Particles containing biologically active substances

Abstract

The present invention generally relates to particles comprising a bioactive substance embedded in a biofilm, wherein the biofilm is embedded in a matrix. More specifically, but not exclusively, it relates to particles comprising biofilm-forming microorganisms embedded in a biofilm, wherein the biofilm is embedded in a matrix. Methods for enhancing the stability of a food or beverage product or a bioactive substance are also provided.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention generally relates to particles comprising a bioactive material embedded in a biofilm, where the biofilm is embedded in a matrix. More specifically, but not exclusively, it relates to particles comprising biofilm-forming microorganisms. Also provided are methods for enhancing the stability of food or beverage products, personal care products, cleaning products, or bioactive materials.

[0002] The particles described herein are useful for extending the shelf life of bioactive materials, such as probiotic microorganisms, at ambient temperatures, particularly in high water activity environments. These particles are particularly useful in ambient-stable, high water activity food and beverage products, such as UHT-processed products, that are stored for extended periods without refrigeration. [Background technology]

[0003] background Consumption of products containing bioactive substances has been associated with a variety of health benefits. Such bioactive substances can include various microorganisms, such as probiotic bacteria.

[0004] However, bioactive substances can suffer from limited stability and / or shelf life. For example, when the bioactive substance is a microorganism, a well-known problem in the art is the stability of the microorganism. Microorganisms tend to become non-viable over time, especially when incorporated into products with high water activity. This can make it difficult to provide products with high levels of active microorganisms.

[0005] Furthermore, if microorganisms in a food or beverage product are subject to uncontrolled growth, this can lead to changes in the characteristics of the product or even spoilage.

[0006] Known methods for improving the stability and / or shelf life of biologically active substances typically include storage at low temperatures (such as by refrigeration) or storage in an environment with very low water activity (such as a dry powder). It is understood that these methods are generally not applicable unless they are useful for incorporating biologically active substances into food or beverage products with high water activity, or into products that should be stored at room temperature.

[0007] Therefore, it would be desirable to have methods and compositions for enhancing the stability and / or shelf life of bioactive materials. This is particularly desirable when the bioactive materials are microorganisms, such as probiotic bacteria; when they are incorporated into food or beverage products that have high water activity; and when the food or beverage products are to be stored at room temperature for extended periods of time. Summary of the Invention [Problem to be solved by the invention]

[0008] It is an object of the present invention to go some way to addressing one or more of these needs, or at least to provide the public with a useful choice. [Means for solving the problem]

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

[0010] In a second aspect, the present invention provides particles comprising a bioactive substance embedded in a biofilm, wherein the biofilm is embedded in a matrix comprising at least 6% by weight of one or more lipids.

[0011] In a third aspect, the present invention provides a food or beverage product comprising particles according to the first or second aspect.

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

[0013] In a fifth aspect, the present invention provides a cleaning product comprising particles according to the first or second aspect.

[0014] In a sixth aspect, the present invention provides a method for enhancing the stability of a biologically active substance, the method comprising the steps of: a. embedding the bioactive substance into the biofilm; b. embedding the biofilm in a matrix having a water vapor transmission rate (WVTR) of 0.1 to 500; and c. forming particles in the matrix; The method includes:

[0015] In a seventh aspect, the present invention provides a method for enhancing the stability of a biologically active substance, the method comprising the steps of: a. embedding the bioactive substance into the biofilm; b. embedding the biofilm in a matrix comprising at least 6% by weight of one or more lipids; and c. forming particles in the matrix; The method includes:

[0016] In an eighth aspect, the present invention provides a method for enhancing the stability of a biologically active substance, the method comprising the steps of: a. embedding the bioactive material in a matrix having a water vapor transmission rate (WVTR) of 0.1 to 500; b. the bioactive agent induces biofilm production; and c. forming particles in the matrix; Including, wherein steps b) and c) may be in any order.

[0017] In some embodiments, step (b) comprises inducing the bioactive material embedded in the matrix to produce a biofilm.

[0018] In a ninth aspect, the present invention provides a method for enhancing the stability of a biologically active agent, comprising: Follow these steps: a. embedding the bioactive agent in a matrix comprising at least 6% by weight of one or more lipids; b. the bioactive agent induces biofilm production; and c. forming particles in the matrix; Including, wherein steps b) and c) may be in any order.

[0019] In some embodiments, step (b) comprises inducing the bioactive material embedded in the matrix to produce a biofilm.

[0020] In some embodiments, the method of the sixth, seventh, eighth, or ninth aspect comprises the steps of: d. combining the particles of step c with a medium having a water activity of at least 0.5, preferably UHT treated yogurt; and e. incubating the medium for 3 to 30 days; Further includes:

[0021] In a further aspect, the present invention provides a method for producing a food or beverage product supplemented with a bioactive substance, comprising the method of the above embodiment and comprising the steps of: f. collecting the particles from the medium of step e, and g. combining the collected particles with a food or beverage product, thereby obtaining a food or beverage product supplemented with the bioactive substance; The method further comprises:

[0022] In a further aspect, the present invention provides a method of producing a food or beverage product supplemented with a biologically active substance, the method comprising the method of the sixth, seventh, eighth or ninth aspect and further comprising the step of combining the particles with a food or beverage product, thereby obtaining a food or beverage product supplemented with a biologically active substance.

[0023] The following embodiments may be optionally applied to the above aspects. In one embodiment, the matrix comprises at least 6% by weight of one or more lipids.

[0024] In one embodiment, the matrix has a density of 0.1 to 500 g / m 2 / 24 hours, for example, 0.1-400, 0.1-300, 0.1-200, 0.1-150, 0.1-100, 0.1-90, 0.1-80, 0.1-70, 0.1-60, 0.1-50, 0.1-40, 0.1-30, 0.1-20, 0.1-10, 0.1-9, 0.1-8, 0.1-7, 0.1-6, 0.1-5, 0.1-4, 0.1-3, 0.5-500, etc., for example, 0.5-400, 0.5-300, 0.5-200, 0.5-150, 0.5-100, 0.5-90, 0.5~80, 0.5~70, 0.5~60, 0.5~50, 0.5~40, 0.5~30, 0.5~20, 0.5~10, 0.5~9, 0.5~8, 0.5~7, 0.5~6, 0.5~5, 0.5~4, 0.5~3, 1~500, for example, 1~400, 1~300, 1~200, 1~150, 1~100, 1~90, 1~80, 1~70, 1~60, 1~50, 1~40, 1~30, 1~20, 1~10, 1~9, 1~8, 1~7, 1~6, 1~5, 1~4, 1~3, 2~50 0, for example, 2-400, 2-300, 2-200, 2-150, 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-20, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-500, for example, 3-400, 3-300, 3-200, 3-150, 3-100, 3-90, 3-80, 3-70, 3-60, 3-50, 3-40, 3-30, 3-20, 3-10, 3-9, 3-8, 3-7, 3-6, 3- 5, 3-4, 4-500, etc., for example, 4-400, 4-300, 4-200, 4-150, 4-100, 4-90, 4-80, 4-70, 4-60, 4-50, 4-40, 4-30, 4-20, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-500, etc., for example, 5-400, 5-300, 5-200, 5-150, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-20, 5-10, 5-9, 5-8, 5-7, or 5-6 g / m 2 / 24 hours. In some embodiments, the matrix has a water vapor transmission rate (WVTR) of 500 g / m 2 / 24 hours, for example, 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 / has a water vapor transmission rate (WVTR) of less than 24 hours.

[0025] In one embodiment, the matrix has a water vapor transmission rate (WVTR) of 0.1-10, for example 1-5. In some embodiments, the matrix has a mass of about 1 to about 350 g / m 2 / 24 hours, 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 to 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 It has a water vapor transmission rate (WVTR) of 24 hours or more.

[0026] 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 2.

[0027] In some embodiments, the particles have a molecular weight 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 1.0 The water activity is 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.

[0028] In one embodiment, the bioactive material comprises a first microorganism. In one embodiment, the biofilm is produced by a first microorganism. In one embodiment, the first microorganism is a probiotic microorganism.

[0029] In one embodiment, the first microorganism is a bacterium of the genus Bacillus, Bifidobacterium, Enterococcus, Lacticaseibacillus, Lactiplantibacillus, Lactobacillus, Lactococcus, Ligilactobacillus, Limosilactobacillus, Lentilactobacillus, Saccharomyces, or Streptococcus. In one embodiment, the first microorganism is Bacillus coagulans, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum, Lacticaseibacillus casei, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum subsp. plantarum. plantarum, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactococcus lactis, Lactococcus lactis subsp.lactis, Lactococcus lactis subsp. lactis biovar diacetylactis, Leuconostoc pseudomesenteroides, Ligilactobacillus salivarius, Limosilactobacillus reuteri, Saccharomyces boulardii, and Streptococcus thermophilus; preferably wherein the first microorganism is Lacticaseibacillus rhamnosus HN001. In some embodiments, the first microorganism is Lacticase Bacillus rhamnosus, Lacticase Bacillus paracasei, preferably Lacticase Bacillus rhamnosus HN001 or Lacticase Bacillus paracasei subsp. paracasei IM514.

[0030] In one embodiment, the particles are 10 to 5×10 12 CFU / g particles, e.g., 10 2 ~5×10 12 , 10 3 ~5×10 12 , 10 4 ~5×10 12 , 10 5 ~5×10 12 , 10 6 ~5×10 12 , 10 7 ~5×10 12 , 10 8 ~5×10 12 , 10 9 ~5×10 12 , 10 10 ~5×10 12 , 10 11 ~5×10 12 , 10 12 ~5×10 12 , 10~10 12 , 10 2 ~1012 、10 3 ~10 12 、10 4 ~10 12 、10 5 ~10 12 、10 6 ~10 12 、10 7 ~10 12 、10 8 ~10 12 、10 9 ~10 12 、10 10 ~10 12 、10 11 ~10 12 、10~10 11 、10 2 ~10 11 、10 3 ~10 11 、10 4 ~10 11 、10 5 ~10 11 、10 6 ~10 11 、10 7 ~10 11 、10 8 ~10 11 、10 9 ~10 11 、10 10 ~10 11 、10~10 10 、10 2 ~10 10 、10 3 ~10 10 、10 4 ~10 10 、10 5 ~10 10 、10 6 ~10 10 、10 7 ~10 10 、10 8 ~10 10 、10 9 ~10 10 、10~10 9 、10 2 ~10 9 、10 3 ~10 9 、10 4 ~10 9 、105 ~10 9 , 10 6 ~10 9 , 10 7 ~10 9 , 10 8 ~10 9 , 10~10 8 , 10 2 ~10 8 , 10 3 ~10 8 , 10 4 ~10 8 , 10 5 ~10 8 , 10 6 ~10 8 , 10 7 ~10 8 , 10~10 7 , 10 2 ~10 7 , 10 3 ~10 7 , 10 4 ~10 7 , 10 5 ~10 7 , 10 6 ~10 7 , 10~10 6 , 10 2 ~10 6 , 10 3 ~10 6 , 10 4 ~10 6 , 10 5 ~10 6 , 10~10 5 , 10 2 ~10 5 , 10 3 ~10 5 , 10 4 ~10 5 , 10~10 4 , 10 2 ~10 4 , 10 3 ~10 4 , 10~10 3 , 10 2 ~10 3 , 10~10 2 The first microorganism is contained in an amount such as CFU / g particles.

[0031] In one embodiment, the particle further comprises a second microorganism that is different from the first microorganism, hi various embodiments, the particle further comprises any number of additional different microorganisms, such as a third, fourth, and / or fifth microorganism.

[0032] 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, all of the microorganisms are probiotic microorganisms. 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.

[0033] In one embodiment, the bioactive material is a non-microbial bioactive material. In one embodiment, the particles further comprise a non-microbial bioactive material. In some embodiments, the non-microbial bioactive material 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 thereof. In a preferred embodiment, the non-microbial bioactive material is a vitamin, prebiotic, postbiotic, or human milk oligosaccharide. In some embodiments, the 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), or vitamin B9 (folate or folic acid). In some embodiments, the prebiotic is a fructooligosaccharide, a galactooligosaccharide, 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 thereof.

[0034] In one embodiment, the biofilm is 360, 430, and / or 530 cm -1 In some embodiments, the Raman spectroscopic data is characterized by peaks in a principal component analysis of the Raman spectroscopic data at, for example, 360, 430, and / or 530 cm -1 The presence of rhamnose is indicated by the presence of a peak in a principal component analysis of the Raman spectroscopic data at 1450 cm. In one embodiment, principal component analysis of the Raman spectroscopic data for the biofilm-containing sample indicates the presence of rhamnose. -1360, 430, and / or 530 cm, which have peak heights higher than the corresponding peak heights in the principal component analysis of Raman spectroscopic data for non-biofilm-containing samples when normalized by the lipid-related peaks at -1 In one embodiment, the peaks are at 360, 430, and / or 530 cm -1 The peak in the principal component analysis of the Raman spectroscopy data at 1450 cm -1 At least 50% of the peak height of the lipid-related peak at, for example, 1450 cm -1 The peak height may be 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% of the peak height of the lipid-associated peak in the sample, although an effective range may 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%).

[0035] In one embodiment, biofilms can be stained with concanavalin A, wheat germ agglutinin, and / or Ulex europaeus agglutinin. In some embodiments, biofilms can be detected by concanavalin A, wheat germ agglutinin, and / or Ulex europaeus agglutinin staining, which extends beyond the bacterial cells.

[0036] In one embodiment, the biofilm comprises glucose, galactose, rhamnose, galactosamine, glucosamine, mannose, and / or fucose.

[0037] In one embodiment, the particles maintain a moisture content of 1% to 60% by weight within the matrix when incorporated into another food product having a water activity of 1.00 over a two week period, preferably a moisture content of 10% to 60%, more preferably a moisture content of 20% to 60%.

[0038] 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 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 between 30°C and 50°C, such as between 32°C and 48°C, between 34°C and 44°C, or between 36°C and 40°C.

[0039] In one embodiment, the lipid comprises or consists of one or more mono-, di-, or triglycerides. In a preferred embodiment, the lipid comprises or consists of one or more triglycerides.

[0040] In one embodiment, the one or more lipids comprise a C10-C22 fatty acid, or a salt or ester thereof. In one embodiment, the one or more lipids comprise a C12-C20 fatty acid, or a salt or ester thereof. In one embodiment, the one or more lipids comprise at least one mono-, di-, or triglyceride. In a preferred embodiment, the one or more lipids comprise at least one triglyceride. In one embodiment, at least one mono-, di-, or triglyceride comprises at least one C12-C20 fatty acid, or a salt or ester thereof. In one embodiment, at least one mono-, di-, or triglyceride comprises at least one fatty acid or ester thereof selected from the group consisting of lauric acid, myristic acid, palmitic acid, stearic acid, and arachidic acid. In one embodiment, each fatty acid or ester thereof of at least one mono-, di-, or triglyceride is selected from the group consisting of lauric acid, myristic acid, palmitic acid, stearic acid, and arachidic acid. In one embodiment, each fatty acid or ester thereof in the one or more lipids is a C12-C20 fatty acid, hi one embodiment, each fatty acid or ester thereof in the one or more lipids is selected from the group consisting of lauric acid, myristic acid, palmitic acid, stearic acid, and arachidic acid.

[0041] In some embodiments, the one or more lipids comprise one or more fatty acids and / or salts and / or esters thereof and comprise at least 1% by weight, such as at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80% fatty acids and / or salts and / or esters thereof. or its salts and / or esters have a carbon chain length of 14 or less, but an effective range can be selected between any of these values ​​(e.g., 1% to 80%, 2% to 80%, 4% to 80%, 6% to 80%, 8% to 80%, 10% to 80%, 20% to 80%, 30% to 80%, 40% to 80%, 1% to 70%, 2% to 70%, 4% to 70%, 6% to 70%, 8% to 70%, 10% to 70%, 20% to 70%, 30% to 70%, or 40% to 70%).

[0042] In some embodiments, the one or more lipids comprise fatty acids and / or salts and / or esters thereof, and comprise at least 30% by weight, such as at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, or at least 90% fatty acids and / or salts and / or esters thereof. The terephthalate has a carbon chain length of 16 or less, although a useful range can be selected between any of these values ​​(e.g., 30% to 90%, 40% to 88%, 50% to 88%, 60% to 88%, 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%).

[0043] In one embodiment, at least 50% by weight of the one or more lipids is saturated fat. In various embodiments, at least 55% by weight of the one or more lipids is saturated fat, for example, 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 at least 99% by weight of the one or more lipids is saturated fat.

[0044] 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 thereof. Preferably, the hydrogenated palm kernel oil is fully hydrogenated.

[0045] In some embodiments, the matrix comprises one or more lipids that are at least 4% by weight of total solids, such as at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, 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 at least 99% by weight of total solids.

[0046] In one embodiment, the matrix comprises 4 to 100% by weight, preferably 30 to 100% by weight, of one or more lipids. In some embodiments, the matrix comprises 4 to 100% by weight of the total solids of one or more lipids, for example, 8 to 100% by weight, 10 to 100% by weight, 20 to 100% by weight, 30 to 100% by weight, 40 to 100% by weight, 50 to 100% by weight, 60 to 100% by weight, 70 to 100% by weight, 80 to 100% by weight, 90 to 100% by weight, 4 to 99.5% by weight, 8 to 99.5% by weight, 10 to 99.5% by weight, 20 to 99.5% by weight, of the total solids. Weight% ~ 99.5%, 30% ~ 99.5%, 40% ~ 99.5%, 50% ~ 99.5%, 60% ~ 99.5%, 70% ~ 99.5%, 80% ~ 99.5%, 90% ~ 99.5%, 4% ~ 95% by weight, 8% to 95% by weight, 10% to 95% by weight, 20% to 95% by weight, 30% to 95% by weight, 40% to 95% by weight, 50% to 95% by weight, 60% to 95% by weight, 70% to 95% by weight, 80% to 95% by weight, 90 Weight% ~ 95%, 10% ~ 90%, 20% ~ 90%, 30% ~ 90%, 40% ~ 90%, 50% ~ 90%, 60% ~ 90%, 70% ~ 90%, 80% ~ 90%, 10% ~ 80% Amount%, 20%~80%, 30%~80%, 40%~80%, 50%~80%, 60%~80%, 70%~80%, 10%~70%, 20%~70%, 30%~70%, 40% and / or 10% to 20% by weight of one or more lipids, such as 10% to 70% by weight, 50% to 70% by weight, 60% to 70% by weight, 10% to 60% by weight, 20% to 60% by weight, 30% to 60% by weight, 40% to 60% by weight, 50% to 60% by weight, 10% to 50% by weight, 20% to 50% by weight, 30% to 50% by weight, 40% to 50% by weight, 10% to 40% by weight, 20% to 40% by weight, 30% to 40% by weight, 10% to 20% by weight, 10% to 30% by weight, or 10% to 20% by weight.

[0047] In one embodiment, the matrix comprises at least 10% by weight, preferably at least 15%, 20%, 25%, or 30% fully hydrogenated coconut oil. In one embodiment, the matrix comprises one or more adjuvants selected from sugars, emulsifiers, milk solids, and salts.

[0048] In some embodiments, the matrix comprises one or more sugars at 0% to 80% by weight of the total solids, e.g., 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 of the total solids. %, 20% to 60% by weight, 30% to 60% by weight, 40% to 60% by weight, 50% to 60% by weight, 0% to 50% by weight, 10% to 50% by weight, 20% to 50% by weight, 30% to 50% by weight, 40% to 50% by weight, 0% to 40% by weight, 10% to 40% by weight, 20% to 40% by weight, 30% to 40% by weight, 0% to 30% by weight, 10% to 30% by weight, 20% to 30% by weight, 0% to 20% by weight, 10% to 20% by weight, or 0% to 10% by weight.

[0049] In some embodiments, the matrix comprises one or more emulsifiers at 0% to 2% by weight of total solids, e.g., 0.2% to 2%, 0.4% to 2%, 0.6% to 2%, 0.8% to 2%, 1.0% to 2%, 1.2% to 2%, 1.4% to 2%, 1.6% to 2%, 1.8% to 2%, 0% to 1.8%, 0.2% to 1.8%, 0.4% by weight of total solids. ~1.8wt%, 0.6wt%~1.8wt%, 0.8wt%~1.8wt%, 1.0wt%~1.8wt%, 1.2wt%~1.8wt%, 1.4wt%~1.8wt%, 1.6wt%~1.8wt%, 0wt%~1. 6wt%, 0.2wt%~1.6wt%, 0.4wt%~1.6wt%, 0.6wt%~1.6wt%, 0.8wt%~1.6wt%, 1.0wt%~1.6wt%, 1.2wt%~1.6wt%, 1.4wt%~1.6wt% Amount%, 0wt%~1.4wt%, 0.2wt%~1.4wt%, 0.4wt%~1.4wt%, 0.6wt%~1.4wt%, 0.8wt%~1.4wt%, 1.0wt%~1.4wt%, 1.2wt%~1.4wt%, 0wt%~1.2wt%, 0.2wt%~1.2wt%, 0.4wt%~1.2wt%, 0.6wt%~1.2wt%, 0.8wt%~1.2wt%, 1.0wt%~1.2wt%, 0wt%~1.0wt%, 0.2wt% The composition may contain one or more emulsifiers, such as 0.4% to 1.0% by weight, 0.4% to 1.0% by weight, 0.8% to 1.0% by weight, 0.6% to 1.0% by weight, 0% to 0.8% by weight, 0.2% to 0.8% by weight, 0.4% to 0.8% by weight, 0.6% to 0.8% by weight, 0.2% to 0.8% by weight, 0.4% to 0.8% by weight, 0.6% to 0.8% by weight, 0.2% to 0.6% by weight, 0.4% to 0.6% by weight, 0.4% to 0.4% by weight, 0.2% to 0.4% by weight, or 0% to 0.2% by weight.

[0050] In some embodiments, the matrix comprises 0% to 30% milk solids by weight of total solids, such as 5% to 30%, 10% to 30%, 15% to 30%, 20% to 30%, 25% to 30%, 0% to 25%, 5% to 25%, 10% to 25%, 15% to 25%, 20% to 25%, 0% to 20%, 5% to 20%, 10% to 20%, 15% to 20%, 0% to 15%, 5% to 15%, 10% to 15%, 0% to 10%, 5% to 10%, or 0% to 5% milk solids by weight of total solids.

[0051] In one embodiment, the matrix comprises, by weight of total solids, one of the following: a. 20-80% sugar, preferably sucrose and / or lactose; b. 0.1 to 20% of an emulsifier, preferably sorbitan tristearate and / or lecithin; and / or c. 10-50% milk solids; Includes.

[0052] In some embodiments, the matrix comprises, by weight of total solids, about 50% to about 70% hydrogenated coconut oil and about 30% to about 50% sucrose; more preferably about 55% to about 65% hydrogenated coconut oil and about 35% to about 45% sucrose; most preferably about 59% hydrogenated coconut oil and about 41% sucrose.

[0053] In some embodiments, the matrix comprises, by weight of total solids, about 20% to about 40% palm stearin, about 20% to about 40% hydrogenated coconut oil, and about 30% to about 50% sucrose; preferably about 25% to about 35% palm stearin, about 25% to about 35% hydrogenated coconut oil, and about 35% to about 45% sucrose; most preferably about 29.5% palm stearin, about 29.5% hydrogenated coconut oil, and about 41% sucrose.

[0054] In some embodiments, the matrix comprises, by weight of total solids, about 0% to about 20% palm stearin, about 0% to about 20% hydrogenated coconut oil, about 30% to about 50% hydrogenated palm kernel stearin, and about 30% to about 50% sucrose; preferably about 5% to about 15% palm stearin, about 5% to about 15% hydrogenated coconut oil, about 35% to about 45% hydrogenated palm kernel stearin, and about 35% to about 45% sucrose; most preferably about 11% palm stearin, about 11% hydrogenated coconut oil, about 37% hydrogenated palm kernel stearin, and about 41% sucrose.

[0055] In some embodiments, the matrix comprises, by weight of total solids, about 90% to about 100% cocoa butter, and optionally about 0.1% to about 1.0% lecithin; preferably about 95% to about 100% cocoa butter, and optionally about 0.1% to about 1.0% lecithin; most preferably about 99.5% cocoa butter and about 0.5% lecithin.

[0056] In some embodiments, the matrix comprises, by weight of total solids, about 65% to about 85% cocoa butter and about 15% to about 35% milk solids; preferably about 70% to about 80% cocoa butter and about 20% to about 30% milk solids; most preferably about 73% cocoa butter and about 26% milk solids.

[0057] In some embodiments, the matrix comprises at least about 80% hydrogenated palm kernel oil, preferably at least about 90%, more preferably at least about 95%, and most preferably about 100% hydrogenated palm kernel oil, by weight of total solids, although a useful range can be selected between any of these values ​​(e.g., about 80% to about 100%, about 90% to about 100%, or about 95% to about 100%).

[0058] In some embodiments, the matrix comprises, by weight of total solids, about 75% to about 95% cocoa butter and about 5% to about 25% lactose; preferably about 80% to about 90% cocoa butter and about 5% to about 15% lactose; more preferably about 89.5% cocoa butter and about 13.5% lactose.

[0059] In some embodiments, the matrix comprises, by weight of total solids, about 75% to about 95% hydrogenated coconut oil and about 5% to about 25% lactose; preferably about 80% to about 90% hydrogenated coconut oil and about 10% to about 20% lactose; more preferably about 86.5% hydrogenated coconut oil and about 13.5% lactose.

[0060] 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 5mm, 100μm~4mm, 100μm~3mm, 100μm~2mm, 100μm~1mm, 100μm~500μm, 200μm~10mm, 200μm~9mm, 200μm~8mm, 20 It has a particle size of 0 μm to 7 mm, 200 μm to 6 mm, 200 μm to 5 mm, 200 μm to 4 mm, 200 μm to 3 mm, 200 μm to 2 mm, 200 μm to 1 mm, 200 μm to 500 μm, etc.

[0061] In one embodiment, the particle further comprises at least one coating layer. In some embodiments, the particle comprises 2, 3, 4, or 5 coating layers. In some embodiments, the coating layer comprises alginate, chitosan, collagen, dextran, pectin, pullulan, gelatin, carrageenan, agar, cellulose, hemicellulose, ethylcellulose, carboxycellulose, or a mixture of any two or more thereof.

[0062] In one embodiment, the food or beverage product comprises: a. protein powders, protein shakes, protein shots, protein gels, or sports nutrition formulas; b. UHT drinks, UHT smoothies, c. infant formula, follow-on formula, growth formula, pediatric formula, human milk fortifier, pediatric food or beverage, maternal supplement, or maternal nutritional formula; d. Gels, such as thermosetting gels, sauces, spreads, jams, jellies, or honey; e. Acid-setting gel, shelf-stable yogurt, stirred yogurt, set yogurt, or drinking yogurt; f. Neutral drinks, acidic drinks, water, juice, milk, smoothies, shakes, shots, alcoholic drinks, soft drinks, kombucha, kefir, or ready-to-mix powders; g. Bars, balls, cakes, cookies, muffins, or bakery products; h. Medical foods, soups, desserts, puddings, custards, enteral formulas, senior or geriatric formulas, tablets, capsules, or supplements; i. Sugar confectionery, gummies, candies, chocolates, fudge, truffles, chewing gum, frozen desserts, ice cream, j. flavorings, toppings, or baking ingredients; k. Cream, cheese, or butter; or l. Livestock feed or pet food; Includes:

[0063] In some embodiments, the food or beverage product includes yogurt, fruit juice such as orange juice, sports shakes, cheese sauce, cream cheese spread, or milk, e.g., non-dairy milk such as oat milk.

[0064] 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. 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.

[0065] In one embodiment, the biologically active material comprises a microorganism, and the decomposition rate of the microorganism measured over 12 months at 30° C. is less than 7 log CFU / g / year, preferably less than 6, 5, 4, 3, 2 or 1 log CFU / g / year.

[0066] In one embodiment, the personal care product is a skin care product, such as a skin cream or sunscreen, a hair care product, such as a shampoo or conditioner, a dental product, such as a toothpaste or a dentifrice, a deodorant, a cosmetic, a beauty product, or a feminine health product.

[0067] In an embodiment of the sixth, seventh, eighth, or ninth aspect, the method further comprises coating the particles with at least one coating layer. In one embodiment of the method according to the sixth, seventh, eighth or ninth aspect, the particles comprise particles according to the first or second aspect. In one embodiment of the sixth, seventh, eighth, or ninth aspect, the method comprises the steps of: d. combining the particles of step c with a medium having a water activity of at least 0.5, preferably UHT treated yogurt; and e. Incubating the medium for at least two weeks; Further includes:

[0068] In one embodiment the food or drink product supplemented with a bioactive substance is a food or drink product according to the third aspect.

[0069] The term "comprising" as used in the present specification and claims means "consisting at least in part of." When interpreting statements in the present specification and claims that include the term "comprising," there may also be other features present in each statement besides the feature that begins with this term. Related terms such as "comprises" and "comprised" should be interpreted in a similar manner.

[0070] Where reference is made herein to patents, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing features of the present invention. Unless specifically stated otherwise, reference to such external documents should not be construed as an admission that such documents or sources of information are prior art or form part of the common general knowledge in the art in any jurisdiction.

[0071] Embodiments of the present invention will now be described with reference to the drawings in which: [Brief explanation of the drawings]

[0072] [Figure 1] FIG. 1 shows contour plots for coating matrices containing palm stearin, fully hydrogenated palm kernel oil, and fully hydrogenated coconut oil, individually and in combination.

[0073] [Figure 2] FIG. 2 shows contour plots for coating matrices containing palm stearin, fully hydrogenated coconut oil, and cocoa powder, individually and in combination.

[0074] [Figure 3] FIG. 3 shows contour plots for coating a matrix containing fully hydrogenated palm kernel oil, fully hydrogenated coconut oil, and cocoa powder, individually and in combination.

[0075] [Figure 4]Figure 4 shows a scanning electron microscope image of exopolysaccharide / biofilm coated Bacillus rhamnosus HN001 in a hydrogenated palm kernel oil matrix at 100x magnification.

[0076] [Figure 5] Figure 5 shows a scanning electron microscope image of exopolysaccharide / biofilm coated Bacillus rhamnosus HN001 in a hydrogenated palm kernel oil matrix at 250x magnification.

[0077] [Figure 6] Figure 6 shows a scanning electron microscope image of exopolysaccharide / biofilm-coated Bacillus rhamnosus HN001 in a hydrogenated palm kernel oil matrix at 1,200x magnification.

[0078] [Figure 7] Figure 7 shows a scanning electron microscope image of exopolysaccharide / biofilm-coated Bacillus rhamnosus HN001 in a hydrogenated palm kernel oil matrix at 4,000x magnification.

[0079] [Figure 8] Figure 8 shows a light microscope image of clusters of B. lactis rhamnosus HN001 attached to the surface of a glass wool strand. The image was taken under oil immersion at 100x magnification and was imaged using crystal violet stain.

[0080] [Figure 9] Figure 9 shows Raman spectroscopy of L. rhamnosus HN001 induced to produce a biofilm. Three distinct peaks (indicated by arrows) in the 350-600 cm spectral region appear to correlate with the presence of EPS components.

[0081] [Figure 10]Figure 10 shows Raman spectroscopy of bead structures incorporating L. rhamnosus HN001. Multivariate curve resolution estimated spectra from analysis of the 120-day data for bead sample AT03 are shown. Arrows indicate the same three EPS-related peaks.

[0082] [Figure 11] Figure 11 shows concanavalin (ConA) staining of sample MVP17 at 90 days of shelf life. Mannose / glucose stained with ConA is shown in orange; proteins are stained green (this stain also stains dead cells); cells are stained light blue with DAPI.

[0083] [Figure 12] Figure 12 shows wheat germ agglutinin (WGA) staining of sample MVP17 at 90 days of shelf life. N-acetylglucosamine stained with WGA is shown in orange; proteins are stained green (this stain also stains dead cells); cells are stained light blue with DAPI.

[0084] [Figure 13] Figure 13 shows U.S. gorse agglutinin (UEAI) staining of sample MVP17 at 90 days of shelf life. UEAI staining is shown in orange; proteins are stained green (this stain also stains dead cells); cells are stained light blue with DAPI. DETAILED DESCRIPTION OF THE INVENTION

[0085] Detailed Description of the Invention This description may refer to material not within the scope of the claims of this application, but that material must be readily ascertainable by one skilled in the art and may aid in practicing the invention as defined by the claims of this application.

[0086] particle Applicants have surprisingly found that particles comprising a biologically active material embedded in a biofilm (wherein the biofilm is embedded in a matrix, where the matrix has a water vapor transmission rate (WVTR) of 0.1 to 500) provide the biologically active material with an improved shelf life, even when stored at room temperature in a high water activity environment.

[0087] Thus, in a first aspect, the present invention provides particles comprising a bioactive material 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. Preferably, the matrix comprises at least 6% by weight of one or more lipids.

[0088] The term "particle" refers to a portion or quantity of a substance, such as a small portion or small amount of a substance. The term may encompass or be used interchangeably with the following terms: pellet, bead, sphere, granule, etc. In some embodiments, the particle may be approximately spherical in shape. Preferably, the particle is edible; that is, the particle preferably does not include any inedible components.

[0089] The term "embedded," as used herein, means "at least partially surrounded by." For example, a bioactive material may be at least partially surrounded by a biofilm, which in turn may be at least partially surrounded by a matrix. The term is not intended to require that the bioactive material or biofilm be completely surrounded on all sides.

[0090] In some embodiments, the bioactive material is encapsulated within the biofilm. In some embodiments, the biofilm is encapsulated within a matrix. The term "encapsulated within," as used herein, means "completely surrounded." In some embodiments, the bioactive material is dispersed throughout the biofilm. In some embodiments, the biofilm is dispersed throughout a matrix.

[0091] The term "matrix" refers to a material or mixture of materials in which a biofilm and / or a bioactive material is embedded. The matrix can be any material or mixture of materials suitable for embedding a bioactive material and forming particles. Preferably, the matrix is ​​a solid at room temperature. In one embodiment, the matrix is ​​edible.

[0092] In some embodiments, the matrix comprises 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 thereof.

[0093] 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, mono-, di-, and triglycerides, and phospholipids. Certain lipids, such as fats, may be saturated or unsaturated. Unsaturated lipids may be converted to saturated lipids by the process of hydrogenation. Hydrogenation may be incomplete or partial (when not all carbon-carbon double bonds are reduced), or complete. The term "fully hydrogenated" and related terms such as "fully hydrogenated" and "100% hydrogenated" are intended to mean that at least 99% of the lipid to which the term is applied does not contain unsaturated carbon-carbon double bonds.

[0094] 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.

[0095] In some embodiments, one or more lipids have a melting temperature of 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, although useful ranges may also be found between any of these 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 ... etc. ~48℃, 5℃~46℃, 10℃~46℃, 15℃~46℃, 20℃~46℃, 25℃~46℃, 30℃~46℃, 32℃~46℃, 34℃~46℃, 36℃~46℃, 5℃~44℃, 10℃~44℃, 15℃~44℃, 20℃~44℃, 25℃~44℃, 30℃~44℃, 32℃~44℃, 34℃~44℃, 36℃~44℃, 5℃ The temperature may be selected from the range of 5°C to 42°C, 10°C to 42°C, 15°C to 42°C, 20°C to 42°C, 25°C 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.

[0096] In some embodiments, the lipid comprises, consists essentially of, or consists of triglycerides, hi some embodiments, the lipid comprises, consists essentially of, or consists of C12-C20 fatty acids, or salts or esters thereof.

[0097] In some embodiments, the one or more lipids comprise one or more fatty acids and / or salts and / or esters thereof, and comprise at least 1% by weight, such as at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80% fatty acids and / or salts thereof. The salts and / or esters thereof have a carbon chain length of 14 or less, but an effective range can be selected between any of these values ​​(e.g., 1% to 80%, 2% to 80%, 4% to 80%, 6% to 80%, 8% to 80%, 10% to 80%, 20% to 80%, 30% to 80%, 40% to 80%, 1% to 70%, 2% to 70%, 4% to 70%, 6% to 70%, 8% to 70%, 10% to 70%, 20% to 70%, 30% to 70%, or 40% to 70%).

[0098] In some embodiments, the one or more lipids comprise fatty acids and / or salts and / or esters thereof, and are at least 30% by weight, such as at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, or at least 90% fatty acids and / or salts and / or esters thereof. has a carbon chain length of 16 or less, although a useful range can be selected between any of these values ​​(e.g., 30% to 90%, 40% to 88%, 50% to 88%, 60% to 88%, 70% to 88%, 74% to 88%, 76% to 88%, 78% to 88%, 78% to 88%, 80% to 88%, 74% to 70% to 86% to 86%, 76% to 86%, 78% to 86%, 78% to 86%, or 80% to 86%).

[0099] In an alternative embodiment, the one or more lipids comprise fatty acids and / or salts and / or esters thereof, and at least 10% by weight, 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%, of the fatty acids and / or salts and / or esters thereof have a carbon chain length of 16 or less, although a useful range is within any of these values. The range may be selected from the ranges (e.g., 10% to 90%, 10% to 80%, 20% to 90%, 20% to 88%, 20% to 86%, 20% to 84%, 25% to 90%, 25% to 88%, 25% to 86%, 25% to 84%, 30% to 90%, 40% to 88%, 50% to 88%, 60% to 88%, 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%).

[0100] The fatty acid esters may include mono-, di-, and / or triglycerides.

[0101] In some embodiments, at least 50% by weight of the one or more lipids is saturated fat. In some embodiments, the lipids may be partially or fully hydrogenated. In some embodiments, the one or more lipids comprise, consist essentially of, or consist of fully hydrogenated palm kernel stearin, palm stearin, fully hydrogenated coconut oil, cocoa butter, or a mixture of any two or more thereof. In preferred embodiments, the one or more lipids comprise, consist essentially of, or consist of fully hydrogenated palm kernel stearin, palm stearin, fully hydrogenated coconut oil, or a mixture of any two or more thereof.

[0102] In some embodiments, the matrix comprises one or more lipids at least 4% by weight of the total solids, such as at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, 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 at least 99% by weight of the total solids. Preferably, the matrix comprises at least 30% by weight of the one or more lipids of the total solids.

[0103] In some embodiments, the matrix comprises one or more lipids that are 4% to 95% by weight of the total solids, e.g., 8% to 95% by weight, 10% to 95% by weight, 20% to 95% by weight, 30% to 95% by weight, 40% to 95% by weight, 50% to 95% by weight, 60% to 95% by weight, 70% to 95% by weight, 80% to 95% by weight, 90% to 95% by weight, 10% to 90% by weight, 20% to 90% by weight, 30% to 90% by weight, 40% to 90% by weight, 50% to 90% by weight, 60% to 90% by weight, 70% to 90% by weight, 80% to 90% by weight, 10% to 80% by weight, 20% to 80% by weight, 30% to 80% by weight, 40% to 90% by weight, %~80wt%, 50wt%~80wt%, 60wt%~80wt%, 70wt%~80wt%, 10wt%~70wt%, 20wt%~70wt%, 30wt%~70wt %, 40% to 70% by weight, 50% to 70% by weight, 60% to 70% by weight, 10% to 60% by weight, 20% to 60% by weight, 30% to 60% by weight, 40% by weight The matrix may comprise one or more lipids in an amount of from 30% to 95% by weight of the total solids, such as from 60% to 60% by weight, from 50% to 60% by weight, from 10% to 50% by weight, from 20% to 50% by weight, from 30% to 50% by weight, from 40% to 50% by weight, from 10% to 40% by weight, from 20% to 40% by weight, from 30% to 40% by weight, from 10% to 20% by weight, or from 10% to 20% by weight. Preferably, the matrix comprises one or more lipids in an amount of from 30% to 95% by weight of the total solids.

[0104] In preferred embodiments, the matrix comprises at least 10% by weight, preferably at least 15%, 20%, 25%, or 30% fully hydrogenated coconut oil.

[0105] 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 useful for providing additional structural integrity to the particles.

[0106] 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 skim 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 skim milk powder).

[0107] In one embodiment, the matrix comprises, consists essentially of, or consists of palm stearin and a sugar (preferably sucrose and / or lactose). In one embodiment, the matrix comprises, consists essentially of, or consists of palm stearin and milk solids (preferably skim milk powder). In one embodiment, the matrix comprises, consists essentially of, or consists of palm stearin, a sugar (preferably sucrose and / or lactose), and milk solids (preferably skim milk powder).

[0108] 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 skim 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 skim milk powder).

[0109] 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 skim 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 skim milk powder).

[0110] 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 skim 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 skim milk powder).

[0111] In some embodiments, the matrix comprises one or more sugars at 0% to 80% by weight of the total solids, e.g., 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 of the total solids. %, 20% to 60% by weight, 30% to 60% by weight, 40% to 60% by weight, 50% to 60% by weight, 0% to 50% by weight, 10% to 50% by weight, 20% to 50% by weight, 30% to 50% by weight, 40% to 50% by weight, 0% to 40% by weight, 10% to 40% by weight, 20% to 40% by weight, 30% to 40% by weight, 0% to 30% by weight, 10% to 30% by weight, 20% to 30% by weight, 0% to 20% by weight, 10% to 20% by weight, 0% to 10% by weight, etc. Preferably, the matrix comprises one or more sugars at 0% to 50% by weight of the total solids. Preferably, the one or more sugars comprise or consist of sucrose and / or lactose.

[0112] In some embodiments, the matrix comprises one or more emulsifiers at 0% to 2% by weight of total solids, e.g., 0.2% to 2%, 0.4% to 2%, 0.6% to 2%, 0.8% to 2%, 1.0% to 2%, 1.2% to 2%, 1.4% to 2%, 1.6% to 2%, 1.8% to 2%, 0% to 1.8%, 0.2% to 1.8%, 0.4% by weight of total solids. ~1.8wt%, 0.6wt%~1.8wt%, 0.8wt%~1.8wt%, 1.0wt%~1.8wt%, 1.2wt%~1.8wt%, 1.4wt%~1.8wt%, 1.6wt%~1.8wt%, 0wt%~1. 6wt%, 0.2wt%~1.6wt%, 0.4wt%~1.6wt%, 0.6wt%~1.6wt%, 0.8wt%~1.6wt%, 1.0wt%~1.6wt%, 1.2wt%~1.6wt%, 1.4wt%~1.6wt% Amount%, 0wt%~1.4wt%, 0.2wt%~1.4wt%, 0.4wt%~1.4wt%, 0.6wt%~1.4wt%, 0.8wt%~1.4wt%, 1.0wt%~1.4wt%, 1.2wt%~1.4wt%, 0wt%~1.2wt%, 0.2wt%~1.2wt%, 0.4wt%~1.2wt%, 0.6wt%~1.2wt%, 0.8wt%~1.2wt%, 1.0wt%~1.2wt%, 0wt%~1.0wt%, 0.2wt% The matrix may comprise one or more emulsifiers in an amount of 0% to 0.5% by weight of total solids, such as sorbitan tristearate and / or lecithin, e.g., sorbitan tristearate and / or lecithin, e.g., soybean lecithin.

[0113] In some embodiments, the matrix comprises 0% to 30% milk solids by weight of total solids, for example, 5% to 30%, 10% to 30%, 15% to 30%, 20% to 30%, 25% to 30%, 0% to 25%, 5% to 25%, 10% to 25%, 15% to 25%, 20% to 25%, 0% to 20%, 5% to 20%, 10% to 20%, 15% to 20%, 0% to 15%, 5% to 15%, 10% to 15%, 0% to 10%, 5% to 10%, or 0% to 5% milk solids by weight of total solids. Preferably, the matrix comprises 20% to 30% milk solids by weight of total solids.

[0114] 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.

[0115] In some embodiments, the particle further comprises an additional coating layer. Such a layer may be used to modify the permeability of the particle, such as water permeability, water vapor transmission rate, or diffusivity. Alternatively or additionally, the coating layer may be used to provide resistance to environmental conditions, such as resistance to gastric fluids.

[0116] In some embodiments, the coating layer comprises agar, alginate, carboxycellulose, carrageenan, cellulose, cellulose acetate phthalate, chitosan, collagen, dextran, ethylcellulose, gelatin, gluco-mannan, hemicellulose, milk protein, shellac, starch, pectin, poly-L-lysine, pullulan, or a mixture of any two or more thereof.

[0117] In some embodiments, the matrix is ​​a solid at 25°C.

[0118] In some embodiments, the matrix has a melting temperature of at least about 30°C, e.g., 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, although a useful range 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).

[0119] Particles can be made to any suitable size using conventional techniques. It is 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 being measured. For example, in one embodiment, particle size is measured by microscopy. This is particularly useful for particles ≧1 mm in diameter. In another embodiment, particle size is measured using a Malvern Mastersizer 3000 or similar analytical device. This is particularly useful for measuring particles <1 mm in diameter.

[0120] In some embodiments, the particles have a diameter of 50 μm to 10 mm, such as 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 (from) 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 200 μm, 75 μm to 10 mm, 75 μm to 9 mm, 75 μm to 8 mm, 75 μm to 7 mm, 75 μm to 6 mm, 75 μm to 5 mm, 75 (from) μm to 4 mm, 75 μm to 3 mm, 75 μm to 2 mm, 75 μm to 1 mm, 75 μm to 900 μm, 75 μm to 800 μm, 75 μm to 700 μm, 75 μm to 600 μm, 75 μm to 500 μm, 75 μm to 400 μm, 75 μm to 300 μm, 75 μm to 200 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 5 mm, 100 (from) μm to 4 mm, 100 μm to 3 mm, 100 μm to 2 mm, 100 μm to 1 mm, 100 μm to 900 μm, 100 μm to 800 μm, 100 μm to 700 μm, 100 μm to 600 μm, 100 μm to 500 μm, 100 μm to 400 μm, 100 μm to 300 μm, 100 μm to 200 μm, 200 μm to 10 mm, 200 μm to 9 mm, 200 μm to 8 mm, 200 μm to 7 mm, 200 μm to 6 mm, 200 μm to 5 mm, 200 (from) μm to 4 mm, 200 μm to 3 mm, 200 μm to 2 mm, 200 μm to 1 mm, 200 μm to 900 μm, 200 μm to 800 μm, 200 μm to 700 μm, 200 μm to 600 μm, 200 μm to 500 μm, 200 μm to 400 μm, or 200 μm to 300 μm, etc.

[0121] Applicants have found that the particles of the present invention are not completely impervious to the ingress of moisture and / or other compounds present in their environment. Applicants have surprisingly found that, contrary to the teachings of the prior art, biologically active substances such as microorganisms can maintain high viability within the particles of the present invention for extended periods of time, even when stored at ambient temperature in a high water activity environment.

[0122] Therefore, in some embodiments, the particles are not impermeable to moisture. Thus, in some embodiments, the matrix has a viscosity of 0.1 to 500, e.g., 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, etc., e.g., 0.5 to 400, 0.5 to 300, 0.5 to 200, 0.5 to 150 , 0.5~100, 0.5~90, 0.5~80, 0.5~70, 0.5~60, 0.5~50, 0.5~40, 0.5~30, 0.5~20, 0.5~10, 0.5~9, 0.5~8, 0.5~7, 0.5~6, 0.5~5, 0.5~4, 0.5~3, 1~500, for example, 1~400, 1~300, 1~200, 1~150, 1~100, 1~90, 1~80, 1~70, 1~60, 1~50, 1~40, 1~30, 1~20, 1~10, 1~9, 1~8, 1~7, 1~6, 1~5, 1~4, 1~3, 2-500, for example, 2-400, 2-300, 2-200, 2-150, 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-20, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-500, for example, 3-400, 3-300, 3-200, 3-150, 3-100, 3-90, 3-80, 3-70, 3-60, 3-50, 3-40, 3-30, 3-20, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4- and 500, for example, 4 to 400, 4 to 300, 4 to 200, 4 to 150, 4 to 100, 4 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 matrix 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 less than about 1.

[0123] Water vapor transmission rate (WVTR) is a measure of the amount of water vapor that passes through a unit area of ​​a test specimen per unit time under specified conditions. Water vapor transmission rate is expressed in grams per square meter per day (g m -2 ·day -1 or g / m 2 / 24 hours). WVTR can be measured using techniques known in the art, for example, a 10 cm 2 The WVTR can be measured using a Versaperm MkV Digital WVTR Meter (Versaperm Ltd., Maidenhead, UK) with a measurement range of 100°C (100°F). In one embodiment, the method for measuring WVTR is based on ISO 15106-3:2003. In a preferred embodiment, WVTR is measured over a 24-hour period at about 20°C and about 75% relative humidity. Relative humidity can be maintained at about 75% by the use of an open-top container with saturated NaCl solution.

[0124] Alternatively, the WVTR can be calculated using the following equation: WVTR=dm / (A dt) WVTR can be calculated by using the formula: where dm = mass of water absorbed during time dt, and A = total surface area of ​​the sample calculated based on particle size measurements. The mass gain should be measured over a period of approximately linear weight gain, e.g., 5 minutes, preferably 7 minutes, after adjusting to 95% relative humidity. WVTR is expressed in g / m 2 Reported in 24 hour increments.

[0125] To measure WVTR, an aliquot of particle sample can be weighed on a specific adsorption microbalance in a dynamic vapor sorption (DVS) instrument (Surface Measurement Systems, Alperton, Middlesex, UK), with measurements performed at 95% relative humidity.

[0126] In some embodiments, the particles maintain a moisture content of 1-60% by weight within the matrix, preferably 10%-60%, and more preferably 20%-60% when incorporated into another food product having a water activity of 0.90-1.00, over a two-week period. Moisture content can be measured by various techniques, such as, for example, Raman spectroscopy.

[0127] In some embodiments, when the bioactive material is a microorganism, the microorganism produces a biofilm. Without wishing to be bound by any theory, it is believed that the physical and / or chemical properties of the particle may induce the microorganism to produce a biofilm, including when the particle is present in a food or beverage product. This may be the result of maintaining a non-zero moisture content within the matrix and / or the influx of other compounds from the environment, such as lactose.

[0128] In some embodiments, the composition of the matrix is ​​selected to favor biofilm formation. For example, in some embodiments, the matrix may include components known to stimulate microorganisms to produce biofilms. In some embodiments, the matrix composition is selected to provide conditions, such as moisture content and / or nutrient ingress, that stimulate microorganisms to produce biofilms.

[0129] In some embodiments, the particle further comprises at least one coating layer. The coating layer can provide the particle with improved or modified properties, such as, for example, enhanced structural integrity. The coating layer can also be used to modify the water permeability of the particle. In some embodiments, multiple coating layers may be used. Preferably, the coating layer is edible.

[0130] Some exemplary coating layers 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 wax, carnauba wax, beeswax, candelilla wax, and polyethylene wax; fatty acids and monoglycerides, such as stearyl alcohol, stearic acid, palmitic acid, mono-, di-, and tri-glycerides; naturally occurring resins, such as wood resins and coumarone-indene; corn zein (α-zein, β ... Proteins, including starches 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; microbially fermented gums, such as xanthan, gellan gum, and chitosan; agar, alginates, seaweed extracts, such as carrageenan and furcellanus; pectin; gluco-mannan; shellac; and poly-L-lysine. Mixtures of these substances may also be used.

[0131] The coating layer may also include one or more plasticizers. Suitable plasticizers include glycols such as polyethylene glycol (PEG), polypropylene glycol (PPG), and lipids such as vegetable oils, mineral oils, medium chain fatty acids, fats, fatty acids, and waxes.

[0132] In one embodiment, the particles are encapsulated by lipospheres. In one embodiment, the biofilm is encapsulated by lipospheres that are embedded within a matrix.

[0133] biologically active substances The term "bioactive substance" refers to any substance that has 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, microbial derivatives, or any combination of two or more thereof. Of course, biological activity need not be directly applied to an organism that consumes the particles of the invention. For example, bioactive substances may include substances that have an effect on the intestinal microflora of an organism, such as, for example, prebiotics.

[0134] In a preferred embodiment, the bioactive material is a microorganism, more preferably a probiotic microorganism, most preferably a probiotic bacterium.

[0135] In some embodiments, the bioactive substance is a derivative of a microorganism, preferably a probiotic microorganism. Derivatives of a microorganism include mutants or homologs of the microorganism, attenuated or killed microorganisms, and / or components of the microorganism that still have useful activity. For example, while the bacterial molecules involved in mediating probiotic activity have not been definitively identified, molecules that have been proposed as potential candidates include bacterial DNA motifs, surface proteins, small organic acids, polysaccharides, and cell wall components, such as lipoteichoic acid and peptidoglycan, which are hypothesized to interact with components of the host immune system and confer 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 cell or non-attenuated) control, although an effective range 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%, about 90 to about 100%).

[0136] In some embodiments, the bioactive material is a non-microbial bioactive material. The term "non-microbial" means that the bioactive material does not contain living microorganisms.

[0137] In some embodiments, the non-microbial bioactive material does not include live or killed microorganisms. In some embodiments, the non-microbial bioactive material does not include microbial derivatives. In some embodiments, the non-microbial bioactive material is a protein, peptide, amino acid, fat, triglyceride, lipid, fatty acid, oligosaccharide, polysaccharide, nucleic acid, nucleotide, nucleoside, vitamin, mineral, or any combination of two or more thereof. In preferred embodiments, the non-microbial bioactive material is a vitamin, prebiotic, postbiotic, or human milk oligosaccharide. In some embodiments, the 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), or vitamin B9 (folate or folic acid). In some embodiments, the prebiotic is a fructooligosaccharide, a galactooligosaccharide, 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 thereof.

[0138] Prebiotics are compounds that induce the growth or activity of beneficial microorganisms.Some prebiotics are complex sugars that cannot be digested by organisms (e.g., mammals) that ingest them, but are digested by beneficial microorganisms.In this way, some prebiotics can be used as a nutrient source by beneficial microorganisms.Some non-limiting examples of prebiotics include fructooligosaccharides, galactooligosaccharides, and inulin.

[0139] Postbiotics are microbially derived products that provide a benefit or benefits to the host. Some postbiotics are secreted by living microorganisms, and others are released after bacterial lysis. Some non-limiting examples of postbiotics include vitamins such as vitamins B and K, short-chain fatty acids, and antimicrobial peptides.

[0140] microorganisms It is anticipated that the particles and methods of the present invention will be useful in providing improved stability and / or shelf life to a variety of microorganisms.

[0141] In one embodiment, the microorganism is a probiotic microorganism. In a preferred embodiment, the microorganism is a probiotic bacterium. In an alternative embodiment, the microorganism is a fungus, such as a yeast. In one embodiment, the microorganism is in a viable form for reproduction.

[0142] The term "probiotic" refers to a microorganism that possesses probiotic activity. The term "probiotic activity" refers to the ability of a particular microorganism to stimulate the immune system. Measurement of the type and activity level 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 assessed by a PBMC cytokine secretion assay.

[0143] In one embodiment, the microorganism is a lactic acid bacterium (LAB). In one embodiment, the microorganism is 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, Lacticaseibacillus casei (formerly Lactobacillus casei), Lacticaseibacillus paracasei (formerly Lactobacillus paracasei), Lacticaseibacillus rhamnosus (formerly Lactobacillus rhamnosus; e.g., L. rhamnosus HN001), Lactibactibacillus plantarum subsp. plantarum (formerly Lactobacillus plantarum), Lactobacillus acidophilus (e.g., Lactobacillus acidophilus (LAVRI-A1)), Lactobacillus delbrueckii, Lactobacillus gasseri, Lactococcus lactis, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis biovar diacetylactis, Leuconostoc pseudomesenteroides, Rigilactobacillus salivarius (formerly Lactobacillus salivarius), Rimosilactobacillus reuteri (formerly Lactobacillus reuteri); for example, L. reuteri ATCC 55730), Saccharomyces boulardii, Streptococcus thermophilus, or a combination of any two or more thereof.

[0144] Preferably, the microorganism is a biofilm-forming microorganism. The ability of a microorganism to form a biofilm can be assessed by means known in the art. For example, the microorganisms may be cultured on a glass fiber or activated carbon surface, and biofilm formation may be measured by their adhesion to the matrix as described in Example 4.

[0145] 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. Additional bioactive substances and / or microorganisms, such as a third and / or fourth microorganism, may also be included.

[0146] Combinations of microorganisms may be selected to have complementary properties. For example, a biofilm producer may be combined with a non-producer. In some embodiments, a microorganism that is a heavy biofilm producer may be combined with a microorganism that is a light biofilm producer. In some embodiments, a particle comprises a first microorganism and a second microorganism, where the biofilm is produced by the first microorganism.

[0147] The one or more microorganisms may also be combined with one or more non-microbial bioactive materials. For example, in some embodiments, the bioactive material comprises a first microorganism, and the particles further comprise a non-microbial bioactive material. Preferably, the non-microbial bioactive material is distinct from any component or product of the first microorganism.

[0148] In some embodiments, 10 to 5×10 12 CFU / g particles, e.g., 10 2 ~5×10 12 , 10 3 ~5×10 12 , 10 4 ~5×10 12 , 10 5 ~5×10 12 , 10 6 ~5×10 12 , 10 7 ~5×10 12 , 10 8 ~5×10 12 , 10 9 ~5×10 12 , 10 10 ~5×1012 、10 11 ~5×10 12 、10 12 ~5×10 12 、10~10 12 、10 2 ~10 12 、10 3 ~10 12 、10 4 ~10 12 、10 5 ~10 12 、10 6 ~10 12 、10 7 ~10 12 、10 8 ~10 12 、10 9 ~10 12 、10 10 ~10 12 、10 11 ~10 12 、10~10 11 、10 2 ~10 11 、10 3 ~10 11 、10 4 ~10 11 、10 5 ~10 11 、10 6 ~10 11 、10 7 ~10 11 、10 8 ~10 11 、10 9 ~10 11 、10 10 ~10 11 、10~10 10 、10 2 ~10 10 、10 3 ~10 10 、10 4 ~10 10 、10 5 ~10 10 、10 6 ~10 10 、10 7 ~10 10 、10 8 ~10 10 、10 9 ~10 10、10~10 9 、10 2 ~10 9 、10 3 ~10 9 、10 4 ~10 9 、10 5 ~10 9 、10 6 ~10 9 、10 7 ~10 9 、10 8 ~10 9 、10~10 8 、10 2 ~10 8 、10 3 ~10 8 、10 4 ~10 8 、10 5 ~10 8 、10 6 ~10 8 、10 7 ~10 8 、10~10 7 、10 2 ~10 7 、10 3 ~10 7 、10 4 ~10 7 、10 5 ~10 7 、10 6 ~10 7 、10~10 6 、10 2 ~10 6 、10 3 ~10 6 、10 4 ~10 6 、10 5 ~10 6 、10~10 5 、10 2 ~10 5 、10 3 ~10 5 、10 4 ~10 5 、10~10 4 、10 2 ~10 4 、10 3 ~10 4 、10~10 3 、102 ~10 3 , 10~10 2 The first microorganism and / or the second microorganism may be present in an amount such as CFU / g particles.

[0149] Biofilm The term "biofilm" refers to the extracellular material produced by and surrounding microorganisms.

[0150] Biofilms are produced by certain microorganisms, e.g., bacteria, in response to specific environmental conditions. The conditions required to stimulate biofilm production may vary between microorganisms, but in some species, biofilm production is triggered by stress. Such stresses include nutrient limitation, osmotic stress, desiccation, exposure to UV light, unfavorable pH or temperature, high pressure, or exposure to certain chemicals, including antimicrobial substances such as antibiotics, or compounds involved in quorum sensing.

[0151] Biofilms typically comprise a slimy outer cell layer surrounding microorganisms. The composition of a biofilm may vary depending on the species of microorganism that produced it. The major portion of a bacterial biofilm is typically a high molecular weight extracellular microbial polymer, such as exopolysaccharides (EPS). It is understood that the term "exopolysaccharide" or "EPS" is a general description that encompasses a variety of different compounds with different chemical and physical properties.

[0152] Biofilms provide many benefits to bacteria, including an enhanced ability to adhere to surfaces and enhanced resistance to stressors.

[0153] Without wishing to be bound by any theory, it is believed that the stability and / or shelf life of a bioactive agent can be improved by embedding the bioactive agent within a biofilm, where the biofilm is embedded within a matrix.

[0154] Biofilms can be detected by many methods known in the art, including Raman spectroscopy, scanning electron microscopy (SEM), and lectin staining. Some exemplary methods for detecting biofilms are provided in Examples 5, 6, and 8.

[0155] In some embodiments, biofilms are detected by Raman spectroscopy. Using Raman spectroscopy, hyperspectral maps can be created that provide structural information about the location of biofilm components within a scanned area of ​​a sample. For biofilm identification, this provides context for where bacterial cells reside and, if any, extracellular components are present around them, providing an indication of biofilm.

[0156] In one embodiment, the biofilm is 360, 430, and / or 530 cm -1 In some embodiments, the Raman spectroscopic data is characterized by peaks in a principal component analysis of the Raman spectroscopic data at, for example, 360, 430, and / or 530 cm -1 The presence of rhamnose is indicated by the presence of peaks in principal component analysis of Raman spectroscopic data at 360, 430, and / or 530 cm. -1 The peak in the principal component analysis of the Raman spectroscopy data at 1450 cm -1 At least 50% of the peak height of the lipid-related peak at, for example, 1450 cm -1 The peak height may be 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% of the peak height of the lipid-associated peak in the sample, although an effective range may 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%).

[0157] For example, the exopolysaccharide (EPS) produced by Bacillus rhamnosus HN001 contains the characteristic sugars glucose, galactose, and rhamnose, along with 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, identification of rhamnose, galactosamine, glucosamine, mannose, and fucose can provide strong evidence of the presence of a biofilm associated with L. rhamnosus HN001 within the particles of the invention. In some embodiments, the biofilm contains glucose, galactose, rhamnose, galactosamine, glucosamine, mannose, and / or fucose.

[0158] In some embodiments, biofilms can be detected by lectin staining. Lectin staining can be used to detect the presence of sugars present in L. rhamnosus HN001 EPS, such as glucose, galactose, rhamnose, galactosamine, glucosamine, mannose, and fucose. For example, biofilm formation can be detected by fluorescence microscopy using fluorescein-conjugated lectins. The present inventors have demonstrated that the presence of mannose, N-acetylglucosamine, and fucose provides strong evidence for the presence of L. rhamnosus HN001-associated biofilms within the particles of the present invention.

[0159] To detect biofilms by lectin staining, particles can be cut into sections approximately 2 mm thick. The cut sections are stained with 5 μl of 0.1 mg / ml fluorescein-tagged lectin dye, 1 μM DAPI, and 0.2% fast green. Different lectin dyes can be used to detect different sugars. In one embodiment, the lectin dye can be selected from concanavalin A, wheat germ agglutinin, and / or gorse agglutinin. Samples are stained for at least 15 minutes before imaging using an inverted confocal fluorescence microscope. A 407 nm laser is used to excite DAPI, a 488 nm laser is used to excite fluorescein-tagged lectins, and 561 nm excitation is used for fast green. The presence of bacterial cells embedded in biofilms can be determined by colocalization of cells stained with DAPI stain and biofilms stained with one or more lectins. Various methods for detecting biofilms by lectin staining are presented in Examples 6, 8, and 11.

[0160] In some embodiments, the biofilm is produced by a bioactive material. For example, in some embodiments, the bioactive material is a first microorganism, and the biofilm is produced by the first microorganism. In some embodiments, the first microorganism is embedded in a matrix, where it produces the biofilm. In other embodiments, the first microorganism is induced to produce a biofilm, which is then embedded in a matrix. The first microorganism may be induced to produce a biofilm by exposure to stress, such as, for example, nutrient limitation, osmotic stress, desiccation, exposure to UV light, unfavorable pH or temperature, high pressure, or exposure to certain chemicals, including, for example, antimicrobial agents such as antibiotics, or compounds involved in quorum sensing.

[0161] In some embodiments, the particle further comprises a second microorganism different from the first microorganism. In some embodiments, the second microorganism is a probiotic microorganism. For example, this can be useful if the first microorganism is included for the purpose of biofilm production and the second microorganism is included for the purpose of providing a benefit, such as a health benefit. Thus, in some embodiments, the particle comprises a first microorganism and a second microorganism, wherein the biofilm is produced by the first microorganism.

[0162] Food and beverage products In a second aspect, the present invention provides a food or beverage product comprising particles of the first aspect.

[0163] The term "food or beverage product" refers to any edible and / or drinkable product or ingredient. The term is intended to encompass products for human consumption and products for non-human consumption, such as, for example, pet food or livestock feed. The term is also intended to encompass products that must undergo further processing before reaching a consumable form, such as, for example, a beverage powder that is dissolved in a liquid to form a beverage. In some embodiments, it is understood that a food or beverage product is an ingredient that is incorporated into other products, such as a baking ingredient.

[0164] It is expected that the particles of the present invention will enable the production of food and / or beverage products with improved stability and / or shelf life.

[0165] In some embodiments, the food or beverage product includes a protein powder, a protein shake, a protein shot, a protein gel, or a sports nutrition formula, a UHT drink, a UHT smoothie, an infant formula, a follow-on formula, a growth formula, a pediatric formula, a human milk fortifier, a pediatric food or beverage, a maternal supplement, or a maternal nutrition formula, a gel such as a thermosetting gel, a sauce, a spread, a jam, a jelly, or honey, an acidic setting gel, a shelf stable yogurt, a stirred yogurt, a set yogurt, or a drinking yogurt, a neutral beverage, an acidic beverage, water, juice, milk , smoothies, shakes, shots, alcoholic beverages, soft drinks, kombucha, kefir or ready to mix powders, bars, balls, cakes, cookies, muffins or bakery products, medical foods, soups, desserts, puddings, custards, enteral formulas, senior or geriatric formulas, tablets, capsules or supplements, confectionery, gummies, candies, chocolates, fudge, truffles, chewing gum, frozen desserts, ice cream, flavorings, toppings or baking ingredients, cream, cheese or butter, or livestock feed or pet food.

[0166] In one embodiment, the food or beverage product comprises an alcoholic beverage. In some embodiments, the alcoholic beverage is beer, wine, spirits, or a ready-to-drink (RTD) beverage. In some embodiments, the food or beverage product comprises a low-alcohol or non-alcoholic beer or wine.

[0167] In one embodiment, the food or beverage product comprises an animal feed, which in some embodiments is a livestock feed, pet food, kibble, pet biscuit, pet treat, animal feed ingredient, livestock forage, livestock supplement, or drench.

[0168] In one embodiment, the food or beverage product comprises a bakery product, hi some embodiments, the bakery product is a bread, a bar, a ball, a cake, a cookie, or a muffin.

[0169] In one embodiment, the food or beverage product comprises a beverage. In some embodiments, the beverage is a protein shake, a protein shot, a UHT beverage, a UHT smoothie, a neutral beverage, an acidic beverage, water, juice, milk, a smoothie, a shake, a shot, an alcoholic beverage, a soft drink, a fermented beverage, kombucha, kefir, a ready-to-drink (RTD) beverage, a coffee beverage, tea, or a creamer. In some embodiments, the food or beverage product comprises a coffee capsule, instant coffee, a tea bag, or a coffee creamer.

[0170] In one embodiment, the food or beverage product comprises a breakfast food, which in some embodiments is cereal, oatmeal, porridge, spreads, jam, jelly, or honey.

[0171] In one embodiment, the food or beverage product comprises a confectionery, which in some embodiments is a hard candy, a candy, a chewing gum, a chocolate bar, a chocolate-coated product, a candy bar, fudge, or a truffle.

[0172] In one embodiment, the food or beverage product includes a dairy product. In one embodiment, the dairy product is yogurt. In some embodiments, the yogurt is refrigerated yogurt, shelf-stable 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 dairy 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 heavy cream or UHT cream. In some embodiments, the dairy product is ice cream.

[0173] In one embodiment, the food or beverage product comprises a dessert, which in some embodiments is ice cream, sorbet, frozen yogurt, pudding, mousse, jelly, fruit puree, or dessert topping.

[0174] In some embodiments, the food or beverage product includes an infant formula, a follow-on formula, a growth formula, a pediatric formula, a human milk fortifier, a pediatric food or beverage, a maternal supplement, or a maternal nutritional formula. In some embodiments, the food or beverage product includes an infant formula powder, a pediatric milk powder, a wet mix infant formula, a baby food, a baby snack, baby rice, baby melting puffs, baby teething rusks, a fruit and / or vegetable puree.

[0175] In one embodiment, the food or beverage product is a food ingredient, hi some embodiments, the food ingredient is a spice, congee (rice porridge), foam cream, instant noodles, oil, pasta, rice, salad dressing, or seasoning.

[0176] 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, a meal replacement shake, a health drink, a soup, a pudding, a nutritional powder, a liquid diet, or a dietary supplement. In some embodiments, the medical food or beverage comprises encapsulated probiotics and / or antibiotics.

[0177] In one embodiment, the food or beverage product comprises a powder, hi some embodiments, the powder is a protein powder, a ready-to-mix beverage powder, or a meal replacement powder.

[0178] In one embodiment, the food or beverage product comprises a snack, which in some embodiments is a baked good, a cheese lollipop, a chip, a bar, a cookie, a dip, dried fruit, nuts, a ball, fruit leather, a high protein baked good, yogurt drops, a grain mix, popcorn, potato chips, a smoothie bar, or veggie chips.

[0179] In some embodiments, the food or beverage product has a long shelf life when stored at ambient temperature. For example, the food or beverage product may have a shelf life of at least 1 month, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months when stored at ambient temperature. The long shelf life of a food or beverage product may be achieved by many techniques known in the art, such as, for example, ultra-high temperature processing (UHT). In one embodiment, the food or beverage product is a UHT food or beverage product.

[0180] In some embodiments, the food or beverage product has a high water activity (a w). The particles described herein are believed to be 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.

[0181] In some embodiments, the biologically active substance 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.

[0182] In embodiments where the biologically active agent is a microorganism, the rate of decomposition 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 over a 12 month period.

[0183] In some embodiments, when the bioactive agent is a microorganism, the food or beverage product may be formulated to allow for the administration of a sufficient amount of the microorganism when ingested to establish a population in the gastrointestinal tract of a subject. The established population may be a transient population or a persistent population.

[0184] Theoretically, one colony-forming unit (cfu) should be sufficient to establish a population of microorganisms in a subject, but in practical situations, a minimum number of units is required to do so. Therefore, for therapeutic mechanisms that rely on a viable, living population of microorganisms, the number of units administered to a subject affects the efficacy of the treatment.

[0185] For example, when the bioactive substance is L. rhamnosus HN001, 6 × 10 per day 9The dose rate of cfu is sufficient (although may not be necessary) to establish a population in the gastrointestinal tract of a human subject. Thus, in one embodiment, the food or beverage product contains at least 6 x 10 cfu per day. 9 cfu of L. rhamnosus HN001.

[0186] Methods for determining the presence of a population of gut flora, such as, for example, L. rhamnosus HN001, in the gastrointestinal tract of a subject are well known in the art. In certain embodiments, the presence of a microbial population, such as, for example, L. rhamnosus HN001, can be determined directly, for example, by analyzing one or more samples obtained from the subject and determining the presence or amount of the microorganism in the sample. In other embodiments, the presence of a microbial population can be determined indirectly, for example, by observing a health effect or a reduction in the number of other gut flora in samples obtained from the subject. Combinations of such methods are also contemplated.

[0187] The method for calculating the appropriate dosage may depend on the nature of the biologically active substance in the food or beverage product. For example, when the product contains live microorganisms, the dosage may be calculated in terms of the number of live microorganisms present. For example, the dosage may be established by reference to the number of colony-forming units (cfu) to be administered per day. In instances where the composition includes one or more derivatives of a microorganism, the dosage may be calculated by reference to the amount or concentration of the microbial derivative present. For example, for a composition including a microbial cell lysate, the dosage may be calculated by reference to the concentration of the cell lysate present in the composition.

[0188] A typical example is approximately 1 x 10 per kg of body weight per day. 6 cfu ~ approx. 1 x 10 12 cfu, preferably about 1 x 10 6 cfu ~ approx. 1 x 10 11 cfu / kg / day, approximately 1 × 10 6 cfu ~ approx. 1 x 10 10 cfu / kg / day, approximately 1 × 10 6 cfu ~ approx. 1 x 109 cfu / kg / day, approximately 1 × 10 6 cfu ~ approx. 1 x 10 8 cfu / kg / day, approximately 1 × 10 6 cfu ~ approx. 5 × 10 7 cfu / kg / day, or approximately 1 × 10 6 cfu ~ approx. 1 x 10 7 Administration of about 5 x 10 cfu / kg / day of microorganisms per kg body weight per day is contemplated. 6 cfu ~ approx. 5 × 10 8 cfu, preferably about 5 x 10 6 cfu ~ approx. 4 × 10 8 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 3 × 10 8 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 2 x 10 8 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 1 x 10 8 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 9 × 10 7 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 8 × 10 7 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 7 x 10 7 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 6 × 10 7 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 5 × 10 7 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 4 × 10 7 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 3 × 10 7 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 2 x 10 7 cfu / kg / day, or approximately 5 × 10 6 cfu ~ approx. 1 x 10 7 Dosing of cfu / kg / day is contemplated.

[0189] In certain embodiments, the regular dosage does not need to be altered based on the subject's weight or other characteristics. In such an example, the daily dosage may be about 1 x 10 6 cfu ~ approx. 1 x 10 13 cfu, preferably about 1 x 10 6 cfu ~ approx. 1 x 10 12 cfu / day, approximately 1 × 10 6 cfu ~ approx. 1 x 10 11 cfu / day, approximately 1 × 10 6 cfu ~ approx. 1 x 10 10 cfu / day, approximately 1 × 10 6 cfu ~ approx. 1 x 10 9 cfu / day, approximately 1 × 10 6 cfu ~ approx. 1 x 10 8 cfu / day, approximately 1 × 10 6 cfu ~ approx. 5 × 10 7 cfu / day, or approximately 1 x 10 6 cfu ~ approx. 1 x 10 7 Administration of about 5 x 10 cfu / day of the microorganism is contemplated. Preferably, about 5 x 10 cfu / day 7 cfu ~ approx. 5 × 10 10 cfu, preferably about 5 x 10 7 cfu ~ approx. 3 × 10 10 cfu / day, approximately 5 × 10 7 cfu ~ approx. 2 x 10 10 cfu / day, approximately 5 × 10 7 cfu ~ approx. 1 x 10 10 cfu / day, approximately 5 × 10 7 cfu ~ approx. 9 × 10 9 cfu / day, approximately 5 × 10 7 cfu ~ approx. 8 × 10 9 cfu / day, approximately 5 × 10 7 cfu ~ approx. 7 x 10 9 cfu / day, approximately 5 × 10 7 cfu ~ approx. 4 × 10 10 cfu / day, approximately 5 × 10 7 cfu ~ approx. 6 × 10 9 cfu / day, approximately 5 × 10 7 cfu ~ approx. 5 × 10 9 cfu / day, approximately 5 × 10 7 cfu ~ approx. 4 × 10 9 cfu / day, approximately 5 × 10 7 cfu ~ approx. 3 × 109 cfu / day, approximately 5 × 10 7 cfu ~ approx. 2 x 10 9 cfu / day, or approximately 5 × 10 7 cfu ~ approx. 1 x 10 9 Dosing of the microorganisms at cfu / day is contemplated.

[0190] For example, in one embodiment, an effective dose of L. rhamnosus HN001 is 6×10 per day 9 It can be cfu.

[0191] It is understood that food or beverage products are preferably formulated to administer an effective dosage of the bioactive substance. The dosage, duration of administration, and general dosing regimen of the administered composition may vary between subjects depending on variables such as the bioactive substance used, the severity of the subject's symptoms, the type of disorder being treated, the method of administration selected, and the subject's age, sex, and / or general health. Furthermore, as noted above, the appropriate dosage may depend on the nature of the bioactive substance in the food or beverage product and the manner of formulation. For example, when the composition contains live microorganisms, the dosage may be calculated in terms of the number of live microorganisms present. For example, as described in the Examples herein, the dosage may be established by reference to the number of colony-forming units (cfu) to be administered per day. In instances where a food or beverage product contains one or more microbial derivatives, the dosage may be calculated by reference to the amount or concentration of bacterial derivatives administered per day. For example, for compositions containing microbial cell lysates, the dosage may be calculated by reference to the concentration of microbial cell lysates present in the composition.

[0192] It is understood that preferred food or beverage products are formulated to provide an effective dosage in a convenient form and amount. In certain embodiments, for example, but not limited to, when the regular dosage does not need to be varied depending on the subject's weight or other characteristics, the product may be formulated for unit administration. It should be understood that administration may include a single daily dose or may include administration of multiple separately divided doses, as appropriate.

[0193] Personal Care Products In one aspect, the present invention provides a personal care product comprising particles according to the first aspect.

[0194] The term "personal care product" refers to any product, article, or preparation used for personal care purposes (such as toiletry purposes), including (but not limited to) products for cleaning or grooming 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, cosmetics, beauty products, and women's health products.

[0195] In one embodiment, the personal care product is a skin care product such as, for example, a skin cream, a sunscreen, a hair care product such as, for example, a shampoo or a conditioner, a dental product such as, for example, a toothpaste or a dentifrice, a deodorant, a cosmetic, a beauty product, or a women's health product.

[0196] In some embodiments, the personal care product is a skin care product. A skin care product is a product that is applied topically to an area of ​​the 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.

[0197] In some embodiments, the personal care product is a hair care product such as, for example, a shampoo, conditioner, hair oil, hair styling gel, hair serum, hair wax, hair clay, pomade, hair mousse, dry powder shampoo, or hair volumizer.

[0198] In some embodiments, the personal care product is a dental product such as, for example, a toothpaste or a dentifrice, a dental gel, a dental chew, a mouthwash, or a mouthwash.

[0199] In some embodiments, the personal care product is a deodorant, such as, for example, a deodorant stick, a roll-on deodorant, or an antiperspirant. In some embodiments, the personal care product is a women's health product.

[0200] cleaning products In one aspect, the present invention provides a cleaning product comprising particles according to the first aspect. In some embodiments, the cleaning product includes a cleaning spray, a surfactant, or a stabilizer.

[0201] Particle manufacturing method Various methods of making the particles of the present invention are envisioned. In some embodiments, the method comprises the following steps: a. contacting a bioactive material with a matrix to form a mixture; and b. forming the mixture into particles; Includes.

[0202] One exemplary method is described below, however, various modifications will be readily apparent to those skilled in the art without departing from the scope of the invention. In some embodiments, to prevent contamination, the particles are prepared under sanitary and / or sterile conditions, such as, for example, using a Biological Safety Cabinet.

[0203] In one embodiment, when the matrix comprises two or more components, the matrix components are combined. Combining can be accomplished by any suitable means known in the art, such as by mixing or blending. In some embodiments, the matrix comprises one or more dry components, which are dry-blended together. In some embodiments, one or more dry components (or dry-blended components) are combined with one or more liquid components. Such liquid components may include components (e.g., one or more lipid components) that have been heated to a temperature sufficient to liquefy them.

[0204] In one embodiment, one or more matrix components are heated to a temperature and for a time sufficient to melt them. The temperature and time required depend on the composition of the matrix component(s). For example, in some embodiments, the matrix component(s) are heated to 65° C. for 30 minutes to melt them. Heating may be by any suitable means known in the art, for example using a hot water or oil bath.

[0205] The heating step may be followed by a tempering step at a lower temperature for a time sufficient for the matrix material to equilibrate at the lower temperature. For example, the components may be maintained at 45°C for a minimum of 30 minutes. The tempering step reduces the temperature of the matrix material to prevent thermal damage to the bioactive material if the bioactive material is susceptible to thermal damage. For example, many microorganisms, such as probiotics, are known to be susceptible to thermal damage, and therefore, a tempering step would typically be used in these cases. For bioactive materials that are less susceptible to thermal damage, a tempering step is not necessary.

[0206] The bioactive agent(s) are then added to the (optionally tempered) liquid matrix component and optionally stirred until homogeneous. Any additional ingredients may also be added at this stage.

[0207] The resulting mixture containing the bioactive agent(s) is then formed into particles and the temperature is reduced to allow the mixture to solidify. The production of particles may be by any suitable means known in the art, such as spray chilling or pelletization. In some embodiments, the particles are produced by pelletization. In other embodiments, the particles are produced by spray chilling. In some embodiments, the mixture is cooled to a temperature below 24°C, such as 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, although a useful range may be selected between any of these values ​​(e.g., 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).

[0208] 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 sterile, sealed container and allowed to cool until the mixture solidifies. For example, the mold can be allowed to cool to 4° C. for 12 hours.

[0209] The particles are optionally washed and / or filtered by size. The particles may optionally be coated with one or more coating layers, which may be applied by techniques known in the art, for example by spray coating. The particles may optionally be dried, for example, by fluidized bed drying, freeze drying, spray drying, or vacuum drying.

[0210] The particles may be incorporated into a product (e.g., a food or beverage product), or they may be stored for future use. Storage may occur at ambient temperature and humidity, or alternatively, under controlled temperature and / or humidity. For example, the particles may be stored refrigerated (e.g., at about 4°C) or frozen (e.g., below 0°C), and / or stored in a sealed container, optionally in a gas-flushed sealed container.

[0211] Method for enhancing stability of biologically active substances - Patents.com In a fifth aspect, the present invention provides a method for enhancing the stability of a biologically active substance, the method comprising the steps of: a. embedding the bioactive substance into the biofilm; b. embedding the biofilm in a matrix; and c. forming particles in the matrix; The method includes:

[0212] In a sixth aspect, the present invention provides a method for enhancing the stability of a biologically active substance, the method comprising the steps of: a. embedding the bioactive material in a matrix; b. the bioactive agent induces biofilm production; and c. forming particles in the matrix; Including, wherein steps b) and c) may be in any order.

[0213] In one embodiment, the bioactive material comprises a first microorganism. In one embodiment, the biofilm is produced by the first microorganism. In some embodiments, the first microorganism is a microorganism described herein. In some embodiments, the method further comprises coating the particle with at least one coating layer, hi some embodiments, the coating layer is a coating layer described herein. In some embodiments, the particles produced in step (c) are particles according to the first aspect.

[0214] In some embodiments, the method comprises the following steps: d. combining the particles of step c with a medium having a water activity of at least 0.5, preferably UHT treated yogurt; and e. Incubating the medium for at least two weeks; Further includes:

[0215] Without wishing to be bound by any theory, it is believed that incubating the particles of step (c) in a medium, such as UHT yogurt, allows for controlled influx of water and / or other compounds present in the medium, such as lactose, into the particles, which may stimulate the production of a biofilm.

[0216] Method for Producing Food or Beverage Products Supplemented with Bioactive Substances - Patent application In a further aspect, the present invention provides a method for producing a food or drink product supplemented with a biologically active substance, comprising the method of the fifth or sixth aspect and comprising the steps of: f. collecting the particles from the medium of step e, and g. combining the collected particles with a food or beverage product, thereby obtaining a food or beverage product supplemented with the bioactive substance; The method further comprises:

[0217] In another aspect, the present invention provides a method of producing a food or beverage product supplemented with a biologically active substance, the method comprising the method of the fifth or sixth aspect and further comprising combining the particles with a food or beverage product, thereby obtaining a food or beverage product supplemented with a biologically active substance.

[0218] In some embodiments, the food or beverage product supplemented with a bioactive agent is a food or beverage product according to the second aspect.

[0219] References Articles, patents, and patent applications mentioned or cited herein; and The contents of all other documents and electronically available information 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 physical and electronic documents.

[0220] The following examples illustrate the invention. [Example]

[0221] 1. Example 1 - Coating matrix for bioactive materials 1.1 Materials and Methods Various vegetable oils, individually and in combination, with and without dietary supplements, were tested for their ability to provide a protective barrier between a probiotic (Lacticaseibacillus rhamnosus HN001) and a fermented dairy product.

[0222] Particles (in the form of beads) were prepared containing a probiotic ingredient (L. rhamnosus HN001) embedded in a matrix containing lipids and / or lipid combinations and auxiliary ingredients. The probiotic ingredient used was a freeze-dried commercial sample.

[0223] Probiotic matrix beads were prepared by first melting the lipid component at 65°C for 30 minutes, followed by tempering at 45°C for a minimum of 30 minutes. The tempering step was to prevent heat damage to the probiotic. To prevent contamination, the particles were prepared in a Biological Safety Cabinet. The probiotic bead component was added to the tempered lipid component and mixed until homogeneous. The dry ingredients were combined by stirring. The resulting probiotic-containing mixture was poured into a pre-sterilized mold, removing all excess material using a sterile spatula. The mold was kept in a sterilized, sealed container and allowed to cool to 4°C for 12 hours. The beads were then removed from the mold and stored in a nitrogen-flushed, heat-sealed bag at 18°C.

[0224] Probiotic-containing particles were prepared in the form of beads (approximately 3-5 mm in diameter as measured by microscopy) using the fats and fat mixtures listed in Table 1. The fat formed a continuous layer throughout the bead. All matrices further contained the following adjunct ingredients (typically 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.

[0225] Table 1. Lipid composition in the coating matrix [Table 1]

[0226] Protective cells were added to thick, solid UHT yogurt formulated for shelf life and stored at 30°C for 12 months to determine their shelf life. Samples were taken at the beginning of the study and monthly and analyzed for HN001 probiotic cell count. Cell degradation rates were measured for each composition. Final samples were taken at 12 months, and degradation rates were calculated for probiotics using each coating matrix.

[0227] 1.2 Results Some particles were more structurally robust than others, depending on the composition; however, most survived the 12-month incubation period in the yogurt matrix. Particles containing the highest 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), and then hydrogenated coconut oil (data not shown).

[0228] A surprising observation was that within the first month of storage, all of the probiotics in the bead particles demonstrated a growth phase followed by the expected degradation. Without wishing to be bound by any theory, we believe this is due to the semi-permeable nature of the beads, which allows some amount of water, lactose, and other nutrients to enter the particle and provide a carbon source for limited growth followed by physiological regulation and quiescence. Since it seems reasonable to assume that the growth and physiological regulation components are separate processes, analysis of degradation and degradation rates within the bead particles was measured one month into the shelf life.

[0229] Some probiotic cells were also observed to be released from the bead particles; however, the majority of the cells remained embedded in the beads.

[0230] The degradation rates for each coating matrix are shown in Table 2. Degradation rates are expressed as log reductions in CFU per gram per year. In other words, the degradation rates measured over 6 and 9 month periods were extended to 1 year for clarity in the table.

[0231] Table 2. Cumulative degradation rates of probiotics encapsulated within a coating matrix (*rates calculated at 9 months; **at 6 months) [Table 2]

[0232] conclusion This example shows that the coating matrix reduces the degradation of probiotic microorganisms in fermented dairy products, such as yogurt, even when stored for extended periods at or above ambient temperatures.

[0233] 2. Example 2 - Additional Coating Matrices for Bioactive Agents Supplementary experiments were performed to test coatings made with fat alone, the effect of lecithin, and the inclusion of an uncoated negative control.

[0234] 2.1 Materials and Methods Probiotic particles were prepared and added to yogurt as described in Example 1, and probiotic survival was monitored over a 12 month period.

[0235] The samples used are: 1. Uncoated lyophilized culture (negative control) 2. 99.5% cocoa butter, 0.5% lecithin 3. 100% Cocoa Butter 4. 99.5% hydrogenated palm kernel oil, 0.5% lecithin 5. 100% hydrogenated palm kernel oil 6. 32% hydrogenated palm kernel oil, and 7. 16% hydrogenated palm kernel oil, 16% cocoa powder, It was.

[0236] 2.2 Results The degradation rate for each coating is shown in Table 3. Degradation rates are expressed as log reductions in CFU per gram per year. Degradation rates measured at 6 and 9 months were extended to 1 year for clarity in the table.

[0237] Table 3. Degradation rate [Table 3]

[0238] The uncoated freeze-dried culture (negative control) did not survive when incubated in yogurt at 30° C. The addition of cocoa powder did not appear to contribute to protecting the probiotics against degradation.

[0239] Interestingly, cocoa butter demonstrated low decay up to six months, followed by degradation at a substantially faster rate after that period.

[0240] Hydrogenated palm kernel oil in combination with adjuvants (at 16% and 32% lipid) demonstrated significantly reduced degradation rates over 12 months of storage (1.9 and 1.8 Log cfu / g / year, respectively).

[0241] Contour plots of the mixtures were used to further visualize the effect of lipids, individually and in different combinations, on probiotic stability. For matrices containing palm stearin, fully hydrogenated palm kernel oil, and fully hydrogenated coconut oil (individually and in combination), the lowest kill was observed when fully hydrogenated and coconut oil were added at the highest levels (Figure 1). Similar results were observed for the combination of palm stearin, fully hydrogenated coconut oil, and cocoa powder (Figure 2).

[0242] The highest mortality was observed when cocoa powder was added at the highest level (Figures 2 and 3), which could 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.

[0243] 2.3 Conclusion This example shows that a coating matrix comprising 100% lipid (i.e., 100% hydrogenated palm kernel oil) reduces the degradation of probiotic microorganisms in a fermented milk product when used alone as a coating matrix. Degradation was further reduced by including the adjuvants described in Example 1. This example also shows that matrices containing fully hydrogenated coconut oil and / or fully hydrogenated palm kernel oil provide very low mortality, palm stearin provides low mortality, and cocoa powder is not very effective.

[0244] 3. Example 3 - Electron Microscopy 3.1 Materials and Methods Bead particles containing a probiotic ingredient (Lacticaseibacillus rhamnosus HN001) and hydrogenated palm kernel oil were prepared and added to yogurt as described in Example 1. In this example, beads were removed after 9 months for electron microscopy analysis.

[0245] The beads were frozen to -20°C and crushed using a razor blade. The beads were embedded with the crushed side facing up, mounted in a scanning electron microscope (SEM TM4000), and cooled to -30°C while the vacuum in the chamber was pumped down. The sample was then ready for observation.

[0246] 3.2 Results A clear phase separation between the yogurt and fat portions of the composition can be seen, as well as (at higher magnification) clear clusters of probiotics that appear embedded in a mucoid coating—either in combination with exopolysaccharides or biofilm material (Figure 4).

[0247] Figure 5 shows a clear phase separation between yogurt and hydrogenated palm kernel oil (HPKO). Clusters of probiotics in the biofilm are demarcated by red borders.

[0248] In Figure 6, the mucoid exopolysaccharide / biofilm network is clearly visible, and in Figure 7, individual rod-shaped bacterial cells are found enmeshed within the exopolysaccharide / biofilm.

[0249] 3.3 Conclusion This example demonstrates that probiotic bacteria encapsulated in a coating matrix are capable of developing a mucoid exopolysaccharide or biofilm layer once embedded in the matrix material.

[0250] 4. Example 4 - Biofilm Formation 4.1 Materials and Methods Bacillus lactis rhamnosus HN001 was demonstrated to produce biofilms by growth and attachment to glass wool or activated carbon surfaces. HN001 cultures were prepared by overnight incubation at 37°C in MRSB medium. The culture (1 ml) was then inoculated into a 500 ml flask containing 100 ml of yogurt. Glass wool (2.5 g) was added to the flask, which was then incubated at 37°C for 18–24 hours in a rotary shaking incubator. The incubated flasks were then stored at 30°C and evaluated for cell count, pH, and EPS or biofilm formation at 4, 7, and 28 days. The same experiment was initiated using 1 g of 0.6–1.1 mm washed granular activated carbon per flask.

[0251] Samples were removed for enumeration of viable probiotic cells at the beginning of storage and at 7 and 28 days. The glass wool (or activated charcoal) was removed and rinsed three times through a mini-sieve with phosphate-buffered water. The rinsed material was placed in a 250 mL Schott bottle containing 20 g of sterile glass beads. Salt-buffered peptone water (9 ml) was added, and the beads and culture were vigorously agitated for 10 minutes to detach the cells from the glass wool or charcoal.

[0252] Serial dilutions were made using 9 ml of salt-buffered peptone water and plated on MRSA agar, followed by incubation at 37°C for 48 hours, after which cells were counted. Cell counts were also made on the remaining yogurt (planktonic or free cells).

[0253] 4.2 Results Table 4 demonstrates that many live HN001 cells remained attached to the glass wool or charcoal surface over the 28-day shelf life and maintained relatively higher levels of viability compared to free cells in yogurt.

[0254] Table 4. Comparison of cell number and pH between cells that remained unattached (planktonic cells) and cells that attached to glass or charcoal surfaces. [Table 4]

[0255] Samples attached to the glass wool were studied using light microscopy under oil immersion at 100x magnification after staining with crystal violet (Figure 8). Clusters of HN001 cells were visible and attached to the surface of the glass wool strands. These attached clusters provide evidence of the ability of HN001 to form a biofilm and sustain cell viability in yogurt.

[0256] 4.3 Conclusion This example demonstrates that Bacillus rhamnosus HN001 can form biofilms and, by doing so, improves cell viability when stored in yogurt for extended periods of time.

[0257] 5. Example 5 - Identification of Biofilm Exopolysaccharide Components Using Raman Spectroscopy Exopolysaccharides (EPS) constitute the main part of the extracellular biofilm matrix and are responsible for anchoring microorganisms to surfaces, a process that initiates biofilm formation.

[0258] The characteristic sugars of EPS produced by Bacillus rhamnosus HN001 EPS are glucose, galactose, and rhamnose, along with traces of galactosamine, glucosamine, mannose, and fucose. Because residual glucose and galactose are likely to be found in yogurt, the identification of rhamnose, galactosamine, glucosamine, mannose, and fucose provides strong evidence for the presence of an HN001-associated biofilm.

[0259] 5.1 Materials and Methods Beads containing L. rhamnosus HN001 were prepared according to Example 1.

[0260] Beads from sample AT03 (see Example 1) were removed from the yogurt and gently rolled on a paper towel to remove excess yogurt. Bead sections were cut to a thickness of approximately 2 mm. The sections were mounted on slides and covered with a coverslip; double-sided tape was used to hold the coverslip down and served as a ~2 mm spacer. The sample was maintained at a temperature of 4°C throughout the measurements. 50 x 10 steps, 75 x 25 μm Raman maps were collected. Each spectrum was collected using a 1 second integration time, a 532 nm excitation wavelength, a 100x oil immersion objective (NA 1.30), and a 50 μm pinhole. At least 10 maps per bead were collected at each time point: 0, 15, 30, 90 / 120, and 218 days.

[0261] As a control, a sample of L. rhamnosus HN001 was cultured using shelf-stable yogurt as the medium and a zinc selenide surface to induce biofilm production.

[0262] 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 removal, baseline setting, and min-max normalization. Raman spectral maps were analyzed using principal component analysis, multivariate curve resolution, and comparison with standards.

[0263] 5.2 Results Principal component analysis of Raman spectroscopic data for L. rhamnosus HN001 cultured using shelf-stable yogurt as a medium and a zinc selenide surface to induce biofilm production revealed a peak at 350–600 cm for the presence of rhamnose. -1 Three distinct peaks in the spectral region were revealed (Figure 9), which may indicate the presence of EPS components.

[0264] Raman spectroscopy of sample AT03 bead structures incorporating L. rhamnosus HN001 revealed similar peaks (Figure 10), while three EPS-related peaks were observed at 360, 430, and 530 cm. -1 appeared in.

[0265] 5.3 Conclusion This example demonstrates that Raman spectroscopy can be used to detect the presence of EPS within probiotic-containing particles.

[0266] 6. Example 6 - Identification of biofilm exopolysaccharides by lectin staining 6.1 Materials and Methods L. rhamnosus HN001-containing beads were prepared according to Example 1.

[0267] The beads were removed from the yogurt and gently rolled on a paper towel to remove excess yogurt. Bead sections were cut to approximately 2 mm thickness. The cut sections were stained with 5 μl of 0.1 mg / ml fluorescein-tagged lectin stain, 1 μM DAPI, and 0.2% fast green. The lectin stains were concanavalin A, wheat germ agglutinin, and gorse agglutinin. Samples were allowed to stain 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 the fluorescein-tagged lectin, and 561 nm excitation was used for fast green.

[0268] 6.2 Results Figures 11-13 show fluorescence microscopy of beads stained with different lectins. The figures are of a single field recorded using a 63x oil immersion objective. One bead was split to stain separate sections with different staining mixtures.

[0269] Lectin staining showed the presence of sugars (glucose, galactose, rhamnose, galactosamine, glucosamine, mannose, and fucose) consistent with the structure of L. rhamnosus HN001 EPS. The presence of mannose (Figure 11), N-acetylglucosamine (Figure 12), and fucose (Figure 13) provided strong evidence for the presence of an HN001-associated biofilm within the bead particles.

[0270] 6.3 Conclusion This example demonstrates that lectin staining and fluorescence microscopy can be used to detect the presence of biofilm within particles.

[0271] 7. Example 7 - Additional Coating Matrices 7.1 Materials and Methods Beads containing Bacillus rhamnosus HN001 were prepared as described in Example 1 using the matrix composition shown in Table 5.

[0272] Table 5. Matrix composition (% w / w) [Table 5] 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 processed skim milk powder); Lac, lactose.

[0273] The beads were added to a lower viscosity UHT yogurt (than the thick set yogurt used in Example 1) and incubated at 25°C or 30°C for 12 months, with samples removed at various time points for cell count analysis.

[0274] 7.2 Results Incubation at 30°C The cell numbers of L. rhamnosus HN001 after incubation at 30°C are shown in Table 6.

[0275] Table 6. Cell counts (log CFU / g particles) of L. rhamnosus HN001 after incubation at 30°C. [Table 6]

[0276] Incubation at 25°C The cell numbers of L. rhamnosus HN001 after incubation at 25°C are shown in Table 7.

[0277] Table 7. Cell counts (log CFU / g particles) of L. rhamnosus HN001 after incubation at 25°C. [Table 7]

[0278] 7.3 Conclusion This example shows that several formulations provide significant stability and that different coating matrices can reduce the degradation of probiotic microorganisms in fermented dairy products.

[0279] 8. Example 8 - Biofilm formation by lectin staining 8.1 Materials and Methods L. rhamnosus HN001-containing beads were prepared according to Example 1 using the matrix composition described in Table 8 with 2.5% HN001.

[0280] Table 8. Matrix composition [Table 8]

[0281] The beads were incubated at 30°C in thick, solid UHT yogurt as described in Example 1 and samples were removed at various time points for analysis. The probiotic-containing beads were removed from the yogurt and the excess yogurt was removed. The particles from time point zero were used before being added to the yogurt.

[0282] Lectins conjugated to fluorophores were used to stain lipid-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.

[0283] Three lectins were selected for use on the beads: wheat germ agglutinin (WGA), concanavalin A (ConA), and gorse 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 were used in combination with DAPI to stain cells, Nile red to stain fat, or fast green to stain proteins.

[0284] The beads were cut to expose the interior surface and a mixture of fluorescent dyes was applied as shown in Table 9. At most 10 μl of the fluorescent dye mixture was applied to the exposed surface of the bead and the bead was allowed to contact the dye for at least 15 minutes and no more than 30 minutes before imaging.

[0285] Table 9. Dye mixture [Table 9]

[0286] Confocal fluorescence images from the stained beads were collected using an inverted Zeiss LSM 800 microscope equipped with a 63x oil immersion objective. The excitation wavelengths and emission collection ranges are listed in Table 10.

[0287] Table 10. Excitation and emission wavelengths [Table 10]

[0288] 8.2 Results AT07 At time zero, differential fluorescence of WGA was observed, indicating the presence of N-acetylglucosamine in the initial composition. WGA bonds were primarily present around the edges of the protein particles. Because cow's milk contains N-acetylglucosamine, this bond may be the result of the incorporation of milk solids into the particles (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).

[0289] After 3 months of incubation, there were clear areas of concentrated WGA fluorescence, and these areas were no longer located at the edges of protein bodies, but instead within cell clusters. At time zero, areas of intense ConA fluorescence were observed that did not correspond to the cell density of the area, which may also be a result of the milk solids included in this matrix composition. After 3 months of incubation, there was a significant increase in ConA fluorescence in areas, and these areas were associated with areas of higher cell density.

[0290] Data from time zero using the UEA-II lectin dye were not collected. After 3 months of incubation, distinct areas of high and low UEA-II fluorescence were present, and areas of high UEA-II fluorescence were largely associated with areas of high cell density. This increase in lectin fluorescence within areas related to cell clusters after incubation was consistent with biofilm formation.

[0291] AT24 No data were collected at time zero using ConA or UEA-II. At time zero, WGA stained the cells. After 3 and 6 months of incubation, WGA fluorescence was observed around the cells; this was most evident after 6 months. After 6 months, ConA fluorescence was evident in the cell-dense areas. Regarding UEA-II fluorescence, there was a small amount of aggregation of UEA-II at 3 and 6 months, however, it did not appear to be closely associated with the cell location. These observations are consistent with biofilm formation.

[0292] AT17-1 The lectin dye used to stain MVP9 has a fluorophore with an excitation maximum at 642 nm or 649 nm, allowing for co-staining with Nile Red, which stains lipids. There was one exception: for the 1-month time point, the UEA-II dye (with an excitation maximum of 495 nm) with a fluorescein fluorophore was used. Nile Red was not used when using this dye due to interference between the dyes; instead, Fast Green was added.

[0293] At time zero, small areas of bacteria were present within the fatty matrix, and WGA stained cells within these areas. After 1 month, more diffuse fluorescence was evident from WGA around the cellular areas, indicating the presence of N-acetylglucosamine extending beyond the pericellular area, consistent with the formation of a biofilm with extracellular polymeric substances containing N-acetylglucosamine.

[0294] At time zero, no differential fluorescence was observed when ConA was used; however, fluorescent regions were observed when the dye accumulated in the fat-free region. There were no areas of weak or strong ConA fluorescence within the cellular region. There were areas of ConA fluorescence that did not appear to be associated with cells. Thus, the dye accumulated within the fat-free region rather than specifically staining mannose or glucose. After one month, differential ConA fluorescence was observed; that is, the fluorescence was due to accumulation within the fat-free region. When WGA was used, irregular and large fluorescent regions associated with cells were observed, consistent with the excretion of extracellular polymeric substances and biofilm formation. Not all cellular regions were stained with ConA; at this time point, there were cellular regions where ConA had accumulated. Furthermore, at one month, areas of strong ConA aggregation were present within the region where cells surrounded the periphery and where ConA had accumulated within but also aggregated within the fat-free region.

[0295] At time zero, UEA-II stained cells within the beads in a manner similar to WGA. These cells were largely confined to cell clusters within the fat matrix. At 1 month, cells had colonized the outer regions of the fat-free areas of the particles, and UEA-II fluorescence was observed beyond the cells, consistent with biofilm formation.

[0296] ATX-1 Only 3-month samples were analyzed. After 3 months, there were areas of significant WGA fluorescence associated with areas of cell density. There were also areas of significant ConA fluorescence and small areas of UEA-II fluorescence associated with areas of cells. All of these observations are consistent with biofilm formation.

[0297] 8.3 Conclusion This example shows that biofilm formation can be detected by fluorescence microscopy using fluorescein-conjugated lectins, and that different matrix compositions are conducive to biofilm formation.

[0298] 9. Example 9 - Stability in various products 9.1 Materials and Methods Probiotic particles containing 0.25% Lacticase I Bacillus rhamnosus HN001 were prepared using the four matrix compositions listed in Table 11.

[0299] Table 11. Matrix composition [Table 11]

[0300] The dry ingredients (sugar, lactose) were weighed into stomacher bags, sealed with a bagger, and stored at room temperature until needed. The lipid ingredients (lipid and lecithin) were weighed into sterile bottles and stored at 3-4°C until needed. L. rhamnosus HN001 at 0.25g / 100g lipid was weighed into a sterile container and stored at 3-4°C until needed. The silicone molds were autoclaved.

[0301] The particles were prepared using the following method: 1. The lipid components were melted in a water bath at 60-70°C. 2. The lipid components were transferred to a 45°C water bath and the lipids were allowed to cool to 45°C (minimum 30 minutes) before adding the probiotics. 3. Probiotics (0.25g) were added to the melted lipid and stirred using a sterile spoon to ensure it was homogenous. 4. Slowly add the remaining dry ingredients, stirring after each addition. 5. Continue stirring until all ingredients are thoroughly combined and give a smooth appearance. 6. The contents were poured into sterile silicone molds. 7. Using a spatula, the mixture was smoothed into the mold cavity and the excess was scraped off. 8. The molds were placed in a pre-sterilized container and stored at 4°C for 24 hours.

[0302] The particles produced each had a diameter of about 5 mm and a weight of about 0.05 g. Five products were evaluated as described in Table 12. The viscosity of the products was measured using a Brookfield viscometer.

[0303] Table 12. Products evaluated [Table 12]

[0304] A 200ml sample of each product was used, and using sterile forceps, three particles were added to each sample, submerging the particles in the top 10-20ml. The samples were then incubated at 30°C.

[0305] At each sampling time point, three particles were removed and placed in a bell tube. 15 ml of MRSB medium pre-warmed to 45°C was added, and the sample was thawed in a 45°C water bath for 5 minutes. The sample was removed from the water bath and gently shaken or vortexed to mix.

[0306] Dilutions were prepared in duplicate. Appropriate sample dilutions (50 μl) were placed on pre-poured agar plates ("drop plate method"). Three dilutions, namely 10 -6 , 10 -7 , and 10 -8 was necessary to obtain a countable range.

[0307] The culture plates were incubated under anaerobic conditions at 37°C for 2-3 days, and the number of colonies was counted to determine the CFU / ml of the original sample.

[0308] 9.2 Results The number of viable L. rhamnosus HN001 at initial incubation and after 1, 2, 3, and 4 months of incubation are shown in Tables 13-17. The 3 and 4 month time points have not yet been tested on cream cheese spread.

[0309] Table 13. Shelf life of orange juice (Log 10 CFU / ml) [Table 13]

[0310] Table 14. Sports Shake Shelf Life (Log 10 CFU / ml) [Table 14]

[0311] Table 15. Shelf life of cheese sauce (Log 10 CFU / ml) [Table 15]

[0312] Table 16. Shelf life of cream cheese spread (Log 10 CFU / ml) [Table 16]

[0313] Table 17. Shelf life (Log) of prebiotic oat milk 10 CFU / ml) [Table 17]

[0314] 9.3 Conclusion This example demonstrates that the coating matrix reduces degradation of probiotic microorganisms in a variety of commercial products, even when stored at or above ambient temperatures for extended periods of time.

[0315] 10. Example 10 - Bacterial Strains 10.1 Materials and Methods Two sets of particles were prepared as described in Example 9, the first containing Bacillus rhamnosus HN001 and the second containing Bacillus paracasei subsp. paracasei IM514.

[0316] The matrix composition contained 86.5% w / w hydrogenated coconut oil (HCO) and 13.5% w / w lactose (formulation ATX-2). An additional cooling step of 1 h at 20°C was applied before adding the bacteria to the melted lipid component. The particles were added to shelf-stable formulated thick, solid UHT yogurt and stored at 30° C. Cell counts of each bacterial strain were monitored periodically.

[0317] 10.2 Results The cell counts of the bacterial strains are shown in Table 18.

[0318] Table 18. Shelf life (Log) of UHT yogurt 10 CFU / ml) [Table 18]

[0319] conclusion This example demonstrates that the particles can provide an extended shelf life under ambient conditions for a variety of bacterial strains.

[0320] 11. Example 11 - Lectin staining for biofilm formation 11.1 Materials and Methods Probiotic beads / particles were prepared using Lacticase Bacillus rhamnosus HN001 or Lacticase Bacillus rhamnosus GG (LGG) with the matrix composition shown in Table 19.

[0321] Table 19. Matrix composition [Table 19]

[0322] Ako Comp ingredients (with 10% probiotics) include: 26g Akosoft36 60g Akofine R 4g Polyglycerol Polyricinoleate (PGPR) 10g freeze-dried probiotic powder Total (100g)

[0323] Ako Comp ingredients (2.5% probiotics added) include: 28.2g Akosoft 36 65g Akofine R 4.3g Polyglycerol Polyricinoleate (PGPR) 2.5g freeze-dried probiotic powder Total (100g)

[0324] Akosoft 36 (hydrogenated palm glycerides) and Akofine R (hydrogenated vegetable oil) are available from IXOM (Melbourne, Australia).

[0325] Samples 1-3 were prepared as described in Example 8, and comparative samples 4-8 were prepared according to the following comparative methods: The fat ingredient(s) 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 pelleted and the pellets were crushed into a powder.

[0326] For all samples, the particles / powder were incubated in thick, solid UHT yogurt for 2 weeks at the temperatures indicated above, as described in Example 1. Particles were removed, cut in half, stained with dye mixtures 1-4, and visualized by confocal fluorescence microscopy as described in Example 8.

[0327] 11.2 Results Illustrative samples (Samples 1-3) For sample 1, areas of intense WGA, ConA, and UEA-I fluorescence were observed extending beyond the cells, consistent with the presence of a biofilm.

[0328] For sample 2, only small areas of intense WGA fluorescence were observed, but no areas of intense ConA fluorescence were observed. Areas of UEA-I fluorescence extending beyond the cells were observed, consistent with the presence of a biofilm.

[0329] For sample 3, strong WGA fluorescence was observed from the cells, and areas extending beyond the cells were also observed. No areas of strong ConA fluorescence were observed. Strong areas of UEA-I fluorescence extending beyond the cells were observed. The spread of WGA and UEA-I fluorescence beyond the cells is consistent with biofilm formation.

[0330] Overall, samples 1-3 showed evidence of biofilm formation with WGA and UEA-I fluorescence extending beyond the cell location defined by DAPI staining, and lectin staining was not just cell shape. ConA staining, consistent with biofilm presence, 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.

[0331] Control samples (samples 4-8) For sample 4, only small areas of strong WGA fluorescence were observed, but no ConA fluorescence was observed, and UEA-I fluorescence that was stronger than background pool levels was only observed in small areas around some cell clusters.

[0332] Due to sample limitations, sample 5 was not stained with Con A. Areas of strong WGA fluorescence were co-located with cells, and only small areas of strong UEA-I fluorescence were observed.

[0333] For sample 6, areas of strong WGA fluorescence were observed that extended across cells; however, not all cell clusters showed strong WGA fluorescence. No strong fluorescence from ConA was observed. Only a few cell regions appeared to be stained by UEA-I in sample 6.

[0334] For sample 7, WGA fluorescence was unexpectedly concentrated around the fat, not necessarily in the same location as the cells. ConA staining was mostly limited to pools in the fat-free region. Small areas of intense UEA-I fluorescence around the cells were observed.

[0335] Sample 8 was identical to Sample 7, except that AD sterilization was used instead of post-pasteurization. Without post-pasteurization, the distribution of WGA fluorescence was different. Strong WGA fluorescence was observed; however, this did not extend beyond the cells. No areas containing many tightly clustered cells were observed. ConA fluorescence was not observed. UEA-I showed variable staining intensity; however, it appeared to stain only the cells, not the extracellular polymeric substances.

[0336] Overall, Samples 4-8 did not show any areas of strong ConA staining, except for one very small area of ​​strong ConA fluorescence, but rather stained for yogurt encapsulation in the beads. Only a small area of ​​strong WGA and UEA fluorescence was observed, which did not extend beyond the cells or associate with any cell clusters in the matrix. Samples 4-8 did not show evidence of extensive biofilm formation, as seen in Samples 1-3.

[0337] 11.3 Conclusion This example demonstrates that beads formed using the matrix and process of the present invention: We showed that lectin staining extended beyond the cells, indicating extensive biofilm formation. In contrast, the comparative particles formed using the comparative process were in most cases lacking lectin staining that extended beyond the cells, suggesting that these particles do not contain the same extensive biofilm as the particles of the present invention.

[0338] 12. Example 12 - Water Vapor Transmission Rate (WVTR) 12.1 Materials and Methods Exemplary particles were prepared as described in Example 9. Comparative particles were also prepared using the comparative method described in Example 11.

[0339] WVTR was measured using an aliquot (20–60 mg) of particle sample at 95% relative humidity by weighing on a specific adsorption microbalance in a dynamic vapor sorption (DVS) apparatus (Surface Measurement Systems, Alperton, Middlesex, UK). WVTR was measured over a 5-minute period (i.e., from the 7th to the 12th minute) 7 minutes after adjusting the relative humidity to 95%.

[0340] WVTR is calculated using the following equation: WVTR=dm / (A dt) WVTR was calculated by using the formula: where dm = mass of water absorbed during time dt, and A = total surface area of ​​the sample calculated based on particle size measurements. WVTR is expressed in g / m 2 Reported in 24 hour increments.

[0341] Water activity was measured by Aqualab 4TE (Addium Inc, Pullman, WA, United States). The particle size of the powders (ranging from 10 to 1,000 microns) was measured using a binocular stereo microscope and subsequently analyzed using "Image pro plus."

[0342] 12.2 Results Water vapor transmission rate (WVTR) and water activity (Aw) were measured for various fat-based matrix compositions (Table 20), all of which contained 0.25% Lacticase Bacillus rhamnosus HN001.

[0343] Table 20. Particle surface area to volume ratio (SA / V), water vapor transmission rate (WVTR), and water activity [Table 20]

[0344] Comparative particles were prepared according to the comparative method described in Example 11 ("Comparative") and compared to particles of the present invention ("Invention").

[0345] Particles containing 2.5% L. rhamnosus HN001 were prepared using the method described in Example 9, and comparative particles containing 10% L. rhamnosus GG were prepared. Two matrix compositions were used - 100% cocoa butter and 100% Ako Comp. The medium particles had a diameter of approximately 5 mm, and the powder particles were crushed to a powder.

[0346] Particles prepared using the method of the present invention had significantly lower WVTR than particles prepared using the comparative method, even though they had similar surface area / volume ratios, regardless of whether medium-sized particles or powders were used (Table 21).

[0347] Table 21. Surface area to volume ratio (SA / V) and water vapor transmission rate (WVTR) of comparative particles [Table 21]

[0348] The WVTR was further compared by varying the probiotic and particle size used. Particles containing L. rhamnosus GG (LGG) had a higher WVTR than those containing L. rhamnosus HN001, while particles produced using the comparative method consistently had a higher WVTR than those produced using the method of the present invention, even though they were of different particle sizes (Table 22).

[0349] Table 22. Comparative Water Vapor Transmission Rates (WVTR) [Table 22]

[0350] 12.3 Conclusion This example shows the WVTR and water activity of particles of the invention and comparative particles.

Claims

1. Particles containing a bioactive substance embedded in a biofilm, wherein the particles have a particle size of 50 μm to 10 mm, the biofilm is embedded in a matrix, the matrix has a water vapor transmission rate (WVTR) of 0.1 to 500 g / m² / 24h, and the matrix contains at least 6% by weight of one or more lipids.

2. The particle according to claim 1, wherein the matrix has a water vapor transmission rate (WVTR) of 0.1 to 10 g / m² / 24h, preferably 1 to 5 g / m² / 24h.

3. The bioactive substance comprises a first microorganism, preferably a probiotic microorganism: a. The biofilm may optionally be produced by the first microorganism; b. Optionally, the first microorganism is a member of the genera Bacillus, Bifidobacterium, Enterococcus, Lacticaseibacillus, Lactiplantybacillus, Lactobacillus, Lactococcus, Ligylactobacillus, Limosilactobacillus, Lentilactobacillus, Saccharomyces, or Streptococcus; preferably, the first microorganism is Bacillus coagulans, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum, Lacticaseibacillus casei, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus , selected from the group consisting of Lactobacillus plantarum subspecies plantarum, Lactobacillus acidophilus, Lactobacillus delbruecki, Lactobacillus gasseri, Lactococcus lactis, Lactococcus lactis subspecies lactis, Lactococcus lactis subspecies diacetylactis, Leuconostoc pseudomecenteroides, Rigilactobacillus salivarius, Rimosilactobacillus reuteri, Saccharomyces blaudii, and Streptococcus thermophilus; more preferably, the first microorganism is Lactocaseibacillus rhamnosus 001; and / or c. The first microorganism is 10 ５ ~10 １２ CFU / g particles, preferably 10 ６ ~10 １０ , more preferably 10 ７ ~10 ９ Most preferably about 10 ８ It exists in the amount of CFU / g; The particle according to claim 1.

4. Furthermore: a. A second microorganism different from the first microorganism, preferably the second microorganism is a probiotic microorganism; and / or b. Non-microbial bioactive substances, preferably vitamins, prebiotics, postbiotics, and / or human milk oligosaccharides; The particles according to claim 3, including the particles described in claim 3.

5. The particles according to claim 1, wherein when the particles are encapsulated in another food product having a water activity of 0.90 to 1.00 for a period of two weeks, they maintain a water content of 1% to 60% by weight, preferably 10% to 60%, more preferably 20% to 60%, within the matrix.

6. The aforementioned one or more lipids meet the following conditions: a. It is solid at 20°C, 25°C, and / or 30°C; b. Having a melting temperature of less than 50°C, preferably between 30°C and 50°C; c. Containing or consisting of triglycerides; d. Containing C12-C20 fatty acids, or their salts or esters; e. One or more fatty acids and / or salts and / or esters thereof: i. At least 1% by weight, preferably 1% to 80% by weight of the fatty acid and / or its salt and / or its ester having a carbon chain length of 14 or less; and / or ii. At least 30% by weight, preferably at least 70% by weight, preferably 30% to 90% by weight, more preferably 70% to 90% by weight of the fatty acid and / or its salt and / or its ester having a carbon chain length of 16 or less; or f. Any combination of two or more of (a) to (e); The particle according to claim 1, satisfying the requirements.

7. The particles according to Claim 1: a. At least 50% by weight of the one or more lipids is saturated fat; b. The one or more lipids include, or consist of, fully hydrogenated palm kernel stearin, palm stearin, fully hydrogenated coconut oil, hydrogenated palm kernel oil, or a mixture of two or more of these; c. The matrix comprises at least 10% by weight, preferably at least 20%, more preferably at least 30%, of fully hydrogenated coconut oil; or d. The matrix contains approximately 90% to approximately 100% cocoa butter based on the weight of total solids; or contains cocoa butter and sugar, substantially consisting of them, or consisting of them; or contains cocoa butter and milk solids, substantially consisting of them, or consisting of them; or contains cocoa butter, sugar, and milk solids, substantially consisting of them, or consisting of them; The particle according to claim 1.

8. The particle according to claim 1, further comprising at least one coating layer.

9. A food or beverage product comprising the particles described in any one of claims 1 to 8.

10. The aforementioned food or beverage product contains the following: a. Protein powder, protein shake, protein shot, protein gel, or sports nutrition preparation, b. UHT beverages, UHT smoothies, c. Infant formulas, follow-on formulas, growth formulas, children's formulas, human milk fortifiers, children's foods or beverages, pregnant and lactating supplements, or pregnant and lactating nutritional formulas. d. Gels such as thermosetting gels, sauces, spreads, jams, jellies, or honey, e. Acid-curing gel, room-temperature stable yogurt, stirred yogurt, solid yogurt, or drinking yogurt, f. Neutral beverages, acidic beverages, water, juice, milk, smoothies, shakes, shots, alcoholic beverages, soft drinks, kombucha, kefir, or ready-to-mix powders. g. Bars, balls, cakes, cookies, muffins, or bread products, h. Medical foods, soups, desserts, puddings, custards, enteral preparations, preparations for seniors or the elderly, tablets, capsules, or supplements. i. Confectionery, gummies, candies, chocolates, fudge, truffles, chewing gum, frozen desserts, or ice cream, j. Flavorings, toppings, or ingredients for bread making, k. Cream, cheese, or butter, or l. Livestock feed or pet food, A food or beverage product according to claim 9, which includes the following:

11. The food or beverage product according to claim 9, wherein the food or beverage product has a water activity of at least 0.2, 0.4, 0.6, 0.8, or 0.9; and / or 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.

12. The food or beverage product according to claim 9, wherein the bioactive substance contains microorganisms, and the degradation rate of the microorganisms, measured at 30°C for 12 months, is less than 7 log CFU / g / year, preferably less than 6, 5, 4, 3, 2, or 1 log CFU / g / year.

13. A method for enhancing the stability of a bioactive substance, comprising the following steps: a. Embed the bioactive substance into a biofilm. b. The biofilm is embedded in a matrix having a water vapor transmission rate (WVTR) of 0.1 to 500 g / m² / 24h, wherein the matrix contains at least 6% by weight of one or more lipids, and c. Forming particles in that matrix; Or follow these steps: i. The bioactive substance is embedded in a matrix having a water vapor transmission rate (WVTR) of 0.1 to 500 g / m² / 24h, wherein the matrix contains at least 6% by weight of one or more lipids. ii. The bioactive substance induces the production of a biofilm, and then, iii. Forming particles in that matrix, The method comprising any of the above, wherein steps ii) and iii) may be in any order.

14. The method according to claim 13, wherein the bioactive substance comprises a first microorganism, and optionally the biofilm is produced by the first microorganism.

15. The method according to claim 13, further comprising the step of coating particles with at least one coating layer.

16. The method according to claim 13, wherein the particles include the particles described in any one of claims 1 to 8.

17. The following steps: d. Combine the particles from 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. The method according to claim 13, further comprising:

18. A method for producing a food or beverage product supplemented with a bioactive substance, comprising the method according to claim 17, and further comprising the following steps: f. Collect the particles from the culture medium in step e, and then, g. Combining the collected particles with food or beverage products to obtain food or beverage products supplemented with bioactive substances. The method further includes the method described above.

19. A method for producing a food or beverage product supplemented with a bioactive substance, comprising the method of claim 13, further comprising the step of combining the particles with a food or beverage product to obtain a food or beverage product supplemented with a bioactive substance.

20. The method according to claim 19, wherein the food or beverage product supplemented with the bioactive substance is the food or beverage product according to claim 9.

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