8, 0.1 to 7, 0.1 to 6, 0.1 to 5, 0.1 to 4, 0.1 to 3, 0.5 to 500, for example 0.5 to 400, 0.5 to 300, 0.5 to 200, 0.5 to 150, 0.5 to 100, 0.5 to 90, 0. .5 to 80, 0.5 to 70, 0.5 to 60, 0.5 to 50, 0.5 to 40, 0.5 to 30, 0.5 to 20, 0.5 to 10, 0.5 to 9, 0.5 to 8, 0.5 to 7, 0.5 to 6, 0.5 to 5, 0.5 to 4, 0.5 to 3, 1 to 500, for example 1 to 400, 1 to 300, 1 to 200, 1 to 150, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 to 3 to 500, for example 2 to 400, 2 to 300, 2 to 200, 3 to 150, 3 to 100, 3 to 90, 2 to 80, 3 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 500, for example 3 to 400, 3 to 300, 3 to 200, 3 to 150, 3 to 100, 3 to 90, 3 to 80, 3 to 70, 3 to 60, 3 to 50, 3 to 40, 3 to 30, 3 to 20, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5 to 400, 5 to 300, 5 to 200, 5 to 150, 5 to 100, 5 to 90, 5 to 80, 5 to 70, 4 to 60, 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 500, for example 5 to 400, 5 to 300, 5 to 200, 5 to 150, 5 to 100, 5 to 90, 5 to 80, 5 to 70, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 20, 5 to 10, 5 to 9, 5 to 8, 5 to 7, or 5 to 6 g / m 2 / 24h water vapor transmission rate (WVTR). In some embodiments, the substrate has a water vapor transmission rate of less than 500 g / m 2 / 24h, such as less than 400, less than 300, less than 200, less than 150, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or about 1 g / m 2 / 24h water vapor transmission rate (WVTR).

[0044] In one embodiment, the substrate has a water vapor transmission rate (WVTR) of 0.1 to 10, such as 1-5.

[0045] In one embodiment, the matrix has a thickness of from about 1 to about 350 g / m 2 / 24h, for example, about 1 to about 325, about 1 to about 300, about 1 to about 275, about 1 to about 250, about 1 to about 225, about 1 to about 200, about 1 to about 175, about 1 to about 150, about 1 to about 125, about 1 to about 100, about 10 to about 350, about 10 to about 325, about 10 to about 300, about 10 to about 275, about 10 to about 250, about 10 to about 225, about 10 g / m 2 or about 200, about 10 to about 175, about 10 to about 150, about 10 to about 125, about 10 to about 100, about 20 to about 350, about 20 to about 325, about 20 to about 300, about 20 to about 275, about 20 to about 250, about 20 to about 225, about 20 to about 200, about 20 to about 175, about 20 to about 150, about 20 to about 125, or about 20 to about 100 g / m 2 or about 200, about 10 to about 175, about 20 to about 150, about 20 to about 125, or about 20 to about 100 g / m 2 / 24h water vapor transmission rate (WVTR).

[0046] In some embodiments, the particles have a surface area to volume ratio of about 1 to about 100, e.g., about 1 to about 75, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, about 1 to about 5, about 1 to about 3, about 1 to about 2, about 1.5 to about 100, about 1.5 to about 75, about 1.5 to about 50, about 1.5 to about 40, about 1.5 to about 30, about 1.5 to about 20, about 1.5 to about 10, about 1.5 to about 5, about 1.5 to about 3, or about 1.5 to about 2.

[0047] In some embodiments, the particles have a water activity of about 0.1 to about 1.0, e.g., about 0.1 to about 0.9, about 0.1 to about 0.8, about 0.1 to about 0.7, about 0.1 to about 0.6, about 0.1 to about 0.5, about 0.2 to about 1.0, about 0.2 to about 0.9, about 0.2 to about 0.8, about 0.2 to about 0.7, about 0.2 to about 0.6, about 0.2 to about 0.5, about 0.3 to about 1.0, about 0.3 to about 0.9, about 0.3 to about 0.8, about 0.3 to about 0.7, about 0.3 to about 0.6, about 0.3 to about 0.5, about 0.4 to about 1.0, about 0.4 to about 0.9, about 0.4 to about 0.8, about 0.4 to about 0.7, about 0.4 to about 0.6, or about 0.4 to about 0.5.

[0048] In one embodiment, the bioactive agent comprises a first microorganism.

[0049] In one embodiment, the biofilm is produced by a first microorganism.

[0050] In one embodiment, the first microorganism is a probiotic microorganism.

[0051] In one embodiment, the first microorganism is Bacillus, Bifidobacterium, Enterococcus, Lacticaseibacillus, Lactiplantibacillus, Lactobacillus, Lactococcus, Ligilactobacillus, Limosilactobacillus, Lentilactobacillus, Saccharomyces, or Streptococcus. In one embodiment, the first microorganism is selected from the group consisting of Bacillus coagulans, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus plantarum subsp. plantarum, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactococcus lactis, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis biovardiacetylactis, Leuconostoc pseudomesentericum, Lactobacillus salivarius, Lactobacillus reuteri, Saccharomyces boulardii and Streptococcus thermophilus; preferably, the first microorganism is Lactobacillus rhamnosus HN001. In some embodiments, the first microorganism is Lacticaseibacillus rhamnosus or Lacticaseibacillus paracasei, preferably Lacticaseibacillus rhamnosus HN001 or Lacticaseibacillus paracasei subsp. paracasei IM514.

[0052] In one embodiment, the particles include an amount of 10 to 5×10 12 CFU / g particle, e.g. 10 2 Up to 5×10 12 , 10 3 Up to 5×10 12 , 10 4 Up to 5×10 12 , 10 5 Up to 5×10 12 , 10 6 Up to 5×10 12 , 10 7 Up to 5×10 12 , 10 8Up to 5×10 12 , 10 9 Up to 5×10 12 , 10 10 Up to 5×10 12 , 10 11 Up to 5×10 12 , 10 12 Up to 5×10 12 , 10 to 10 12 , 10 2 Up to 10 12 , 10 3 Up to 10 12 , 10 4 Up to 10 12 , 10 5 Up to 10 12 , 10 6 Up to 10 12 , 10 7 Up to 10 12 , 10 8 Up to 10 12 , 10 9 Up to 10 12 , 10 10 Up to 10 12 , 10 11 Up to 10 12 , 10 to 10 11 , 10 2 Up to 10 11 , 10 3 Up to 10 11 , 10 4 Up to 10 11 , 10 5 Up to 10 11 , 10 6 Up to 10 11 , 10 7 Up to 10 11 , 10 8 Up to 10 11 , 10 9 Up to 10 11 , 10 10 Up to 10 11 , 10 to 10 10 , 10 2 Up to 10 10 , 10 3 Up to 10 10 , 10 4 Up to 10 10 , 10 5 Up to 10 10 , 10 6 Up to 10 10 , 10 7 Up to 1010 , 10 8 Up to 10 10 , 10 9 Up to 10 10 , 10 to 10 9 , 10 2 Up to 10 9 , 10 3 Up to 10 9 , 10 4 Up to 10 9 , 10 5 Up to 10 9 , 10 6 Up to 10 9 , 10 7 Up to 10 9 , 10 8 Up to 10 9 , 10 to 10 8 , 10 2 Up to 10 8 , 10 3 Up to 10 8 , 10 4 Up to 10 8 , 10 5 Up to 10 8 , 10 6 Up to 10 8 , 10 7 Up to 10 8 , 10 to 10 7 , 10 2 Up to 10 7 , 10 3 Up to 10 7 , 10 4 Up to 10 7 , 10 5 Up to 10 7 , 10 6 Up to 10 7 , 10 to 10 6 , 10 2 Up to 10 6 , 10 3 Up to 10 6 , 10 4 Up to 10 6 , 10 5 Up to 10 6 , 10 to 10 5 , 10 2 Up to 10 5 , 10 3 Up to 10 5 , 10 4 Up to 10 5 , 10 to 10 4 , 102 Up to 10 4 , 10 3 Up to 10 4 , 10 to 10 3 , 10 2 Up to 10 3 , or 10 to 10 2 CFU / g of particles of the first microorganism.

[0053] In one embodiment, the particle further comprises a second microorganism different from the first microorganism.In various embodiments, the particle further comprises any number of additional different microorganisms, such as a third, fourth and / or fifth microorganism.

[0054] In one embodiment, the second microorganism is a probiotic microorganism. In some embodiments, the third, fourth and / or fifth microorganism is a probiotic microorganism. In one embodiment, each microorganism is a probiotic microorganism. In one embodiment, the first microorganism is a biofilm-producing microorganism and the second microorganism is a probiotic microorganism. In another embodiment, the first microorganism is a probiotic microorganism and the second microorganism is a biofilm-producing microorganism.

[0055] In one embodiment, the bioactive agent is a non-microorganism bioactive agent. In one embodiment, the particle also includes a non-microorganism bioactive agent. In some embodiments, the non-microorganism bioactive agent is a protein, peptide, amino acid, fat, triglyceride, lipid, fatty acid, fatty acid salt, oligosaccharide, polysaccharide, nucleic acid, nucleotide, nucleoside, vitamin, mineral or any combination of two or more of these. In a preferred embodiment, the non-microorganism bioactive agent is vitamin, prebiotic, postbiotic or human milk oligosaccharide. In some embodiments, vitamin is vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B6 (pyridoxine), vitamin B12 (cyanocobalamin), vitamin B5 (pantothenic acid), vitamin B7 (biotin), vitamin B9 (folate or folic acid). In some embodiments, prebiotic is oligofructose, oligogalactose or inulin. In some embodiments, the human milk oligosaccharide is 2'-fucosyllactose (2FL), 3'-fucosyllactose (3FL), 3'-sialyllactose (3SL), 6'-sialyllactose (6SL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), or any combination of two or more of these.

[0056] In one embodiment, the biofilm is characterized by the presence of ions at 360, 430 and / or 530 cm in a principal component analysis of the Raman spectroscopy data. -1In some embodiments, the Raman spectral data indicates the presence of rhamnose, for example, by peaks at 360, 430, and / or 530 cm in a principal component analysis of the Raman spectral data. -1 In one embodiment, the principal component analysis of the Raman spectroscopy data of the sample containing the biofilm is performed at 360, 430 and / or 530 cm -1 There is a peak at 1450cm -1 When normalized to the fat-related peak at 360, 430 and / or 530 cm, the peak height is higher than the corresponding peak height in the principal component analysis of the Raman spectroscopy data of the sample without biofilm. In one embodiment, the principal component analysis of the Raman spectroscopy data at 360, 430 and / or 530 cm -1 The peak height at 1450 cm -1 At least 50% of the peak height of the fat-related peak at 1450 cm -1 The peak height of the fat-related peak at is at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100%, and useful ranges can be selected between any of these values ​​(e.g., 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 50% to 90%, 60% to 90%, 70% to 90%, or 80% to 90%).

[0057] In one embodiment, the biofilm can be stained with Concanavalin A, wheat germ agglutinin and / or Ulex europaeus agglutinin. In some embodiments, the biofilm can be detected by staining with Concanavalin A, wheat germ agglutinin and / or Ulex europaeus agglutinin that extends beyond the bacterial cells.

[0058] In one embodiment, the biofilm comprises glucose, galactose, rhamnose, galactosamine, glucosamine, mannose and / or fucose.

[0059] In one embodiment, the particles maintain a moisture content of 1 wt% to 60 wt%, preferably 10 wt% to 60 wt%, more preferably 20 wt% to 60 wt% moisture in the matrix when included in another food having a water activity of 1.00 for 2 weeks.

[0060] In one embodiment, the one or more lipids are solid at 5°C, 10°C, 15°C, 20°C or 25°C. In some embodiments, the one or more lipids are solid at 35°C, such as solid at 34°C, 33°C, 32°C, 31°C, 30°C, 29°C, 28°C, 27°C or 26°C. In one embodiment, the one or more lipids have a melting temperature of 50°C or less, such as 48°C or less, 46°C or less, 44°C or less, 42°C or less, 40°C or less, 38°C or less, 36°C or less, 34°C or less, 32°C or less, or 30°C or less. In one embodiment, the one or more lipids have a melting temperature of 30°C to 50°C, for example, 32°C to 48°C, 34°C to 44°C or 36°C to 40°C.

[0061] In one embodiment, the one or more lipids comprise or consist of: a monoglyceride, a diglyceride or a triglyceride. In a preferred embodiment, the one or more lipids comprise or consist of a triglyceride.

[0062] In one embodiment, one or more lipids include C10-C22 fatty acids or their salts or esters. In one embodiment, one or more lipids include C12-C20 fatty acids or their salts or esters. In one embodiment, one or more lipids include at least one monoglyceride, diglyceride or triglyceride. In a preferred embodiment, one or more lipids include at least one triglyceride. In one embodiment, at least one monoglyceride, diglyceride or triglyceride includes at least one C12-C20 fatty acid or its salt or ester. In one embodiment, at least one monoglyceride, diglyceride or triglyceride includes at least one fatty acid or its ester selected from the group consisting of the following: lauric acid, myristic acid, palmitic acid, stearic acid and arachidic acid. In one embodiment, each fatty acid or its ester of at least one monoglyceride, diglyceride or triglyceride is selected from lauric acid, myristic acid, palmitic acid, stearic acid and arachidic acid. In one embodiment, each fatty acid or its ester of one or more lipids is C12-C20 fatty acids. In one embodiment, each fatty acid or its ester of one or more lipids is selected from the group consisting of the following: lauric acid, myristic acid, palmitic acid, stearic acid and arachidic acid.

[0063] In some embodiments, the one or more lipids include one or more fatty acids and / or salts and / or esters thereof, and at least 1 wt% of the fatty acids and / or salts and / or esters thereof have a carbon chain length of 14 or less, such as at least 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 12 wt%, 14 wt%, 16 wt%, 18 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, 65 wt%, 70 wt%, 75 wt%, or at least 80 wt%, and useful The range can be selected between any of these values ​​(e.g., 1 wt% to 80wt%, 2wt% to 80wt%, 4wt% to 80wt%, 6wt% to 80wt%, 8wt% to 80wt%, 10wt% to 80wt%, 20wt% to 80wt%, 30wt% to 80wt%, 40wt% to 80wt%, 1wt% to 70wt%, 2wt% to 70wt%, 4wt% to 70wt%, 6wt% to 70wt%, 8wt% to 70wt%, 10wt% to 70wt%, 20wt% to 70wt%, 30wt% to 70wt%, or 40wt% to 70wt%).

[0064] In some embodiments, the one or more lipids include fatty acids and / or salts and / or esters thereof, and at least 30 wt% of the fatty acids and / or salts and / or esters thereof have a carbon chain length of 16 or less, such as at least 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, 65 wt%, 70 wt%, 72 wt%, 74 wt%, 76 wt%, 78 wt%, 80 wt%, 82 wt%, 84 wt%, 86 wt%, 88 wt%, or at least 90 wt%, and useful ranges can be selected between any of these values. % to 88wt%, 78wt% to 88wt%, 78wt% to 88wt%, 80wt% to 88wt%, 70wt% to 88wt% from74wt% to 86wt%, 76wt% to 88wt%, 78wt% to 88wt%, 78wt% to 88wt%, 80wt% to 88wt%, 70wt% to 86wt% from74wt% to 86wt%, 76wt% to 86wt%, 78wt% to 86wt%, 78wt% to 86wt%, or 80wt% to 86wt%.

[0065] In one embodiment, at least 50wt% of one or more lipids are saturated fats. In various embodiments, at least 55wt% of one or more lipids are saturated fats, for example at least 60wt%, at least 65wt%, at least 70wt%, at least 75wt%, at least 80wt%, at least 85wt%, at least 90wt%, at least 95wt% or at least 99wt% of one or more lipids are saturated fats.

[0066] In one embodiment, the one or more lipids comprise or consist of fully hydrogenated palm kernel stearin, palm stearin, fully hydrogenated coconut oil, hydrogenated palm kernel oil, or a mixture of any two or more of these. Preferably, the hydrogenated palm kernel oil is fully hydrogenated.

[0067] In some embodiments, the matrix includes at least 4 wt% of one or more lipids based on the total solid weight, for example, at least 8 wt%, at least 10 wt%, at least 15 wt%, at least 20 wt%, at least 25 wt%, at least 30 wt%, at least 35 wt%, at least 40 wt%, at least 45 wt%, at least 50 wt%, at least 55 wt%, at least 60 wt%, at least 65 wt%, at least 70 wt%, at least 75 wt%, at least 80 wt%, at least 85 wt%, at least 90 wt%, at least 95 wt%, or at least 99 wt% of one or more lipids based on the total solid weight.

[0068] In one embodiment, the matrix comprises 4 to 100 wt%, preferably 30 to 100% of one or more lipids. In some embodiments, the matrix comprises 4 to 100 wt% of one or more lipids by weight of the total solids, for example 8 wt% to 100 wt%, 10 wt% to 100 wt%, 20 wt% to 100 wt%, 30 wt% to 100 wt%, 40 wt% to 100 wt%, 50 wt% to 100 wt%, 60 wt% to 100 wt%, 70 wt% to 100 wt%, 80 wt% to 100 wt%, 90 wt% to 100 wt%, 4 wt% to 99.5 wt%, 8 wt% to 99.5 wt%, 10 wt% to 99.5 wt%, 20 wt% to 99.5 ... 9.5wt%, 30wt% to 99.5wt%, 40wt% to 99.5wt%, 50wt% to 99.5wt%, 60wt% to 99.5wt%, 70wt% to 99.5wt%, 80wt% to 99.5wt%, 90wt% to 99.5wt%, 4wt% to 95wt%, 8wt% to 95wt%, 10wt% to 95wt%, 20wt% to 95wt%, 30wt% to 95wt%, 40wt% to 95wt%, 50wt% to 95wt%, 60wt% to 95wt%, 70wt% to 95wt%, 80wt% to 95wt%, 90 % to 95wt%, 10wt% to 90wt%, 20wt% to 90wt%, 30wt% to 90wt%, 40wt% to 90wt%, 50wt% to 90wt%, 60wt% to 90wt%, 70wt% to 90wt%, 80wt% to 90wt%, 10wt% to 80wt%, 20wt% to 80wt%, 30wt% to 80wt%, 40wt% to 80wt%, 50wt% to 80wt%, 60wt% to 80wt%, 70wt% to 80wt%, 10wt% to 70wt%, 20wt% to 70wt%, 30wt% to 70wt% %, 40wt% to 70wt%, 50wt% to 70wt%, 60wt% to 70wt%, 10wt% to 60wt%, 20wt% to 60wt%, 30wt% to 60wt%, 40wt% to 60wt%, 50wt% to 60wt%, 10wt% to 50wt%, 20wt% to 50wt%, 30wt% to 50wt%, 40wt% to 50wt%, 10wt% to 40wt%, 20wt% to 40wt%, 30wt% to 40wt%, 10wt% to 20wt%, 10wt% to 30wt%, or 10wt% to 20wt% of one or more lipids.

[0069] In one embodiment the matrix comprises at least 10 wt%, preferably at least 15, 20, 25 or 30 wt% fully hydrogenated coconut oil.

[0070] In one embodiment, the matrix comprises one or more adjuvants selected from sugars, emulsifiers, milk solids and salts.

[0071] In some embodiments, the matrix includes 0% to 80% by weight of one or more sugars, for example, 10% to 80% by weight, 20% to 80% by weight, 30% to 80% by weight, 40% to 80% by weight, 50% to 80% by weight, 60% to 80% by weight, 70% to 80% by weight, 0% to 70% by weight, 10% to 70% by weight, 20% to 70% by weight, 30% to 70% by weight, 40% to 70% by weight, 50% to 70% by weight, 60% to 70% by weight, 0% to 60% by weight, 10% to 60% by weight, %, 20wt% to 60wt%, 30wt% to 60wt%, 40wt% to 60wt%, 50wt% to 60wt%, 0wt% to 50wt%, 10wt% to 50wt%, 20wt% to 50wt%, 30wt% to 50wt%, 40wt% to 50wt%, 0wt% to 40wt%, 10wt% to 40wt%, 20wt% to 40wt%, 30wt% to 40wt%, 0wt% to 30wt%, 10wt% to 30wt%, 20wt% to 30wt%, 0wt% to 20wt%, 10wt% to 20wt%, or 0wt% to 10wt% of one or more sugars.

[0072] In some embodiments, the matrix includes 0 to 2 wt% of one or more emulsifiers, based on the total solid weight, for example, 0.2 to 2 wt%, 0.4 to 2 wt%, 0.6 to 2 wt%, 0.8 to 2 wt%, 1.0 to 2 wt%, 1.2 to 2 wt%, 1.4 to 2 wt%, 1.6 to 2 wt%, 1.8 to 2 wt%, 0 to 1.8 wt%, 0.2 to 1.8 wt%, 0.4 to 1.8 wt%, 0.6 to 2 wt%, 0.8 to 2 wt%, 1.0 to 2 wt%, 1.2 to 2 wt%, 1.4 to 2 wt%, 1.6 to 2 wt%, 1.8 to 2 wt%, 0 to 1.8 wt%, 0.2 to 1.8 wt%, 0.4 to 1.8 wt%, 0.6 to 2 % to 1.8wt%, 0.8wt% to 1.8wt%, 1.0wt% to 1.8wt%, 1.2wt% to 1.8wt%, 1.4wt% to 1.8wt%, 1.6wt% to 1.8wt%, 0wt% to 1.6wt%, 0.2wt% to 1.6wt%, 0.4wt% to 1.6wt%, 0.6wt% to 1.6wt%, 0.8wt% to 1.6wt%, 1.0wt% to 1.6wt%, 1.2wt% to 1.6wt%, 1.4wt% to 1.6wt%, 0wt % to 1.4wt%, 0.2wt% to 1.4wt%, 0.4wt% to 1.4wt%, 0.6wt% to 1.4wt%, 0.8wt% to 1.4wt%, 1.0wt% to 1.4wt%, 1.2wt% to 1.4wt%, 0wt% to 1.2wt%, 0.2wt% to 1.2wt%, 0.4wt% to 1.2wt%, 0.6wt% to 1.2wt%, 0.8wt% to 1.2wt%, 1.0wt% to 1.2wt%, 0wt% to 1.0wt%, 0.2wt% to 1.2wt%. % to 1.0wt%, 0.4wt% to 1.0wt%, 0.6wt% to 1.0wt%, 0.8wt% to 1.0wt%, 0wt% to 0.8wt%, 0.2wt% to 0.8wt%, 0.4wt% to 0.8wt%, 0.6wt% to 0.8wt%, 0wt% to 0.6wt%, 0.2wt% to 0.6wt%, 0.4wt% to 0.6wt%, 0wt% to 0.4wt%, 0.2wt% to 0.4wt%, or 0wt% to 0.2wt% of one or more emulsifiers.

[0073] In some embodiments, the matrix includes 0wt% to 30wt% dairy solids by weight of total solids, for example, 5wt% to 30wt%, 10wt% to 30wt%, 15wt% to 30wt%, 20wt% to 30wt%, 25wt% to 30wt%, 0wt% to 25wt%, 5wt% to 25wt%, 10wt% to 25wt%, 15wt% to 25wt%, 20wt% to 25wt%, 0wt% to 20wt%, 5wt% to 20wt%, 10wt% to 20wt%, 15wt% to 20wt%, 0wt% to 15wt%, 5wt% to 15wt%, 10wt% to 15wt%, 0wt% to 10wt%, 5wt% to 10wt%, or 0wt% to 5wt% dairy solids by weight of total solids.

[0074] In one embodiment, the matrix further comprises, by total solids weight:

[0075] a. 20-80% sugar, preferably sucrose and / or lactose;

[0076] b. 0.1-20% emulsifier, preferably sorbitan tristearate and / or lecithin; and / or

[0077] c. 10-50% milk solids.

[0078] In some embodiments, the matrix comprises about 50 wt % to about 70 wt % hydrogenated coconut oil and about 30 wt % to about 50 wt % sucrose, based on the total solid weight; more preferably about 55 wt % to about 65 wt % hydrogenated coconut oil, and about 35 wt % to about 45 wt % sucrose; most preferably about 59 wt % hydrogenated coconut oil and about 41 wt % sucrose.

[0079] In some embodiments, the matrix includes about 20 wt % to about 40 wt % palm stearin, about 20 wt % to about 40 wt % hydrogenated coconut oil, and about 30 wt % to about 50 wt % sucrose, based on the total solid weight; preferably about 25 wt % to about 35 wt % palm stearin, about 25 wt % to about 35 wt % hydrogenated coconut oil, and about 35 wt % to about 45 wt % sucrose; most preferably about 29.5 wt % palm stearin, about 29.5 wt % hydrogenated coconut oil, and about 41 wt % sucrose.

[0080] In some embodiments, the matrix includes about 0 wt % to about 20 wt % palm stearin, about 0 wt % to about 20 wt % hydrogenated coconut oil, about 30 wt % to about 50 wt % hydrogenated palm kernel stearin and about 30 wt % to about 50 wt % sucrose based on the total solid weight; preferably about 5 wt % to about 15 wt % palm stearin, about 5 wt % to about 15 wt % hydrogenated coconut oil, about 35 wt % to about 45 wt % hydrogenated palm kernel stearin, and about 35 wt % to about 45 wt % sucrose; most preferably about 11 wt % palm stearin, about 11 wt % hydrogenated coconut oil, about 37 wt % hydrogenated palm kernel stearin and about 41 wt % sucrose.

[0081] In some embodiments, the matrix comprises, based on total solid weight, about 90 wt % to about 100 wt % cocoa butter and optionally about 0.1 wt % to about 1.0 wt % lecithin; preferably about 95 wt % to about 100 wt % cocoa butter, and optionally about 0.1 wt % to about 1.0 wt % lecithin; most preferably about 99.5 wt % cocoa butter and about 0.5 wt % lecithin.

[0082] In some embodiments, the matrix comprises, based on total solids weight, about 65 wt % to about 85 wt % cocoa butter and about 15 wt % to about 35 wt % milk solids; preferably about 70 wt % to about 80 wt % cocoa butter and about 20 wt % to about 30 wt % milk solids; most preferably about 73 wt % cocoa butter and about 26 wt % milk solids.

[0083] In some embodiments, the matrix comprises at least about 80 wt % hydrogenated palm kernel oil, preferably at least about 90 wt %, more preferably at least about 95 wt %, and most preferably about 100 wt % hydrogenated palm kernel oil, based on the total solid weight, and a useful range can be selected between any of these values ​​(e.g., about 80 wt % to about 100 wt %, about 90 wt % to about 100 wt %, or about 95 wt % to about 100 wt %).

[0084] In some embodiments, the matrix comprises, based on total solid weight, about 75 wt % to about 95 wt % cocoa butter and about 5 wt % to about 25 wt % lactose; preferably about 80 wt % to about 90 wt % cocoa butter and about 5 wt % to about 15 wt % lactose; more preferably about 89.5 wt % cocoa butter and about 13.5 wt % lactose.

[0085] In some embodiments, the matrix comprises about 75 wt % to about 95 wt % hydrogenated coconut oil and about 5 wt % to about 25 wt % lactose, based on the total solid weight; preferably about 80 wt % to about 90 wt % hydrogenated coconut oil and about 10 wt % to about 20 wt % lactose; more preferably about 86.5 wt % hydrogenated coconut oil and about 13.5 wt % lactose.

[0086] In one embodiment, the particles have a diameter of 50 μm to 10 mm, for example 50 μm to 9 mm, 50 μm to 8 mm, 50 μm to 7 mm, 50 μm to 6 mm, 50 μm to 5 mm, 50 μm to 4 mm, 50 μm to 3 mm, 50 μm to 2 mm, 50 μm to 1 mm, 50 μm to 500 μm, 100 μm to 10 mm, 100 μm to 9 mm, 100 μm to 8 mm, 100 μm to 7 mm, 100 μm to 6 mm, 100 μm to 4 mm, 50 μm to 3 mm, 50 μm to 2 mm, 50 μm to 1 mm, 50 μm to 500 μm, 100 μm to 10 mm, 100 μm to 9 mm, 100 μm to 8 mm, 100 μm to 7 mm, 100 μm to 6 mm, 100 μm to To 5mm, 100μm to 4mm, 100μm to 3mm, 100μm to 2mm, 100μm to 1mm, 100μm to 500μm, 200μm to 10mm, 200μm to 9mm, 200μm to 8mm, 200μm to 7mm, 200μm to 6mm, 200μm to 5mm, 200μm to 4mm, 200μm to 3mm, 200μm to 2mm, 200μm to 1mm, or 200μm to 500μm.

[0087] In one embodiment, the particle also includes at least one coating. In some embodiments, the particle includes two, three, four or five coatings. In some embodiments, the coating includes alginate, chitosan, collagen, dextran, pectin, pullulan, gelatin, carrageenan, agar, cellulose, hemicellulose, ethyl cellulose, carboxycellulose or any two or more mixtures of these.

[0088] In one embodiment, the food or beverage product comprises:

[0089] a. Protein powder, protein shake, protein pill, protein gel or sports nutrition preparation,

[0090] b.UHT drinks, UHT smoothies,

[0091] c. Infant formula, follow-on formula, growing-up formula, pediatric formula, human milk fortifier, children's food or beverage, maternal supplement or maternal nutritional preparation,

[0092] d. Gels, such as heat-set gels, sauces, spreads, jams, jellies or honey;

[0093] e. Acid-coagulated gel, room temperature stable yogurt, stirred yogurt, set yogurt or drinking yogurt,

[0094] f. Neutral beverages, acidic beverages, water, juice, milk, smoothies, milkshakes, pills, alcoholic beverages, soft drinks, kombucha, kefir or instant mix powder,

[0095] g. bars, balls, cakes, cookies, muffins or baked goods,

[0096] h. Medical foods, soups, desserts, puddings, custards, enteral preparations, preparations for the elderly, tablets, capsules or supplements,

[0097] i. Candy, gummies, candies, chocolates, soft candies, truffles, chewing gum, frozen desserts, ice cream

[0098] j. Seasonings, toppings or baking ingredients,

[0099] k.Cream, cheese or butter, or

[0100] l. Livestock food or pet food.

[0101] In some embodiments, the food or beverage product includes yogurt, juice (such as orange juice), sports shake, yogurt sauce, cream cheese sauce, or milk, for example, non-dairy milk such as oat milk.

[0102] In one embodiment, the food or beverage product has a water activity of at least 0.2, 0.4, 0.6, 0.8 or 0.9.

[0103] In one embodiment, the food or beverage product is stable at 30°C for at least 30 days, preferably at least 60 days, more preferably at least 6 months.

[0104] In one embodiment, the bioactive agent comprises a microorganism and the degradation rate of the microorganism measured at 30°C over 12 months is less than 7 log CFU / g / y, preferably less than 6, 5, 4, 3, 2 or 1 log CFU / g / y.

[0105] In one embodiment, the personal care product is a skin care product such as a skin cream, a sunscreen, a hair care product (such as a shampoo or conditioner), a dental product (such as a toothpaste or dentifrice), a deodorant, a make-up product, a beauty product, or a feminine care product.

[0106] In one embodiment of the sixth, seventh, eighth or ninth aspect, the method further comprises the step of coating the particles in at least one coating layer.

[0107] In one embodiment of the method of the sixth, seventh, eighth or ninth aspect, the particle comprises the particle of the first or second aspect.

[0108] In one embodiment of the sixth, seventh, eighth or ninth aspect, the method further comprises the steps of:

[0109] d. combining the granules of step c with a medium having a water activity of at least 0.5, preferably UHT treated yogurt, and

[0110] e. Incubate the culture medium for at least 2 weeks.

[0111] In one embodiment, the bioactive agent-supplemented food or beverage product is a food or beverage product according to the third aspect.

[0112] The term "comprising" as used in this specification and claims means "consisting at least in part of. When interpreting statements in this specification and claims that include the term "comprising," other features may be present in addition to the features preceded by the term in each statement. The related terms "comprise" and "comprised" are to be interpreted in a similar manner.

[0113] In this specification where reference has been made to patent specifications, other external documents or other sources of information, this is generally to provide a context for discussing the features of the invention. Unless expressly stated otherwise, reference to such external documents should not be construed as an admission that such documents or such sources of information are prior art or form part of the common general knowledge in the art in any area of ​​rights. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] Embodiments of the present invention will now be described with reference to the accompanying drawings, in which:

[0115] Figure 1 Contour plots of coating matrices including palm stearin, fully hydrogenated palm kernel oil, and fully hydrogenated coconut oil, alone and in combination, are shown.

[0116] Figure 2 Contour plots of coating matrices including palm stearin, fully hydrogenated coconut oil and cocoa powder, alone and in combination, are shown.

[0117] Figure 3 Contour plots of coating matrices including fully hydrogenated palm kernel oil, fully hydrogenated coconut oil and cocoa powder, alone and in combination, are shown.

[0118] Figure 4 Shown is a scanning electron microscope image of exopolysaccharide / biofilm coated Lactobacillus rhamnosus HN001 in a matrix with hydrogenated palm kernel oil at 100x magnification.

[0119] Figure 5 Shown is a scanning electron microscopy image of exopolysaccharide / biofilm coated Lactobacillus rhamnosus HN001 at 250x magnification in a matrix with hydrogenated palm kernel oil.

[0120] Figure 6Shown is a scanning electron microscopy image of exopolysaccharide / biofilm coated Lactobacillus rhamnosus HN001 at 1,200x magnification in a matrix with hydrogenated palm kernel oil.

[0121] Figure 7 Shown is a scanning electron microscopy image of exopolysaccharide / biofilm coated Lactobacillus rhamnosus HN001 at 4,000x magnification in a matrix with hydrogenated palm kernel oil.

[0122] Figure 8 Light microscopy images of Lactobacillus rhamnosus HN001 clusters attached to the surface of a glass wool strip are shown. Images were taken under oil immersion at 100x magnification and imaged with crystal violet stain.

[0123] Fig. 9 The Raman spectrum of the biofilm induced by Lactobacillus rhamnosus HN001 is shown. 350-600 cm -1 Three distinct peaks in the spectral region (indicated by arrows) appear to be associated with the presence of EPS components.

[0124] Fig.10 Raman spectra of bead structures containing Lactobacillus rhamnosus HN001 are shown. Multivariate curve resolution estimated spectra from day 120 data analysis of bead sample AT03 are shown. Arrows indicate the same three EPS-related peaks.

[0125] Fig.11 Concanavalin (Con A) staining is shown for sample MVP17 at shelf life day 90. Mannose / glucose stained with ConA is shown in orange; proteins are stained green (this stain will also stain dead cells); cells are stained with DAPI in light blue.

[0126] Fig.12 Shown is wheat germ agglutinin (WGA) staining of sample MVP17 at shelf life day 90. N-acetylglucosamine stained with WGA appears orange; proteins are stained green (this stain will also stain dead cells); cells are stained with DAPI in light blue.

[0127] Fig.13 Shown is Ulex oleraceus agglutinin (UEAI) staining of sample MVP17 at day 90 of shelf life. UEAI staining is shown in orange; proteins are stained green (this stain will also stain dead cells); cells are stained with DAPI in light blue. DETAILED DESCRIPTION

[0128] In the description of this specification, reference may be made to subject matter that is not within the scope of the claims of this application. This subject matter should be easily recognized by those skilled in the art and can help practice the invention as defined in the claims of this application.

[0129] Particles

[0130] Applicants have surprisingly discovered that particles comprising a bioactive agent embedded in a biofilm, wherein the biofilm is embedded in a matrix, wherein the matrix has a water vapor transmission rate (WVTR) of 0.1 to 500, provide the bioactive agent with an improved shelf life even when stored in a high water activity environment at ambient temperature.

[0131] Thus, in a first aspect, the invention provides a particle comprising a bioactive agent entrapped in a biological membrane, wherein the biological membrane is embedded in a matrix, wherein the matrix has a water vapour transmission rate (WVTR) of 0.1 to 500. Preferably, the matrix comprises at least 6 wt% of one or more lipids.

[0132] The term "particle" refers to a portion or amount of a material, such as a small portion or amount of a material. The term includes or can be used interchangeably with: pellets, beads, spheres, pellets, etc. In some embodiments, the particles can be generally spherical. Preferably, the particles are edible. That is, preferably the particles do not include any non-edible ingredients.

[0133] As used herein, the term "encapsulated" means "at least partially surrounded by..." For example, the bioactive agent is at least partially surrounded by the biofilm, and the biofilm is at least partially surrounded by the matrix. The term is not intended to require that the bioactive agent or the biofilm be completely surrounded on all sides.

[0134] In some embodiments, the bioactive agent is encapsulated within the biofilm. In some embodiments, the biofilm is encapsulated within the matrix. As used herein, the term "encapsulated within" means "completely surrounded by". In some embodiments, the bioactive agent is dispersed in the biofilm. In some embodiments, the biofilm is dispersed in the matrix.

[0135] The term "matrix" refers to a substance or mixture of substances in which the biofilm and / or bioactive agent is embedded. The matrix can be any substance or mixture of substances suitable for embedding the bioactive agent and forming particles. Preferably, the matrix is ​​solid at room temperature. In one embodiment, the matrix is ​​edible.

[0136] In some embodiments, the matrix includes one or more lipids, one or more carbohydrates, one or more sugars, one or more proteins, one or more minerals, or any combination of two or more of these.

[0137] The term "lipid" refers to a class of organic compounds that are soluble in non-polar solvents. Some examples of lipids include, but are not limited to, fats, fatty acids, waxes, monoglycerides, diglycerides and triglycerides and phospholipids. Some lipids such as fats can be saturated or unsaturated. Unsaturated lipids can be converted into saturated lipids by hydrogenation. Hydrogenation can be incomplete or partial (if not all carbon-carbon double bonds are reduced), or it can be complete. The term "completely hydrogenated" and related terms such as "all hydrogenated" and "100wt% hydrogenated" refer to at least 99wt% of the lipid to which the term is applied that does not contain unsaturated carbon-carbon double bonds.

[0138] In some embodiments, the matrix comprises, consists essentially of, or consists of one or more lipids.Preferably, the one or more lipids are solid at 25°C.

[0139] In some embodiments, the melting temperature of one or more lipids is 50°C or less, e.g., 48°C or less, 46°C or less, 44°C or less, 42°C or less, 40°C or less, 38°C or less, 36°C or less, 34°C or less, 32°C or less, 30°C or less, 25°C or less, 20°C or less, 15°C or less, 10°C or less, or 5°C or less, and useful ranges can be in this range. any of the following values ​​(e.g., 5°C to 50°C, 10°C to 50°C, 15°C to 50°C, 20°C to 50°C, 25°C to 50°C, 30°C to 50°C, 32°C to 50°C, 34°C to 50°C, 36°C to 50°C, 5°C to 48°C, 10°C to 48°C, 15°C to 48°C, 20°C to 48°C, 25°C to 48°C, 30°C to 48°C, 32°C to 48°C, 34°C to 48°C , 36℃ to 48℃, 5℃ to 46℃, 10℃ to 46℃, 15℃ to 46℃, 20℃ to 46℃, 25℃ to 46℃, 30℃ to 46℃, 32℃ to 46℃, 34℃ to 46℃, 36℃ to 46℃, 5℃ to 44℃, 10℃ to 44℃, 15℃ to 44℃, 20℃ to 44℃, 25℃ to 44℃, 30℃ to 44℃, 32℃ to 44℃, 34℃ to 44℃, 36℃ to to 42°C, 30°C to 42°C, 32°C to 42°C, 34°C to 42°C, 36°C to 42°C, 5°C to 40°C, 10°C to 40°C, 15°C to 40°C, 20°C to 40°C, 25°C to 40°C, 30°C to 40°C, 32°C to 40°C, 34°C to 40°C, or 36°C to 40°C).

[0140] In some embodiments, the lipid comprises, consists essentially of, or consists of triglycerides. In some embodiments, the lipid comprises, consists essentially of, or consists of C12-C20 fatty acids or salts or esters thereof.

[0141] In some embodiments, the one or more lipids include one or more fatty acids and / or salts and / or esters thereof, and at least 1 wt% of the fatty acids and / or salts and / or esters thereof have a carbon chain length of 14 or less, such as at least 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 12 wt%, 14 wt%, 16 wt%, 18 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, 65 wt%, 70 wt%, 75 wt%, or at least 80 wt%, and useful The range can be selected between any of these values ​​(e.g., 1 wt% to 80wt%, 2wt% to 80wt%, 4wt% to 80wt%, 6wt% to 80wt%, 8wt% to 80wt%, 10wt% to 80wt%, 20wt% to 80wt%, 30wt% to 80wt%, 40wt% to 80wt%, 1wt% to 70wt%, 2wt% to 70wt%, 4wt% to 70wt%, 6wt% to 70wt%, 8wt% to 70wt%, 10wt% to 70wt%, 20wt% to 70wt%, 30wt% to 70wt%, or 40wt% to 70wt%).

[0142] In some embodiments, the one or more lipids include fatty acids and / or salts and / or esters thereof, and at least 30 wt% of the fatty acids and / or salts and / or esters thereof have a carbon chain length of 16 or less, such as at least 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, 65 wt%, 70 wt%, 72 wt%, 74 wt%, 76 wt%, 78 wt%, 80 wt%, 82 wt%, 84 wt%, 86 wt%, 88 wt%, or at least 90 wt%, and useful ranges can be selected between any of these values. % to 88wt%, 78wt% to 88wt%, 78wt% to 88wt%, 80wt% to 88wt%, 70wt% to 88wt% from74wt% to 86wt%, 76wt% to 88wt%, 78wt% to 88wt%, 78wt% to 88wt%, 80wt% to 88wt%, 70wt% to 86wt% from74wt% to 86wt%, 76wt% to 86wt%, 78wt% to 86wt%, 78wt% to 86wt%, or 80wt% to 86wt%.

[0143] In alternative embodiments, the one or more lipids include fatty acids and / or salts and / or esters thereof, and at least 10 wt % of the fatty acids and / or salts and / or esters thereof have a carbon chain length of 16 or less, such as at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, or at least 90%, and useful ranges can be selected between any of these values ​​(e.g., 10% to 90%, 10% to 120%, 120% to 150%, 120% to 16 ... 8%, 70% to 88%, 74% to 88%, 76% to 88%, 78% to 88%, 78% to 88%, 80% to 88%, 70% to 86%, 74% to 86%, 76% to 86%, 78% to 86%, 78% to 86%, or 80% to 86%).

[0144] The fatty acid esters may include monoglycerides, diglycerides and / or triglycerides.

[0145] In some embodiments, at least 50wt% of one or more lipids are saturated fats. In some embodiments, lipids can be partially or completely hydrogenated. In some embodiments, one or more lipids include, are essentially composed of or are composed of: fully hydrogenated palm kernel stearin, palm stearin, fully hydrogenated coconut oil, cocoa butter, or any two or more of these mixtures. In a preferred embodiment, one or more lipids include, are essentially composed of or are composed of: fully hydrogenated palm kernel stearin, palm stearin, fully hydrogenated coconut oil, or any two or more of these mixtures.

[0146] In some embodiments, the matrix comprises at least 4wt% of one or more lipids by weight of the total solids, for example at least 8wt%, at least 10wt%, at least 15wt%, at least 20wt%, at least 25wt%, at least 30wt%, at least 35wt%, at least 40wt%, at least 45wt%, at least 50wt%, at least 55wt%, at least 60wt%, at least 65wt%, at least 70wt%, at least 75wt%, at least 80wt%, at least 85wt%, at least 90wt%, at least 95wt% or at least 99wt% of one or more lipids by weight of the total solids. Preferably, the matrix comprises at least 30% of one or more lipids by weight of the total solids.

[0147] In some embodiments, the matrix comprises 4% to 95% of one or more lipids by weight of total solids, for example, at least 8% to 95%, 10% to 95%, 20% to 95%, 30% to 95%, 40% to 95%, 50% to 95%, 60% to 95%, 70% to 95%, 80% to 95%, 90% to 95%, 10% to 90%, 20% to 90%, 30% to 90%, 40% to 90%, 50% to 90%, 60% to 90%, 70% to 90%, 80% to 90%, 10% to 80%, 20% to 80%, 30% to 80% by weight of total solids. %, 40% to 80%, 50% to 80%, 60% to 80%, 70% to 80%, 10% to 70%, 20% to 70%, 30% to 70%, 40% to 70%, 50% to 70%, 60% to 70%, 10% to 60%, 20% to 60%, 30% to 60%, 40% to 60%, 50% to 60%, 10% to 50%, 20% to 50%, 30% to 50%, 40% to 50%, 10% to 40%, 20% to 40%, 30% to 40%, 10% to 20%, 10% to 30%, or 10% to 20% of one or more lipids. Preferably, the matrix comprises 30% to 95% of one or more lipids by weight of the total solids.

[0148] In a preferred embodiment, the matrix comprises at least 10 wt%, preferably at least 15, 20, 25 or 30% fully hydrogenated coconut oil.

[0149] In some embodiments, the matrix includes one or more adjuvants. Examples of adjuvants include sugars, emulsifiers, milk solids, and salts. In some embodiments, adjuvants can be used to provide additional structural integrity to the particles.

[0150] In one embodiment, the matrix comprises, consists essentially of, or consists of hydrogenated palm kernel stearin (preferably fully hydrogenated palm kernel stearin) and sugar (preferably sucrose and / or lactose). In one embodiment, the matrix comprises, consists essentially of, or consists of hydrogenated palm kernel stearin (preferably fully hydrogenated palm kernel stearin) and milk solids (preferably skimmed milk powder). In one embodiment, the matrix comprises, consists essentially of, or consists of hydrogenated palm kernel stearin (preferably fully hydrogenated palm kernel stearin), sugar (preferably sucrose and / or lactose) and milk solids (preferably skimmed milk powder).

[0151] In one embodiment, the matrix comprises, consists essentially of, or consists of palm stearin and sugar (preferably sucrose and / or lactose). In one embodiment, the matrix comprises, consists essentially of, or consists of palm stearin and milk solids (preferably skimmed milk powder). In one embodiment, the matrix comprises, consists essentially of, or consists of palm stearin, sugar (preferably sucrose and / or lactose) and milk solids (preferably skimmed milk powder).

[0152] In one embodiment, the matrix comprises, consists essentially of, or consists of hydrogenated coconut oil (preferably fully hydrogenated coconut oil) and sugar (preferably sucrose and / or lactose). In one embodiment, the matrix comprises, consists essentially of, or consists of hydrogenated coconut oil (preferably fully hydrogenated coconut oil) and milk solids (preferably skimmed milk powder). In one embodiment, the matrix comprises, consists essentially of, or consists of hydrogenated coconut oil (preferably fully hydrogenated coconut oil), sugar (preferably sucrose and / or lactose) and milk solids (preferably skimmed milk powder).

[0153] In one embodiment, the matrix comprises, consists essentially of, or consists of: hydrogenated palm kernel oil and sugar (preferably sucrose and / or lactose). In one embodiment, the matrix comprises, consists essentially of, or consists of: hydrogenated palm kernel oil and milk solids (preferably skimmed milk powder). In one embodiment, the matrix comprises, consists essentially of, or consists of: hydrogenated palm kernel oil, sugar (preferably sucrose and / or lactose) and milk solids (preferably skimmed milk powder).

[0154] In one embodiment, the matrix comprises, consists essentially of, or consists of cocoa butter and sugar (preferably sucrose and / or lactose). In one embodiment, the matrix comprises, consists essentially of, or consists of cocoa butter and milk solids (preferably skimmed milk powder). In one embodiment, the matrix comprises, consists essentially of, or consists of cocoa butter, sugar (preferably sucrose and / or lactose) and milk solids (preferably skimmed milk powder).

[0155] In some embodiments, the matrix includes 0% to 80% by weight of one or more sugars, for example, 10% to 80% by weight, 20% to 80% by weight, 30% to 80% by weight, 40% to 80% by weight, 50% to 80% by weight, 60% to 80% by weight, 70% to 80% by weight, 0% to 70% by weight, 10% to 70% by weight, 20% to 70% by weight, 30% to 70% by weight, 40% to 70% by weight, 50% to 70% by weight, 60% to 70% by weight, 0% to 60% by weight, 10% to 60% by weight, %, 20wt% to 60wt%, 30wt% to 60wt%, 40wt% to 60wt%, 50wt% to 60wt%, 0wt% to 50wt%, 10wt% to 50wt%, 20wt% to 50wt%, 30wt% to 50wt%, 40wt% to 50wt%, 0wt% to 40wt%, 10wt% to 40wt%, 20wt% to 40wt%, 30wt% to 40wt%, 0wt% to 30wt%, 10wt% to 30wt%, 20wt% to 30wt%, 0wt% to 20wt%, 10wt% to 20wt%, or 0wt% to 10wt% of one or more sugars. Preferably, the matrix includes one or more sugars of total solid weight 0% to 50%. Preferably, the one or more sugars include sucrose and / or lactose or are composed of sucrose and / or lactose.

[0156] In some embodiments, the matrix includes 0 to 2 wt% of one or more emulsifiers, based on the total solid weight, for example, 0.2 to 2 wt%, 0.4 to 2 wt%, 0.6 to 2 wt%, 0.8 to 2 wt%, 1.0 to 2 wt%, 1.2 to 2 wt%, 1.4 to 2 wt%, 1.6 to 2 wt%, 1.8 to 2 wt%, 0 to 1.8 wt%, 0.2 to 1.8 wt%, 0.4 to 1.8 wt%, 0.6 to 2 wt%, 0.8 to 2 wt%, 1.0 to 2 wt%, 1.2 to 2 wt%, 1.4 to 2 wt%, 1.6 to 2 wt%, 1.8 to 2 wt%, 0 to 1.8 wt%, 0.2 to 1.8 wt%, 0.4 to 1.8 wt%, 0.6 to 2 % to 1.8wt%, 0.8wt% to 1.8wt%, 1.0wt% to 1.8wt%, 1.2wt% to 1.8wt%, 1.4wt% to 1.8wt%, 1.6wt% to 1.8wt%, 0wt% to 1.6wt%, 0.2wt% to 1.6wt%, 0.4wt% to 1.6wt%, 0.6wt% to 1.6wt%, 0.8wt% to 1.6wt%, 1.0wt% to 1.6wt%, 1.2wt% to 1.6wt%, 1.4wt% to 1.6wt%, 0wt % to 1.4wt%, 0.2wt% to 1.4wt%, 0.4wt% to 1.4wt%, 0.6wt% to 1.4wt%, 0.8wt% to 1.4wt%, 1.0wt% to 1.4wt%, 1.2wt% to 1.4wt%, 0wt% to 1.2wt%, 0.2wt% to 1.2wt%, 0.4wt% to 1.2wt%, 0.6wt% to 1.2wt%, 0.8wt% to 1.2wt%, 1.0wt% to 1.2wt%, 0wt% to 1.0wt%, 0.2wt% to 1.2wt%. % to 1.0wt%, 0.4wt% to 1.0wt%, 0.6wt% to 1.0wt%, 0.8wt% to 1.0wt%, 0wt% to 0.8wt%, 0.2wt% to 0.8wt%, 0.4wt% to 0.8wt%, 0.6wt% to 0.8wt%, 0wt% to 0.6wt%, 0.2wt% to 0.6wt%, 0.4wt% to 0.6wt%, 0wt% to 0.4wt%, 0.2wt% to 0.4wt%, or 0wt% to 0.2wt% of one or more emulsifiers. Preferably, the matrix includes 0% to 0.5% of one or more emulsifiers by weight of the total solids. Preferably, the one or more emulsifiers include or consist of sorbitan tristearate and / or lecithin (such as soy lecithin).

[0157] In some embodiments, the matrix comprises 0 to 30 wt% of milk solids by weight of the total solids, for example 5 to 30 wt%, 10 to 30 wt%, 15 to 30 wt%, 20 to 30 wt%, 25 to 30 wt%, 0 to 25 wt%, 5 to 25 wt%, 10 to 25 wt%, 15 to 25 wt%, 20 to 25 wt%, 0 to 20 wt%, 5 to 20 wt%, 10 to 20 wt%, 15 to 20 wt%, 0 to 15 wt%, 5 to 15 wt%, 10 to 15 wt%, 0 to 10 wt%, 5 to 10 wt%, or 0 to 5 wt% of milk solids by weight of the total solids. Preferably, the matrix comprises 20 to 30 wt% of milk solids by weight of the total solids.

[0158] In a preferred embodiment, the matrix comprises, by weight of total solids: 20-80% sugar, preferably sucrose; 0.1-20% emulsifier, preferably sorbitan tristearate and / or lecithin; and / or 10-50% milk solids.

[0159] In some embodiments, the particles further comprise an additional coating. Such a layer can be used to alter the permeability of the particles, such as water permeability, water vapor transmission rate, or diffusivity. Alternatively or in addition, the coating can be used to provide resistance to environmental conditions, such as resistance to gastric fluid.

[0160] In some embodiments, the coating comprises agar, alginate, carboxycellulose, carrageenan, cellulose, cellulose acetate phthalate, chitosan, collagen, dextran, ethylcellulose, gelatin, glucomannan, hemicellulose, lactoprotein, shellac, starch, pectin, poly-L-lysine, pullulan, or a mixture of any two or more of these.

[0161] In some embodiments, the matrix is ​​solid at 25°C.

[0162] In some embodiments, the melting temperature of the matrix is ​​at least about 30°C, such as at least about 32°C, 34°C, 35°C, 36°C, 38°C, 40°C, 42°C, 44°C, 45°C, 46°C, 48°C, 50°C, 52°C, 54°C, or at least about 55°C, and useful ranges can be selected between any of these values ​​(e.g., about 30°C to about 55°C, about 35°C to about 50°C, or about 40°C to about 48°C).

[0163] The particles can be made into any suitable size using conventional techniques. It should be understood that different applications may require particles of different sizes. Particle size can be measured using a variety of techniques known in the art. The technique used to measure particle size can vary depending on the size of the particle to be measured. For example, in one embodiment, particle size is measured by microscopy. This is particularly useful for particles with a diameter of ≥1 mm. In another embodiment, particle size is measured using a Malvern Mastersizer 3000 or similar analyzer. This is particularly useful for measuring particles with a diameter <1 mm.

[0164] In some embodiments, the particles have a diameter of 50 μm to 10 mm, for example, 50 μm to 9 mm, 50 μm to 8 mm, 50 μm to 7 mm, 50 μm to 6 mm, 50 μm to 5 mm, 50 μm to 4 mm, 50 μm to 3 mm, 50 μm to 2 mm, 50 μm to 1 mm, 50 μm to 900 μm, 50 μm to 800 μm, 50 μm to 700 μm, 50 μm to 600 μm, 50 μm to 500 μm, 50 μm to 400 μm, 50 μm to 300 μm, 50 μm to 2 mm. 00μm, 75μm to 10mm, 75μm to 9mm, 75μm to 8mm, 75μm to 7mm, 75μm to 6mm, 75μm to 5mm, 75μm to 4mm, 75μm to 3mm, 75μm to 2mm, 75μm to 1mm, 75μm to 900μm, 75μm to 800μm, 75μm to 700μm, 75μm to 600μm, 75μm to 500μm, 75μm to 400μm, 75μm to 300μm, 75μm to 200μm, 100μm to 10mm, 100μm to 9mm, 100μm to 8mm, 100μm to 7mm, 100μm to 6mm, 100μm to 5mm, 100μm to 4mm, 100μm to 3mm, 100μm to 2mm, 100μm to 1mm, 100μm to 900μm, 100μm to 800μm, 100μm to 700μm, 100μm to 600μm, 100μm to 500μm, 100μm to 400μm, 100μm to 300μm, 100μm to 200μm , 200μm to 10mm, 200μm to 9mm, 200μm to 8mm, 200μm to 7mm, 200μm to 6mm, 200μm to 5mm, 200fromμm to 4mm, 200μm to 3mm, 200μm to 2mm, 200μm to 1mm, 200μm to 900μm, 200μm to 800μm, 200μm to 700μm, 200μm to 600μm, 200μm to 500μm, 200μm to 400μm, or 200μm to 300μm in diameter.

[0165] Applicants have discovered that the particles of the present invention are not completely impermeable to the ingress of moisture and / or other compounds present in their environment. Applicants have surprisingly discovered that, contrary to the teachings of the prior art, biologically active agents such as microorganisms are able to maintain high viability for extended periods of time within the particles of the present invention, even when stored in a high water activity environment at ambient temperature.

[0166] Thus, in some embodiments, the particles are impermeable to moisture. Thus, in some embodiments, the matrix has a molecular weight of 0.1 to 500, such as 0.1 to 400, 0.1 to 300, 0.1 to 200, 0.1 to 150, 0.1 to 100, 0.1 to 90, 0.1 to 80, 0.1 to 70, 0.1 to 60, 0.1 to 50, 0.1 to 40, 0.1 to 30, 0.1 to 20, 0.1 to 10, 0.1 to 9, 0.1 to 8, 0.1 to 7, 0.1 to 6, 0.1 to 5, 0.1 to 4, 0.1 to 3, 0.5 to 500, such as 0.5 to 400, 0.5 to 300, 0.5 to 200, 0.5 to 150, 0.5 to 1 to 100, 0.5 to 90, 0.5 to 80, 0.5 to 70, 0.5 to 60, 0.5 to 50, 0.5 to 40, 0.5 to 30, 0.5 to 20, 0.5 to 10, 0.5 to 9, 0.5 to 8, 0.5 to 7, 0.5 to 6, 0.5 to 5, 0.5 to 4, 0.5 to 3, 1 to 500, for example 1 to 400, 1 to 300, 1 to 200, 1 to 150, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3. 2 to 500, for example 2 to 400, 2 to 300, 2 to 200, 2 to 150, 2 to 100, 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3. 3 to 500, for example 3 to 400, 3 to 300, 3 to 200, 3 to 150, 3 to 100, 3 to 90, 3 to 80, 3 to 70, 3 to 60, 3 to 50, 3 to 40, 3 to 30, 3 to 20, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 5 to 8, 5 to 7, 4 to 6, 4 to 500, for example, 5 to 400, 5 to 300, 5 to 200, 5 to 150, 5 to 100, 5 to 90, 4 to 80, 4 to 70, 4 to 60, 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 500, for example, 5 to 400, 5 to 300, 5 to 200, 5 to 150, 5 to 100, 5 to 90, 5 to 80, 5 to 70, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 20, 5 to 10, 5 to 9, 5 to 8, 5 to 7, or 5 to 6.In some embodiments, the substrate has a water vapor transmission rate (WVTR) of less than 500, e.g., less than 400, less than 300, less than 200, less than 150, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or about 1.

[0167] Water vapor transmission rate (WVTR) is a measure of the amount of water vapor that passes through a unit area of ​​a sample per unit time under specific conditions. Water vapor transmission rate is expressed in grams per square meter per day (g·m -2 ·sky -1 or g / m 2 / 24h). WVTR can be measured using techniques known in the art, such as using a 10cm 2 A Versaperm MkV Digital WVTR Meter from Versaperm Ltd., Maidenhead, UK, equipped with an electrolytic detection sensor, is used to measure the area. In one embodiment, the method for measuring WVTR is based on ISO 15106-3:2003. In a preferred embodiment, WVTR is measured at about 20°C and about 75% relative humidity for 24 hours. The relative humidity can be maintained at about 75% by using an open container containing a saturated sodium chloride solution.

[0168] Alternatively, WVTR can be calculated using the following equation:

[0169] WVTR=dm / (A·dt)

[0170] Where dm = the mass of water absorbed during time dt, and A = the total surface area of ​​the sample calculated based on particle size measurements. The mass increase should be measured over a period of approximately linear weight increase, for example, over a period of 5 minutes, preferably 7 minutes, after adjusting the relative humidity to 95%. WVTR is expressed in g / m 2 / Report in 24h units.

[0171] To measure WVTR, an aliquot of the particle sample can be weighed in an intrinsic sorption microbalance in a dynamic vapor sorption (DVS) instrument (Surface Measurement Systems, Alperton, Middlesex, UK) and measured at 95% relative humidity.

[0172] In some embodiments, when included in another food having a water activity of 0.90 to 1.00 for 2 weeks, the particles maintain a moisture content of 1 to 60 wt % within the matrix, preferably a moisture content of 10 to 60%, more preferably a moisture content of 20 to 60%. Moisture content can be measured by a variety of techniques, such as by Raman spectroscopy.

[0173] In some embodiments, when the bioactive agent is a microorganism, the microorganism produces a biofilm. Without wishing to be bound by theory, it is believed that the physical and / or chemical properties of the particles (including when the particles are present in a food or beverage product) can induce the microorganism to produce a biofilm. This may be a result of maintaining a non-zero moisture content within the matrix, and / or a result of the influx of other compounds (e.g., lactose) from the environment.

[0174] In some embodiments, the composition of the matrix is ​​selected to facilitate biofilm formation. For example, in some embodiments, the matrix can include components known to stimulate microorganisms to produce biofilm. In some embodiments, the matrix composition is selected to provide conditions that stimulate microorganisms to produce biofilm, such as moisture and / or nutrients to enter.

[0175] In some embodiments, the particles further include at least one coating. The coating can provide improved or altered properties for the particles, such as increased structural integrity. The coating can also be used to change the water permeability of the particles. In some embodiments, multiple coatings can be used. Preferably, the coating is edible.

[0176] Some exemplary coatings include certain polysaccharides, including cellulose and its derivatives, such as cellulose acetate phthalate, hemicellulose, methylcellulose, ethylcellulose, carboxycellulose, carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC), hydroxypropylcellulose, hydroxypropylmethylcellulose (HPMC), and microcrystalline cellulose; lipids and resins, including waxes and oils, such as paraffin, carnauba wax, beeswax, candelilla wax, and polyethylene wax; fatty acids and monoglycerides, such as stearyl alcohol, stearic acid, palmitic acid, monoglycerides, diglycerides, and triglycerides; naturally occurring resins, such as wood resins and coumarone-indene; proteins, including zein (α-zein protein The invention also includes the following materials: starch and derivatives, such as raw starch, modified starch, pregelatinized starch, dextrin, dextran, maltodextrin, corn syrup, sucrose, dextrose / fructose, and sugar polyols; extruded gums, such as gum arabic, gum ghatti, gum karaya, and gum tragacanth; seed gums, such as guar gum and locust bean gum; microbial fermented gums, such as xanthan gum, gellan gum, and chitosan; seaweed extracts, such as agar, alginates, carrageenan, and furcellaran; pectin; gluco-mannan; shellac; and poly-L-lysine. Mixtures of these materials may also be used.

[0177] The coating may also include one or more plasticizers. Suitable plasticizers include glycols such as polyethylene glycol (PEG), polypropylene glycol (PPG), etc., lipids such as vegetable oils, mineral oils, medium chain triglycerides, fats, fatty acids, waxes, etc.

[0178] In one embodiment, the particles are encapsulated in lipid globules. In one embodiment, the biomembrane is encapsulated in lipid globules embedded in a matrix.

[0179] Bioactive Agents

[0180] The term "bioactive agent" refers to any material having biological activity, including but not limited to proteins, peptides, amino acids, fats, triglycerides, lipids, fatty acids, oligosaccharides, polysaccharides, nucleic acids, nucleotides, nucleosides, vitamins, minerals, microorganisms, derivatives of microorganisms, or any combination of two or more of these. It should be understood that the biological activity need not be directly applied to the organism that has consumed the particles of the present invention. For example, the bioactive agent can include materials that have an effect on the intestinal microbial flora of the organism, such as prebiotics.

[0181] In a preferred embodiment, the bioactive agent is a microorganism, more preferably a probiotic microorganism, most preferably a probiotic bacterium.

[0182] In some embodiments, bioactive agent is a derivative of microorganism, preferably a probiotic microorganism. The derivative of microorganism can include a mutant or homologue of microorganism, attenuated or killed microorganism, and / or a microbial component still having useful activity. For example, although there is no clear identification of the bacterial molecule responsible for mediating probiotic activity, it has been proposed that the molecule as a possible candidate includes bacterial DNA motifs, surface proteins, small organic acids, polysaccharides and cell wall components such as lipoteichoic acid and peptidoglycan. It has been assumed that these interact with the components of the host immune system to produce immunomodulatory effects. Preferably, the activity retained is at least about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100% of the activity of an untreated (i.e., live or non-attenuated) control, and useful ranges can be selected between any of these values ​​(e.g., about 35 to about 100%, about 50 to about 100%, about 60 to about 100%, about 70 to about 100%, about 80 to about 100%, and about 90 to about 100%).

[0183] In some embodiments, the bioactive agent is a non-microbial bioactive agent. The term "non-microbial" means that the bioactive agent does not include living microorganisms.

[0184] In some embodiments, non-microorganism bioactive agent does not include live or killed microorganisms. In some embodiments, non-microorganism bioactive agent does not include microbial derivatives. In some embodiments, non-microorganism bioactive agent is protein, peptide, amino acid, fat, triglyceride, lipid, fatty acid, oligosaccharide, polysaccharide, nucleic acid, nucleotide, nucleoside, vitamin, mineral or any combination of two or more of these. In a preferred embodiment, non-microorganism bioactive agent is vitamin, prebiotic, postbiotic or human milk oligosaccharide. In some embodiments, vitamin is vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B6 (pyridoxine), vitamin B12 (cyanocobalamin), vitamin B5 (pantothenic acid), vitamin B7 (biotin), vitamin B9 (folate or folic acid). In some embodiments, prebiotic is oligofructose, oligogalactose or inulin. In some embodiments, the human milk oligosaccharide is 2'-fucosyllactose (2FL), 3'-fucosyllactose (3FL), 3'-sialyllactose (3SL), 6'-sialyllactose (6SL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), or any combination of two or more of these.

[0185] Prebiotics are compounds that induce the growth or activity of beneficial microorganisms. Some prebiotics are complex carbohydrates that cannot be digested by the organism (e.g., mammal) that ingests them, but can be digested by beneficial microorganisms. In this way, beneficial microorganisms can use some prebiotics as a source of nutrition. Some non-limiting examples of prebiotics include oligofructose, oligogalactose, and inulin.

[0186] Postbiotics are products from microorganisms that provide a benefit to the host. Some postbiotics are secreted by living microorganisms, while others are released after bacterial lysis. Some non-limiting examples of prebiotics include vitamins such as vitamin B and K, short-chain fatty acids, and antimicrobial peptides.

[0187] microorganism

[0188] It is expected that the particles and methods of the present invention can be used to provide improved stability and / or shelf life to a variety of microorganisms.

[0189] In one embodiment, the microorganism is a probiotic microorganism. In a preferred embodiment, the microorganism is a probiotic bacterium. In another embodiment, the microorganism is a fungus, such as a yeast. In one embodiment, the microorganism is a reproductively active form.

[0190] The term "probiotic" refers to a microorganism that has probiotic activity. The term "probiotic activity" refers to the ability of certain microorganisms to stimulate the immune system. Measuring the type and level of activity of probiotic microorganisms is known to those skilled in the art; see, for example, Mercenier et al. (2004), Leyer et al. (2004), or Cummings et al. (2004). Preferably, probiotic activity is evaluated by a PBMC cytokine secretion assay.

[0191] In one embodiment, the microorganism is lactic acid bacteria (LAB). In one embodiment, the microorganism is selected from the group consisting of Bacillus coagulans, Bifidobacterium animalis (e.g., Bifidobacterium animalis subsp. lactis strain HN019 or Bifidobacterium animalis subsp. lactis strain BB12), Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum, Lactobacillus casei (formerly known as Lactobacillus casei), Lactobacillus paracasei (formerly known as Lactobacillus paracasei), Lactobacillus rhamnosus (formerly known as Lactobacillus rhamnosus; e.g., Lactobacillus rhamnosus HN001), Lactobacillus plantarum subsp. plantarum (formerly known as Lactobacillus plantarum), Lactobacillus acidophilus (e.g., Lactobacillus acidophilus (LAVRI-A1)), Lactobacillus delbrueckii, Lactobacillus gasseri, Lactococcus lactis, Lactococcus lactis lactis), Lactococcus lactis subsp. lactis biovar diacetylactis, Leuconostoc pseudomesentericum, Ligilactobacillus salivarius (formerly known as Lactobacillus salivarius), Lactobacillus reuteri (formerly known as Lactobacillus reuteri; e.g., Lactobacillus reuteri ATCC 55730), Saccharomyces boulardii, Streptococcus thermophilus, or a combination of any two or more thereof.

[0192] Preferably, the microorganism is a biofilm forming microorganism. The ability of a microorganism to form a biofilm can be evaluated by methods known in the art. For example, as described in Microbial Example 4, microorganisms can grow in the presence of glass fibers or activated carbon surfaces and determine the formation of biofilm by their adhesion to substrates.

[0193] In some embodiments, the particles include a second microorganism that is different from the first microorganism. In some embodiments, the second microorganism is a probiotic microorganism. In some embodiments, the second microorganism is selected from any of those listed above. Other bioactive agents and / or microorganisms, such as the third and / or fourth microorganisms, may also be included.

[0194] Combinations of microorganisms with complementary properties can be selected. For example, a biofilm-producing microorganism can be combined with a non-producing microorganism. In some embodiments, a microorganism that is a prolific producer of biofilm can be combined with a microorganism that is a less prolific producer of biofilm. In some embodiments, the particle includes a first microorganism and a second microorganism, wherein the biofilm is produced by the first microorganism.

[0195] One or more microorganisms can also be combined with one or more non-microorganism bioactive agents. For example, in some embodiments, the bioactive agent includes a first microorganism, and the particles also include a non-microorganism bioactive agent. Preferably, the non-microorganism bioactive agent is different from any component or product of the first microorganism.

[0196] In some embodiments, the particles contain 10 to 5×10 12 CFU / g particle, e.g. 10 2 Up to 5×10 12 , 10 3 Up to 5×10 12 , 10 4 Up to 5×10 12 , 10 5 Up to 5×10 12 , 10 6 Up to 5×10 12 , 10 7 Up to 5×10 12 , 10 8 Up to 5×10 12 , 10 9 Up to 5×10 12 , 10 10 Up to 5×10 12 , 10 11 Up to 5×10 12 , 10 12 Up to 5×10 12 , 10 to 10 12 , 10 2 Up to 10 12 , 10 3 Up to 10 12 , 10 4 Up to 10 12 , 10 5 Up to 10 12 , 10 6 Up to 10 12 , 10 7 Up to 10 12 , 10 8 Up to 10 12 , 10 9 Up to 10 12 , 10 10 Up to 1012 , 10 11 Up to 10 12 , 10 to 10 11 , 10 2 Up to 10 11 , 10 3 Up to 10 11 , 10 4 Up to 10 11 , 10 5 Up to 10 11 , 10 6 Up to 10 11 , 10 7 Up to 10 11 , 10 8 Up to 10 11 , 10 9 Up to 10 11 , 10 10 Up to 10 11 , 10 to 10 10 , 10 2 Up to 10 10 , 10 3 Up to 10 10 , 10 4 Up to 10 10 , 10 5 Up to 10 10 , 10 6 Up to 10 10 , 10 7 Up to 10 10 , 10 8 Up to 10 10 , 10 9 Up to 10 10 , 10 to 10 9 , 10 2 Up to 10 9 , 10 3 Up to 10 9 , 10 4 Up to 10 9 , 10 5 Up to 10 9 , 10 6 Up to 10 9 , 10 7 Up to 10 9 , 10 8 Up to 10 9 , 10 to 10 8 , 10 2 Up to 10 8 , 10 3 Up to 10 8 , 10 4 Up to 10 8 , 10 5Up to 10 8 , 10 6 Up to 10 8 , 10 7 Up to 10 8 , 10 to 10 7 , 10 2 Up to 10 7 , 10 3 Up to 10 7 , 10 4 Up to 10 7 , 10 5 Up to 10 7 , 10 6 Up to 10 7 , 10 to 10 6 , 10 2 Up to 10 6 , 10 3 Up to 10 6 , 10 4 Up to 10 6 , 10 5 Up to 10 6 , 10 to 10 5 , 10 2 Up to 10 5 , 10 3 Up to 10 5 , 10 4 Up to 10 5 , 10 to 10 4 , 10 2 Up to 10 4 , 10 3 Up to 10 4 , 10 to 10 3 , 10 2 Up to 10 3 , or 10 to 10 2 CFU / g of particles of the first and / or second microorganism.

[0197] Biofilm

[0198] The term "biofilm" refers to a layer of extracellular material produced by and including microorganisms.

[0199] Biofilms are produced by certain microorganisms, such as bacteria, in response to certain environmental conditions. The conditions required to stimulate biofilm production may vary depending on the microorganism, but in some species, biofilm production may be induced by stress. Such stresses may include nutrient limitation, osmotic stress, desiccation, exposure to UV light, unfavorable pH or temperature, high pressure, or exposure to certain chemical compounds, including antimicrobial agents such as antibiotics, and compounds involved in quorum sensing.

[0200] Biofilms typically include an outer layer of slime cells that include microorganisms. The composition of a biofilm can vary depending on the species of microorganism that produces it. The main components of bacterial biofilms are typically high molecular weight extracellular microbial polymers, such as exopolysaccharides (EPS). It should be understood that the term "exopolysaccharide" or "EPS" is a general description of a variety of different compounds that cover different chemical and physical properties.

[0201] Biofilms provide many benefits to bacteria, including increased ability to adhere to surfaces and increased resistance to stressors.

[0202] Without wishing to be bound by theory, it is believed that the stability and / or shelf life of a bioactive agent may be improved by encapsulating the bioactive agent in a biofilm, wherein the biofilm is embedded in a matrix.

[0203] Biofilms can be detected by a variety of methods known in the art. These methods include Raman spectroscopy, scanning electron microscopy (SEM) and lectin staining. Some exemplary methods for detecting biofilms are given in Examples 5, 6 and 8.

[0204] In some embodiments, biofilms can be detected by Raman spectroscopy. Using Raman spectroscopy, a hyperspectral image can be generated that provides structural information about the location of biofilm components within the sample scan area. In terms of biofilm identification, this provides contextual information about where bacterial cells are and whether there are indeed exopolymer components around them that indicate a biofilm.

[0205] In one embodiment, the biofilm is characterized by the presence of ions at 360, 430 and / or 530 cm in a principal component analysis of the Raman spectroscopy data. -1 In some embodiments, the Raman spectral data indicates the presence of rhamnose, for example, by peaks at 360, 430, and / or 530 cm in a principal component analysis of the Raman spectral data. -1 In one embodiment, the peaks at 360, 430 and / or 530 cm in the principal component analysis of the Raman spectroscopy data are -1 The peak height at 1450 cm -1 At least 50% of the peak height of the fat-related peak at 1450 cm -1 The peak height of the fat-related peak at is at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100%, and useful ranges can be selected between any of these values ​​(e.g., 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 50% to 90%, 60% to 90%, 70% to 90%, or 80% to 90%).

[0206] For example, the exopolysaccharide (EPS) produced by Lactobacillus rhamnosus HN001 contains the characteristic sugars of glucose, galactose and rhamnose, as well as trace amounts of galactosamine, glucosamine, mannose and fucose. Because residual glucose and galactose may be present in the growth medium and / or food or beverage composition, the identification of rhamnose, galactosamine, glucosamine, mannose and fucose can provide strong evidence of the presence of a biofilm associated with Lactobacillus rhamnosus HN001 in the particles of the present invention. In some embodiments, the biofilm includes glucose, galactose, rhamnose, galactosamine, glucosamine, mannose and / or fucose.

[0207] In some embodiments, biofilm can be detected by lectin staining. Lectin staining can be used to detect the presence of sugars in the Lactobacillus rhamnosus HN001 EPS, such as glucose, galactose, rhamnose, galactosamine, glucosamine, mannose and fucose. For example, lectins connected with fluorescein can be used to detect biofilm formation by fluorescence microscopy. The inventors have demonstrated that the presence of mannose, N-acetylglucosamine and fucose provides strong evidence for the presence of biofilms associated with the Lactobacillus rhamnosus HN001 in the granules of the present invention.

[0208] In order to detect biofilm by lectin staining, the particles can be cut into slices of about 2 mm thickness. The slices are stained with 5 μl of 0.1 mg / ml fluorescein-labeled lectin dye, 1 μM DAPI and 0.2% fast green. Different lectin stains can be used to detect different sugars. In one embodiment, the lectin stain can be selected from concanavalin A, wheat germ agglutinin and / or olea oleifera agglutinin. The sample is stained for at least 15 minutes and then imaged using an inverted confocal fluorescence microscope. A 407 nm laser is used to excite DAPI, a 488 nm laser is used to excite fluorescein-labeled lectin, and a 561 nm excitation is used for fast green. The presence of bacterial cells embedded in the biofilm can be determined by the co-localization of cells stained with DAPI stain and biofilms stained with one or more lectins. Various methods for detecting biofilm by lectin staining are given in Examples 6, 8 and 11.

[0209] In some embodiments, biofilm is produced by a bioactive agent. For example, in some embodiments, the bioactive agent is a first microorganism, and biofilm is produced by the first microorganism. In some embodiments, the first microorganism is embedded in a matrix, wherein it produces a biofilm. In other embodiments, the first microorganism is induced to produce a biofilm, which is then embedded in a matrix. The first microorganism can be exposed to stress such as nutritional limitation, osmotic stress, drying, exposure to UV light, unfavorable pH or temperature, high pressure, or exposure to some chemical compounds (including antimicrobial agents such as antibiotics, and compounds related to quorum sensing) to induce the first microorganism to produce a biofilm.

[0210] In some embodiments, the particle also includes a second microorganism that is different from the first microorganism. In some embodiments, the second microorganism is a probiotic microorganism. This may be useful, for example, when the first microorganism is included for the purpose of biofilm production, and when the second microorganism is included for the purpose of providing a benefit such as a health benefit. Therefore, in some embodiments, the particle includes a first microorganism and a second microorganism, wherein the biofilm is produced by the first microorganism.

[0211] Food and beverage products

[0212] In a second aspect, the present invention provides a food or beverage product comprising the particles of the first aspect.

[0213] The term "food or beverage product" refers to any edible and / or drinkable product or ingredient. The term is intended to include products for human consumption and products for non-human consumption, such as pet food or livestock food. The term is also intended to include products that must undergo further processing before being in a consumable form, such as a beverage powder that is dissolved in a liquid to form a beverage. It should be understood that in some embodiments, the food or beverage product will be an ingredient for incorporation into other products, such as a baked food ingredient.

[0214] It is expected that the particles of the present invention allow the production of food and / or beverage products with improved stability and / or shelf life.

[0215] In some embodiments, the food or beverage product comprises a protein powder, a protein shake, a protein pill, a protein gel, or a sports nutrition formulation; a UHT beverage, a UHT smoothie; an infant formula, a follow-on formula, a growing-up formula, a pediatric formula, a human milk fortifier, a children's food or beverage, a maternal supplement, or a maternal nutrition formulation; a gel, such as a heat-set gel, a sauce, a spread, a jam, a jelly, or honey; an acid-set gel, an ambient-stable yogurt, a stirred yogurt, a set yogurt, or a drinking yogurt; Neutral beverages, acidic beverages, water, juice, milk, smoothies, milkshakes, pills, alcoholic beverages, soft drinks, kombucha, kefir, or instant mixes; bars, balls, cakes, cookies, muffins, or baked goods; medical foods, soups, desserts, puddings, custards, enteral preparations, preparations for the elderly, tablets, capsules, or supplements; candies, gummies, candies, chocolates, soft candies, truffles, chewing gum, frozen desserts, ice cream; seasonings, toppings, or baking ingredients; cream, cheese, or butter, or livestock or pet food.

[0216] In one embodiment, food or beverage products include alcoholic beverages. In some embodiments, alcoholic beverages are beer, wine, spirits or ready-to-drink (RTD) beverages. In some embodiments, food or beverage products include low-alcohol or non-alcoholic beer or wine.

[0217] In one embodiment, the food or beverage product comprises an animal food. In some embodiments, the animal food is a livestock food, a pet food, a kibble, a pet biscuit, a pet treat, an animal feed ingredient, a livestock feed, a livestock supplement, or a gavage.

[0218] In one embodiment, the food or beverage product comprises a baked food. In some embodiments, the baked food is bread, a bar, a ball, a cake, a cookie or a muffin.

[0219] In one embodiment, the food or beverage product comprises a beverage. In some embodiments, the beverage is a protein shake, a protein pill, a UHT beverage, a UHT smoothie, a neutral beverage, an acidic beverage, water, fruit juice, milk, smoothie, a milkshake, a pill, an alcoholic beverage, a soft drink, a fermented beverage, kombucha, kefir, a ready-to-drink (RTD) beverage, a coffee beverage, tea, or creamer. In some embodiments, the food or beverage product comprises a coffee capsule, instant coffee, a tea bag, or a coffee creamer.

[0220] In one embodiment, the food or beverage product comprises a breakfast food. In some embodiments, the breakfast food is cereal, oats, porridge, spreads, jams, jelly or honey.

[0221] In one embodiment, the food or beverage product comprises a confectionery. In some embodiments, the confectionery is a hard candy, a candy, a chewing gum, a chocolate bar, a chocolate coated product, a confectionery bar, a soft candy, or a truffle.

[0222] In one embodiment, the food or beverage product comprises a dairy product. In one embodiment, the dairy product is yogurt. In some embodiments, the yogurt is refrigerated yogurt, room temperature yogurt, stirred yogurt, set yogurt or drinking yogurt. In one embodiment, the dairy product is milk. In some embodiments, the milk is fresh milk, UHT milk, fortified milk, flavored milk or a fermented milk product such as kefir. In one embodiment, the dairy product is cheese. In some embodiments, the cheese is block cheese, cream cheese or processed cheese. In some embodiments, the dairy product is cream, such as fresh cream or UHT cream. In some embodiments, the dairy product is ice cream.

[0223] In one embodiment, the food or beverage product comprises a dessert. In some embodiments, the dessert is ice cream, sherbet, frozen yogurt, pudding, custard, jelly, puree, or dessert topping.

[0224] In some embodiments, the food or beverage product comprises an infant formula, a follow-on formula, a growing-up formula, a pediatric formula, a human milk fortifier, a children's food or beverage, a maternal supplement, or a maternal nutritional preparation. In some embodiments, the food or beverage product comprises an infant formula, a children's milk powder, a wet blended infant formula, a baby food, a baby snack, a baby rice, a baby melting puff, a baby teething biscuit, a fruit and / or vegetable puree.

[0225] In one embodiment, the food or beverage product is a dietary ingredient. In some embodiments, the dietary ingredient is a condiment, porridge (rice porridge), foam cream, instant noodles, oil, pasta, rice, salad dressing or seasoning.

[0226] In one embodiment, the food or beverage product comprises a medical food or beverage. In some embodiments, the medical food or beverage is a meal replacement product, a meal replacement product milkshake, a nutritional beverage, a soup, a pudding, a nutritional powder, a liquid meal or a dietary supplement. In some embodiments, the medical food or beverage comprises encapsulated probiotics and / or antibiotics.

[0227] In one embodiment, the food or beverage product comprises a powder. In some embodiments, the powder is a protein powder, a drink mix powder, or a meal replacement powder.

[0228] In one embodiment, the food or beverage product comprises a snack. In some embodiments, the snack is a baked good, a cheese lollipop, a slice, a bar, a cookie, a dip, dried fruit, nuts, a ball, a fruit peel, a high protein baked good, sour cream, a cereal mix, popcorn, potato chips, a smoothie bar, or a wavy vegetable potato chip.

[0229] In some embodiments, when stored at ambient temperature, food or beverage products have the shelf life of extension.For example, when stored at ambient temperature, food or beverage products can have at least 1 month, preferably at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months of shelf life.The shelf life of the extension of food or beverage products can be realized by multiple technologies known in the art, such as ultra-high thermal treatment (UHT).In one embodiment, food or beverage products are UHT food or beverage products.

[0230] In some embodiments, the food or beverage product has a high water activity (a w ). It is believed that the particles described herein are particularly useful in high water activity products. In various embodiments, the food or beverage product has a water activity of at least 0.1, preferably at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 0.95. In a preferred embodiment, the food or beverage product has a water activity of about 1.0.

[0231] In some embodiments, the bioactive agent in the food or beverage product remains active for at least 2 weeks, preferably at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months when stored at 30°C.

[0232] In embodiments where the bioactive agent is a microorganism, the degradation rate of the microorganism in the food or beverage product may be less than 7 log cfu / g / year, preferably less than 6, 5, 4, 3, 2 or 1 log cfu / g / year when measured at 30°C for 12 months.

[0233] In some embodiments, when the bioactive agent is a microorganism, the food or beverage product can be formulated to allow administration of a sufficient amount of the microorganism to establish a population in the gastrointestinal tract of a subject upon ingestion. The established population can be a transient or permanent population.

[0234] In theory, one colony forming unit (CFU) should be sufficient to establish a microbial population in a subject, but in practice, a minimum number of units is required to do so. Therefore, for therapeutic mechanisms that rely on a population of live, viable microorganisms, the number of units administered to a subject will affect the efficacy of the treatment.

[0235] For example, when the bioactive agent is Lactobacillus rhamnosus HN001, 6×10 9 The dosage rate of cfu is sufficient (but may not be necessary) to establish a population in the gastrointestinal tract of a human subject. Thus, in one embodiment, a food or beverage product can be formulated to provide at least about 6×10 cfu per day. 9 cfu Lactobacillus rhamnosus HN001.

[0236] Methods for determining whether a subject's gastrointestinal tract contains intestinal flora (e.g., Lactobacillus rhamnosus HN001) are well known in the art. In certain embodiments, the presence of a Lactobacillus rhamnosus HN001 colony can be determined directly, for example, by analyzing one or more samples obtained from the subject and determining the presence or amount of microorganisms in the sample. In other embodiments, the presence of a microbial colony can be determined indirectly, for example, by observing a health effect, or a reduction in the number of other intestinal flora in a sample obtained from the subject. Combinations of these methods are also contemplated.

[0237] The method for calculating the appropriate dosage may depend on the nature of the bioactive agent in the food or beverage product. For example, when the product contains live microorganisms, the dosage can be calculated with reference to the number of live microorganisms present. For example, the dosage can be determined with reference to the colony forming units (cfu) administered daily. In the example where the composition includes one or more derivatives of microorganisms, the dosage can be calculated with reference to the amount or concentration of the microbial derivatives present. For example, for a composition including a microbial cell lysate, the dosage can be calculated with reference to the concentration of the cell lysate present in the composition.

[0238] As a general example, consider administration of approximately 1×10 6 cfu is about 1×10 12 cfu of microorganisms, preferably about 1×10 6 cfu to 1×10 11 cfu / kg / day, about 1×10 6 cfu to 1×10 10 cfu / kg / day, about 1×10 6 cfu to 1×10 9 cfu / kg / day, about 1×10 6 cfu to 1×10 8 cfu / kg / day, about 1×10 6 cfu to 5×10 7 cfu / kg / day, or about 1×10 6 cfu to 1×10 7 cfu / kg / day. Preferably, about 5×10 cfu / kg body weight per day is considered to be administered 6 cfu is about 5×10 8 cfu of microorganisms, preferably about 5×10 6 cfu is about 4×10 8 cfu / kg / day, about 5×10 6 cfu is about 3×10 8 cfu / kg / day, about 5×10 6 cfu is about 2×10 8 cfu / kg / day, about 5×10 6 cfu is about 1×10 8 cfu / kg / day, about 5×10 6 cfu is about 9×10 7 cfu / kg / day, about 5×10 6 cfu is about 8×10 7 cfu / kg / day, about 5×10 6 cfu is about 7×10 7 cfu / kg / day, about 5×10 6cfu is about 6×10 7 cfu / kg / day, about 5×10 6 cfu is about 5×10 7 cfu / kg / day, about 5×10 6 cfu is about 4×10 7 cfu / kg / day, about 5×10 6 cfu is about 3×10 7 cfu / kg / day, about 5×10 6 cfu is about 2×10 7 cfu / kg / day, or about 5×10 6 cfu is about 1×10 7 cfu / kg / day.

[0239] In certain embodiments, the periodic dosage need not vary with the subject's weight or other characteristics. In such an example, consider administering about 1×10 6 cfu is about 1×10 13 cfu of microorganisms, preferably about 1×10 6 cfu is about 1×10 12 cfu / day, about 1×10 6 cfu is about 1×10 11 cfu / day, about 1×10 6 cfu is about 1×10 10 cfu / day, about 1×10 6 cfu is about 1×10 9 cfu / day, about 1×10 6 cfu is about 1×10 8 cfu / day, about 1×10 6 cfu is about 5×10 7 cfu / day, or about 1×10 6 cfu is about 1×10 7 cfu / day. Preferably, administration of about 5×10 7 cfu is about 5×10 10 cfu of microorganisms, preferably about 5×10 7 cfu is about 4×10 10 cfu / day, about 5×10 7 cfu is about 3×10 10 cfu / day, about 5×10 7 cfu is about 2×10 10 cfu / day, about 5×10 7 cfu is about 1×10 10 cfu / day, about 5×10 7 cfu is about 9×109 cfu / day, about 5×10 7 cfu is about 8×10 9 cfu / day, about 5×10 7 cfu is about 7×10 9 cfu / day, about 5×10 7 cfu is about 6×10 9 cfu / day, about 5×10 7 cfu is about 5×10 9 cfu / day, about 5×10 7 cfu is about 4×10 9 cfu / day, about 5×10 7 cfu is about 3×10 9 cfu / day, about 5×10 7 cfu is about 2×10 9 cfu / day, or about 5×10 7 cfu is about 1×10 9 cfu / day.

[0240] For example, in one embodiment, the effective dose of Lactobacillus rhamnosus HN001 may be 6×10 9 cfu.

[0241] It should be understood that it is preferred to prepare food or beverage products to allow the administration of an effective dose of a bioactive agent. The dosage of the composition administered, the time period of administration, and the general administration regimen may be different between subjects, depending on variables such as the following: the severity of the bioactive agent used, the symptoms of the subject, the type of the disorder to be treated, the selected mode of administration, and the age, sex, and / or general health of the subject. In addition, as described above, suitable dosage may depend on the properties and formulation of the bioactive agent in the food or beverage product. For example, when the composition includes live microorganisms, the dosage may be calculated with reference to the number of live microorganisms present. For example, as described in the embodiments herein, the dosage may be determined with reference to the colony forming units (cfu) number administered every day. In the example where the food or beverage product includes one or more microbial derivatives, the dosage may be calculated with reference to the amount or concentration of the microbial derivative administered every day. For example, for a composition including a microbial cell lysate, the dosage may be calculated with reference to the concentration of the microbial cell lysate present in the composition.

[0242] It should be understood that preferred food or beverage products are formulated to provide effective doses in convenient forms and amounts. In certain embodiments, such as but not limited to those embodiments where periodic doses do not need to vary with the subject's weight or other characteristics, the product can be formulated into unit doses. It should be understood that administration can include the administration of a single daily dose or appropriate multiple discrete divided doses.

[0243] Personal Care Products

[0244] In one aspect, the present invention provides a personal care product comprising the particles of the first aspect.

[0245] The term "personal care product" refers to any product, preparation, or formulation used for personal care purposes (e.g., cosmetic purposes), including (but not limited to) products used to clean or groom oneself, products used for personal hygiene, and products used for beautification. Some examples of personal care products include, but are not limited to, skin care products, hair care products, dental products, deodorants, makeup, beauty products, and feminine health products.

[0246] In one embodiment, the personal care product is a skin care product such as a skin cream, a sunscreen, a hair care product (such as a shampoo or conditioner), a dental product (such as a toothpaste or dentifrice), a deodorant, a make-up product, a beauty product, or a feminine care product.

[0247] In some embodiments, the personal care product is a skin care product. Skin care products are products that are applied topically to an area of ​​skin. Some examples of skin care products include moisturizers, lotions, creams, cleansers, and serums. In some embodiments, the skin care product is an eczema cream, lotion, or sunscreen.

[0248] In some embodiments, the personal care product is a hair care product, such as a shampoo, conditioner, hair oil, hair gel, hair serum, hair wax, hair clay, pomade, hair mousse, dry shampoo, or hair volume enhancer.

[0249] In some embodiments, the personal care product is a dental product, such as a toothpaste, dentifrice, dental gel, dental chew, mouthwash, or mouth rinse.

[0250] In some embodiments, the personal care product is a deodorant, such as a deodorant stick, a roll-on deodorant, or an antiperspirant.

[0251] In some embodiments, the personal care product is a feminine care product.

[0252] Cleaning Products

[0253] In one aspect, the invention provides a cleaning product comprising the particle of the first aspect.

[0254] In some embodiments, the cleaning product includes a cleaning spray, a detergent, or a disinfectant.

[0255] Method of making pellets

[0256] Various methods of making the particles of the present invention are contemplated. In some embodiments, the method comprises the following steps:

[0257] a. contacting the bioactive agent with the matrix to form a mixture, and

[0258] b. Granulate the mixture.

[0259] An exemplary method is described below, but various modifications will be apparent to those skilled in the art without departing from the scope of the invention.

[0260] In some embodiments, to prevent contamination, particles are prepared under sanitary and / or sterile conditions, such as using a Biological Safety Cabinet.

[0261] In one embodiment, when matrix comprises more than one composition, matrix components are merged.Combination can be by any suitable means known in the art, for example, by mixing or blending.In some embodiments, matrix comprises one or more dry ingredients, and they are dry mixed together.In some embodiments, one or more dry ingredients (or dry mixed ingredients) are combined with one or more liquid ingredients.Such liquid ingredients can include the composition (for example, one or more lipid components) that has been heated to the temperature that is enough to liquefy them.

[0262] In one embodiment, one or more matrix components are heated to a temperature sufficient to melt them for a period of time. The required temperature and time will depend on the composition of the matrix components. For example, in some embodiments, the matrix components are heated to 65° C. for 30 minutes to melt them.

[0263] Heating may be performed by any suitable means known in the art, such as using a heated water or oil bath.

[0264] The heating step may be followed by a tempering step at a lower temperature for a time sufficient to allow the matrix material to equilibrate at a lower temperature. For example, the composition may be kept at 45°C for a minimum of 30 minutes. In the case where the bioactive agent is susceptible to thermal damage, the tempering step reduces the temperature of the matrix material to prevent thermal damage to the bioactive agent. It is known that many microorganisms such as probiotics are sensitive to thermal damage, so a tempering step is typically used in these cases. For bioactive agents that are not susceptible to thermal damage, a tempering step is unnecessary.

[0265] The bioactive agent is then added to the (optionally tempered) liquid matrix component and optionally stirred until homogeneous. Any additional ingredients may also be added at this stage.

[0266] Then the obtained mixture containing bioactive agent is formed into particles, and the temperature is reduced to solidify the mixture. The production of particles can be by any suitable method known in the art, for example, by spray cooling or granulation. In some embodiments, particles are prepared by granulation. In other embodiments, particles are produced by spray cooling. In some embodiments, the mixture is cooled to a temperature lower than 24°C, for example 22°C, 20°C, 18°C, 16°C, 14°C, 12°C, 10°C, 8°C, 6°C, 4°C, 2°C or about 0°C, and a useful range can be selected between any one of these values ​​(for example, 0°C to 24°C, 0°C to 10°C, 0°C to 8°C, 0°C to 6°C, 0°C to 4°C, 0°C to 2°C, 2°C to 24°C, 2°C to 10°C, 2°C to 8°C, 2°C to 6°C or 2°C to 4°C).

[0267] In one embodiment, the mixture is poured into a pre-sterilized mold and any excess material is removed, for example, using a sterile spatula. The mold is then kept in a sterilized and sealed container and cooled to solidify the mixture. For example, the mold can be cooled to 4° C. for 12 h.

[0268] The particles are optionally washed and / or filtered by size.

[0269] The particles may optionally be coated with one or more coatings. The coatings may be applied by techniques known in the art, for example by spraying.

[0270] The granules may optionally be dried, for example by fluid bed drying, freeze drying, spray drying or vacuum drying.

[0271] The particles can be incorporated into a product (e.g., a food or beverage product), or they can be stored for future use. Storage can be at ambient temperature and humidity, or can be at controlled temperature and / or humidity. For example, the particles can be stored refrigerated (e.g., at about 4° C.) or frozen (e.g., at less than 0° C.), and / or can be stored in a sealed container, optionally a gas-flushed sealed container.

[0272] Methods for increasing the stability of bioactive agents

[0273] In a fifth aspect, the present invention provides a method for increasing the stability of a bioactive agent, the method comprising the steps of:

[0274] a. Encapsulation of bioactive agents in biofilms,

[0275] b. embedding the biofilm in a matrix, and

[0276] c. Granulate the matrix.

[0277] In a sixth aspect, the present invention provides a method for increasing the stability of a bioactive agent, the method comprising the steps of:

[0278] a. embedding the bioactive agent in the matrix,

[0279] b. Inducing bioactive agents to produce biofilm, and

[0280] c. forming the matrix into particles,

[0281] Steps b) and c) may be performed in any order.

[0282] In one embodiment, the bioactive agent comprises a first microorganism. In one embodiment, the biofilm is produced by a first microorganism. In some embodiments, the first microorganism is a microorganism as described herein.

[0283] In some embodiments, the method further comprises the step of coating the particles in at least one coating. In some embodiments, the coating is a coating as described herein.

[0284] In some embodiments, the particle produced in step (c) is a particle according to the first aspect.

[0285] In some embodiments, the method further comprises the following steps:

[0286] d. combining the granules of step c with a medium having a water activity of at least 0.5, preferably UHT treated yogurt, and

[0287] e. Incubate the culture medium for at least 2 weeks.

[0288] Without wishing to be bound by theory, it is believed that incubating the particles of step (c) in a culture medium such as UHT treated yoghurt may allow a controlled influx of water and / or other compounds present in the culture medium such as lactose into the particles, stimulating the production of biofilm.

[0289] Method for producing a bioactive agent-supplemented food or beverage product

[0290] In another aspect, the present invention provides a method for producing a bioactive agent-supplemented food or beverage product, the method comprising the method of the fifth or sixth aspect, and further comprising the following steps:

[0291] f. harvesting the particles from the culture medium of step e, and

[0292] g. Combining the harvested particles with a food or beverage product to obtain a bioactive agent-supplemented food or beverage product.

[0293] In another aspect, the present invention provides a method for producing a food or beverage product supplemented with a bioactive agent, the method comprising the method of the fifth or sixth aspect, and further comprising the step of combining the particles with the food or beverage product to obtain a food or beverage product supplemented with a bioactive agent.

[0294] In some embodiments, the bioactive agent-supplemented food or beverage product is a food or beverage product according to the second aspect.

[0295] Incorporated by Reference

[0296] The contents of the articles, patents, and patent applications, and all other literature and electronically available information mentioned or cited herein are incorporated herein by reference in their entirety, to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic files.

[0297] The following examples illustrate the invention.

[0298] Example

[0299] 1. Example 1 - Coating Matrix for Bioactive Agents

[0300] 1.1 Materials and methods

[0301] Various vegetable oils (alone or in combination with food ingredient adjuvants) were tested for their ability to provide a protective barrier between the probiotic bacteria (Lactobacillus rhamnosus HN001) and fermented dairy products.

[0302] Granules (in the form of beads) containing a probiotic ingredient (Lactobacillus rhamnosus HN001) embedded in a matrix consisting of fat and / or a combination of fat and a supporting ingredient were prepared. The probiotic ingredient used was a freeze-dried commercial sample.

[0303] The probiotic matrix beads were prepared by first melting the lipid component at 65°C for 30 minutes and then tempering at 45°C for a minimum of 30 minutes. The tempering step was to prevent heat damage to the probiotics. To prevent contamination, the particles were prepared in a biological safety cabinet. The probiotic bead component was added to the tempered lipid component and stirred until homogeneous. The dry ingredients were combined under stirring. The resulting mixture containing the probiotics was poured into a pre-sterilized mold and any excess material was removed using a sterile spatula. The mold was kept in a sterilized and sealed container and cooled to 4°C for 12h. The beads were then removed from the mold and stored in a nitrogen-purged and heat-sealed bag at -18°C.

[0304] The probiotic-containing particles were prepared in the form of beads (diameter of about 3 to 5 mm as determined by microscopy) using the fats and fat mixtures described in Table 1. The fat formed a continuous layer throughout the beads. All matrices additionally contained the following auxiliary ingredients (commonly found in confectionery) as a percentage of total solids: 40.7% sucrose, 0.3% sorbitan tristearate (emulsifier 492), 0.5% soy lecithin, 26% milk solids and 0.12% sodium.

[0305] Table 1. Composition of fat in the coating matrix.

[0306]

[0307] The protected cells were added to thick UHT yogurt formulated for ambient storage and stored at 30°C for 12 months to determine their shelf life. Samples were taken at the beginning of the trial and monthly and analyzed for cell counts of the HN001 probiotic. The cell degradation rate of each composition was determined. Final samples were taken at the 12-month elapsed time and the degradation rate of the probiotic was calculated using each coating matrix.

[0308] 1.2 Results

[0309] Some particles were more structurally robust than others, depending on the composition, however, most particles survived the duration of incubation in the yogurt matrix for 12 months. Pellets comprising a high amount of fully hydrogenated palm kernel stearin were the most robust, followed by hydrogenated palm kernel oil (as described in Example 2), then cocoa butter (as described in Example 2), then hydrogenated coconut oil (data not shown).

[0310] A surprising observation was that within the first month of storage, all probiotics in the bead granules exhibited a growth phase followed by the expected degradation. Without wishing to be bound by theory, it is believed that this may be due to the semi-permeability of the beads, which allows some amount of water, lactose and other nutrients to enter the granules and provide a carbon source for limited growth, followed by physiological regulation and a stagnant phase.

[0311] Analysis of bead degradation and degradation rates was determined starting from one month of storage, as it is reasonable to assume that the growth and physiological regulation components are separate processes.

[0312] It was also observed that some probiotic cells were released from the bead particles, however the majority of cells remained entrapped in the beads.

[0313] The degradation rates for each coating matrix are shown in Table 2. The degradation rates are expressed as log reductions in CFU / g / year. In other words, for clarity in the table, the degradation rates measured over the 6 and 9 month time periods were extrapolated to 1 year.

[0314] Table 2. Cumulative degradation rates of probiotics encapsulated in coating matrices. (* Rates calculated at 9 months elapsed time; ** 6 months elapsed time)

[0315]

[0316] 1.3 Conclusion

[0317] This example demonstrates that the coating matrix can reduce the degradation of probiotic microorganisms in fermented milk products such as yogurt, even when stored for extended periods of time at or above ambient temperature.

[0318] 2. Example 2 - Additional Coating Matrices for Bioactive Agents

[0319] Supplementary experiments were performed to test the effect of a coating made entirely of fat, lecithin, and to include an uncoated negative control.

[0320] 2.1 Materials and methods

[0321] Probiotic granules were prepared as described in Example 1, added to yogurt, and probiotic survival was monitored over a 12 month period.

[0322] The samples used are:

[0323] 1. Uncoated lyophilized culture (negative control),

[0324] 2.99.5% cocoa butter, 0.5% lecithin,

[0325] 3.100% cocoa butter,

[0326] 4.99.5% hydrogenated palm kernel oil, 0.5% lecithin,

[0327] 5.100% hydrogenated palm kernel oil,

[0328] 6.32% hydrogenated palm kernel oil, and

[0329] 7.16% hydrogenated palm kernel oil, 16% cocoa powder.

[0330] 2.2 Results

[0331] The degradation rates of each coating are shown in Table 3. The degradation rates are expressed as log reductions in CFU / g / year. The degradation rates measured over the 6-month and 9-month time periods were extrapolated to 1 year.

[0332] Table 3. Degradation rate

[0333]

[0334] The freeze-dried cultures without coating (negative control) did not survive when incubated in yogurt at 30° C. The addition of cocoa powder did not appear to help protect the probiotics from degradation.

[0335] Interestingly, cocoa butter showed low decay until 6 months, but degraded at a significantly higher rate after this period.

[0336] The combination of hydrogenated palm kernel oil with adjuvant (16% and 32% fat) demonstrated further significantly reduced degradation rates over 12 months storage (1.9 and 1.8 Log cfu / g / y respectively).

[0337] Mixture contour plots were used to further demonstrate the effect of fat on probiotic stability, both individually and in different combinations. For matrices including palm stearin, fully hydrogenated palm kernel oil, and fully hydrogenated coconut oil (alone and in combination), the lowest mortality was observed when fully hydrogenated coconut oil was added at the highest level ( Figure 1 Similar results were observed for palm stearin, fully hydrogenated coconut oil and cocoa powder combinations ( Figure 2 ).

[0338] The highest mortality was observed when cocoa powder was added at the highest level ( Figure 2 and 3 ). This can be removed from the formulation, leaving fully hydrogenated palm kernel oil, fully hydrogenated coconut oil, palm stearin, or a combination thereof as a potential protective matrix.

[0339] 2.3 Conclusion

[0340] This example shows that a coating matrix comprising 100% fat (ie 100% hydrogenated palm kernel oil) can reduce the degradation of probiotic microorganisms in a fermented dairy product when used alone as a coating matrix. The degradation can be further reduced by including an adjuvant as described in Example 1.

[0341] This example also shows that a matrix comprising fully hydrogenated coconut oil and / or fully hydrogenated palm kernel oil provided very low mortality, palm stearin provided low mortality, and cocoa powder was less effective.

[0342] 3. Example 3 - Electron Microscopy

[0343] 3.1 Materials and methods

[0344] Beaded particles containing a probiotic ingredient (Lactobacillus rhamnosus HN001) and hydrogenated palm kernel oil were prepared and added to yogurt as described in Example 1. In this example, the beads were removed after 9 months for electron microscopy analysis.

[0345] The beads were frozen to -20°C and fractured using a razor blade. The beads were mounted fracture side up, loaded in a scanning electron microscope (SEM TM4000) and cooled to -30°C while the chamber was evacuated. The samples were then ready for observation.

[0346] 3.2 Results

[0347] A clear phase separation between the yogurt and fat portions of the composition was visible, as well as distinct clusters of probiotics which, at higher magnification, appeared to be embedded in a mucus-like coating - either exopolysaccharides or in combination with biofilm material ( Figure 4 ).

[0348] Figure 5 Clear phase separation between yogurt and hydrogenated palm kernel oil (HPKO) is shown. Probiotic clusters in the biofilm are demarcated by red boundaries.

[0349] exist Figure 6 In the , the mucoid exopolysaccharide / biofilm network is clearly visible and Figure 7 In the figure, individual rod-shaped bacterial cells can be seen embedded in the exopolysaccharide / biofilm.

[0350] 3.3 Conclusion

[0351] This example shows that probiotics encapsulated in a coating matrix can form a mucus-like exopolysaccharide or biofilm layer once embedded in the matrix material.

[0352] 4. Example 4 - Biofilm Formation

[0353] 4.1 Materials and methods

[0354] It was demonstrated that Lactobacillus rhamnosus HN001 produces biofilm by growing and attaching to glass wool or activated carbon surfaces. Cultures of HN001 were prepared by incubating overnight in MRSB medium at 37°C. The culture (1ml) was then inoculated into a 500ml flask containing 100ml yogurt. Glass wool (2.5g) was added to the flask, which was subsequently incubated at 37°C in a rotary shaking incubator for 18-24 hours. The incubated flasks were then stored at 30°C and cell count, pH and EPS or biofilm formation were evaluated at 4, 7 and 28 days. The same experiment was performed on the flasks with 1g of 0.6-1.1mm washed granular activated carbon.

[0355] Samples were taken at the beginning of storage and on the 7th and 28th days for the count of viable probiotic cells. The glass wool (or activated charcoal) was removed and rinsed 3 times through a microsieve with phosphate buffered water. The rinsed material was placed in a 250 mL Schott bottle containing 20 g of sterile glass beads. Salted buffered peptone water (9 ml) was added and the beads and culture were vigorously stirred for 10 minutes to separate the cells from the glass wool or charcoal.

[0356] Serial dilutions were made using 9 ml of saline buffered peptone water and plated on MRSA agar, followed by incubation at 37° C. for 48 hours before counting the cells. Cell counts were also performed on residual yogurt (planktonic or free cells).

[0357] 4.2 Results

[0358] Table 4 shows that a large number of viable HN001 cells remained attached to the glass wool or charcoal surfaces during the 28-day shelf life, maintaining a relatively high survival level compared to free cells in yogurt.

[0359] Table 4. Cell counts and pH of cells attached to glass or charcoal surfaces compared to cells that remained unattached (planktonic cells).

[0360]

[0361] After staining with crystal violet, samples attached to glass wool were studied using an optical microscope at 100× magnification under oil immersion ( Figure 8 ). Clusters of HN001 cells were visible, attached to the surface of the glass wool strips. These attached clusters provided evidence of the ability of HN001 to form biofilms and maintain cell viability in yogurt.

[0362] 4.3 Conclusion

[0363] This example shows that Lactobacillus rhamnosus HN001 is able to form biofilms and that doing so improves the viability of the cells when stored in yogurt for extended periods of time.

[0364] 5. Example 5 - Identification of exopolysaccharide components of biofilms using Raman spectroscopy

[0365] Exopolysaccharides (EPS) constitute the major component of the extracellular biofilm matrix and have the function of anchoring microorganisms to surfaces, a process that initiates biofilm formation.

[0366] The characteristic sugars of EPS produced by Lactobacillus rhamnosus HN001 EPS are glucose, galactose and rhamnose and trace amounts of galactosamine, glucosamine, mannose and fucose. Because residual glucose and galactose may be found in yogurt, the identification of rhamnose, galactosamine, glucosamine, mannose and fucose provides strong evidence for the presence of biofilm associated with HN001.

[0367] 5.1 Materials and methods

[0368] Beads containing Lactobacillus rhamnosus HN001 were prepared according to Example 1.

[0369] The beads of sample AT03 (see Example 1) were removed from the yogurt and gently rolled on a paper towel to remove excess yogurt. The beads were sliced ​​to a thickness of about 2 mm. The slices were placed on a slide and covered with a cover slip; the cover slip was fixed down using double-sided tape and acted as a spacer of about 2 mm. The sample was kept at a temperature of 4°C throughout the measurement. A Raman spectrum of 50×10 steps and 75×25 μm was collected. Each spectrum was collected using a 1 s integration time, 532 nm excitation wavelength, 100x oil immersion objective (NA1.30), and 50 μm pinhole. At least 10 spectra were collected for each bead at each time point: 0, 15, 30, 90 / 120, 218 days.

[0370] As a control, Lactobacillus rhamnosus HN001 samples were cultured using environmental yogurt as the culture medium and zinc selenide surfaces to induce biofilm production.

[0371] Raman data files were converted to text files for subsequent analysis in R (version 4.1.1) and R Studio (version 1.4.1717). Spectra were preprocessed using cosmic ray suppression, baseline, and min-max normalization. Raman spectra were analyzed using principal component analysis, multivariate curve analysis, and comparison with reference materials.

[0372] 5.2 Results

[0373] Principal component analysis of the Raman spectroscopy data of Lactobacillus rhamnosus HN001 grown on environmental yogurt as a medium and ZnSe surfaces to induce biofilm production showed that the 350–600 cm -1 There are three distinct peaks in the spectral region ( Fig. 9 ). These can indicate the presence of EPS components.

[0374] The Raman spectrum of the sample AT03 bead structure containing Lactobacillus rhamnosus HN001 showed similar peaks ( Fig.10 ). Similarly, three EPS-related peaks appear at 360, 430, and 530 cm -1 Place.

[0375] 5.3 Conclusion

[0376] This example shows that Raman spectroscopy can be used to detect the presence of EPS in granules containing probiotics.

[0377] 6. Example 6 - Identification of biofilm exopolysaccharides by lectin staining

[0378] 6.1 Materials and methods

[0379] Beads containing Lactobacillus rhamnosus HN001 were prepared according to Example 1.

[0380] The beads were removed from the yogurt and gently rolled on a paper towel to remove excess yogurt. The beads were sliced ​​to approximately 2 mm thick. The slices were stained with 5 μl of 0.1 mg / ml fluorescein-labeled lectin dye, 1 μM DAPI, and 0.2% Fast Green. The lectin dyes were Concanavalin A, wheat germ agglutinin, and Ulex oleraceus agglutinin. The samples were stained for at least 15 minutes and then imaged using an inverted Zeiss LSM800 confocal fluorescence microscope. A 407 nm laser was used to excite DAPI, a 488 nm laser was used to excite fluorescein-labeled lectin, and a 561 nm excitation was used for Fast Green.

[0381] 6.2 Results

[0382] Figure 11-13 Fluorescence microscopy of beads stained with different lectins is shown. These images are one field recorded using a 63x oil immersion objective. One bead was split to allow different sections to be stained with different stain mixtures.

[0383] Lectin staining showed the presence of sugars consistent with the structure of Lactobacillus rhamnosus HN001 EPS (glucose, galactose, rhamnose, galactosamine, glucosamine, mannose, and fucose). Fig.11 )、N-acetylglucosamine( Fig.12 ) and fucose ( Fig.13 ) provides strong evidence for the presence of HN001-associated biofilm in the bead particles.

[0384] 6.3 Conclusion

[0385] This example shows that lectin staining and fluorescence microscopy can be used to detect the presence of biofilm in particles.

[0386] 7. Example 7 - Additional Coating Matrix

[0387] 7.1 Materials and methods

[0388] Beads comprising Lactobacillus rhamnosus HN001 were prepared as described in Example 1 using the matrix formulations shown in Table 5.

[0389] Table 5. Matrix composition (% w / w).

[0390]

[0391]

[0392] PS, palm stearin; HCO, fully hydrogenated coconut oil; CP, cocoa powder; HPKS, fully hydrogenated palm kernel stearin; HPKO, hydrogenated palm kernel oil; CB, cocoa butter; Suc, sucrose; 492, sorbitan tristearate (emulsifier 492); Lec, soy lecithin; SMP, milk solids (medium temperature skimmed milk powder); Lac, lactose.

[0393] The beads were added to less viscous UHT yoghurt (rather than the viscous yoghurt used in Example 1) and incubated at 25 or 30°C for 12 months, with samples removed at different time points for cell count analysis.

[0394] 7.2 Results

[0395] Incubate at 30 °C

[0396] The cell counts of Lactobacillus rhamnosus HN001 after incubation at 30°C are shown in Table 6.

[0397] Table 6. Cell counts (log CFU / g particles) of Lactobacillus rhamnosus HN001 after incubation at 30°C.

[0398]

[0399] * Fewer than 100 colonies per plate, below the level for accurate determination.

[0400] Incubate at 25 °C

[0401] The cell counts of Lactobacillus rhamnosus HN001 after incubation at 25°C are shown in Table 7.

[0402] Table 7. Cell counts (log CFU / g particles) of Lactobacillus rhamnosus HN001 after incubation at 25°C.

[0403]

[0404] 7.3 Conclusion

[0405] This example demonstrates that the coating matrix can reduce the degradation of probiotic microorganisms in fermented milk products, with some formulations resulting in significant stabilization.

[0406] 8. Example 8 - Biofilm formation by lectin staining

[0407] 8.1 Materials and methods

[0408] According to Example 1, beads containing Lactobacillus rhamnosus HN001 were prepared using the matrix formulation described in Table 8, wherein the HN001 content was 2.5%

[0409] Table 8. Matrix compositions.

[0410]

[0411] As described in Example 1, the beads were incubated at 30°C in thick UHT yoghurt and samples were removed at different time points for analysis.

[0412] Remove the probiotic-containing beads from the yogurt and discard the excess yogurt. Use Time Point Zero Grains before filling the yogurt.

[0413] Lectins conjugated to fluorophores were used to stain fat-based beads for confocal fluorescence microscopy. Fluorescently labeled lectins have previously been used to study extracellular polymeric substances in biofilms because lectins bind to sugar residues present in the polysaccharides of extracellular polymeric substances.

[0414] Three lectins were chosen for the beads: wheat germ agglutinin (WGA), concanavalin A (ConA), and Ulex oleraceus agglutinin I (UEA-II). WGA is specific for N-acetylglucosamine, ConA is specific for α-mannose and α-glucose, and UEA-II is specific for α-fucose. These are combined with DAPI for staining cells, Nile red for staining fats, or Fast Green for staining proteins.

[0415] The beads were cut to expose the inner surface, and a mixture of fluorescent dyes was applied as shown in Table 9. A maximum of 10 μl of the fluorescent dye mixture was applied to the exposed surface of the beads, and the beads were allowed to contact the dye for at least 15 minutes and no more than 30 minutes before imaging.

[0416] Table 9. Dye mixtures.

[0417]

[0418] Confocal fluorescence images were collected from the stained beads using an inverted Zeiss LSM 800 microscope with a 63x oil immersion objective. The excitation wavelengths and emission collection ranges are listed in Table 10.

[0419] Table 10. Excitation and emission wavelengths.

[0420]

[0421] 8.2 Results

[0422] AT07

[0423] At time zero, differential fluorescence of WGA was observed, indicating that N-acetylglucosamine was present in the initial composition. WGA binding was mainly around the edges of the protein bodies. This binding may be a result of including milk solids in the particles due to the presence of N-acetylglucosamine in cow milk (Li et al. (2023) Quantification of cow milk in adulterated goat milk by HPLC-MS / MS using N-acetylglucosamine as a reliable biomarker of cow milk, Journal of Food Composition and Analysis, 105583).

[0424] After 3 months of incubation, there were clear areas of concentrated WGA fluorescence that were no longer at the edges of protein bodies but rather within the cell clusters.

[0425] At time zero, dense areas of cone fluorescence were observed and these areas did not coincide with areas of cell density. This may also be a result of the milk solids included in the matrix formulation.

[0426] After 3 months of incubation, the areas of perceptible ConA fluorescence increased and these areas correlated with areas of higher cell density.

[0427] No data were collected from time zero using the UEA-II lectin dye. After 3 months of incubation, there were clear areas of high and low UEA-II fluorescence, with areas of high UEA-II fluorescence primarily associated with densely packed areas of cells. The increase in lectin fluorescence areas surrounding cell clusters after incubation was consistent with biofilm formation.

[0428] AT24

[0429] No data were collected from time zero using ConA or UEA-II dye.

[0430] At time zero, WGA stained cells. After 3 and 6 months of incubation, WGA fluorescence was observed around the cells; this was most obvious after 6 months. After 6 months, ConA fluorescence was shown in densely populated areas of cells. For UEA-II fluorescence, there was a small amount of UEA-II aggregation at 3 and 6 months, however, it did not seem to be closely associated with cell location. These observations are consistent with biofilm formation.

[0431] AT17-1

[0432] The lectin dyes used to stain MVP9 have fluorophores with excitation maxima at 642 or 649 nm, allowing for simultaneous staining with Nile red, which stains fat. For the one-month time point, there is one exception - the UEA-II dye with a fluorescein fluorophore, which has an excitation maximum at 495 nm, is used. When this dye is used, Nile red is not used and fast green is added instead due to interference between dyes.

[0433] At time zero, small areas of bacteria were present in the adipose matrix, and WGA stained cells in these areas. One month later, more extensive fluorescence from WGA surrounding the cellular areas was evident, indicating the presence of N-acetylglucosamine extending beyond the immediate area of ​​the cells, consistent with the formation of a biofilm of extracellular polymeric substances containing N-acetylglucosamine.

[0434] At time zero, no differential fluorescence was observed with ConA, but areas of fluorescence were seen where the dye was aggregated in non-fat areas. Within the cellular regions, there were no areas of weak and strong ConA fluorescence. There were areas of ConA fluorescence that showed no association with the cells. Therefore, the dye was aggregated in non-fat areas, rather than specifically staining mannose or glucose. One month later, differential fluorescence of ConA was observed, i.e., the fluorescence was not simply due to aggregation in non-fat areas. As with WGA, areas of uneven and extensive fluorescence around the cells were observed, consistent with excretion of extracellular polymeric substances and formation of biofilms. Not all cellular areas were stained with ConA; at this time point, there were cellular areas of freshly aggregated ConA. In addition, at one month, there were areas of dense ConA aggregation in areas around the edges of the cells, and ConA aggregated inside, but also within the non-fat areas.

[0435] At time zero, UEA-II stained cells in the beads in a manner similar to WGA. These cells were primarily confined to clusters of cells in the fatty matrix. At one month, cells colonized the outer regions of the non-fatty regions of the pellet, and fluorescence due to UEA-II fluorescence was observed outside of the cells, consistent with biofilm formation.

[0436] ATX-1

[0437] Only samples from three months were analyzed.

[0438] After 3 months, there were significant areas of WGA fluorescence associated with areas of cell density. There were also significant areas of ConA fluorescence (also associated with cells) and small areas of UEA-II fluorescence. All of these observations were consistent with biofilm formation.

[0439] 8.3 Conclusion

[0440] This example shows that biofilm formation can be detected by fluorescence microscopy using fluorescein-linked lectins and that various matrix compositions facilitate biofilm formation.

[0441] 9. Example 9 - Stability in various products

[0442] 9.1 Materials and methods

[0443] Probiotic granules including 0.25% Lactobacillus rhamnosus HN001 were prepared using four matrix formulations as described in Table 11.

[0444] Table 11. Matrix compositions.

[0445]

[0446] The dry ingredients (sugar, lactose) were weighed into a homogenizer bag, sealed with a bag sealer, and stored at room temperature until needed. The lipid ingredients (fats and lecithin) were weighed into a sterile bottle and stored at 3-4°C until needed. 0.25 g / 100 g of lipid from Lactobacillus rhamnosus HN001 was weighed into a sterile container and stored at 3-4°C until needed. The silicon mold was autoclaved.

[0447] Prepare the particles using the following method:

[0448] 1. Melt the lipid components in a 60-70°C water bath.

[0449] 2. Transfer the lipid component to a 45°C water bath, ensuring that the lipids and fats are cooled to 45°C (minimum 30 minutes) before adding the probiotics.

[0450] 3. Add probiotics (0.25 g) to the melted lipids and stir with a sterilized spoon to ensure uniformity.

[0451] 4. Slowly add remaining dry ingredients, stirring between each addition.

[0452] 5. Keep stirring throughout the process to ensure all ingredients are incorporated and a smooth finish is achieved.

[0453] 6. Pour the contents into a sterilized silicon mold.

[0454] 7. Using a spatula, smooth the mixture into the mold cavity and scrape off excess mixture.

[0455] 8. Place the mold in a pre-sterilized container and store at 4°C for 24 hours.

[0456] The resulting particles had a diameter of about 5 mm and a weight of about 0.05 g each.

[0457] Five products were evaluated as described in Table 12. The viscosity of the products was measured using a Brookfield viscometer.

[0458] Table 12. Products evaluated.

[0459]

[0460] Using 200 ml samples of each product, three pellets were added to each sample using sterile forceps, soaking the pellets in the top 10-20 ml. The samples were then incubated at 30°C.

[0461] At each sampling time point, three pellets were recovered and placed in a bell tube. 15 ml of MRSB medium preheated to 45 °C was added and the samples were melted in a 45 °C water bath for 5 min. The samples were removed from the water bath and gently shaken / vortexed to mix.

[0462] Prepare dilutions in duplicate. Plate the appropriate sample dilution (50 μl) on pre-inoculated agar plates ("drop plate method"). Three dilutions are required, i.e. 10 -6 , 10 -7 and 10 -8 , to get a countable range.

[0463] The plates were incubated at 37°C under anaerobic conditions for 2-3 days and the colonies were counted to determine the CFU / ml in the original sample.

[0464] 9.2 Results

[0465] The viable Lactobacillus rhamnosus HN001 counts at initial set-up and after 1, 2, 3 and 4 months of incubation are shown in Tables 13 - 17. The 3 and 4 month time points for the cream cheese spread have not been tested.

[0466] Table 13. Shelf life in orange juice (Log 10 CFU / ml).

[0467]

[0468] Table 14. Shelf life in sports shakes (Log 10 CFU / ml).

[0469]

[0470] Table 15. Shelf life in cheese sauce (Log 10 CFU / ml).

[0471]

[0472] Table 16. Shelf life in cream cheese sauce (Log 10CFU / ml).

[0473]

[0474] Table 17. Shelf life (Log 10 CFU / ml).

[0475]

[0476] 9.3 Conclusion

[0477] This example demonstrates that the coating matrix can reduce the degradation of probiotic microorganisms in a variety of products even when stored at or above ambient temperature for extended periods of time.

[0478] 10. Example 10 - Bacterial strains

[0479] 10.1 Materials and methods

[0480] Two sets of particles were prepared as described in Example 9, the first set comprising Lactobacillus rhamnosus HN001 and the second set comprising Lacticaseibacillus paracasei subsp. paracasei IM514.

[0481] The matrix composition contained 86.5% w / w hydrogenated coconut oil (HCO) and 13.5% w / w lactose (formulation ATX-2).Before adding the bacteria to the molten lipid component, a further cooling step of 1 hour at 20°C was applied.

[0482] The particles were added to thick UHT yogurt formulated for ambient storage and stored at 30° C. Cell counts were performed regularly for each bacterial strain.

[0483] 10.2 Results

[0484] The cell counts of the bacterial strains are shown in Table 18.

[0485] Table 18. Shelf life (Log 10 CFU / ml).

[0486]

[0487] 10.3 Conclusion

[0488] This example demonstrates that the particles can provide extended shelf life under ambient conditions for a variety of bacterial strains.

[0489] 11. Example 11 - Lectin staining for biofilm formation

[0490] 11.1 Materials and methods

[0491] Probiotic beads / granules were prepared using Lactobacillus rhamnosus HN001 or Lactobacillus rhamnosus GG (LGG) and the matrix formulations shown in Table 19.

[0492] Table 19. Matrix formulations.

[0493]

[0494] Ako Comp ingredients (10% probiotic load) include:

[0495] 26g Akosoft 36

[0496] 60g Akofine R

[0497] 4g Polyglyceryl Ricinoleate (PGPR)

[0498] 10g freeze-dried probiotic powder

[0499] Total (100g)

[0500] Ako Comp ingredients (2.5% probiotic load) include:

[0501] 28.2g Akosoft 36

[0502] 65g Akofine R

[0503] 4.3g Polyglyceryl Ricinoleate (PGPR)

[0504] 2.5g freeze-dried probiotic powder

[0505] Total (100g)

[0506] Akosoft 36 (hydrogenated coconut glycerides) and Akofine R (hydrogenated vegetable oil) were purchased from IXOM (Melbourne, Australia).

[0507] Samples 1-3 were prepared as described in Example 8, and comparative samples 4-8 were prepared according to the following comparative method:

[0508] The fat component and emulsifier (PGPR or soy lecithin) were mixed at 75°C with continuous stirring until all ingredients were combined. The freeze-dried probiotic powder was added and stirred for 10-20 seconds. The suspension was then granulated and the granules were ground into powder.

[0509] For all samples, the granules / powders were incubated as described in Example 1 for two weeks at the above temperatures in thick UHT yoghurt.

[0510] The pellet was removed, cut in half, stained with dye mixture 1-4, and visualized by confocal fluorescence microscopy as described in Example 8.

[0511] 11.2 Results

[0512] Exemplary samples (Samples 1-3)

[0513] For sample 1, strong regions of WGA, ConA, and UEA-I fluorescence were observed that extended beyond the cells. This is consistent with the presence of a biofilm.

[0514] Only a small area of ​​strong WGA fluorescence and no areas of strong ConA fluorescence were observed for sample 2. An area of ​​UEA-I fluorescence was observed that extended beyond the cells, consistent with the presence of a biofilm.

[0515] For sample 3, strong WGA fluorescence was observed from the cells, and regions extending beyond the cells were also observed. No strong regions of ConA fluorescence were observed. Strong regions of UEA-I extending beyond the cells were observed. The spread of WGA and UEA-I fluorescence outside the cells is consistent with biofilm formation.

[0516] In summary, samples 1-3 showed evidence of biofilm formation due to WGA and UEA-I fluorescence that extended beyond the cell location defined by DAPI staining, and lectin staining was not limited to cell shape. ConA staining consistent with the presence of biofilm was only observed in particles with HN001 cells (sample 1). ConA is specific for α-mannose and α-glucose, so this difference may be due to differences in biofilm composition between HN001 and LGG.

[0517] Comparative samples (samples 4-8)

[0518] For sample 4, only small areas of strong WGA fluorescence were observed, no ConA fluorescence was observed, and UEA-I fluorescence stronger than the background aggregation level was observed only in small areas around some cell clusters.

[0519] Due to sample limitations, sample 5 was not stained with Con A. Strong areas of WGA fluorescence were consistent with cells, and only small areas of strong UEA-I fluorescence were observed.

[0520] For sample 6, the area of ​​strong WGA fluorescence was observed to extend beyond the cells, however not all cell clusters showed strong WGA fluorescence. No strong fluorescence from Con A was observed. Only some cell areas in sample 6 were stained with UEA-I.

[0521] For sample 7, WGA fluorescence was unexpectedly concentrated at the edge of fat, which is not necessarily consistent with cells. ConA staining is usually limited to being concentrated in non-fat areas. Small areas of strong UEA-I fluorescence were observed around cells.

[0522] Sample 8 is the same as sample 7, except that AD sterilization is used instead of pasteurization. Without pasteurization, the distribution of WGA fluorescence is different. Strong WGA fluorescence is observed, but this does not extend beyond cells. Areas where a large number of cells are not tightly aggregated are observed. ConA fluorescence is not observed. UEA-I shows variable staining intensity, but it seems to be only staining cells, rather than extracellular polymeric substances.

[0523] Overall, sample 4-8 showed no strong ConA staining areas except for a very small area of ​​strong ConA fluorescence, but stained the yogurt included in the beads. Only small areas of strong WGA and UEA fluorescence were observed, which did not extend beyond the cells or were not associated with each cell cluster in the matrix. Sample 4-8 did not show evidence of extensive biofilm formation observed in samples 1-3.

[0524] 11.3 Conclusion

[0525] This example shows that beads formed using the matrices and methods of the invention exhibit lectin staining that extends beyond the cells, indicating extensive biofilm formation.

[0526] In contrast, control particles formed using the control method generally lacked lectin staining extending beyond the cells, indicating that these particles did not contain the same extensive biofilm as the particles of the present invention.

[0527] 12. Example 12 - Water Vapor Transmission Rate (WVTR)

[0528] 12.1 Materials and methods

[0529] Exemplary particles were prepared as described in Example 9. Comparative particles were also prepared using the comparative method described in Example 11.

[0530] WVTR was measured using aliquots (20-60 mg) of the particle sample by weighing in an intrinsic sorption microbalance of a dynamic vapor sorption (DVS) instrument (Surface Measurement Systems, Alperton, Middlesex, UK) at 95% relative humidity. WVTR was measured over a period of 5 minutes, 7 minutes after the relative humidity was adjusted to 95% (i.e., from 7 minutes to 12 minutes).

[0531] Calculate WVTR by using the following equation:

[0532] WVTR=dm / (A·dt)

[0533] Where dm = the mass of water absorbed during time dt, and A = the total surface area of ​​the sample calculated based on particle size measurements. WVTR is expressed in g / m 2 / Report in 24h units.

[0534] Water activity was determined by Aqualab 4TE (Addium Inc, Pullman, WA, USA). The particle size of the powder (ranging from 10 to 1,000 microns) was measured using a stereo microscope and subsequently analyzed using "Image pro plus".

[0535] 12.2 Results

[0536] The water vapor transmission rate (WVTR) and water activity (Aw) of various fat-based matrix formulations were measured (Table 20). All included 0.25% Lactobacillus rhamnosus HN001.

[0537] Table 20. Surface area to volume ratio (SA / V), water vapor transmission rate (WVTR) and water activity of particles.

[0538]

[0539] Comparative granules were prepared according to the comparative method described in Example 11 ("Comparative") and compared with granules of the present invention ("Invention").

[0540] Granules containing 2.5% Lactobacillus rhamnosus HN001 and control granules containing 10% Lactobacillus rhamnosus GG were prepared using the method described in Example 9. Two matrix preparations were used - 100% cocoa butter and 100% AkoComp. The medium granules had a diameter of about 5 mm and the powder granules were ground into powder.

[0541] The particles prepared using the process of the present invention have significantly lower WVTR than particles prepared using the comparative process, whether using medium-sized particles or powders, even with similar surface area / volume ratios (Table 21).

[0542] Table 21. Surface area to volume ratio (SA / V) and water vapor transmission rate (WVTR) of comparative particles.

[0543]

[0544] The WVTR was further compared by varying the probiotics used and the particle size. Granules including Lactobacillus rhamnosus GG (LGG) had a higher WVTR than granules including Lactobacillus rhamnosus HN001, and even at different particle sizes, granules prepared using the comparative method consistently had a higher WVTR than granules produced using the inventive method (Table 22).

[0545] Table 22. Comparative Water Vapor Transmission Rate (WVTR).

[0546]

[0547] 12.3 Conclusion

[0548] This example shows the WVTR and water activity of particles according to the invention and comparative particles.

Claims

1. A particle comprising a bioactive agent embedded in a biomembrane, wherein the biomembrane is embedded in a matrix, wherein the matrix has a water vapor transmission rate (WVTR) of 0.1 to 500.

2. Particles according to claim 1, wherein the matrix comprises at least 6 wt%, preferably 10-100 wt%, more preferably 30-100% of one or more lipids.

3. The particle according to claim 1 or 2, wherein the matrix has a water vapor transmission rate (WVTR) of 0.1 to 10, preferably 1 to 5.

4. The particle according to any one of claims 1 to 3, wherein the bioactive agent comprises a first microorganism, preferably a probiotic microorganism.

5. The particle of claim 4, wherein the biofilm is produced by the first microorganism.

6. The particle according to claim 4 or 5, wherein the first microorganism is a Bacillus, Bifidobacterium, Enterococcus, Lactaseibacillus, Lactiplantibacillus, Lactobacillus, Lactococcus, Ligilactobacillus, Limosilactobacillus, Lentilactobacillus, Saccharomyces or Streptococcus; preferably, wherein the first microorganism is selected from the group consisting of Bacillus coagulans, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactiplantibacillus plantarum subsp. plantarum, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactococcus lactis, Lactococcus lactis lactis), Lactococcus lactis subsp. lactis biovardiacetylactis, Leuconostoc pseudomesentericum, Lactobacillus salivarius, Lactobacillus reuteri, Saccharomyces boulardii and Streptococcus thermophilus; more preferably, the first microorganism is Lactobacillus rhamnosus HN001.

7. The particle according to any one of claims 4 to 6, comprising the first microorganism in an amount of 10% of the particle. 5 Up to 10 12 CFU / g, preferably 10 6 Up to 10 10 CFU / g, more preferably 10 7 Up to 10 9 CFU / g, most preferably about 10 8 CFU / g.

8. The particle according to any one of claims 4 to 7, further comprising a second microorganism different from the first microorganism, preferably wherein the second microorganism is a probiotic microorganism.

9. The particle according to any one of claims 1 to 8, further comprising a non-microbial bioactive agent, preferably a vitamin, a prebiotic, a postbiotic and / or a human milk oligosaccharide.

10. A particle according to any one of claims 1 to 9, wherein the biofilm is characterized by a peak at 360, 430 and / or 530 cm in a principal component analysis of Raman spectroscopy data. -1 The peak of .

11. The particle according to any one of claims 1 to 10, wherein the biofilm is stainable by Concanavalin A, wheat germ agglutinin and / or Ulex europaeus agglutinin.

12. The particle according to any one of claims 1 to 11, wherein the particle maintains a moisture content of 1 wt% to 60 wt%, preferably 10 wt% to 60 wt%, more preferably 20 wt% to 60 wt% moisture in the matrix when included in another food having a water activity of 0.90 to 1.00 for 2 weeks.

13. The particle of any one of claims 2 to 12, wherein the one or more lipids: a. solid at 20°C, 25°C and / or 30°C; b. having a melting temperature of 50°C or less, preferably 30°C to 50°C; c. including or consisting of triglycerides; d. comprising C12-C20 fatty acids or their salts or esters; or e. Any combination of any two or more of (a) to (d).

14. A particle according to any one of claims 2 to 13, wherein the one or more lipids comprise one or more fatty acids and / or salts and / or esters thereof, and wherein: a. at least 1wt% of fatty acids and / or salts and / or esters thereof having a carbon chain length of 14 or less, preferably 1wt% to 80wt%; and / or b. At least 30 wt% of the fatty acids and / or salts and / or esters thereof have a carbon chain length of 16 or less, preferably at least 70 wt%, preferably 30 wt% to 90 wt%, more preferably 70 wt% to 90 wt%.

15. A particle according to any one of claims 2 to 14, wherein at least 50 wt% of the one or more lipids are saturated fats.

16. A particle according to any one of claims 2 to 15, wherein the one or more lipids comprise or consist of: Fully hydrogenated palm kernel stearin, palm stearin, fully hydrogenated coconut oil, hydrogenated palm kernel oil, or a mixture of any two or more of these.

17. A particle according to any one of claims 1 to 16, wherein the matrix comprises at least 10 wt%, preferably at least 20 wt%, more preferably at least 30 wt% fully hydrogenated coconut oil.

18. The particle according to any one of claims 1 to 17, wherein the particle has a particle size of 50 μm to 10 mm.

19. The particle according to any one of claims 1 to 18, further comprising at least one coating.

20. A food or beverage product comprising particles as claimed in any preceding claim.

21. The food or beverage product of claim 20, wherein the food or beverage product comprises: a. Protein powder, protein shake, protein pill, protein gel or sports nutrition preparation, b.UHT drinks, UHT smoothies, c. Infant formula, follow-on formula, growing-up formula, pediatric formula, human milk fortifier, children's food or beverage, maternal supplement or maternal nutritional preparation, d. Gels, such as heat-set gels, sauces, spreads, jams, jellies or honey; e. Acid-coagulated gel, room temperature stable yogurt, stirred yogurt, set yogurt or drinking yogurt, f. Neutral beverages, acidic beverages, water, juice, milk, smoothies, milkshakes, pills, alcoholic beverages, soft drinks, kombucha, kefir or instant mix powder, g. bars, balls, cakes, cookies, muffins or baked goods, h. Medical foods, soups, desserts, puddings, custards, enteral preparations, preparations for the elderly, tablets, capsules or supplements, i. candy, gummies, candies, chocolates, fudge, truffles, chewing gum, frozen desserts, or ice cream, j. Seasonings, toppings or baking ingredients, k.Cream, cheese or butter, or l. Livestock food or pet food.

22. The food or beverage product of claim 20 or 21, wherein the food or beverage product has a water activity of at least 0.2, 0.4, 0.6, 0.8 or 0.

9.

23. The food or beverage product according to any one of claims 20 to 22, wherein the food or beverage product is stable at 30°C for at least 30 days, preferably at least 60 days, more preferably at least 6 months.

24. A food or beverage product according to any one of claims 20 to 23, wherein the bioactive agent comprises a microorganism, and wherein the degradation rate of the microorganism measured at 30°C over 12 months is less than 7 log CFU / g / y, preferably less than 6, 5, 4, 3, 2 or 1 log CFU / g / y.

25. A method for increasing the stability of a bioactive agent, the method comprising the steps of: a. embedding the bioactive agent in the biofilm, b. embedding the biofilm in a matrix having a water vapor transmission rate (WVTR) of 0.1 to 500, and c. forming the matrix into particles.

26. A method for increasing the stability of a bioactive agent, the method comprising the steps of: a. embedding the bioactive agent in a matrix having a water vapor transmission rate (WVTR) of 0.1 to 500, b. inducing the bioactive agent to produce a biofilm, and c. forming said matrix into particles, Steps b) and c) can be performed in any order.

27. The method of claim 25 or 26, wherein the matrix comprises at least 6 wt% of one or more lipids.

28. The method of any one of claims 25 to 27, wherein the bioactive agent comprises a first microorganism.

29. The method of claim 28, wherein the biofilm is produced by the first microorganism.

30. A method according to any one of claims 25 to 29, further comprising the step of coating the particles in at least one coating layer.

31. The method of any one of claims 25 to 30, wherein the particles comprise particles of any one of claims 1 to 19.

32. The method according to any one of claims 25 to 31, further comprising the steps of: d. combining the particles of step c with a culture medium having a water activity of at least 0.5, preferably UHT treated yogurt, and e. Incubate the culture medium for 3 to 30 days.

33. A method of producing a bioactive agent-supplemented food or beverage product, the method comprising the method according to claim 32, and further comprising the steps of: f. harvesting the particles from the culture medium of step e, and g. combining the harvested particles with a food or beverage product to obtain the bioactive agent-supplemented food or beverage product.

34. A method for producing a bioactive agent-supplemented food or beverage product, the method comprising the method according to any one of claims 25 to 32, and further comprising the step of combining the particles with a food or beverage product to obtain the bioactive agent-supplemented food or beverage product.

35. The method of claim 33 or 34, wherein the bioactive agent-supplemented food or beverage product is a food or beverage product according to any one of claims 20 to 24.

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