TW202416850A - Particles comprising bioactive agents - Google Patents

Abstract

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

Description

Particles containing biologically active agents

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

The particles described herein can be used to increase the shelf life of bioactive agents, such as probiotic microorganisms, particularly in high water activity environments at room temperature. The particles are particularly useful for high water activity foods and beverages in stable environments, such as UHT processed products, which are stored for long periods of time without refrigeration.

Consuming products containing bioactive agents has many health benefits. Such bioactive agents may include various microorganisms, such as probiotics.

However, bioactive agents have limited stability and/or shelf life. For example, when the bioactive agent is a microorganism, a well-known problem in the art is the stability of the microorganism. Microorganisms tend to become non-viable over time, especially when added to products with high water activity. This makes it difficult to provide products with high levels of active microorganisms.

Furthermore, if microorganisms in food or beverages grow unchecked, this may lead to changes in product characteristics or even spoilage.

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

Therefore, methods and compositions for increasing the stability and/or shelf life of bioactive agents are desirable. This is particularly desirable when the bioactive agent is a microorganism (e.g., a probiotic), when it is incorporated into a food or beverage with a high water activity, and when the food or beverage is stored at room temperature for a long time.

It is an object of the present invention to go some way toward addressing one or more of these pressing needs, or at least to provide the public with a useful choice.

In a first aspect, the present invention provides a particle comprising a bioactive agent embedded in a biofilm, wherein the biofilm is embedded in a matrix, wherein the water vapor transmission rate (WVTR) of the matrix is 0.1-500.

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

In a third aspect, the present invention provides a food or drink comprising the particles of the first or second aspect.

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

In a fifth aspect, the present invention provides a cleaning product comprising the particles of the first or second aspect.

In a sixth aspect, the present invention provides a method for increasing the stability of a bioactive agent, the method comprising the following steps: a. embedding the bioactive agent in a biofilm, b. embedding the biofilm in a matrix having a water vapor transmission rate (WVTR) of 0.1 to 500, and c. forming the matrix into particles.

In a seventh embodiment, the present invention provides a method for increasing the stability of a bioactive agent, the method comprising the following steps: a. embedding the bioactive agent in a biofilm, b. embedding the biofilm in a matrix comprising at least 6% by weight of one or more lipids, and c. forming the matrix into particles.

In an eighth aspect, the present invention provides a method for increasing the stability of a bioactive agent, the method comprising the following steps: a. embedding the bioactive agent in a matrix having a water vapor transmission rate (WVTR) of 0.1-500, b. inducing the bioactive agent to produce a biofilm, and c. forming the matrix into particles, wherein steps b) and c) can be in any order.

In some embodiments, step (b) comprises inducing the matrix-entrapped bioactive agent to form a biofilm.

In the ninth aspect, the present invention provides a method for increasing the stability of a bioactive agent, the method comprising the following steps: a. embedding the bioactive agent in a matrix comprising at least 6% by weight of one or more lipids, b. inducing the bioactive agent to produce a biofilm, and c. forming the matrix into particles, wherein steps b) and c) can be in any order.

In some embodiments, step (b) comprises inducing the matrix-entrapped bioactive agent to form a biofilm.

In some embodiments, the method of the sixth, seventh, eighth or ninth embodiment further comprises the following steps: d. combining the granules of step c with a culture medium having a water activity of at least 0.5 (preferably UHT-treated yogurt), and e. culturing the culture medium for 3 to 30 days.

In another embodiment, the present invention provides a method for producing a food or drink supplemented with a bioactive agent, the method comprising the method of the above-mentioned embodiment, and further comprising the following steps: f. harvesting particles from the culture medium of step e, and g. combining the harvested particles with a food or drink to obtain a food or drink supplemented with a bioactive agent.

In another aspect, the present invention provides a method for producing a food or drink supplemented with a biologically active agent, the method comprising the method of the sixth, seventh, eighth or ninth aspect, and further comprising the step of combining the particles with the food or drink to obtain the food or drink supplemented with the biologically active agent.

The following implementation aspects may be applied to any of the above aspects.

In one embodiment, the matrix comprises at least 6% by weight of one or more lipids.

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

In one embodiment, the water vapor transmission rate (WVTR) of the substrate is 0.1 to 10, such as 1 to 5.

In some embodiments, the water vapor transmission rate (WVTR) of the substrate is about 1 to about 350 g/m ² /24h, such as 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 g/m ² /24h.

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

In some embodiments, the water activity of the particles is from about 0.1 to about 1.0, e.g., from about 0.1 to about 0.9, from about 0.1 to about 0.8, from about 0.1 to about 0.7, from about 0.1 to about 0.6, from about 0.1 to about 0.5, from about 0.2 to about 1.0, from about 0.2 to about 0.9, from about 0.2 to about 0.8, from about 0.2 to about 0.7, from about 0.2 to about 0.6, from about 0.2 to about 0.5, about 0.3 to about 1.0, about 0.3 to about 0.9, about 0.3 to about 0.8, about 0.3 to about 0.7, about 0.3 to about 0.6, about 0.3 to about 0.5, about 0.4 to about 1.0, about 0.4 to about 0.9, about 0.4 to about 0.8, about 0.4 to about 0.7, about 0.4 to about 0.6, or about 0.4 to about 0.5.

In one embodiment, the bioactive agent 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.

In one embodiment, the first microorganism is Bacillus , Bifidobacterium , Enterococcus , Lacticaseibacillus , Lactiplantibacillus , Lactobacillus , Lactococcus, Ligilactobacillus , Limosilactobacillus , Lentilactobacillus , Saccharomyces , or Streptococcus . In one embodiment, the first microorganism is selected from the group consisting of: Bacillus coagulans , Bifidobacterium animalis , Bifidobacterium breve , Bifidobacterium bifidum , Bifidobacterium longum , Lacticaseibacillus casei , Lacticaseibacillus paracasei , Lacticaseibacillus rhamnosus , Lactiplantibacillus plantarum subsp. 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, the first microorganism is Lactococcus rhamnosus HN001. In some embodiments, the first microorganism is Lactobacillus rhamnosus or Lactobacillus paracasei, preferably Lactobacillus rhamnosus HN001 or Lactobacillus paracasei subsp. paracasei IM514.

In one embodiment, the granules contain the first microorganism in an amount of 10 to 5×10 ¹² CFU/gram of granules, for example, 10 ² to 5×10 ¹² , 10 ³ to 5×10 ¹² , 10 ⁴ to 5×10 ¹² , 10 ⁵ to 5×10 ¹² , 10 ⁶ to 5×10 ¹² , 10 ⁷ to 5×10 ¹² , 10 ⁸ to 5×10 ¹² , 10 ⁹ to 5×10 ¹² , 10 ¹⁰ to 5×10 ¹² , 10 ¹¹ to 5×10 ¹² , 10 ¹² to 5×10 ¹² , 10 to 10 ¹² , 10 ² to 10 ¹² , 10 ³ to 10 ¹² , 10 ⁴ to 10 ¹² , 10 ⁵ to 10 ^{10 6} to 10 ¹² ,10 ⁷ to 10 ¹² ,10 ⁸ to 10 ¹² ,10 ⁹ to 10 ¹² ,10 ¹⁰ to 10 ¹² ,10 ¹¹ to 10 ¹² ,10 to 10 ¹¹ ,10 ² to 10 ¹¹ ,10 ³ to 10 ¹¹ ,10 ⁴ to 10 ¹¹ ,10 ⁵ to 10 ¹¹ ,10 ⁶ to 10 ¹¹ ,10 ⁷ to 10 ¹¹ ,10 ⁸ to 10 ¹¹ ,10 ⁹ to 10 ¹¹ ,10 ¹⁰ to 10 ¹¹ ,10 to 10 ¹⁰ ,10 ² to 10 ¹⁰ ,10 ³ to 10 ¹⁰ ,10 ⁴ to 10 ¹⁰ ,10 ⁵ to 10 11 ⁸ ,10 ³ to 10 ⁸ ,10 4 to 10 8 ,10 ^{5 to 10 8 ,10 6 to 10 8 ,} 10 ⁷ to 10 ⁸ ,10 ⁸ to 10 ¹⁰ ,10 9 to 10 ¹⁰ ,10 to 10 9 ,10 ² to 10 ⁹ ,10 3 to 10 ⁹ ,10 ⁴ to 10 ⁹ ,10 ⁵ to 10 ⁹ ,10 ⁶ to 10 ⁹ ,10 ⁷ to 10 ⁹ ,10 ⁸ to 10 ⁹ ,10 to 10 ⁸ ,10 ² to 10 ⁸ ,10 ³ to 10 ⁸ ,10 ⁴ to 10 ⁸ ,10 ⁵ to 10 ⁸ ,10 ⁶ to 10 ⁸ ,10 ⁷ to 10 ⁸ ,10 to 10 ⁷ ,10 ² to 10 ⁷ ,10 ³ to 10 ⁷ ,10 ⁴ to 10 ⁷ , 10 ⁵ to 10 ⁷ , 10 ⁶ to 10 ⁷ , 10 to 10 ⁶ , 10 ² to 10 ⁶ , 10 ³ to 10 ⁶ , 10 ⁴ to 10 ⁶ , 10 ⁵ to 10 ⁶ , 10 to 10 ⁵ , 10 ² to 10 ⁵ , 10 ³ to 10 ⁵ , 10 ⁴ to 10 ⁵ , 10 to 10 ⁴ , 10 ² to 10 ⁴ , 10 ³ to 10 ⁴ , 10 to 10 ³ , 10 ² to 10 ³ , or 10 to 10 ² CFU/gram particle.

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

In one embodiment, the second microorganism is a probiotic microorganism. In some embodiments, the third, fourth and/or fifth microorganisms are probiotic microorganisms. In one embodiment, each microorganism is a probiotic microorganism. In one embodiment, the first microorganism is a microorganism that produces biofilms, and the second microorganism is a probiotic microorganism. In another embodiment, the first microorganism is a probiotic microorganism, and the second microorganism is a microorganism that produces biofilms.

In one embodiment, the bioactive agent is a non-microbial bioactive agent. In one embodiment, the particles further comprise a non-microbial bioactive agent. In some embodiments, the non-microbial bioactive agent is a protein, peptide, amino acid, fat, triglyceride, lipid, fatty acid, fatty acid salt, oligosaccharide, polysaccharide, nucleic acid, nucleotide, nucleoside, vitamin, mineral or any combination of two or more of the foregoing. In a preferred embodiment, the non-microbial bioactive agent is a vitamin, a prebiotic, a 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), vitamin B9 (folate or folic acid). In some embodiments, the prebiotic is fructooligosaccharide, 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 of the foregoing.

In one embodiment, the biofilm is characterized by peaks at 360 cm ^-1 , 430 cm ^-1 , and/or 530 cm ^{- 1} in a principal component analysis of the Raman spectroscopy data. In some embodiments, the Raman spectroscopy data indicates the presence of rhamnose, for example, by the presence of peaks at 360 cm ^-1 , 430 cm-1, and/or 530 cm- ¹ in a principal component analysis of the Raman spectroscopy data. In one embodiment, the principal component analysis of the Raman spectroscopy data of the biofilm-containing sample has peaks at 360 cm ^-1 , 430 cm ^-1 , and/or 530 cm ^-1 , and when normalized with the fat-related peak at 1450 cm ^-1 , the peak height is higher than the corresponding peak height in the principal component analysis of the Raman spectroscopy data of the non-biofilm-containing sample. In one embodiment, the peak heights of the peaks at 360 cm ^-1 , 430 cm ^-1 and/or 530 cm- ¹ in the principal component analysis of the Raman spectral data are at least 50% of the peak height of the fat-related peak at 1450 cm ^{-1 ,} for example, 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 fat-related peak at 1450 cm-1, and a useful range can be selected arbitrarily between 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%).

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

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

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

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

In one embodiment, one or more lipids comprise or consist of monoglycerides, diglycerides or triglycerides. In a preferred embodiment, one or more lipids comprise or consist of triglycerides.

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

In some embodiments, the one or more lipids comprise one or more fatty acids and/or salts and/or esters thereof, and at least 1% by weight of the fatty acids and/or salts and/or esters thereof have a carbon chain length of 14 or less, for example, 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%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 240%, 241%, 242%, 243%, 244%, 245%, 246%, 247%, 248%, 249%, 250%, or at least 80%, and a useful range can be arbitrarily selected between 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%).

In some embodiments, one or more lipids comprise fatty acids and/or salts and/or esters thereof, and at least 30% by weight of the fatty acids and/or salts and/or esters thereof have a carbon chain length of 16 or less, for example, at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, or at least 90%, and any useful range can be selected between 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%, 80% to 88%, 70% to 86%, 74% to 86%, 76% to 86%, 78% to 86%, or 80% to 86%).

In one embodiment, at least 50% by weight of the one or more lipids are saturated fats. In various embodiments, at least 55% by weight of the one or more lipids are saturated fats, such as at least 60% by weight, at least 65% by weight, at least 70% by weight, at least 75% by weight, at least 80% by weight, at least 85% by weight, at least 90% by weight, at least 95% by weight, or at least 99% by weight of the one or more lipids are saturated fats.

In one embodiment, one or more lipids include or are composed of fully hydrogenated palm kernel stearin, palm stearin, fully hydrogenated coconut oil, hydrogenated palm kernel oil, or any two or more of the foregoing mixtures. Preferably, hydrogenated palm kernel oil is fully hydrogenated.

In some embodiments, the matrix comprises at least 4% of one or more lipids by weight of total solids, for example, 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% of one or more lipids by weight of total solids.

In one embodiment, the matrix comprises 4-100% by weight of one or more lipids, preferably 30-100% by weight. In some embodiments, the matrix comprises 4% to 100% of one or more lipids by weight of the total solids, for example, 8% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 4% to 99.5%, 8% to 99.5 ... 10% to 99.5%, 20% to 99.5%, 30% to 99.5%, 40% to 99.5%, 50% to 99.5%, 60% to 99.5%, 70% to 99.5%, 80% to 99.5%, 90% to 99.5%, 4% to 95%, 8% to 95%, 10% to 95%, 20% to 95%, 30% to 95%, 40% to 95%, 50% to 95%, 60% to 95%, 70% to 9 5%, 80% to 95%, 90% to 95%, 10% to 90%, 20% to 90%, 30% to 90%, 40% to 90%, 50% to 90%, 60% to 90%, 70% to 90%, 80% to 90%, 10% to 80%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 60% to 80%, 70% to 80%, 10% to 70%, 20% to 70%, 30 % to 70%, 40% to 70%, 50% to 70%, 60% to 70%, 10% to 60%, 20% to 60%, 30% to 60%, 40% to 60%, 50% to 60%, 10% to 50%, 20% to 50%, 30% to 50%, 40% to 50%, 10% to 40%, 20% to 40%, 30% to 40%, 10% to 20%, 10% to 30%, or 10% to 20% of one or more lipids.

In one embodiment, the matrix comprises at least 10% by weight of fully hydrogenated coconut oil, preferably at least 15, 20, 25, or 30% by weight.

In one embodiment, the matrix comprises one or more auxiliary agents selected from the group consisting of sugar, emulsifier, milk solids and salt.

In some embodiments, the matrix comprises 0% to 80% of one or more sugars, based on the weight of the total solids, for example, 10% to 80%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 60% to 80%, 70% to 80%, 0% to 70%, 10% to 70%, 20% to 70%, 30% to 70%, 40% to 70%, 50% to 70%, 60% to 70%, 0% to 60% %, 10% to 60%, 20% to 60%, 30% to 60%, 40% to 60%, 50% to 60%, 0% to 50%, 10% to 50%, 20% to 50%, 30% to 50%, 40% to 50%, 0% to 40%, 10% to 40%, 20% to 40%, 30% to 40%, 0% to 30%, 10% to 30%, 20% to 30%, 0% to 20%, 10% to 20%, or 0% to 10% of one or more sugars.

In some embodiments, the matrix comprises 0% to 2% of one or more emulsifiers, based on the weight of the total solids, for example, 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% to 1.8%, 0.6% to 1.8%, 0.8% to 1.8%, 1.0% to 1.8%, 1.2% to 1.8%, 1.4% to 1.8%, 1.6% to 1.8%, 0% to 1.6%, 0.2% to 1.6%, 0.4% to 1.6%, 0.6% to 1.6%, 0.8% to 1.6%, 1.0% to 1.6%, 1.2% to 1.6%, 1.4% to 1.6%, 0% to 1.4%, 0.2% to 1.4%, 0.4% to 1.4%, 0.6% to 1.4%, 0.8% to 1.4%, 1.0% to 1.4%, 1.2% to 1.4%, 0% to 1.2%, 0.2% to 1.2%, 0.4% to 1.2%, 0.6% to 1.2%, 0.8% to 1.2%, 1.0% to 1.2%, 0% to 1.0%, 0.2% to 1.0%, 0.4% to 1.0%, 0.6% to 1.0%, 0.8% to 1.0%, 0% to 0.8%, 0.2% to 0.8%, 0.4% to 0.8%, 0.6% to 0.8%, 0% to 0.6%, 0.2% to 0.6%, 0.4% to 0.6%, 0% to 0.4%, 0.2% to 0.4%, or 0% to 0.2% of one or more emulsifiers.

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.

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

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; and most preferably, about 59% hydrogenated coconut oil and about 41% sucrose.

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

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; and most preferably, about 11% palm stearin, about 11% hydrogenated coconut oil, about 37% hydrogenated palm kernel stearin, and about 41% sucrose.

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; and most preferably, about 99.5% cocoa butter and about 0.5% lecithin.

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; and most preferably about 73% cocoa butter and about 26% milk solids.

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, based on the weight of the total solids, and any useful range can be selected between these values (e.g., about 80% to about 100%, about 90% to about 100%, or about 95% to about 100%).

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; and more preferably about 89.5% cocoa butter and about 13.5% lactose.

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; and more preferably about 86.5% hydrogenated coconut oil and about 13.5% lactose.

In one embodiment, the particle size is 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 10 ... micrometers to 5 mm, 100 micrometers to 4 mm, 100 micrometers to 3 mm, 100 micrometers to 2 mm, 100 micrometers to 1 mm, 100 micrometers to 500 micrometers, 200 micrometers to 10 mm, 200 micrometers to 9 mm, 200 micrometers to 8 mm, 200 micrometers to 7 mm, 200 micrometers to 6 mm, 200 micrometers to 5 mm, 200 micrometers to 4 mm, 200 micrometers to 3 mm, 200 micrometers to 2 mm, 200 micrometers to 1 mm, or 200 micrometers to 500 micrometers.

In one embodiment, the particles further comprise at least one coating. In some embodiments, the particles comprise two, three, four or five coatings. In some embodiments, the coating comprises alginate, chitosan, collagen, dextran, pectin, pullulan, gelatin, carrageenan, agar-agar, cellulose, hemicellulose, ethyl cellulose, carboxy cellulose, or a mixture of any two or more of the foregoing.

In one embodiment, the food or beverage comprises: a. protein powder, protein shake, protein shot, protein gel or sports nutritional formulation, b. UHT drink, UHT smoothie, c. infant formula, follow-on formula, growing formula, pediatric formula, human milk fortifier, children's food or drink, maternal supplement or maternal nutritional formulation, d. gel, such as heat-set gel, sauce, spread, jam, jelly, or honey, e. acid-set gel gel), ambient stable yoghurt, stirred yoghurt, set yoghurt, or drinking yoghurt, f.   Neutral beverages, acidic beverages, water, juice, milk, smoothies, milkshakes, concentrated drinks (shots), alcoholic beverages, soft drinks, kombucha, kefir, or ready-to-eat powdered mixes, g.   Bars, balls, cakes, cookies, muffins, or baked goods, h.   Medical foods, soups, desserts, puddings, custards, enteral preparations, preparations for senior or aged populations, tablets, capsules, or supplements, i.   Preserves, gummy candies, candies, chocolates, fudge, truffles, chewing gum, frozen desserts or ice cream, j.   Condiments, toppings or baking ingredients, k.   Cream, cheese or butter, or l.   Livestock food or pet food.

In some embodiments, the food or beverage comprises yogurt, juice (e.g., orange juice), a sports shake, cheese sauce, cream cheese spread, or milk (e.g., non-dairy milk such as oat milk).

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

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

In one embodiment, the personal care product is a skin care product (e.g., skin cream, sunscreen), a hair care product (e.g., shampoo or conditioner), a dental product (e.g., toothpaste or toothpaste), a deodorant, a cosmetic, a beauty product, or a feminine health product.

In an implementation of the sixth, seventh, eighth or ninth aspect, the method further comprises the step of coating the particles in at least one coating layer.

In one embodiment of the method of the sixth, seventh, eighth or ninth aspect, the particles comprise particles of the first or second aspect.

In an embodiment of the sixth, seventh, eighth or ninth aspect, the method further comprises the following steps: d. combining the granules of step c with a culture medium having a water activity of at least 0.5 (preferably UHT-treated yogurt), and e. culturing the culture medium for at least 2 weeks.

In one embodiment, the food or drink supplemented with a bioactive agent is the food or drink according to the third aspect.

The term "comprising" as used in this specification and the claims means "consisting at least in part of." When interpreting statements in this specification and the claims that include the term "comprising," additional features may be present in addition to the features that begin with the term in each statement. Related terms such as "comprise" and "comprised" are to be interpreted in a similar manner.

In this specification, reference is made to patent specifications, other external documents or other sources of information, generally for the purpose of providing a context for discussing features of the invention. Unless expressly stated otherwise, reference to these external documents should not be construed as an admission that these documents or these sources of information are prior art or form part of the common general knowledge in the art in any jurisdiction.

In the description of this specification, reference may be made to subject matter that is not within the scope of the present application. Such subject matter should be easily recognizable by a person having ordinary knowledge in the art to which the present invention belongs and may be helpful in implementing the invention as defined in the scope of the present application.

Particles ( Particle ）

The applicant unexpectedly discovered that particles comprising a bioactive agent embedded in a biofilm, wherein the biofilm is embedded in a matrix, wherein the matrix has a water vapor transmission rate (WVTR) of 0.1 to 500, provide an enhanced shelf life for the bioactive agent even when stored at ambient temperature in a high water activity environment.

Thus, in a first aspect, the present invention provides a particle comprising a bioactive agent embedded in a biomembrane, wherein the biomembrane is embedded in a matrix, wherein the matrix has a water vapor transmission rate (WVTR) of 0.1 to 500. Preferably, the matrix comprises at least 6% by weight of one or more lipids.

The term "particle" refers to a portion or amount of a material, such as a small portion or a small amount of a material. The term includes or may be used interchangeably with: pill, bead, ball, granule, etc. In some embodiments, the particle may be generally spherical. Preferably, the particle is edible. That is, the particle preferably does not contain any inedible components.

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

In some embodiments, the bioactive agent is encapsulated within the biofilm. In some embodiments, the biofilm is encapsulated within a matrix. As used herein, the term "encapsulated within" means "entirely surrounded by". In some embodiments, the bioactive agent is dispersed in the biofilm. In some embodiments, the biofilm is dispersed in the matrix.

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

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 of the foregoing.

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

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.

In some embodiments, the melting temperature of one or more lipids is 50°C or lower, for example, 48°C or lower, 46°C or lower, 44°C or lower, 40°C or lower, 38°C or lower, 36°C or lower, 32°C or lower, 30°C or lower, 25°C or lower, 20°C or lower, 15°C or lower, 10°C or lower, or 5°C or lower, and any useful range can be selected between 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°C, 36°C to 48°C, 5°C to 46°C, 1 0°C to 46°C, 15°C to 46°C, 20°C to 46°C, 25°C to 46°C, 30°C to 46°C, 32°C to 46°C, 34°C to 46°C, 36°C to 46°C, 5°C to 44°C, 10°C to 44°C, 15°C to 44°C, 20°C to 44°C, 25°C to 44°C, 30°C to 44°C, 32°C to 44°C, 34°C to 44°C, 36°C to 44°C, 5°C to 42 °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).

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

In some embodiments, the one or more lipids comprise one or more fatty acids and/or salts and/or esters thereof, and at least 1% by weight of the fatty acids and/or salts and/or esters thereof have a carbon chain length of 14 or less, for example, 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, and a useful range can be selected arbitrarily between these values (for example: 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%).

In some embodiments, the one or more lipids comprise fatty acids and/or salts and/or esters thereof, and at least 30% by weight of the fatty acids and/or salts and/or esters thereof have a carbon chain length of 16 or less, for example, at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, or at least 90%, (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%).

In alternative embodiments, one or more lipids comprise fatty acids and/or salts and/or esters thereof, and at least 10% by weight of the fatty acids and/or salts and/or esters thereof have a carbon chain length of 16 or less, for example, at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, or at least 90%, and a useful range can be selected arbitrarily between these values (e.g., 10% to 100%. between 15% and 20% of the total dose (i.e., between 15% and 25% of the total dose), between 15% and 25% of the total dose, between 15% and 25% of the total dose, between 15% and 25% of the total dose, between 15% and 25% of the total dose, between 15% and 25% of the total dose, between 15% and 25% of the total dose, between 15% and 25% of the total dose, between 15% and 25% of the total dose, between 15% and 25% of the total dose, between 15% and 25% of the total dose, between 15% and 25% of the total dose, between 15% and 25% of the total dose, between 15% and 25% of the total dose, between 15% and 25% of the total dose, between 15% and 25% of the total dose,

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

In some embodiments, at least 50% by weight of one or more lipids are saturated fats. In some embodiments, the lipids may be partially or completely hydrogenated. In some embodiments, the one or more lipids are comprised of, essentially consisting of, or consisting of: fully hydrogenated palm kernel stearin, palm stearin, fully hydrogenated coconut oil, cocoa butter, or a mixture of any two or more of the foregoing. In preferred embodiments, the one or more lipids are comprised of, essentially consisting of, or consisting of: fully hydrogenated palm kernel stearin, palm stearin, fully hydrogenated coconut oil, or a mixture of any two or more of the foregoing.

In some embodiments, the matrix comprises at least 4% of one or more lipids by weight of the total solids, for example, 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% of one or more lipids by weight of the total solids. Preferably, the matrix comprises at least 30% of one or more lipids by weight of the total solids.

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

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

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

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

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

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

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

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

In some embodiments, the matrix comprises 0% to 80% of one or more sugars, based on the weight of the total solids, for example, 10% to 80%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 60% to 80%, 70% to 80%, 0% to 70%, 10% to 70%, 20% to 70%, 30% to 70%, 40% to 70%, 50% to 70%, 60% to 70%, 0% to 60% Preferably, the matrix comprises 0% to 50% of the one or more sugars by weight of the total solids. Preferably, the one or more sugars comprise or consist of sucrose and/or lactose.

In some embodiments, the matrix comprises 0% to 2% of one or more emulsifiers, based on the weight of the total solids, for example, 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% to 1.8%, 0.6% to 1.8%, 0.8% to 1.8%, 1.0% to 1.8%, 1.2% to 1.8%, 1.4% to 1.8%, 1.6% to 1.8%, 0% to 1.6%, 0.2% to 1.6%, 0.4% to 1.6%, 0.6% to 1.6%, 0.8% to 1.6%, 1.0% to 1.6%, 1.2% to 1.6%, 1.4% to 1.6%, 0% to 1.4%, 0.2% to 1.4%, 0.4% to 1.4%, 0.6% to 1.4%, 0.8% to 1.4%, 1.0% to 1.4%, 1.2% to 1.4%, 0% to 1.2%, 0.2% to 1.2%, 0.4% to 1.2%, 0.6% to 1.2%, 0.8% to 1.2%, 1.0% to 1.2%, 0% to 1.0%, Preferably, the matrix comprises one or more emulsifiers in an amount of 0% to 0.5% by weight of the total solids. Preferably, the one or more emulsifiers comprise or consist of sorbitan tristearate and/or lecithin (e.g., soy lecithin).

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.

In a preferred embodiment, the matrix comprises, based on the 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.

In some embodiments, the particles further include additional coating layers. These layers can be used to modify the permeability of the particles, such as water permeability, water vapor transmission rate, or diffusion rate. Alternatively or additionally, the coating layer can be used to provide resistance to environmental conditions, such as resistance to gastric fluid.

In some embodiments, the coating layer comprises agar, alginate, carboxycellulose, carrageenan, cellulose, cellulose acetate phthalate, chitosan, collagen, dextran, ethylcellulose, gelatin, gluco-mannans, hemicellulose, lactoprotein, worm glue, starch, pectin, poly-L-lysine, polytriglucose, or a mixture of any two or more of the foregoing.

In some embodiments, the matrix is solid at 25°C.

In some embodiments, the matrix has a melting temperature of at least about 30°C, for example, at least about 32°C, 34°C, 35°C, 36°C, 38°C, 40°C, 42°C, 44°C, 45°C, 46°C, 48°C, 50°C, 52°C, 54°C, or at least about 55°C, and any useful range between these values can be selected (e.g., about 30°C to about 55°C, about 35°C to about 50°C, or about 40°C to about 48°C).

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

In some embodiments, the diameter of the particles is 50 μm to 10 mm, for example, 50 μm to 9 mm, 50 μm to 8 mm, 50 μm to 7 mm, 50 μm to 6 mm, 50 μm to 5 mm, 50 μm to 4 mm, 50 μm to 3 mm, 50 μm to 2 mm, 50 μm to 1 mm, 50 μm to 900 μm, 50 μm to 800 μm, 50 μm to 700 μm, 50 μm to 600 μm, 50 μm to 500 μm, 50 μm to 400 μm, 50 μm to 300 μm, 50 μm to 400 μm, 50 μm to 500 μm, 50 μm to 600 μm, 50 μm to 700 μm, 50 μm to 800 μm, 50 μm to 900 μm, 50 μm to 800 μm, 50 μm to 900 μm, 50 μm to 800 μm, 50 μm to 800 μm, 50 μm to 800 μm, 50 μm to 900 μm, 50 μm to 10 ... 75 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 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 100 microns to 10 mm, 100 microns to 9 mm, 100 microns to 8 mm, 100 microns to 7 mm, 100 microns to 6 mm, 100 microns to 5 mm, 100 microns to 4 mm, 100 microns to 3 mm, 100 microns to 2 mm, 100 microns to 1 mm, 100 microns to 900 microns, 100 microns to 800 microns, 100 microns to 700 microns, 100 microns to 600 microns, 100 microns to 500 microns, 100 microns to 400 microns, 100 microns to 300 microns, 100 microns to 20 0 microns to 10 mm, 200 microns to 9 mm, 200 microns to 8 mm, 200 microns to 7 mm, 200 microns to 6 mm, 200 microns to 5 mm, 200 microns to 4 mm, 200 microns to 3 mm, 200 microns to 2 mm, 200 microns to 1 mm, 200 microns to 900 microns, 200 microns to 800 microns, 200 microns to 700 microns, 200 microns to 600 microns, 200 microns to 500 microns, 200 microns to 400 microns, or 200 microns to 300 microns.

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

Thus, in some embodiments, the particles are not impermeable to water. Thus, in some embodiments, the water vapor transmission rate (WVTR) of the matrix is 0.1 to 500, such as 0.1 to 400, 0.1 to 300, 0.1 to 200, 0.1 to 150, 0.1 to 100, 0.1 to 90, 0.1 to 80, 0.1 to 70, 0.1 to 60, 0.1 to 50, 0.1 to 40, 0.1 to 30, 0.1 to 20, 0.1 to 10, 0.1 to 9, 0.1 to 8, 0.1 to 7, 0.1 to 6, 0.1 to 5, 0.1 to 4, 0.1 to 3, 0.5 to 500, such as 0.5 to 400, 0.5 to 300, 0.5 to 200, 1 to 300, 1 to 200, 1 to 150, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 0.5 to 50, 0.5 to 40, 0.5 to 30, 0.5 to 20, 0.5 to 10, 0.5 to 9, 0.5 to 8, 0.5 to 7, 0.5 to 6, 0.5 to 5, 0.5 to 4, 0.5 to 3, 1 to 500, for example 1 to 400, 1 to 300, 1 to 200, 1 to 150, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 to 500, for example 2 to 400, 2 to 300, 2 to 200, 2 to 150, 2 to 100, 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 500, for example 3 to 400, 3 to 300, 3 to 200, 3 to 150, 3 to 100, 3 to 90, 3 to 80, 3 to 70, 3 to 60, 3 to 50, 3 to 40, 3 to 30, 3 to 20, 3 to 10, 3 to 9, 3 to 8, 3 5 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 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 water vapor transmission rate (WVTR) of the substrate is less than 500, e.g., less than 400, less than 300, less than 200, less than 150, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or about 1.

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

Alternatively, WVTR can be calculated using the following formula: WVTR = dm⁄(A‧dt) Where dm = mass of water absorbed in time dt, A = total surface area of the sample calculated based on particle size measurement. The mass increase should be measured over a period of approximately linear weight gain, e.g. within 5 minutes, preferably within 7 minutes after adjusting the relative humidity to 95%. WVTR is reported in g/ ^m2 /24h.

To measure WVTR, a sample of particles was weighed in an intrinsic sorption microbalance in a dynamic vapor sorption (DVS) instrument (Surface Measurement Systems, Alperton, Middlesex, UK), where the measurement was performed at 95% relative humidity.

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

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

In some embodiments, the composition of the substrate is selected to facilitate biofilm formation. For example, in some embodiments, the substrate may include ingredients known to stimulate microorganisms to produce biofilms. In some embodiments, the substrate composition is selected to provide conditions (e.g., water and/or nutrient ingress) that stimulate microorganisms to produce biofilms.

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

Some exemplary coatings include: certain polysaccharides including cellulose and its derivatives, such as cellulose acetate phthalate, hemicellulose, methylcellulose, ethylcellulose, carboxycellulose, carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (hydroxypropyl cellulose), hydroxypropyl methylcellulose (HPMC) and microcrystalline cellulose; lipids and resins including waxes and oils, such as paraffin, carnauba wax, beeswax, candelilla wax, wax) and polyethylene wax; fatty acids and monoglycerides, such as stearyl alcohol, stearic acid, palmitic acid, monoglycerides, diglycerides and triglycerides; natural resins, such as wood resin and coumarone-indene; proteins, including corn zein (α-zein, β-zein and/or γ-zein), milk protein, wheat gluten, soy protein, peanut protein, keratin, collagen, gelatin, casein, and whey protein; starch and derivatives, such as raw starch, modified starch, pregelatinized starch, dextrin, dextran, maltodextrin, corn syrup, sucrose, glucose/fructose and sugar polyols; extruded gum, such as gum arabic, gum gandhi, and sucrose; ghatti), gum karaya, and gum tragacanth; seed gums such as guar gum and locust bean gum; microbial fermented gums such as xanthan gum, gellan gum, and chitan; seaweed extracts such as agar, alginates, carrageenan, and furcellaran; pectin; glucomannan; wormwood; and poly-L-lysine. Mixtures of these materials may also be used.

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

In one embodiment, the particles are encapsulated in lipospheres. In one embodiment, the biomembrane is encapsulated in lipospheres embedded in a matrix.

Bioactive Agents

The term "biologically active agent" refers to any substance with biological activity, including but not limited to proteins, peptides, amino acids, fats, triglycerides, lipids, fatty acids, oligosaccharides, polysaccharides, nucleic acids, nucleotides, nucleosides, vitamins, minerals, microorganisms, derivatives of microorganisms, or any combination of two or more of the foregoing. It should be understood that biological activity does not need to be directly applied to the organism that has consumed the particles of the present invention. For example, the biologically active agent may include substances that have an effect on the intestinal microbiota of the organism, such as prebiotics.

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

In some embodiments, the bioactive agent is a derivative of a microorganism (preferably a probiotic microorganism). The derivative of the microorganism may include a mutant or homolog of the microorganism, an attenuated or killed microorganism, and/or a microbial component that still has useful activity. For example, although the bacterial molecules responsible for regulating the activity of probiotics have not been clearly identified, molecules that have been proposed as possible candidates include bacterial DNA motifs, surface proteins, small organic acids, polysaccharides, and cell wall components (e.g., lipoteichoic acid and peptidoglycan). It is speculated that it interacts with components of the host immune system to produce an immunomodulatory effect. Preferably, the activity retained is at least about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% of the activity of an untreated (i.e., live or non-attenuated) control, and any useful range can be selected between these values (e.g., about 35 to about 100%, about 50 to about 100%, about 60 to about 100%, about 70 to about 100%, about 80 to about 100%, and about 90 to about 100%).

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

In some embodiments, the non-microbial bioactive agent does not include live or killed microorganisms. In some embodiments, the non-microbial bioactive agent does not include a derivative of a microorganism. In some embodiments, the non-microbial bioactive agent is a protein, a peptide, an amino acid, a fat, a triglyceride, a lipid, a fatty acid, an oligosaccharide, a polysaccharide, a nucleic acid, a nucleotide, a nucleoside, a vitamin, a mineral, or any combination of two or more of the foregoing. In a preferred embodiment, the non-microbial bioactive agent is a vitamin, a prebiotic, a postbiotic, or a 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), vitamin B9 (folic acid or folic acid). In some embodiments, the prebiotic is fructooligosaccharide, galacto-oligosaccharide, 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.

Prebiotics are compounds that induce the growth or activity of beneficial microorganisms. Some prebiotics are complex carbohydrates that cannot be digested by the organisms that ingest them (such as mammals), but can be digested by beneficial microorganisms. In this way, some prebiotics can be used as a nutrient source for beneficial microorganisms. Some non-limiting examples of prebiotics include fructooligosaccharides, galacto-oligosaccharides, and inulin.

Postbiotics are products of microorganisms that provide benefits to the host. Some postbiotics are secreted by living microorganisms, while others are released after bacterial lysis. Some non-limiting examples of postbiotics include vitamins (e.g., vitamin B and K), short-chain fatty acids, and antimicrobial peptides.

microorganism

It is contemplated that the particles and methods of the present invention may be used to provide improved stability and/or shelf life to a variety of microorganisms.

In one embodiment, the microorganism is a probiotic microorganism. In a preferred embodiment, the microorganism is a probiotic. In another embodiment, the microorganism is a fungus, such as a yeast. In one embodiment, the microorganism is a reproductively viable form.

The term "probiotic" refers to microorganisms that have probiotic activity. The term "probiotic activity" refers to the ability of certain microorganisms to stimulate the immune system. Measuring the types and activity levels 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.

In one embodiment, the microorganism is lactic acid bacteria (LAB). In one embodiment, the microorganism is selected from Bacillus coagulans, Bifidobacterium animalis subsp. lactis (e.g., Bifidobacterium animalis subsp . lactis strain HN019 or Bifidobacterium animalis subsp. lactis strain BB12), Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum, Lactobacillus casei (formerly Lactobacillus casei ), Lactobacillus paracasei (formerly Lactobacillus paracasei ), Lactobacillus rhamnosus (formerly Lactobacillus rhamnosus ; for example, Lactobacillus rhamnosus HN001), Lactobacillus plantarum subsp. Lactiplantibacillus plantarum subsp. plantarum , formerly Lactobacillus plantarum ), Lactobacillus acidophilus (e.g., Lactobacillus acidophilus (LAVRI-A1)), Lactobacillus de Bieber, Lactobacillus gasseri, Lactococcus lactis, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis diacetyl mutant, Leuconostoc pseudoenteroides, Ligilactobacillus salivarius (formerly Lactobacillus salivarius ), Limosilactobacillus reuteri (formerly Lactobacillus reuteri ; e.g., Lactobacillus reuteri ATCC 55730), Saccharomyces boulardii, Streptococcus thermophilus, or a combination of any two or more of the foregoing.

Preferably, the microorganism is a biofilm-forming microorganism. The ability of a microorganism to form a biofilm can be evaluated by methods known in the art. For example, the microorganism can be grown in the presence of a glass fiber or activated carbon surface, and the formation of a biofilm is determined by its adhesion to the substrate, as described in Example 4.

In some embodiments, the particles include a second microorganism 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 microorganism listed above. Other bioactive agents and/or microorganisms may also be included, such as a third and/or fourth microorganism.

Combinations of microorganisms may be selected that have complementary properties. For example, a microorganism that produces biofilm may be combined with a microorganism that does not produce biofilm. In some embodiments, a microorganism that is a prolific producer of biofilm may be combined with a microorganism that is a less prolific producer of biofilm. In some embodiments, the particle comprises a first and a second microorganism, wherein the biofilm is produced by the first microorganism.

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

In some embodiments, the particles comprise the first and/or second microorganisms in an amount of 10 to 5×10 ¹² CFU/gram of particles, for example, 10 ² to 5×10 ¹² , 10 ³ to 5×10 ¹² , 10 ⁴ to 5×10 ¹² , 10 ⁵ to 5×10 ¹² , 10 ⁶ to 5×10 ¹² , 10 ⁷ to 5×10 ¹² , 10 ⁸ to 5×10 ¹² , 10 ⁹ to 5×10 ¹² , 10 ¹⁰ to 5×10 ¹² , 10 ¹¹ to 5×10 ¹² , 10 ¹² to 5×10 ¹² , 10 to 10 ¹² , 10 ² to 10 ¹² , 10 ³ to 10 ¹² , 10 ⁴ to 10 ¹² , 10 ⁵ to 10 ¹² 、10 ⁶ to 10 ¹² 、10 ⁷ to 10 ¹² 、10 ⁸ to 10 ¹² 、10 ⁹ to 10 ¹² 、10 ¹⁰ to 10 ¹² 、10 ¹¹ to 10 ¹² 、10 to 10 ¹¹ 、10 ² to 10 ¹¹ 、10 ³ to 10 ¹¹ 、10 ⁴ to 10 ¹¹ 、10 ⁵ to 10 ¹¹ 、10 ⁶ to 10 ¹¹ 、10 ⁷ to 10 ¹¹ 、10 ⁸ to 10 ¹¹ 、10 ⁹ to 10 ¹¹ 、10 ¹⁰ to 10 ¹¹ 、10 to 10 ¹⁰ 、10 ² to 10 ¹⁰ 、10 ³ to 10 ¹⁰ 、10 ⁴ to 10 ¹⁰ 、10 ⁵ to 10 ¹⁰ 、10 ⁶ to 10 ¹⁰ 、10 ⁷ to 10 ¹⁰ 、10 ⁸ to 10 ¹⁰ 、10 ⁹ to 10 ¹⁰ 、10 to 10 ⁹ 、10 ² to 10 ⁹ 、10 ³ to 10 ⁹ 、10 ⁴ to 10 ⁹ 、10 ⁵ to 10 ⁹ 、10 ⁶ to 10 ⁹ 、10 ⁷ to 10 ⁹ 、10 ⁸ to 10 ⁹ 、10 to 10 ⁸ 、10 ² to 10 ⁸ 、10 ³ to 10 ⁸ 、10 ⁴ to 10 ⁸ 、10 ⁵ to 10 ⁸ 、10 ⁶ to 10 ⁸ 、10 ⁷ to 10 ⁸ 、10 to 10 ⁷ 、10 ² to 10 ⁷ 、10 ³ to 10 ⁷ , 10 ⁴ to 10 ⁷ , 10 ⁵ to 10 ⁷ , 10 ⁶ to 10 ⁷ , 10 to 10 ⁶ , 10 ² to 10 ⁶ , 10 ³ to 10 ⁶ , 10 ⁴ to 10 ⁶ , 10 ⁵ to 10 ⁶ , 10 to 10 ⁵ , 10 ² to 10 ⁵ , 10 ³ to 10 ⁵ , 10 ⁴ to 10 ⁵ , 10 to 10 ⁴ , 10 ² to 10 ⁴ , 10 ³ to 10 ⁴ , 10 to 10 ³ , 10 ² to 10 ³ , or 10 to 10 ² CFU/gram particle.

Biofilm

The term "biofilm" refers to the layer of extracellular material produced by and surrounding microorganisms.

Biofilms are produced by certain microorganisms (e.g., bacteria) in response to certain environmental conditions. The conditions required to stimulate biofilm production may vary from microorganism to microorganism, but in some species, biofilm production can be induced by stress. Such stresses may include nutrient limitation, osmotic stress, drying, exposure to ultraviolet (UV) light, adverse pH or temperature, high pressure, or exposure to certain compounds (including antimicrobials (e.g., antibiotics) and compounds involved in quorum sensing).

Biofilms typically consist of a mucilaginous extracellular layer surrounding the microorganisms. The composition of the biofilm may vary depending on the type of microorganism that produces the biofilm. The major components of bacterial biofilms are usually high molecular weight extracellular microbial polymers, such as exopolysaccharides (EPS). It should be understood that the term "exopolysaccharides" or "EPS" is a general description that covers a variety of different compounds with different chemical and physical properties.

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

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

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

In some embodiments, biofilms can be detected by Raman spectroscopy. A hyperspectral map can be generated using Raman spectroscopy to provide structural information about the location of biofilm components within the sample scan area. For biofilm identification, this provides contextual information about where bacterial cells are located and whether exopolymeric components that indicate biofilms are actually present around them.

In one embodiment, the biofilm is characterized by peaks at 360 cm ^-1 , 430 cm ^-1 , and/or 530 cm ^-1 in a principal component analysis of the Raman spectroscopy data. In some embodiments, the Raman spectroscopy data indicates the presence of rhamnose, for example by the presence of peaks at 360 cm ^-1 , 430 cm ^-1 , and/or 530 cm ^-1 in a principal component analysis of the Raman spectroscopy data. In one embodiment, the peak heights of the peaks at 360 cm ^-1 , 430 cm ^-1 and/or 530 cm-1 in the principal component analysis of the Raman spectral data are at least 50% of the peak height of the fat-related peak at 1450 cm ^{-1 ,} for example, 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 fat-related peak at 1450 cm ^-1 , and a useful range can be selected arbitrarily between 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%).

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

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

To detect biofilms by lectin staining, pellets can be cut into sections approximately 2 mm thick. Sections are stained with 5 μL of 0.1 mg/mL fluorescein-labeled lectin stain, 1 μM DAPI, and 0.2% fast green. Different lectin stains can be used to detect different sugars. In one embodiment, the lectin stain can be selected from concanavalin A, wheat germ agglutinin, and/or lentin. Samples are allowed to stain 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-labeled lectin, and a 561 nm excitation is used for fast green. The presence of bacterial cells embedded in a biofilm can be determined by the co-localisation of DAPI-stained cells and one or more lectin-stained biofilms. Various methods for detecting biofilms by lectin staining are shown in Examples 6, 8 and 11.

In some embodiments, the biofilm is produced by a bioactive agent. For example, in some embodiments, the bioactive agent is a first microorganism, and the biofilm is produced by the first microorganism. In some embodiments, the first microorganism is embedded in a matrix, wherein the first microorganism produces a biofilm. In other embodiments, the first microorganism is induced to produce a biofilm, which is then embedded in a matrix. The first microorganism can be induced to produce a biofilm by exposure to stress, such as: nutrient limitation, osmotic pressure, drying, exposure to ultraviolet light, unfavorable pH or temperature, high pressure, or exposure to certain compounds (including antimicrobial agents such as antibiotics, and compounds involving quorum sensing).

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. This may be useful, for example, when the first microorganism is included for the purpose of biofilm production, and when the second microorganism is included for the purpose of providing a benefit (e.g., a health benefit). Thus, in some embodiments, the particle comprises the first and second microorganisms, wherein the biofilm is produced by the first microorganism.

Food and Drinks

In a second aspect, the present invention provides a food or drink comprising the particles of the first aspect.

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

It is expected that the granules of the invention allow the production of food and/or beverages with improved stability and/or shelf life.

In some embodiments, the food or beverage comprises protein powder, protein shake, protein concentrate, protein gel or sports nutrition preparation, UHT drink, UHT smoothie, infant formula, follow-on formula, growing formula, pediatric formula, human milk fortifier, children's food or drink, maternal supplement or maternal nutrition preparation, gel (e.g., heat-set gel), sauce, spread, jam, jelly or honey, acid-set gel, room temperature stable yogurt, stirred yogurt, set yogurt or yogurt, neutral beverage, acid sexual beverages, water, juice, milk, smoothies, milkshakes, concentrated beverages, alcoholic beverages, soft drinks, kombucha, kefir or premixed powders, bars, balls, cakes, cookies, muffins or baked goods, medical foods, soups, desserts, puddings, custard, enteral preparations, preparations for the elderly, tablets, capsules or supplements, candies, gummies, candies, chocolate, fudge, brownies, chewing gum, frozen desserts, ice cream, seasonings, sauces or baking ingredients, whipped cream, cheese or butter, or livestock or pet food.

In one embodiment, the food or beverage comprises an alcoholic beverage. In some embodiments, the alcoholic beverage is beer, wine, spirits, or ready-to-drink (RTD) beverages. In some embodiments, the food or beverage comprises low-alcohol or alcohol-free beer or wine.

In one embodiment, the food or beverage comprises animal food. In some embodiments, the animal food is livestock food, pet food, kibble, pet cookies, pet treats, animal feed ingredients, livestock forage, livestock supplements, or drench.

In one embodiment, the food or beverage comprises a baked good. In some embodiments, the baked good is bread, a stick, a ball, a cake, a cookie or a muffin.

In one embodiment, the food or beverage comprises a beverage. In some embodiments, the beverage is a protein shake, protein concentrate, UHT beverage, UHT smoothie, neutral beverage, acidic beverage, water, juice, milk, smoothie, milkshake, concentrated beverage, alcoholic beverage, soft drink, fermented beverage, kombucha, kefir, ready-to-drink (RTD) beverage, coffee beverage, tea, or creamer. In some embodiments, the food or beverage comprises coffee capsules, instant coffee, tea bags, or coffee creamer.

In one embodiment, the food or beverage comprises breakfast food. In some embodiments, the breakfast food is cereal, oatmeal, porridge, spread, jam, jelly or honey.

In one embodiment, the food or beverage comprises a confectionery. In some embodiments, the confectionery is a boiled sweet, a candy, a chewing gum, a chocolate bar, a chocolate coated product, a candy bar, a fudgy candy, or a crispy chocolate.

In one embodiment, the food or beverage comprises a dairy product. In one embodiment, the dairy product is yogurt. In some embodiments, the yogurt is chilled yoghurt, room temperature yogurt, stirred yogurt, solidified yogurt or yogurt. In one embodiment, the dairy product is milk. In some embodiments, the milk is fresh milk, UHT milk, fortified milk, flavored milk, or fermented milk product (e.g., 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 fresh cream, such as fresh cream or UHT cream. In some embodiments, the dairy product is ice cream.

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

In some embodiments, the food or beverage comprises infant formula, follow-on formula, growing formula, pediatric formula, human milk fortifier, children's food or beverage, maternal supplement or maternal nutritional preparation. In some embodiments, the food or beverage comprises infant formula, children's milk powder, wet blend infant formula, baby food, baby snack, baby rice, baby melting puff, baby teething rusk, fruit and/or vegetable puree.

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

In one embodiment, the food or beverage comprises a medical food or beverage. In some embodiments, the medical food or beverage is a meal replacement, a meal replacement shake, a nutritional 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.

In one embodiment, the food or beverage comprises a powder. In some embodiments, the powder is a protein powder, an instant drink powder, or a meal replacement powder.

In one embodiment, the food or beverage comprises a snack. In some embodiments, the snack is a baked good, a cheese lollipop, chips, a bar, a cookie, a dip, dried fruit, nuts, a ball, a fruit leather, a high-protein baked good, a yoghurt drop, a cereal mix, popcorn, potato chips, a smoothie bar, or a vegetable chip.

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

In some embodiments, the food or beverage has a high water activity ( _aw ). It is believed that the particles described herein are particularly useful in high water activity products. In various embodiments, the food or beverage 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 preferred embodiments, the food or beverage has a water activity of about 1.0.

In some embodiments, when stored at 30°C, the bioactive agent in the food or beverage 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.

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

In some embodiments, when the bioactive agent is a microorganism, the food or beverage can be formulated to allow administration of a sufficient amount of the microorganism to establish a population in the gastrointestinal tract of an individual upon ingestion. The established population can be a transient population or a resident population.

In theory, one colony forming unit (cfu) should be sufficient to establish a microbial colony in an individual, but in practice a minimum number of units is required to achieve this. Therefore, for therapeutic mechanisms that rely on viable, reproducible microbial colonies, the number of units given to an individual will affect the efficacy of the treatment.

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

Methods for determining the presence of a population of intestinal flora (e.g., Lactobacillus rhamnosus HN001) in the gastrointestinal tract of an individual are well known in the art. In certain embodiments, the presence of a population of microorganisms (e.g., Lactobacillus rhamnosus HN001) can be determined directly, for example, by analyzing one or more samples obtained from the individual and determining the presence or amount of the microorganism in the sample. In other embodiments, the presence of a population of microorganisms can be determined indirectly, for example, by observing health effects or a decrease in the number of other intestinal flora in a sample obtained from the individual. Combinations of these methods are also contemplated.

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

As a general example, it is contemplated that about 1×10 ⁶ cfu to about 1×10 ¹² cfu of a microorganism is administered per kilogram of body weight per day, preferably about 1×10 ⁶ cfu/kg/day to about 1×10 ¹¹ cfu/kg/day, about 1×10 ⁶ cfu/kg/day to about 1×10 ¹⁰ cfu/kg/day, about 1×10 ⁶ cfu/kg/day to about 1×10 ⁹ cfu/kg/day, about 1×10 ⁶ cfu/kg/day to about 1×10 ⁸ cfu/kg/day, about 1×10 ⁶ cfu/kg/day to about 5×10 ⁷ cfu/kg/day, or about 1×10 ⁶ cfu/kg/day to about 1×10 ⁷ cfu/kg/day. Preferably, it is contemplated that about 5×10 ⁶ cfu to about 5×10 ⁸ cfu of the microorganism is administered per kilogram of body weight per day, preferably about 5×10 ⁶ cfu/kg/day to about 4×10 ⁸ cfu/kg/day, about 5×10 ⁶ cfu/kg/day to about 3×10 ⁸ cfu/kg/day, about 5×10 ⁶ cfu/kg/day to about 2×10 ⁸ cfu/kg/day, about 5×10 6 cfu/kg/day to about 1×10 ⁸ cfu/kg/day, about 5×10 ⁶ cfu/kg/day to about 9×10 ⁷ cfu/kg/day, about 5×10 6 cfu/kg/day to about 8×10 ⁷ cfu/kg/day, about 5×10 ⁶ cfu/kg/day to about 7×10 ⁷ cfu/kg/day, about 5×10 6 cfu/kg/day to about 8×10 ⁷ cfu/kg/day, about 5×10 ⁶ cfu/kg/day to about 9×10 ⁷ cfu/kg/day cfu/kg/day to about 6×10 ⁷ cfu/kg/day, about 5×10 ⁶ cfu/kg/day to about 5×10 ⁷ cfu/kg/day, about 5×10 ⁶ cfu/kg/day to about 4×10 ⁷ cfu/kg/day, about 5×10 ⁶ cfu/kg/day to about 3×10 ⁷ cfu/kg/day, about 5×10 ⁶ cfu/kg/day to about 2×10 ⁷ cfu/kg/day, or about 5×10 ⁶ cfu/kg/day to about 1×10 ⁷ cfu/kg/day.

In certain embodiments, the periodic dose does not need to vary with the weight or other characteristics of the individual. In these examples, it is contemplated that about 1×10 ⁶ cfu to about 1×10 ¹³ cfu of a microorganism is administered daily, preferably about 1×10 ⁶ cfu/day to about 1×10 ¹² cfu/day, about 1×10 ⁶ cfu/day to about 1×10 ¹¹ cfu/day, about 1×10 ⁶ cfu/day to about 1×10 ¹⁰ cfu/day, about 1×10 ⁶ cfu/day to about 1×10 ⁹ cfu/day, about 1×10 ⁶ cfu/day to about 1×10 ⁸ cfu/day, about 1×10 ⁶ cfu/day to about 5×10 ⁷ cfu/day, or about 1×10 ⁶ cfu/day to about 1×10 ⁷ cfu/day. Preferably, it is contemplated that about 5×10 ⁷ cfu to about 5×10 ¹⁰ cfu of microorganisms are administered daily, preferably about 5×10 ⁷ cfu/day to about 4×10 ¹⁰ cfu/day, about 5×10 ⁷ cfu/day to about 3×10 ¹⁰ cfu/day, about 5×10 ⁷ cfu/day to about 2×10 ¹⁰ cfu/day, about 5×10 ⁷ cfu/day to about 1×10 ¹⁰ cfu/day, about 5×10 ⁷ cfu/day to about 9×10 ⁹ cfu/day, about 5×10 ⁷ cfu/day to about 8×10 ⁹ cfu/day, about 5×10 ⁷ cfu/day to about 7×10 ⁹ cfu/day, about 5×10 ⁷ cfu/day to about 6×10 ⁹ cfu/day, about 5×10 ⁷ cfu/day to about cfu/day to about 5×10 ⁹ cfu/day, about 5×10 ⁷ cfu/day to about 4×10 ⁹ cfu/day, about 5×10 ⁷ cfu/day to about 3×10 ⁹ cfu/day, about 5×10 ⁷ cfu/day to about 2×10 ⁹ cfu/day, or about 5×10 ⁷ cfu/day to about 1×10 ⁹ cfu/day.

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

It should be understood that food or beverage is preferably formulated to allow the administration of an effective dose of the bioactive agent. The dosage, administration cycle and general administration regimen of the composition administered may vary from individual to individual, depending on variables such as the bioactive agent used, the severity of individual symptoms, the type of disease to be treated, the selected mode of administration, and the age, sex and/or general health of the individual. In addition, as described above, the appropriate dosage may depend on the nature and formulation of the bioactive agent in the food or beverage. For example, when the composition comprises live microorganisms, the dosage may be calculated with reference to the number of live microorganisms present. For example, as described in the embodiments herein, the dosage may be determined by reference to the number of colony forming units (cfu) administered daily. In the case where the food or beverage contains one or more derivatives of microorganisms, the dosage can be calculated by reference to the amount or concentration of the derivative of the microorganism administered per day. For example, for a composition containing a microbial cell lysate, the dosage can be calculated by reference to the concentration of the microbial cell lysate present in the composition.

It should be understood that the preferred food or beverage is formulated to provide an effective dose in a convenient form and amount. In certain embodiments (for example but not limited to those where the periodic dose does not need to vary with the individual's weight or other characteristics), the product can be formulated as a unit dose. It should be understood that administration can include a single daily dose or multiple discrete divided doses, as appropriate.

Personal Care Products

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

The term "personal care product" means any product, preparation, or formulation used for personal care purposes (e.g., toilet purposes), including, but not limited to, products used to clean or groom oneself, products used for personal hygiene, and products used for beauty. 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.

In one embodiment, the personal care product is a skin care product (e.g., skin cream), sunscreen, a hair care product (e.g., shampoo or conditioner), a dental product (e.g., toothpaste or toothpaste), a deodorant, a cosmetic, a beauty product, or a feminine health product.

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.

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

In some embodiments, the personal care product is a dental product, such as toothpaste, toothpaste, dental gel, dental chew, mouthwash, or gargle.

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

In some embodiments, the personal care product is a feminine health product.

Cleaning Products

In one aspect, the present invention provides a cleaning product comprising the particles of the first aspect.

In some embodiments, the cleaning product comprises a cleaning spray, a detergent, or a disinfectant.

Method for producing granules

Various methods of making the particles of the present invention are contemplated. In some embodiments, the method comprises the following steps: a. contacting the bioactive agent and the matrix to form a mixture; and b. forming the mixture into particles.

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

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

In one embodiment, when the substrate comprises more than one component, the substrate components are combined. The combination can be performed by any suitable means known in the art, such as by mixing or blending. In some embodiments, the substrate comprises one or more dry components and is dry-blended together. In some embodiments, one or more dry components (or dry-blended components) are combined with one or more liquid components. The liquid components may include components (e.g., one or more lipid components) that have been heated to a temperature sufficient to liquefy them.

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

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

The heating step may be followed by a tempering step at a lower temperature and for a period of time sufficient to allow the matrix material to equilibrate at the lower temperature. For example, the composition may be held at 45°C for at least 30 minutes. In cases where the biologically active agent is susceptible to thermal damage, the tempering step reduces the temperature of the matrix material to prevent thermal damage to the biologically active agent. Many microorganisms (e.g., probiotics) are known to be susceptible to thermal damage, so in such cases, a tempering step is typically used. For biologically active agents that are not susceptible to thermal damage, a tempering step is optional.

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

The resulting mixture containing the biologically active agent is then formed into granules, and the temperature is reduced to solidify the mixture. The granules can be produced by any suitable method known in the art, such as by spray chilling or prilling. In some embodiments, the granules are produced by granulation. In other embodiments, the granules are produced by spray chilling. In some embodiments, the mixture is cooled to a temperature below 24°C, for example, 22°C, 20°C, 18°C, 16°C, 14°C, 12°C, 10°C, 8°C, 6°C, 4°C, 2°C, or about 0°C, and any useful range can be selected between 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).

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 stored in a sterilized and sealed container and cooled to solidify the mixture. For example, the mold can be cooled to 4°C for 12 hours.

The particles are optionally washed and/or filtered according to size.

The particles may be coated with one or more coatings as desired. The coatings may be applied by techniques known in the art, such as by spraying.

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

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

Method for increasing the stability of biologically active agents

In a fifth embodiment, the present invention provides a method for increasing the stability of a bioactive agent, the method comprising the following steps: a. embedding the bioactive agent in a biofilm; b. embedding the biofilm in a matrix; and c. forming the matrix into particles.

In a sixth aspect, the present invention provides a method for increasing the stability of a bioactive agent, the method comprising the following steps: a. embedding the bioactive agent in a matrix; b. inducing the bioactive agent to produce a biofilm, and c. forming the matrix into particles, wherein steps b and c can be in any order.

In one embodiment, the bioactive agent 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 the step of coating the particles in at least one coating layer. In 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.

In some embodiments, the method further comprises the following steps: d. mixing the granules of step c with a culture medium having a water activity of at least 0.5 (preferably UHT-treated yogurt), and e. culturing the culture medium for at least 2 weeks.

Without wishing to be bound by theory, it is believed that in culture medium such as UHT treated yogurt, the granules of step (c) may allow a controlled influx of water and/or other compounds (e.g. lactose) present in the medium into the granules, stimulating the formation of biofilm.

Method for producing food or beverage supplemented with biologically active agents

In another embodiment, the present invention provides a method for producing a food or beverage supplemented with a bioactive agent, the method comprising the method of the fifth or sixth embodiment, and further comprising the following steps: f. harvesting particles from the culture medium of step e, and g. combining the harvested particles with a food or beverage product to obtain a food or beverage product supplemented with a bioactive agent.

In another aspect, the present invention provides a method for producing a food or drink supplemented with a biologically active agent, the method comprising the method of the fifth or sixth aspect, and further comprising the step of combining the particles with the food or drink to obtain the food or drink supplemented with the biologically active agent.

In some embodiments, the food or drink supplemented with a bioactive agent is a food or drink according to the second aspect.

Incorporated by reference

The contents of the articles, patents and patent applications, and all other documents and electronically available information mentioned or cited herein are incorporated herein by reference as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other paper and electronic documents.

The following examples illustrate the present invention.

Embodiment

1. Embodiment 1 - Bioactive Agent Encapsulation Matrix

1.1 Materials and Methods

Various vegetable oils (alone or in combination, with or without food ingredient adjuvants) were tested for their ability to provide a protective barrier between a probiotic bacterium (Lactobacillus rhamnosus HN001) and a fermented dairy product.

Particles (in the form of beads) containing a probiotic ingredient (Lactobacillus rhamnosus HN001) embedded in a matrix comprising fat and/or a combination of fat and auxiliary ingredients were prepared. The probiotic ingredient used was a freeze-dried commercial sample.

The probiotic matrix beads are prepared by first melting the lipid component at 65ºC for 30 minutes and then tempering at 45ºC for at least 30 minutes. The tempering step is to prevent heat damage to the probiotics. To prevent contamination, the beads are prepared in a biosafety cabinet. The probiotic beads component is added to the tempered lipid component and stirred until homogeneous. Stir to combine the dry ingredients. The resulting mixture containing the probiotics is poured into a pre-sterilized mold and any excess material is removed with a sterile spatula. The mold is kept in a sterilized and sealed container and cooled to 4ºC for 12 hours. The beads are then removed from the mold and stored in a nitrogen-flushed heat-sealed bag at -18ºC.

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

Table 1. Composition of fat in the coating matrix Sample ID Fully Hydrogenated Palm Kernel Stearin Palm stearin Fully Hydrogenated Coconut Oil cocoa powder AT01 32 0 0 0 AT02 0 32 0 0 AT03 0 0 32 0 AT04 16 16 0 0 AT05 16 0 16 0 AT06 16 0 0 16 AT07 0 16 16 0 AT08 0 16 0 16 AT09 0 0 16 16 AT10 10.7 10.7 10.7 0 AT11 10.7 10.7 0 10.7 AT12 10.7 0 10.7 10.7 AT13 0 10.7 10.7 10.7 AT14 8 8 8 8 AT15 20 4 4 4 AT16 4 20 4 4 AT17 4 4 20 4

The protected cells were added to thickened UHT yogurt formulated for ambient storage and stored at 30ºC for 12 months to determine its shelf life. Samples were taken at the beginning of the trial and monthly and analyzed for the cell count of the HN001 probiotic. The cell degradation rate was determined for each composition. A final sample was taken after a period of 12 months and the degradation rate of the probiotic using each encapsulation matrix was calculated.

1.2 result

Some particles were more robust in structure than others, depending on the composition, however, most particles were still viable after 12 months of incubation in the yogurt matrix. The particles containing a high amount of fully hydrogenated palm kernel stearin were the most robust, followed by hydrogenated palm kernel oil (as described in Example 2), then cocoa butter (as described in Example 2), then hydrogenated coconut oil (data not shown).

A surprising observation was that all probiotics in the beads showed growth during the first month of storage, followed by the expected degradation. Without wishing to be bound by theory, it is believed that this may be due to the semipermeability of the beads, which allows a certain amount of water, lactose and other nutrients to enter the beads and provide a carbon source for limited growth, followed by a physiological regulation and stasis phase.

Analysis of degradation and degradation rates in the beads was determined starting from 1 month of storage, as it was reasonably assumed that growth and physiological regulation were partially independent processes.

It was also observed that some probiotic cells were released from the beads, but most of the cells remained entrapped in the beads.

The degradation rates of the various coating substrates are shown in Table 2. The degradation rates are expressed as the logarithmic reduction of CFU per gram per year. In other words, for the sake of clarity in the table, the degradation rates measured in the 6-month and 9-month time periods are extrapolated to 1 year.

Table 2. Cumulative degradation rate of probiotics encapsulated in the coating matrix (* rate calculated after 9 months; ** after 6 months) Sample ID Probiotic degradation rate ( log CFU/g/y ) 6 months 9 months 12 months AT01 4.1 2.4 2.9 AT02 1.7 3.4 3.8 AT03 1.2 1.8 0.88 AT04 2.5 2.9 2.9* AT05 1.7 1.8 1.8* AT06 2.2 4.5 6.6 AT07 3.9 3.6 2 AT08 4.5 8.5 5.5 AT09 2.6 2.6 1.9 AT10 4.2 2.04 2.04 AT11 2.5 4.1 6.1 AT12 3.0 3.03 3.0** AT13 3.1 4.2 3.2 AT14 2.7 3.1 6 AT15 3.6 1.5 3.4* AT16 2.6 1.9 4.1 AT17 2.6 2.6 3

1.3 Conclusion

This example shows that a coating matrix can reduce the degradation of probiotic microorganisms in fermented dairy products (e.g., yogurt) even during long-term storage at or above ambient temperature.

2. Embodiment 2 - Other coating matrices for bioactive agents

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

2.1 Materials and Methods

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

The samples used were: 1. Uncoated freeze-dried 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.

2.2 result

The degradation rates of the various coatings are shown in Table 3. The degradation rates are expressed as log reductions in CFU per gram per year. The degradation rates measured over the 6-month and 9-month time periods were extrapolated to 1 year.

surface 3. Degradation rate Coating Degradation rate ( log CFU/g/y ） 6 Month 9 Month 12 Month Unwrapped Not alive Not alive Not alive 99.5% cocoa butter , 0.5% lecithin 2.3 2.04 4 100% Coco fat 2.5 7.3 8.9 99.5% Hydrogenated Palm Kernel Oil , 0.5% Lecithin 3.1 2.4 2.5 100% Hydrogenated Palm Kernel Oil 2.7 2.5 3.2 32% Hydrogenated Palm Kernel Oil 2.9 1.8 1.8 16% Hydrogenated Palm Kernel Oil , 16% Cocoa Powder 2.3 1.9 1.9

Uncoated freeze-dried cultures (negative control) did not survive in yogurt incubated at 30ºC. The addition of cocoa powder did not appear to help protect the probiotics from degradation.

Interestingly, cocoa butter showed a lower degree of decay up to 6 months, but degraded at a significantly higher rate after that period.

The combination of HPO and adjuvant (16% and 32% fat content) significantly reduced the degradation rate over a 12-month storage period (1.9 and 1.8 log CFU/g/y, respectively).

Mixture contour plots were used to further investigate the effects of fats (alone and in different combinations) on probiotic stability. For the matrix containing palm stearin, fully hydrogenated palm kernel oil, and fully hydrogenated coconut oil (alone and in combination), the lowest die-off was observed when fully hydrogenated coconut oil was added at the highest level (Figure 1). Similar results were observed for the combination of palm stearin, fully hydrogenated coconut oil, and cocoa powder (Figure 2).

The highest mortality was observed when cocoa powder was added at the highest level (Figures 2 and 3). This could be removed from the formulation, leaving fully hydrogenated palm kernel oil, fully hydrogenated coconut oil, palm stearin, or a combination of the foregoing as potential protective matrices.

2.3 Conclusion

This example shows that when a coating matrix comprising 100% fat (i.e., 100% hydrogenated palm kernel oil) is used alone as a coating matrix, it can reduce the degradation of probiotic microorganisms in a fermented milk product. The degradation can be further reduced by adding the adjuvant described in Example 1.

This example also shows that a matrix comprising fully hydrogenated coconut oil and/or fully hydrogenated palm kernel oil provides very low mortality, palm stearin provides low mortality, and cocoa powder is less effective.

3. Embodiment 3 — Electron microscopy

3.1 Materials and Methods

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

The beads were frozen to -20ºC and fractured using a razor blade. The beads were packaged with the fracture side facing up, loaded in a scanning electron microscope (SEM TM4000), and cooled to -30ºC while the chamber was evacuated. The samples were then prepared for observation.

3.2 result

Clear phase separation was seen between the yogurt and fat portions of the composition, as well as distinct probiotic populations that appeared to be entrapped in a mucus coating—either exopolysaccharides or in combination with biofilm material—at higher magnification (Figure 4).

Figure 5 shows a clear phase separation between yogurt and hydrogenated palm kernel oil (HPKO). The probiotic population in the biofilm is demarcated by the red boundary.

In Figure 6, the mucus exopolysaccharide/biofilm network is clearly visible, and in Figure 7, individual rod-shaped bacterial cells can be seen encased in the exopolysaccharide/biofilm.

3.3 Conclusion

This example shows that once embedded in the matrix material, the probiotics encapsulated in the coating matrix can form a mucus exopolysaccharide or biofilm layer.

4. Embodiment 4 — Biofilm formation

4.1 Materials and Methods

Lactobacillus rhamnosus HN001 has been shown to produce biofilm by growing and attaching to glass wool or activated carbon surfaces. Cultures of HN001 were prepared by incubation in MRSB medium at 37ºC overnight. The culture (1 ml) was then inoculated into a 500 ml flask containing 100 ml yogurt. Glass wool (2.5 g) was added to the flask, followed by incubation at 37ºC in a rotary incubator for 18 to 24 hours. The culture flasks were then stored at 30ºC and evaluated for cell number, pH, and extracellular polysaccharides (EPS) or biofilm formation on days 4, 7, and 28. The same experiment was performed on the flask using 1 gram of 0.6 to 1.1 mm washed granular activated carbon.

Remove samples at the beginning of storage, and on days 7 and 28 for viable probiotic cell counts. Remove the glass wool (or activated charcoal) and rinse three times with phosphate-buffered water through a mini-sieve. Place the rinsed material in a 250-mL Schott bottle containing 20 g of sterile glass beads. Add salted buffered peptone water (9 mL) and vigorously stir the beads and culture for 10 minutes to separate the cells from the glass wool or charcoal.

Serial dilutions were made in 9 ml of saline-buffered protein water and plated onto MRSA agar, followed by incubation at 37°C for 48 hours and cell counts. Cell counts were also performed on the remaining yogurt (planktonic or free cells).

4.2 result

Table 4 shows that a large number of live HN001 cells remained attached to the surface of glass wool or charcoal during the 28-day shelf life, maintaining a relatively higher survival rate than the free cells in yogurt.

Table 4. Comparison of cell counts and pH values of cells attached to glass or carbon surfaces and cells that remained unattached (planktonic cells) Sample Time (days) 0 7 28 HN001 ( log cfu/g ) Glass wool (biofilm) 8.76 8.06 8.08 Carbon (biofilm) 7.26 8.47 8.93 Glass wool (floating) Not applicable 7.19 6.07 Carbon (floating) Not applicable 7.07 7.06 pH Yogurt with glass wool 4.22 3.85 3.49 Yogurt with charcoal 4.22 4.02 3.64

The samples attached to the glass wool were stained with crystal violet and examined under oil immersion with an optical microscope at 100x magnification (Figure 8). Clusters of HN001 cells were seen attached to the surface of the glass wool. These attached clusters provided evidence of the ability of HN001 to form biofilms, maintaining the viability of the cells in the yogurt.

4.3 Conclusion

This example shows that Lactobacillus rhamnosus HN001 is able to form a biofilm and this can improve cell viability when stored in yogurt for a long time.

5. Embodiment 5 — Identification of exopolysaccharide components in biofilm cells using Raman spectroscopy

Extracellular polysaccharides (EPS) are the main components of the extracellular biofilm matrix and have the function of anchoring microorganisms to the surface, which is the process that initiates biofilm formation.

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

5.1 Materials and Methods

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

The beads of sample AT03 (see Example 1) were removed from the yogurt and gently rolled on a paper towel to remove excess yogurt. Sections of the beads were cut to approximately 2 mm thick. The sections were placed on a glass slide and covered with a cover slip; double-sided tape was used to hold the cover slip down and act as a spacer of approximately 2 mm. The sample temperature was maintained at 4°C throughout the measurement. Raman spectra were collected with a 50×10 step, 75×25 μm. Each spectrum was collected using a 1 s integration time, 532 nm excitation wavelength, 100x oil immersion objective (NA 1.30), and a 50 μm pinhole. At least 10 spectra were collected for each bead at each time point: 0, 15, 30, 90/120, 218 days.

As a control, Lactobacillus rhamnosus HN001 samples were grown using room temperature yogurt as the culture medium and ZnSe surfaces to induce biofilm formation.

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 pre-processed using cosmic ray rejection, baselining and minimum-maximum normalisation. Raman spectra were analysed using principal component analysis, multivariate curve resolution and comparison with reference materials.

5.2 result

Principal component analysis of the Raman spectroscopy data of Lactobacillus rhamnosus HN001 grown using ambient yogurt as a culture medium and ZnSe surfaces to induce biofilm production showed three distinct peaks in the 350-600 cm ^-1 spectral region associated with the presence of rhamnose (Figure 9). This may indicate the presence of EPS components.

The Raman spectrum of the AT03 bead structure doped with Lactobacillus rhamnosus HN001 showed similar peaks (Figure 10). Similarly, three EPS-related peaks appeared at 360, 430, and 530 cm ^-1 .

5.3 Conclusion

This example shows that Raman spectroscopy can be used to detect the presence of EPS in particles containing probiotics.

6. Embodiment 6 — Identification of biofilm exopolysaccharides by lectin staining

6.1 Materials and Methods

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

A bead was removed from the yogurt and gently rolled on a paper towel to remove excess yogurt. Sections of the beads were cut to approximately 2 mm thick. Sections were stained with 5 μL of 0.1 mg/mL fluorescein-tagged lectin stain, 1 μM DAPI, and 0.2% fast green. Lectin stains were concanavalin A, wheat germ agglutinin, and lentin. Samples were stained for at least 15 minutes before imaging using an inverted Zeiss LSM800 confocal fluorescence microscope. 407 nm laser was used for excitation of DAPI, 488 nm laser for excitation of fluorescein-tagged lectin, and 561 nm for excitation of fast green.

6.2 result

Figures 11 to 13 show fluorescent micrographs of beads stained with different lectins. The images are from one field recorded using a 63x oil immersion objective. One bead was split to allow different sections to be stained with different staining mixtures.

Lectin staining revealed the presence of sugars (glucose, galactose, rhamnose, galactosamine, glucosamine, mannose, and fucose) consistent with the EPS structure of Lactobacillus rhamnosus HN001. The presence of mannose (Figure 11), N-acetylglucosamine (Figure 12), and fucose (Figure 13) provided strong evidence for the presence of HN001-associated biofilm in the beads.

6.3 Conclusion

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

7. Embodiment 7 — Other coating substrates

7.1 Materials and Methods

Beads containing Lactobacillus rhamnosus HN001 were prepared as described in Example 1 using the matrix formulation shown in Table 5.

Table 5. Matrix composition (% w/w) Sample PS HCO CP HPKS HPKO CB Suc 492 Lec SMP NaCl Lac AT03 32 41 0.3 0.5 26 0.12 AT03-1 74 26 AT03-2 59 41 AT07 16 16 41 0.3 0.5 26 0.12 AT07-1 50 50 AT07-2 37 37 26 AT07-3 29.5 29.5 41 AT17 4 20 4 4 41 0.3 0.5 26 0.12 AT17-1 19 62 19 AT17-2 14 46 14 26 AT17-3 11 11 37 41 AT21 99.5 0.5 AT21-1 73.5 0.5 26 AT24 100 ATX-1 86.5 13.5 ATX-2 86.5 13.5 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 heat skim milk powder); Lac, lactose.

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

7.2 result

exist 30°C Lower cultivation

After incubation at 30°C, the cell counts of Lactobacillus rhamnosus HN001 are shown in Table 6.

Table 6. Cell counts of Lactobacillus rhamnosus HN001 after incubation at 30ºC ( log CFU /g particles) Sample Cultivation time (months) 0 0.5 1 2 4 6 9 12 AT03 9.08 10.37 9.65 9.26 9.10 8.71 * * AT03-1 8.99 9.62 9.37 8.01 8.53 4.46 * * AT03-2 8.97 9.64 9.44 9.09 8.24 7.51 4.23 * AT07 8.74 9.86 9.78 9.30 8.07 7.46 2.40 2.40 AT07-1 8.74 9.05 9.80 8.59 7.05 6.46 * * AT07-2 8.47 9.17 9.83 8.92 7.57 6.90 * * AT07-3 8.87 9.65 9.89 9.17 9.22 8.57 6.90 2.48 AT17 9.09 10.60 9.76 9.40 9.18 8.67 2.24 2.24 AT17-1 8.90 8.57 8.92 8.07 7.12 6.48 2.40 2.40 AT17-2 9.10 9.80 9.53 8.58 7.13 6.15 4.40 2.48 AT17-3 8.97 9.20 9.45 8.75 8.47 8.07 4.80 4.91 AT21 8.96 9.21 9.67 9.32 9.29 8.74 7.81 7.63 AT21-1 9.08 10.16 9.95 9.65 9.52 8.02 6.19 6.49 AT24 8.98 8.74 9.01 8.75 8.42 6.96 6.45 4.26 ATX-1 9.08 9.53 9.39 9.22 9.25 8.61 8.11 8.10 ATX-2 8.90 8.30 9.30 8.10 8.95 7.63 6.91 5.52 *Less than 100 colonies per plate, too low for accurate determination.

exist 25°C Lower cultivation

After incubation at 25ºC, the cell counts of Lactobacillus rhamnosus HN001 are shown in Table 7.

surface 7. exist 25°C After culturing under HN001 Cell counts ( log CFU / gram particles) Sample Cultivation time (months) 0 0.5 1 2 4 6 9 12 AT03 8.95 10.11 10.16 9.32 9.35 9.13 7.33 7.27 AT03-1 9.04 8.96 8.72 8.71 7.31 5.80 5.77 3.44 AT03-2 9.00 8.79 9.56 8.98 7.23 8.87 6.62 6.57 AT07 8.97 9.88 9.78 9.43 9.05 8.15 8.10 6.52 AT07-1 9.11 7.93 8.52 8.53 8.98 6.36 6.98 6.75 AT07-2 8.67 8.70 9.04 8.90 6.16 7.62 6.36 6.61 AT07-3 8.87 8.83 9.54 9.06 8.98 8.43 7.36 6.81 AT17 8.92 10.25 10.41 9.45 9.45 9.47 8.60 7.25 AT17-1 8.93 8.88 8.95 8.90 7.09 7.59 6.52 5.11 AT17-2 9.03 9.53 9.50 9.43 7.98 7.49 6.71 5.91 AT17-3 9.09 9.01 9.55 8.93 9.04 7.09 6.39 6.41 AT21 8.57 9.25 9.41 9.05 7.74 8.39 8.69 5.84 AT21-1 8.98 9.67 9.70 9.13 7.88 8.13 8.66 4.56 AT24 9.01 8.55 8.59 8.82 6.05 7.45 7.04 6.62 ATX-1 8.89 9.53 9.67 9.03 9.58 9.49 9.16 8.35 ATX-2 9.01 8.18 8.12 7.87 8.35 8.02 7.18 6.45

7.3 Conclusion

This example shows that various coating matrices can reduce the degradation of probiotic microorganisms in fermented milk products, and some formulations produce significant stability.

8 . Embodiment 8 - Biofilm formation detection by lectin staining

8.1 Materials and Methods

According to Example 1, beads containing Lactobacillus rhamnosus HN001 were prepared using the matrix formulation described in Table 8, wherein 2.5% of HN001

surface 8. Matrix composition formula Matrix composition ( w/w ） AT07 16% hydrogenated coconut oil, 16% palm stearin, 41% sucrose, 0.3% sorbitan tristearate, 0.5% soy lecithin, 26% milk solids, 0.12% sodium chloride AT24 100% Hydrogenated Palm Kernel Oil AT17-1 62% Hydrogenated Coconut Oil, 19% Palm Stearin, 19% Hydrogenated Palm Kernel Stearin ATX-1 86.5% cocoa butter, 13.5% lactose

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

Remove the beads containing probiotics from the yogurt and discard the excess yogurt. Use the beads from time point zero before filling the yogurt.

Lectins conjugated to fluorophores are used to stain lipid-based beads for conjugate fluorescence microscopy. Because lectins bind to sugar residues in polysaccharides present in exopolymers, fluorescently labeled lectins have been previously used to study exopolymers in biological membranes.

Three lectins were selected for use on the beads: wheat germ agglutinin (WGA), concanavalin A (ConA), and UEA-I-I. WGA is specific for N-acetylglucosamine, ConA is specific for α-mannose and α-glucose, and UEA-I-I is specific for α-fucose. These were used in conjunction with DAPI to stain cells, Nile red to stain fats, or Fast Green to stain proteins.

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

surface 9. Dye mixture Dye mixture dye 1 dye 2 dye 3 1 DAPI 0.1 mg/ml WGA-640 0.1 mg/ml Nile Red 0.5% w/v 2 DAPI 0.1 mg/ml ConA-640 0.1 mg/ml Nile Red 0.5% w/v 3 DAPI 0.1 mg/ml UEA-I-I-640 0.1 mg/ml Nile Red 0.5% w/v 4 DAPI 0.3 mg/ml Fast Green 0.2% w/v Nile Red 0.5% w/v 5 DAPI 0.1 mg/ml WGA-Fl 0.1 mg/ml Fast Green 0.2% w/v 6 DAPI 0.1 mg/ml ConA-Fl 0.1 mg/ml Fast Green 0.2% w/v 7 DAPI 0.1 mg/ml UEA-I-I-Fl 0.1 mg/ml Fast Green 0.2% w/v

Confocal fluorescence images were collected from the stained beads using an inverted Zeiss LSM 800 microscope with a 63x oil immersion objective. Table 10 lists the excitation wavelength and emission collection range.

Table 10. Excitation and emission wavelengths dye Excitation (Nano) Emission and collection range (nanometers) DAPI 405 400-595 WGA-640R 640 656-700 ConA-640R 640 656-700 UEA-II Dylight 649 640 656-700 Fast Green 640 656-700 Nile Red 561 570-700 WGA- Luciferin 488 410-530 ConA- luciferin 488 410-530 UEA-II- fluorescein 488 410-530

8.2 result

AT07

At time zero, differential fluorescence of WGA was observed, indicating the presence of N-acetylglucosamine in the initial composition. WGA binding was mainly at the edges of protein bodies. This binding may be a result of the inclusion of milk solids in the particles, as cow milk contains N-acetylglucosamine (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).

After three months of culture, there were clear areas of WGA fluorescence concentration, which were no longer located at the edges of protein bodies but were located within the cell clusters.

At time zero, dense areas of ConA fluorescence were observed that did not coincide with areas of cell density. This may also be a result of the presence of milk solids in the matrix formulation.

After three months in culture, the areas of perceptible ConA fluorescence increased and correlated with areas of higher cell density.

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

AT24

No data were collected from time point zero using ConA or UEA-I-I dye.

At time point zero, cells were stained with WGA. After 3 and 6 months of culture, WGA fluorescence was observed around cells; this was most pronounced after 6 months. After 6 months, ConA fluorescence appeared in areas of dense cell populations. For UEA-I-I fluorescence, there was a small amount of UEA-I-I aggregation at 3 and 6 months, however, this did not appear to be closely related to cell location. These observations are consistent with biofilm formation.

AT17-1

The lectin dyes used to stain sample MVP9 have fluorophores with excitation maxima at 642 or 649 nm, allowing for simultaneous staining with Nile red, which stains fat. With one exception, for the one-month time point, the UEA-I-I dye was used, which has a fluorophore with an excitation maximum at 495 nm. When this dye was used, Nile red was not used and Fast Green was added instead due to interference between the dyes.

At time point zero, small areas of bacteria were present within the fatty matrix, and WGA stained cells in these areas. One month later, more extensive fluorescence from WGA surrounding the cell area was evident, indicating that the presence of N-acetylglucosamine extended beyond the immediate vicinity of the cell, consistent with the formation of a biofilm of extracellular polymeric substances containing N-acetylglucosamine.

At time point zero, no differential fluorescence was observed with ConA, but zones of fluorescence were seen where the dye was concentrated in non-fat areas. Within the cell region, there were no zones of weak and strong ConA fluorescence. The zones of ConA fluorescence did not appear to be associated with the cell. Therefore, the dye was concentrated in non-fat areas rather than specifically staining mannose or glucose. One month later, differential fluorescence was observed for ConA, i.e., the fluorescence was not due solely to concentration in non-fat areas. As with WGA, heterogeneous and widespread zones of fluorescence were observed around the cells, consistent with secretion of extracellular polymeric substances and biofilm formation. Not all cell regions were stained with ConA; at this time point, there were cell regions where ConA had just concentrated. In addition, at one month, there were areas of high ConA accumulation around the cell border, and ConA accumulated in the interior but also in the non-fat areas.

At time point zero, UEA-I-I stained cells in the beads in a manner similar to WGA. The cells were primarily confined to clusters in the adipose stroma. At one month, cells had migrated to the outer regions of the beads that were not adipose, and fluorescence due to UEA-I-I fluorescence was observed outside the cells, consistent with biofilm formation.

ATX-1

Only three months of samples were analyzed.

After three months, there were prominent areas of WGA fluorescence associated with areas of cell density. There were also prominent areas of ConA fluorescence associated with cells, and small areas of UEA-I-I fluorescence. All of these observations are consistent with biofilm formation.

8.3 Conclusion

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

9. Embodiment 9 - Stability of various products

9.1 Materials and Methods

Probiotic granules containing 0.25% Lactobacillus rhamnosus HN001 were prepared using the four matrix formulations described in Table 11.

Table 11. Matrix composition formula Matrix composition ( w/w ) AT07-3 29.5% Palm Stearin, 29.5% Hydrogenated Coconut Oil, 41% Sucrose AT21 99.5% cocoa butter, 0.5% soy lecithin AT24 100% Hydrogenated Palm Kernel Oil ATX-2 86.5% Hydrogenated Coconut Oil, 13.5% Lactose

Weigh the dry ingredients (sugar, lactose) into a Stomacher homogenizing bag, seal with a bag sealer and store at room temperature until needed. Weigh the lipid components (fats and lecithin) into a sterile bottle and store at 3-4°C until needed. Weigh 0.25 g/100 g lipid of Lactobacillus rhamnosus HN001 into a sterile container and store at 3-4°C until needed. Autoclave the silicon mold.

Prepare the granules using the following method: 1. Melt the lipid component in a 60-70ºC water bath. 2. Transfer the lipid component to a 45ºC water bath, ensuring that the lipid and fat cool to 45ºC before adding the probiotics (minimum 30 minutes). 3. Add the probiotics (0.25 g) to the melted lipid, stirring with a sterile spoon to ensure uniformity. 4. Slowly add the remaining dry ingredients, stirring between each addition. 5. Keep stirring at all times to ensure that all ingredients are combined to produce a smooth appearance. 6. Pour the contents into a sterilized silicone mold. 7. Using a spatula, smooth the mixture into the mold cavity and scrape off the excess. 8. Place the molds in a pre-sterilized container and store at 4ºC for 24 hours.

The resulting particles are approximately 5 mm in diameter and weigh approximately 0.05 g each.

Five products were evaluated as shown in Table 12. The viscosity of the products was measured using a Brookfield viscometer.

Table 12. Evaluated products Product Type Product Name Parameters Acidic drinks Keri® Orange Juice pH: 3.71 Viscosity: 3.16 cP Neutral drinks Fonterra® Sports Shake pH: 6.74 Viscosity: 26.8 cP food Dolmio® Cheese Sauce pH: 3.88 Viscosity: 2130 cP food Bega® Cream Cheese Spread pH: 5.49 Viscosity: 1557 cP High water activity products Vitasoy® Prebiotic Oat Milk pH: 6.49 Viscosity: 4.68 cP

A 200 ml sample of each product was used and three pellets were added to each sample using sterile tweezers, soaking the pellets in the top 10 to 20 ml. The samples were then incubated at 30ºC.

At each sampling time point, recover three pellets and place in a bell tube. Add 15 ml of MRSB medium preheated to 45°C and thaw the sample in a 45°C water bath for 5 minutes. Remove the sample from the water bath and gently shake/vortex to mix.

Prepare duplicate dilutions. Place the appropriate sample dilution (50 μl) on a pre-poured agar plate ("drop plate method"). Three dilutions (i.e. 10 ^-6 , 10 ^-7 and 10 ^-8 ) are required to obtain a countable range.

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

9.2 result

Tables 13-17 show the number of surviving Lactobacillus rhamnosus HN001 at the initial setup and after 1, 2, 3, and 4 months of culture.

Table 13. Shelf life in orange juice (Log ₁₀ CFU/mL) formula Time (month) 0 1 2 3 4 AT07-3 8.41 8.49 7.93 6.33 3.13 AT21 8.54 8.27 5.78 ＜10 ^-3 2.56 AT24 8.87 7.93 6.40 4.30 ＜10 ^-2 ATX-2 9.23 8.52 7.13 ＜10 ^-3 2.23

Table 14. Shelf life in sports shakes (Log ₁₀ CFU/mL) formula Time (month) 0 1 2 3 4 6 AT07-3 8.41 9.12 8.36 7.03 7.94 7.27 AT21 8.54 8.75 7.08 6.28 6.85 6.76 AT24 8.87 8.48 6.48 5.81 6.47 6.17 ATX-2 9.23 9.35 8.37 8.07 8.00 7.57

Table 15. Shelf life in cheese sauce (Log ₁₀ CFU/mL) formula Time (month) 0 1 2 3 4 6 AT07-3 8.41 9.35 9.13 8.72 9.18 8.54 AT21 8.54 9.34 8.80 7.79 8.96 8.70 AT24 8.87 9.23 8.41 6.56 8.18 6.68 ATX-2 9.23 9.17 7.77 5.53 7.12 7.22

Table 16. Shelf life in cream cheese spread (Log ₁₀ CFU/mL) formula Time (month) 0 1 2 4 AT07-3 9.79 9.85 9.14 9.16 AT21 9.63 9.20 8.66 8.16 AT24 8.60 8.51 8.37 7.81 ATX-2 8.52 9.30 8.99 8.93

Table 17. Shelf life in prebiotic oat milk (Log ₁₀ CFU/mL) formula Time (month) 0 1 2 3 4 6 AT07-3 8.41 8.64 6.19 5.53 ＜10 ^-2 ＜10 ^-2 AT21 8.54 7.62 7.47 ＜10 ^-3 ＜10 ^-2 ＜10 ^-2 AT24 8.87 7.58 5.78 ＜10 ^-3 2.12 ＜10 ^-2 ATX-2 9.23 7.83 5.60 7.65 6.33 5.03

9.3 Conclusion

This example shows that a coating matrix can reduce the degradation of probiotic microorganisms in a variety of products, even when stored at or above ambient temperature for long periods of time.

10. Embodiment 10 — Bacterial strains

10.1 Materials and Methods

Two groups of granules were prepared as described in Example 9, the first group comprising Lactobacillus rhamnosus HN001 and the second group comprising Lactobacillus paracasei subsp. casei IM514.

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 one hour at 20ºC was performed before adding the bacteria to the melted lipid component.

The granules were added to thick UHT yogurt formulated for ambient storage and stored at 30ºC. Cell counts were performed regularly for each bacterial strain.

10.2 result

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

Table 18. Shelf life in UHT yogurt (Log ₁₀ CFU/mL) germ Time (month) 0 0.5 1 2 4 Lactobacillus rhamnosus HN001 8.38 9.06 9.36 8.29 7.47 Lactobacillus paracasei subsp. casei IM514 9.10 7.11 7.04 7.68 7.23

10.3 Conclusion

This example shows that particles can provide extended shelf life for multiple bacterial strains under ambient conditions.

11. Embodiment 11 — Lectin staining for biofilm formation

11.1 Materials and Methods

Probiotic beads/granules were prepared using Lactobacillus rhamnosus HN001 or Lactobacillus rhamnosus GG (LGG), and the matrix formula was shown in Table 19.

Table 19. Matrix formula Sample Probiotic content ( w/w ) Matrix composition ( w/w ) Particle size Cultivation Sterilization 1 2.5% HN001 100% Ako Comp About 5 mm beads 30°C Sterile addition 2 2.5% LGG 100% Ako Comp About 5 mm beads 30°C Sterile addition 3 2.5% LGG 99.5% cocoa butter, 0.5% soy lecithin About 5 mm beads 30°C Sterile addition 4 10% HN001 100%Ako Comp 2-3 mm small particles 25°C Post-pasteurization 5 10% LGG 100%Ako Comp 2-3 mm small particles 25°C Post-pasteurization 6 10% HN001 99.5% cocoa butter, 0.5% soy lecithin 2-3 mm small particles 25°C Sterile addition 7 10% LGG 99.5% cocoa butter, 0.5% soy lecithin 2-3 mm small particles 25°C Post-pasteurization 8 10% LGG 99.5% cocoa butter, 0.5% soy lecithin 2-3 mm small particles 25°C Sterile addition

Ako Comp ingredients (10% probiotic content) include: 26g Akosoft 36 60g Akofine R 4g polyglycerol polyricinoleate (PGPR) 10g freeze-dried probiotic powder Total (100g)

Ako Comp ingredients (2.5% probiotic content) include: 28.2g Akosoft 36 65g Akofine R 4.3g Polyglycerol Polyricinoleate (PGPR) 2.5g Freeze-dried probiotic powder Total (100g)

Akosoft 36 (hydrogenated coco-glyceride) and Akofine R (hydrogenated vegetable oil) are available from IXOM (Melbourne, Australia).

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

The fat ingredients and emulsifier (PGPR or soy lecithin) are mixed at 75ºC with continuous stirring until all ingredients are combined. Add the freeze-dried probiotic powder and stir for 10-20 seconds. The suspension is then granulated and the granules are ground into powder.

For all samples, the granules/powders were incubated in thick UHT yogurt at the above temperature for two weeks as described in Example 1.

As described in Example 8, the pellets were removed, cut in half, dyed with dye mixture 1-4, and observed by confocal fluorescence microscopy.

11.2 result

Exemplary samples (Samples 1–3)

For sample 1, intense areas of fluorescence for WGA, ConA, and UEA-I were observed that extended outside the cells, which is consistent with the presence of a biofilm.

For sample 2, only a small area of strong WGA fluorescence was observed, and no area of strong ConA fluorescence was observed. Areas of UEA-I fluorescence extending outside the cells were observed, consistent with the presence of a biofilm.

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

Overall, samples 1-3 showed evidence of biofilm formation, as WGA and UEA-I fluorescence extended beyond the cell location defined by DAPI staining, and lectin staining was not limited to the shape of the cells. ConA staining, consistent with the presence of a biofilm, was only observed in pellets 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.

Comparison samples (samples 4-8)

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

Due to sample limitations, ConA staining was not performed on sample 5. The areas of intense WGA fluorescence were consistent with cells, and only small areas of intense UEA-I fluorescence were observed.

For sample 6, strong WGA fluorescence areas extending outside the cells were observed, however, not all cell clusters showed strong WGA fluorescence. Strong fluorescence from ConA was not observed. In sample 6, only some cell areas were stained with UEA-I.

For sample 7, WGA fluorescence was unexpectedly concentrated at the fat rim which does not necessarily coincide with the cells. ConA staining is usually restricted to accumulation in non-fat areas. Small areas of intense UEA-I fluorescence were observed around the cells.

Sample 8 was the same as sample 7 except that AD sterilization was used instead of post-pasteurization. The distribution of WGA fluorescence was different when post-pasteurization was not performed. Intense WGA fluorescence was observed, but this did not extend beyond the cells. Areas with large numbers of loosely aggregated cells were observed. ConA fluorescence was not observed. UEA-I showed variable staining intensity, however it appeared to stain only cells, not extracellular polymeric substances.

Overall, samples 4-8 showed no areas of intense ConA staining, except for a very small area of intense ConA fluorescence, which stained the yogurt inclusions in the beads. Only small areas of intense WGA and UEA fluorescence were observed, which did not extend beyond the cells or were not associated with every cell cluster in the matrix. Samples 4–8 showed no evidence of extensive biofilm formation as seen in samples 1–3.

11.3 Conclusion

This example shows that beads formed using the matrix and methods of the present invention show lectin staining that extends beyond the cells, indicating widespread biofilm formation.

In contrast, comparative particles formed using comparative methods generally lacked lectin staining that extended beyond the cells, indicating that such particles did not contain the same extensive biofilm as the particles of the present invention.

12. Embodiment 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.

WVTR was measured using aliquots of pellet samples (20–60 mg) at 95% relative humidity by weighing in an intrinsic sorption microbalance in a dynamic vapour sorption (DVS) instrument (Surface Measurement Systems, Appleton, Middlesex, UK). WVTR was measured within a 5-min period (i.e., from 7 to 12 min) 7 min after adjusting the relative humidity to 95%.

WVTR is calculated using the following formula: WVTR=dm⁄(A‧dt) Where dm = the mass of water absorbed in time dt, A = the total surface area of the sample calculated based on particle size measurement. WVTR is reported in g/m ² /24h.

Water activity was determined by Aqualab 4TE (Addium Inc, Pullman, WA, USA). Particle size of the powders was measured using a stereo microscope (range 10 to 1000 μm) and then analyzed using “Image pro plus”.

12.2 result

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

Table 20. Surface area to volume ratio (SA/V), water vapor transmission rate (WVTR) and water activity of particles Sample formula Particle size SA/V WVTR ( g/ ^m2 /24h ) Water activity AT03-1 74% HCO, 26% skim milk powder 3–5 mm 1.45 98.4 0.68 AT03-2 59% HCO, 41% sucrose 3–5 mm 1.45 103.8 0.55 AT21 99.5% CB, 0.5% lecithin 3–5 mm 1.45 53.7 0.49 AT21-1 73.5% CB, 26% skimmed milk powder, 0.5% soy lecithin 3–5 mm 1.45 168.2 0.65 ATX-1 86.5% CB, 13.5% lactose 3–5 mm 1.45 35.8 0.44 AT07-3 29.5% PS, 29.5% HCO, 41% sucrose 2–3 mm 1.98 152.8 0.49 AT24 100% HPKO 2–3 mm 1.83 41.7 0.43 ATX-2 86.5% HCO, 13.5% lactose 2–3 mm 1.77 65.1 0.42

Comparative example particles ("Comparative Example") were prepared according to the comparative example method described in Example 11 and compared with the particles of the present invention ("the present invention").

Granules containing 2.5% Lactobacillus rhamnosus HN001 and comparative granules containing 10% Lactobacillus rhamnosus GG were prepared using the method described in Example 9. Two base formulations were used - 100% cocoa butter and 100% Ako Comp. The medium granules had a diameter of approximately 5 mm and the powder granules were ground into a powder.

Whether using medium-sized particles or powders (even with similar surface area/volume ratios), the particles prepared using the method of the present invention have significantly lower WVTRs compared to the particles prepared using the comparative method (Table 21).

Table 21. Surface area to volume ratio (SA/V) and water vapor transmission rate (WVTR) of comparative particles Particle size Technology Base SA/V WVTR ( g/ ^m2 /24h ) 2–3 mm The invention Coco fat 1.8 119.4 Ako Comp 1.8 149 Comparison Example Coco fat 1.8 416 Ako Comp 1.9 381 10–1000 μm The invention Coco fat 48 twenty three Ako Comp 32 62 Comparison Example Coco fat 33 121 Ako Comp twenty three 196

The WVTR was further compared by varying the probiotic used and the particle size. Particles containing Lactobacillus rhamnosus GG (LGG) had a higher WVTR than those containing Lactobacillus rhamnosus HN001, and even at different particle sizes, particles produced using the comparative method always had a higher WVTR than particles produced using the method of the present invention (Table 22).

surface twenty two. Comparison of water vapor transmission rate ( WVTR ） Technology Composition Particle size WVTR （ g/ ^m2 /24h ） HN001 LGG Comparison Example Coco fat 2–3 mm 143.2 415.50 The invention Coco fat 3–5 mm 119.4 373.92 Comparison Example Ako Comp 2–3 mm 169.2 380.57 The invention Ako Comp 1–1.6 mm 148.9 133.67

12.3 Conclusion

This example shows the WVTR and water activity of the granules of the present invention and comparative examples.

without

The embodiments of the present invention will now be described with reference to the accompanying drawings, in which:

FIG. 1 is a contour plot showing coating matrices comprising palm stearin, fully hydrogenated palm kernel oil, and fully hydrogenated coconut oil, alone and in combination.

FIG. 2 is a contour plot showing coating matrices comprising palm stearin, fully hydrogenated coconut oil and cocoa powder alone and in combination.

FIG. 3 shows contour plots of coating matrices comprising fully hydrogenated palm kernel oil, fully hydrogenated coconut oil and cocoa powder, alone and in combination.

Figure 4 shows a scanning electron microscopy image of exopolysaccharide (EPS)/biofilm-encapsulated Lactobacillus rhamnosus HN001 in a matrix containing hydrogenated palm kernel oil at 100 times magnification.

FIG5 shows a scanning electron microscopy image of exopolysaccharide/biofilm-coated Lactobacillus rhamnosus HN001 in a matrix containing hydrogenated palm kernel oil at 250 times magnification.

FIG6 is a scanning electron microscopy image of exopolysaccharide/biofilm-coated Lactobacillus rhamnosus HN001 in a matrix containing hydrogenated palm kernel oil at 1,200 times magnification.

FIG. 7 shows a scanning electron microscopy image of exopolysaccharide/biofilm-coated Lactobacillus rhamnosus HN001 in a matrix containing hydrogenated palm kernel oil at a magnification of 4,000 times.

Figure 8 shows an optical microscope image of Lactobacillus rhamnosus HN001 clusters attached to the surface of glass wool strands. The image was taken at 100x magnification under oil immersion and stained with crystal violet.

Figure 9 shows the Raman spectrum of Lactobacillus rhamnosus HN001 induced to produce biofilm. Three distinct peaks (indicated by arrows) in the spectral region of 350 cm ^-1 to 600 cm ^-1 appear to be associated with the presence of EPS components.

Figure 10 shows the Raman spectra of bead structures doped with Lactobacillus rhamnosus HN001. The multivariate curve resolution estimated spectrum from the 120-day data analysis of bead sample AT03 is shown. The arrows indicate the same three EPS-related peaks.

Figure 11 shows the concanavalin (Con A) staining of sample MVP17 at day 90 of shelf life. Mannose/glucose stained with ConA appear orange; proteins are stained green (this staining also stains dead cells); and cells are stained light blue with DAPI.

Figure 12 shows the Wheat Germ Agglutinin (WGA) staining of sample MVP17 at day 90 of storage. N-acetylglucosamine stained with WGA appears orange; proteins are stained green (this staining also stains dead cells); and cells are stained light blue with DAPI.

Figure 13 shows the Ulex Europaeus Agglutinin (UEAI) staining of sample MVP17 at day 90 of shelf life. UEAI staining is shown in orange; proteins are stained green (this staining also stains dead cells); and cells are stained light blue with DAPI.

Claims (35)

A particle comprising a bioactive agent embedded in a biofilm, wherein the biofilm is embedded in a matrix, wherein the water vapor transmission rate (WVTR) of the matrix is 0.1 to 500. The particles as described in claim 1, wherein the matrix comprises at least 6% by weight of one or more lipids, preferably 10-100% by weight, and more preferably 30-100%. The particles as described in claim 1 or 2, wherein the WVTR of the matrix is 0.1 to 10, preferably 1 to 5. The particle as described in any one of claims 1 to 3, wherein the bioactive agent comprises a first microorganism, preferably a probiotic microorganism. The particle as described in claim 4, wherein the biofilm is produced by the first microorganism. The granules as claimed in claim 4 or 5, wherein the first microorganism is Bacillus , Bifidobacterium , Enterococcus , Lacticaseibacillus , Lactiplantibacillus , Lactobacillus , Lactococcus, Ligilactobacillus, Limosilactobacillus, Lentilactobacillus , Saccharomyces or Streptococcus ; preferably , the first microorganism is selected from the group consisting of: Bacillus coagulans ), Bifidobacterium animalis , Bifidobacterium breve , Bifidobacterium bifidum , Bifidobacterium longum , Lacticaseibacillus casei , Lacticaseibacillus paracasei , Lacticaseibacillus rhamnosus , Lactiplantibacillus plantarum subsp. plantarum , Lactobacillus acidophilus , Lactobacillus delbrueckii , Lactobacillus gasseri , Lactococcus lactis lactis ), Lactococcus lactis subsp. lactis , Lactococcus lactis subsp. lactis biovar diacetylactis , Leuconostoc pseudomesenteroides , Ligilactobacillus salivarius , Limosilactobacillus reuteri , Saccharomyces boulardii and Streptococcus thermophilus ; more preferably, the first microorganism is Lactococcus rhamnosus HN001. The granule as claimed in any one of claims 4 to 6, comprising the first microorganism in an amount of 10 ⁵ to 10 ¹² CFU per gram of the granule, preferably 10 ⁶ to 10 ¹⁰ CFU/gram, more preferably 10 ⁷ to 10 ⁹ CFU/gram, and most preferably about 10 ⁸ CFU/gram. The particle as described in any one of claims 4 to 7 further comprises a second microorganism different from the first microorganism, preferably, wherein the second microorganism is a probiotic microorganism. The particle as described in any one of claims 1 to 8 further comprises a non-microbial bioactive agent, preferably comprising vitamins, prebiotics, postbiotics and/or human milk oligosaccharides. The particle of any one of claims 1 to 9, wherein the biofilm is characterized by peaks at 360 cm ^-1 , 430 cm ^-1 and/or 530 cm ^-1 in principal component analysis of Raman spectroscopy data. The particle of any one of claims 1 to 10, wherein the biofilm is capable of being stained by concanavalin A, wheat germ agglutinin and/or Ulex europaeus agglutinin. The particles as described in any one of claims 1 to 11, wherein when the particles are contained in another food having a water activity of 0.90 to 1.00 for 2 weeks, the particles maintain a water content of 1% to 60% by weight in the matrix, preferably a water content of 10% to 60% by weight, and more preferably a water content of 20% to 60% by weight. A particle as described in any one of claims 2 to 12, wherein the one or more lipids: a. are solid at 20ºC, 25ºC and/or 30ºC; b. have a melting temperature of 50ºC or lower, preferably 30ºC to 50ºC; c. contain or consist of triglycerides; d. contain C12-C20 fatty acids or salts or esters thereof; or e. any combination of any two or more of (a) to (d). A particle as described in any one of claims 2 to 13, wherein the one or more lipids comprise one or more fatty acids and/or salts and/or esters thereof, and wherein: a. At least 1% by weight of the fatty acids and/or salts and/or esters thereof have a carbon chain length of 14 or less, preferably 1% by weight to 80% by weight; and/or b. At least 30% by weight of the fatty acids and/or salts and/or esters thereof have a carbon chain length of 16 or less, preferably at least 70% by weight, preferably 30% by weight to 90% by weight, and more preferably 70% by weight to 90% by weight. The particle of any one of claims 2 to 14, wherein at least 50% by weight of the one or more lipids are saturated fats. The particles as described in any one of claims 2 to 15, wherein the one or more lipids comprise or consist of: fully hydrogenated palm kernel stearin, palm stearin, fully hydrogenated coconut oil, hydrogenated palm kernel oil, or a mixture of any two or more of the foregoing. The particles as described in any one of claims 1 to 16, wherein the matrix comprises at least 10 wt % of fully hydrogenated coconut oil, preferably at least 20 wt %, and more preferably at least 30 wt %. The particle of any one of claims 1 to 17, wherein the particle has a particle size of 50 μm to 10 mm. The particle as described in any one of claims 1 to 18 further comprises at least one coating layer. A food or drink comprising the particles described in any of the preceding claims. A food or drink as claimed in claim 20, wherein the food or drink comprises: a. a protein powder, a protein shake, a protein shot, a protein gel or a sports nutritional formulation, b. a UHT drink, a UHT smoothie, c. an infant formula, a follow-on formula, a growing formula, a pediatric formula, a human milk fortifier, a children's food or drink, a maternal supplement or a maternal nutritional formulation, d. a gel, such as a heat-set gel, a sauce, a spread, a jam, a jelly, or honey, e. an acid-set gel, such as a gel), ambient stable yoghurt, stirred yoghurt, set yoghurt, or drinking yoghurt, f. neutral beverages, acidic beverages, water, juice, milk, smoothies, milkshakes, concentrated beverages, alcoholic beverages, soft drinks, kombucha, kefir, or ready-to-eat powdered mixes, g. bars, balls, cakes, cookies, muffins, or baked goods, h. medical foods, soups, desserts, puddings, custards, enteral preparations, preparations for senior or aged populations, tablets, capsules, or supplements, i. Preserves, gummy candies, candies, chocolates, fudge, truffles, chewing gum, frozen desserts or ice cream, j. seasonings, toppings or baking ingredients, k. cream, cheese or butter, or l. livestock food or pet food. The food or drink of claim 20 or 21, wherein the water activity of the food or drink is at least 0.2, 0.4, 0.6, 0.8 or 0.9. The food or drink as claimed in any one of claims 20 to 22, wherein the food or drink is stable at 30°C for at least 30 days, preferably at least 60 days, and more preferably at least 6 months. The food or drink as claimed in any one of claims 20 to 23, wherein the bioactive agent comprises a microorganism, and wherein the degradation rate of the microorganism measured at 30°C within 12 months is less than 7 log CFU/gram/year (log CFU/g/y), preferably less than 6, 5, 4, 3, 2 or 1 log CFU/g/y. A method for increasing the stability of a bioactive agent, the method comprising the following steps: a. embedding the bioactive agent in a biofilm, b. embedding the biofilm in a matrix having a WVTR of 0.1 to 500, and c. forming the matrix into particles. A method for increasing the stability of a bioactive agent, the method comprising the following steps: a. embedding the bioactive agent in a matrix with a WVTR of 0.1 to 500, b. inducing the bioactive agent to form a biofilm, and c. forming the matrix into particles, wherein steps b) and c) can be in any order. The method of claim 25 or 26, wherein the matrix comprises at least 6% by weight of one or more lipids. The method of any one of claims 25 to 27, wherein the bioactive agent comprises a first microorganism. The method of claim 28, wherein the biofilm is produced by the first microorganism. The method as described in any one of claims 25 to 29 further comprises the step of coating the particle in at least one coating layer. A method as described in any one of claims 25 to 30, wherein the particles comprise particles as described in any one of claims 1 to 19. The method as claimed in any one of claims 25 to 31, further comprising the following steps: d. combining the granules of step c with a culture medium having a water activity of at least 0.5 (preferably UHT-treated yogurt), and e. culturing the culture medium for 3 to 30 days. A method for producing a food or drink supplemented with a biologically active agent, the method comprising the method as described in claim 32, and further comprising the following steps: f. harvesting the particles from the culture medium of step e, and g. combining the harvested particles with a food or drink to obtain a food or drink supplemented with a biologically active agent. A method for producing a food or drink supplemented with a biologically active agent, the method comprising the method as described in any one of claims 25 to 32, and further comprising the step of combining the particles with a food or drink to obtain a food or drink supplemented with a biologically active agent. The method as described in claim 33 or 34, wherein the food or drink supplemented with the biologically active agent is the food or drink as described in any one of claims 20 to 24.