CA3079993C

CA3079993C - Composition and method for germinative compounds in probiotic food and beverage products for human consumption

Info

Publication number: CA3079993C
Application number: CA3079993A
Authority: CA
Inventors: Jordan E. Church; Gabriel F.K. Everett; Charles J. Greenwald; Jr. Lester LEVY
Original assignee: NCH Corp
Current assignee: NCH Corp
Priority date: 2017-11-03
Filing date: 2018-11-02
Publication date: 2022-09-20
Anticipated expiration: 2038-11-02
Also published as: WO2019090065A1; WO2019090065A8; EP3703721A4; CN111295197A; CA3079993A1; CO2020006844A2; PH12020550820A1; MX2020004473A; EP3703721A1; BR112020008712A2

Abstract

A composition and method for activating probiotic spores in food and beverage products, such as steeped teas, coffee, soups, and sauces. A nutrient-germinant composition comprises one or more L-amino acids, optionally one or more buffers to maintain the pH of the composition when added to water in a range of around 6-8, optionally D-glucose, D-fructose, or both D-glucose and D-fructose, and optionally, an osmoprotectant. The nutrient-germinant composition, one or more species of Bacillus spores, and a food or beverage product may be pre-mixed in any combination. Water is added to the mixture and heated to a temperature range of 42 to 100°C to germinate the probiotic spores prior to being consumed.

Description

COMPOSITION AND METHOD FOR GERMINATIVE COMPOUNDS
IN PROBIOTIC FOOD AND BEVERAGE PRODUCTS FOR HUMAN CONSUMPTION
[0001]
BACKGROUND

[0002] Recent scientific advances have shed light on the significance of the relationship between human gut bacteria (the microbiome) and general health.
The human microbiome is a product of our genetics, our diets, and our environment.

Probiotics may be used to alter the human microbiome. Probiotics for human consumption are available as capsules and as food products (e.g. yogurt, beverages, etc.). Although there are examples of probiotic tea on the market, there are none that include an activating component to germinate probiotic spores for improved efficacy.
Here, an invention is described that includes the provision of activating compounds with Bacillus bacterial spores for human probiotic consumption in tea, soups, gravies, sauces, hot chocolate, and other food and beverage products, particularly heated liquids and fluids.

Date recue / Date received 2021-11-30 BRIEF DESCRIPTION OF THE FIGURES

[0003] The system and method of the invention are further described and explained in relation to the following drawing wherein:
FIG. 1 shows photographs of Bacillus species in a nutrient germinant composition according to a preferred embodiment at time zero (when hot water is initially added) and after steeping in hot water for 60 minutes at various temperatures according to preferred methods of the invention, the darker spots indicate germinated bacteria and the lighter/brighter spot indicate non-germinated spores;
FIG. 2 shows photographs of Bacillus species and a nutrient germinant composition according to a preferred embodiment in steeped tea (black and green tea) according to a preferred method of the invention, the darker spots indicate germinated bacteria and the lighter/brighter spots indicate non-germinated spores.

DETAILED DESCRIPTION

[0004] Recent scientific advances have shed light on the significance of the relationship between human gut bacteria (the microbiome) and overall health.
The human microbiome is a product of our genetics, our diets, and our environment.

Probiotics may be used to alter the human microbiome in order to regulate the digestive system and bolster the immune system. Generally, probiotics are non-pathogenic bacteria that may include species of lactic acid bacteria (Lactobacillus), Bifidobacterium, and Propionibacterium. Species of Bacillus are also common as probiotics.
Probiotics have been widely used in animals as an alternative to hormones and antibiotics, in plants to enhance certain growth characteristics, and in humans as a digestive supplement. Probiotics for human consumption are available as capsules and as food products (e.g. yogurt, juice, etc.). Probiotics delivered in tea are also on the market.
Probiotic tea products contain probiotic bacteria packaged with tea in a porous bag. Tea bags are steeped for a period of time in hot water and are consumed.
Alternatively, Kombucha tea is a type of probiotic drink in which a symbiotic culture of bacteria and yeast (SCOBY) is added to tea containing sugar. The SCOBY then acts to ferment the sugar in the tea to produce vitamins and acids. In this case, the sugar acts as a substrate and the drink does not contain amino acids that are known to activate bacteria.

[0005] In the case of Bacillus species as probiotics (e.g. B. coagulans), the bacteria are supplied as spores. Spores represent a unique life stage of specific species of bacteria in that these species have the ability to transition into a dormant spore stage if environmental conditions are unfavorable. Spores are naturally dehydrated, are stable for extended periods of time, and are resistant to environmental stresses (e.g. heat, salt, pH, etc.). In the case of Bacillus spores in tea products, the spores are preferably more resistant to the high temperatures of steeping water.

[0006] In order for spores to return to a live, vegitatively growing state, they must first germinate. Spore germination is a multistep process in which spores are revived from the dormant state. The first step is one by which spores are activated and are induced to germinate, typically by an environmental signal called a germinant.
This signal can be a nutrient such as an L-amino acid, potassium ions (e.g. KCI), and/or natural osmoprotectant compounds (e.g. ectoine). Nutrient germinants bind to receptors in the inner-membrane of the spore to initiate germination. Additionally, sugars have been shown to increase the binding affinity of L-amino acids for their cognate receptors.

[0007] The germ inant signal initiates an irreversible cascade of events, also known as commitment. Commitment is fast and is typically >90% complete in 2 minutes.
As germination progresses, the spore rapidly takes on water. As the spore absorbs water it consequently loses its refractivity. This loss of refractivity towards the end of the first phase in the germination process allows spore germination to be observed via phase-contrast microscopy. The initial phase is typically complete within 30 to 60 minutes depending on the temperature. The second phase of germination is an outgrowth step in which the spore's metabolic, biosynthetic, and DNA
replication/repair pathways initiate. The outgrowth period includes a ripening period in which no morphological changes (such as cell growth) occur, but the spore's molecular machinery (e.g. transcription factors, translation machinery, biosynthesis machinery, etc.) is activated. This period can vary in length based on the initial resources that are packaged with the spore during the process of sporulation.

[0008] It is known that spores can be induced to germinate via heat-activation.
Spores of various Bacillus species have been heat-activated at strain specific temperatures (e.g. B. subtilis spores can be heat-activated at 75 C for 30 minutes while B. licheniformis spores can be heat-activated at 65 C for 20 minutes). The heat-activation is believed to cause a transient, reversible unfolding of spore proteins. If the spore proteins are not presented with a nutrient germ inant, such as L-alanine, they will refold and effectively reverse the germination process.

[0009] The present invention describes a composition and method for activating probiotic spores in in food and beverage products, such as steeped teas, soups, gravies, sauces, hot chocolate, coffee, and other products, particularly heated liquids and fluids. The method includes the provision of nutrient germinants with Bacillus spores that will be heat activated and subsequently germinated with nutrient germ inants while the food or beverage product is being heated, such as while tea is steeping in hot water. According to one preferred embodiment, the two probiotic components (bacterial spores and nutrient germinants) may be provided as dry components in an automatic coffee maker pod (e.g. Keurig , Nespresso , Tassimo , etc.) containing tea or another food or beverage product. According to another preferred embodiment, the ingredients may be provided as mixed ingredients (nutrient germinant composition and spores) in a single serve packet to be added to tea or another food or beverage product, mixed components (nutrient germinant composition and spores) in a single bag containing tea or another food or beverage product, or as separated components in separate tea bags (e.g. two bags containing any combination of the nutrient germinant composition, spores, and tea) or other container for other food or beverage products. According to one preferred embodiment, spores can be heat-activated in the presence of nutrient germinants in a method that is described in U.S. Patent Application No 15/479,773.

[0010] Nutrient Compositions

[0011] Described herein are preferred embodiments of nutrient compositions that may contain one or more L-amino acids, D-glucose, 0-fructose, a biological buffer, a potassium ion source, and/or a natural osmoprotectant. All components in the nutrient formulation must be Generally Regarded as Safe (GRAS) for human consumption by the United States Food and Drug Administration. At the time of this writing, most, although not all, of the components described herein have been deemed GRAS.

[0012] According to one preferred embodiment, the nutrient germinant composition can include one or more L-amino acids. Preferred L-amino acid(s) included in the nutrient formulation are L-alanine, L-asparagine, L- valine, and/or L-cysteine. The choice of L-amino acids is determined by the species of Bacillus used in the probiotic formulation. The L-amino acids can be provided in the form of any suitable source, such as their pure forms and/or a hydrolysate of soy protein.

[0013] According to another preferred embodiment, the nutrient germinant composition can optionally contain an amount of 0-glucose and/or 0-fructose.
The choice of sugar is determined by the species of Bacillus used in the probiotic formulation.

[0014] According to another preferred embodiment, the nutrient germinant composition can optionally contain one or more sources of potassium ions.
Preferably, KCI can be included as a source of potassium ions in the nutrient formulation.
The Date recue / Date received 2021-11-30 inclusion of potassium ions is determined by the species of Bacillus used in the probiotic formulation.

[0015] According to another preferred embodiment, the nutrient germinant composition can optionally contain one or more biological buffers. The biologic buffer is a buffer that can buffer the nutrient formulation and/or nutrient-spore formulation, to maintain the formulation at the proper pH for spore germination (about pH 6-8).
Preferred biologic buffers include, but are not limited to, a phosphate buffer or a HEPES
sodium salt. According to another preferred embodiment, monosodium phosphate and disodium phosphate, preferably used together, can be included in the nutrient formulation as buffers. According to another preferred embodiment, a HEPES
buffer may be used.

[0016] According to another preferred embodiment, the nutrient solution can optionally contain an osmoprotectant compound. Preferably, ectoine, a natural osmoprotectant produced by some species of bacteria, may be included. The inclusion of an osmoprotectant is determined by the species of Bacillus used in the probiotic formulation.

[0017] According to another preferred embodiment, the nutrient composition is any one of the nutrient compositions described in U.S. Patent Application No 15/479,773.

[0018] According to yet another preferred embodiment, a nutrient composition may contain ingredients in one or more of the above-reference categories of ingredients and may contain one or more ingredients from within those categories.

[0019] Spore Formulations

[0020] Preferred embodiments of spore formulations include one or more Bacillus species of spores, including but not limited to, Bacillus ficheniformis, Bacillus subtiffis, Bacillus amyloliquiefaciens, Bacillus polymyxa, Bacillus thuringiensis, Bacillus megaterium, Bacillus coagulans, Bacillus lentus, Bacillus clausii, Bacillus circulans, Bacillus firmus, Bacillus lactis, Bacillus laterosporus, Bacillus laevolacticus, Bacillus polymyxa, Bacillus pumilus, Bacillus simplex, Bacillus sphaericus, Bacillus sonorensis, Bacillus, homeckiae, Bacillus axarquiensis, Bacillus mucilaginosus, Bacillus olivae, and any combinations thereof. All strains in the probiotic formulation must be Generally Regarded as Safe (GRAS) for human consumption by the United States Food and Drug Administration. At the time of filing this application, several, although not all, of the above mentioned strains have been deemed GRAS. Any Bacillus species subsequently determined to be GRAS are included here as suitable for use with embodiments of the invention.

[0021] Preferred spore formulations can contain 1-20 or more species of Bacillus spores. According to one preferred embodiment, a spore formulation can contain 3 strains of Bacillus bacteria. According to another preferred embodiment, 2 strains of the Bacillus bacteria can each be a different strain of the species Bacillus licheniformis and the third strain is a species of Bacillus subtilis.
According to another preferred embodiment, about 80% of the formulation can be Bacillus licheniformis (40%
of each strain) and 20% of the spores in the spore formulation can be is Bacillus subtilis.
According to another preferred embodiment, the spores of the strain(s) included in the spore formulation can be mixed with water or other suitable carrier and/or organic salts.

[0022] Most preferably, the Bacillus species that can be contained in the spore formulations can produce and/or be capable of producing one or more enzymes including, but not limited to, proteases, amylases, lipases, glycosidases, cellulases, esterases, and xylanases. Tests and assays for determining the production of such enzymes from a Bacillus species are generally known in the art and to one of ordinary skill in the art.

[0023] According to one preferred embodiment, the spore formulation can contain about 0.1% to 90% by weight spores, along with salt or other suitable carrier, such as sodium bicarbonate, or maltodextrin, or a combination thereof. In some embodiments, the spore formulation contains about 5% by weight spores. The spore formulation can be and/or include a powder or other dry form (e.g. spray-dried form of a liquid spore concentrate, or lyophilized spore formulation) containing spores.
According to another preferred embodiment, the total concentration of spores in the spore formulation can range from about 1 x 105 CFU/mL or spores/g to 1 x 1014 CFU/mL
or spores/g or any specific concentration or range therein. The total preferred concentration of spores in the spore formulation can be about 1, 1.125, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5, or 9.75 x 105, 106, 107, 108, 109, 1019, 10" , 1012, 1013, or 1014 CFU/mL or spores/g or any range or other value therein.
According to another preferred embodiment, any one specific spore species can be present in the spore formulation at a concentration that can range from about 1 x 105 CFU/mL to 1 x 1014 CFU/mL or any specific range therein. The preferred concentration of any one specific spore species present in the preferred embodiments of spore formulations according to the invention can be about 1, 1.125, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5, or 9.75 x 105, 106, 107, 108, 109, 1019, 1011, 1012, 1013, or 1014 CFU/mL or spores/g or any range or other value therein.
Preferably, the spore formulation is biodegradable. According to another preferred embodiment, the concentrated spore formulation can contain about 1-9 x 109 or 101 CFU/mL or spores/g.
According to another preferred embodiment, the concentrated spore formulation can contain about 1010 CFU/mL or spores/g.

[0024] According to one preferred embodiment of a probiotic tea composition, the composition comprises a nutrient composition, a spore formulation, and tea.
According to another preferred embodiment or a probiotic composition for human consumption, the composition comprises a nutrient composition, a spore formulation, and a food or beverage product, such as soups, gravies, sauces, hot chocolate.
Most preferably, the nutrient composition and spore formulation in these embodiments are one of the above described embodiments, both in dry powdered or other dry solid form.
Any variety of tea or other food or beverage product may be used. Most preferably, the food or beverage product is one that is heated before being consumed.

[0025] According to one preferred method of providing probiotic tea for human consumption, a nutrient composition and spore formulation are added to any variety of tea. Most preferably, the nutrient composition and spore formulation are one of the above described embodiments. According to one preferred embodiment, the nutrient composition, spore formulation, and tea or other food or beverage product are combined into a mixture prior to steeping in hot water or otherwise heating the food or beverage product. According to other preferred embodiments, the nutrient composition, spore formulation, and tea may be separate components that are added together to hot water to prepare the probiotic tea or added to the other food or beverage product to be heated or already heated, or any combination of two of the components may be pre-mixed together and added to the third component prior to or after adding hot water to prepare the probiotic tea or prior to or after heating the other food or beverage product.
The order of addition of the three components and the hot water or hot food or beverage is not critical and any order may be used. When used for tea, after mixing or adding the three components, individually or as a pre-mixed combination, with hot water, the mixture is allowed to steep, preferably for 2 to 10 minutes prior to being consumed by a human. Two minutes is the preferred minimum amount of steeping time, but the total time may vary based on the type of tea used and the strength of the tea desired by the person who will consume the probiotic tea. For example, green tea is typically steeped for 2-4 minutes while Oolong tea is typically steeped for 5-8 minutes.
Consumption is preferably by drinking or otherwise orally ingesting the probiotic tea. Most preferably, the hot water is at a temperature in the range of 42 to 100 C when initially added to the other components. The hot water temperature is preferably not below 42 C, and the range may vary based on the type of tea being used. For example, green tea is typically steeped in the range of 65-80 C and black tea is usually steeped in the range of 80-100 C.

[0026] Various compositions according to preferred embodiments of the invention were tested according to preferred methods of the invention. The compositions, methods, and results are described below.

[0027] EXAMPLE 1 ¨ A probiotic blend of Bacillus spores (B. subtilis and B.
licheniformis) at a final concentration of approx. 4 x 106 CFU/mL, where CFU
stands for colony forming unit, and a nutrient germinant composition according to a preferred embodiment of the invention comprising L-alanine (0.21g), monosodium phosphate (0.14g), and disodium phosphate (0.5g), were added to approx. 8 fluid ounces of tap water at various temperatures.

[0028] The amount of spores added represents a "dose" of 109 CFU of probiotic bacteria, as recommended by a Harvard Health study. The amount of nutrient germinants used represents the minimum concentration of germinants necessary to initiate germination. The temperatures used represent a range of tea steeping temperatures.

[0029] Spores from each reaction were observed at time 0 and after 60 minutes using phase contrast microscopy. Slides were prepared using standard procedures.
Spores were viewed on an Olympus BX41 microscope (100X oil emersion objective) and imaged using an Olympus UC30 camera controlled by the cellSens Dimension software package.

[0030] FIG. 1 shows representative images from these tests at time 0 (i.e.
immediately after water addition) and after 60 minutes in the water. The darker spots show germinated spores, the lighter spots show non-germinated spores. The images in group A represent spores that had been germinated using a nutrient-germinant composition and heated during the incubation period at 42 C according to a preferred composition and preferred method of the invention. The images in group B
represent spores that had been germinated using a nutrient-germinant composition and heated during the incubation period at 65 C according to a preferred composition and preferred method of the invention. The images in group C represent spores that had been germinated using a nutrient-germinant composition and heated during the incubation period at 75 C according to a preferred composition and preferred method of the invention. The images in group D represent spores that had been germinated using a nutrient-germinant composition and heated during the incubation period at 85 C

according to a preferred composition and preferred method of the invention.
The images in group E represent represents spores that had been germinated using a nutrient-germinant composition and heated during the incubation period at 100 C
according to a preferred composition and preferred method of the invention.

[0031] Taken together, these images show that Bacillus spores are germinated in the compositions and conditions of preferred embodiments of the present invention.

[0032] EXAMPLE 2 ¨ A probiotic blend of Bacillus spores (B. subtilis and B.
licheniformis) at a final concentration of approx. 4 x 106 CFU/mL and a nutrient germinant composition, according to a preferred embodiment of the invention, comprising L-alanine (0.21g), monosodium phosphate (0.14g), and disodium phosphate (0.5g), were added to approx. 8 fluid ounces of tap water at 85 C. A tea bag containing the indicated variety of tea (black or green as shown in FIG. 2) was steeped in the water for approx. 3 minutes, as recommended by the tea manufacturer. After steeping, the tea bags were removed while the spores remained in the mixture to be ingested as a probiotic dose.

[0033] Spores from each reaction were observed after 60 minutes using phase contrast microscopy. Slides were prepared using standard procedures. Spores were viewed on an Olympus BX41 microscope (100X oil emersion objective) and imaged using an Olympus UC30 camera controlled by the cellSens Dimension software package.

[0034] FIG. 2 shows representative images from these tests after 60 minutes in the water. The darker spots show germinated spores, the lighter/brighter spots show non-germinated spores. The top images represent spores germinated in steeped black tea (Lipton 1M) while the bottom images represent spores germinated in steeped green tea (Bigelow ).

[0035] Taken together, these images show that Bacillus spores are germinated in the compositions and conditions of preferred embodiments of the present invention and that compounds that are extracted from tea during steeping do not abate the germination process.

[0036] Although the above examples are in reference to use of probiotic compositions with tea, the compositions and methods of the invention may be used with other types of food or beverage products, such as soups, gravies, or hot chocolate.
Most preferably, such food and beverage products are liquids or fluids and are consumed after heating, but other types of food and beverage products may also be used.

[0037] All amounts for ingredients or ratios of ingredients indicated herein as a range include each individual amount or ratio within those ranges and any and all subset combinations within ranges, including subsets that overlap from one preferred range to a more preferred range. Those of ordinary skill in the art will appreciate upon reading this specification, including the examples contained herein, that modifications and alterations to the composition and methodology for making the composition may be made within the scope of the invention and it is intended that the scope of the invention disclosed herein be limited only by the broadest interpretation of the appended claims to which the inventor is legally entitled.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A probiotic food or beverage composition for human consumption, the composition comprising:
one or more Bacillus species in spore form;
one or more L-amino acids;
any variety of food or beverage product;
one or more phosphate buffers to maintain the pH of the composition when added to water in a range of around 6-8;
wherein the composition is heated to a temperature in a range of 42 C. to 100 C.;
and wherein the food or beverage product is any variety of tea, coffee, hot chocolate, soup, gravy, or sauce.

2. The probiotic food or beverage composition according to claim 1 wherein the one or more the L-amino acids is L-alanine, L-asparagine, L-valine, L-cysteine, a hydrolysate of soy protein, or a combination thereof.

3. The probiotic food or beverage composition according to claim 2 wherein the composition further comprises at least one of the following: (1) D-glucose, (2) D-fructose, (3) an osmoprotectant or (4) a source of potassium ions.

4. The probiotic food or beverage composition according to claim 1 wherein the composition further comprises at least two of the following: (1) D-glucose, (2) D-fructose, (3) an osmoprotectant and (4) a source of potassium ions.

5. The probiotic food or beverage composition according to claim 2 further comprising potassium chloride.

6. The probiotic food or beverage composition according to claim 2 wherein the Bacillus species are one or more of Bacillus licheniformis, Bacillus subtillis, Bacillus Date Recue/Date Received 2022-03-25 amyloliquiefaciens, Bacillus polymyxa, Bacillus thuringiensis, Bacillus megaterium, Bacillus coagulans, Bacillus lentus, Bacillus clausii, Bacillus circulans, Bacillus firmus, Bacillus lactis, Bacillus laterosporus, Bacillus laevolacticus, Bacillus pumilus, Bacillus simplex, Bacillus sphaericus, Bacillus sonorensis, Bacillus, horneckiae, Bacillus axarquiensis, Bacillus mucilaginosus, Bacillus olivae.

7. The probiotic food or beverage composition according to claim 2 wherein the Bacillus species are Bacillus licheniformis, or Bacillus subtilis, or a combination thereof.

8. The probiotic food or beverage composition according to claim 7 wherein the Bacillus species are two different strains of Bacillus licheniformis and at least one strain of Bacillus subtilis.

9. The probiotic food or beverage composition according to claim 8 wherein the Bacillus species comprises around 40% of a first strain of Bacillus licheniformis, around 40% of a second strain of Bacillus licheniformis, and around 20% of the at least one strain of Bacillus subtilis.

10. The composition of claim 2 further comprising a source of potassium ions.

11. The probiotic food or beverage composition according to claim 1 wherein the composition further comprises an osmoprotectant.

12. The probiotic food or beverage composition according to claim 11 wherein the food or beverage product is a soup.

13. The probiotic food or beverage composition according to claim 11 wherein the food or beverage product is a gravy or sauce.

14. The probiotic food or beverage composition according to claim 7 wherein the composition further comprises an osmoprotectant.

Date Recue/Date Received 2022-03-25

15. The probiotic food or beverage composition according to claim 14 wherein the composition further comprises a source of potassium ions.

16. The probiotic food or beverage composition according to claim 8 wherein the composition further comprises an osmoprotectant.

17. The probiotic food or beverage composition according to claim 1 further comprising a preservative or germination inhibitor.

18. The probiotic food or beverage composition according to claim 17 wherein the preservative or germination inhibitor comprises propylparaben, methylparaben, NaCI, D-alanine, or a combination thereof.

19. The probiotic food or beverage composition according to claim 18 wherein the composition is heated to a temperature in a range of 44 C. to 60 C.

20. A method of preparing a probiotic food or beverage for human consumption, the method comprising:
providing a probiotic composition in a dry or powdered form;
heating (1) the probiotic composition or (2) at least one component of the probiotic composition if at least one component is separate from one or more other components, to a temperature in a range of 42 C to 100 C;
wherein the probiotic composition comprises the following components: one or more Bacillus species in spore form, one or more L-amino acids, one or more buffers, and any food or beverage product;
wherein the heating step comprises adding a hot liquid comprising water to the probiotic composition or the at least one component of the probiotic composition; and wherein the one or more buffers maintain the pH of the probiotic composition in a range of around 6-8 when the water is added to the probiotic composition.

21. The method of claim 20 wherein the hot liquid consists essentially of water.
Date Recue/Date Received 2022-03-25

22. The method of claim 20 wherein the food or beverage product comprises any variety of tea and the probiotic composition is in a water permeable bag;
wherein the heating step comprises placing the water permeable bag in a container of the hot liquid; and wherein the method further comprises:
producing a drinkable tea by steeping the probiotic composition in the hot water.

23. The method of claim 22 wherein the probiotic composition steeps in the hot water for 2 to 10 minutes prior to being consumed by a human.

24. The method of claim 20 wherein at least one of the components of the probiotic composition is separate from one or more other components of the probiotic composition, the method further comprising mixing all of the components to form the probiotic composition.

25. The method of claim 20 further comprising placing the probiotic composition or components of the probiotic composition in a water permeable bag;
wherein the heating step comprises placing the water permeable bag in a container of the hot liquid.

26. The method of claim 20 wherein the probiotic composition is heated for 2 to 10 minutes prior to being consumed by a human.

27. The method of claim 21 wherein the food or beverage product is any variety of tea, hot chocolate, coffee, soup, gravy, or sauce.

28. The method of claim 20 wherein the one or more the L-amino acids is L-alanine, L-asparagine, L-valine, L-cysteine, a hydrolysate of soy protein, or a combination thereof.

29. The method of claim 28 wherein the probiotic composition further comprises at least one of the following: (1) D-glucose, (2) D-fructose, (3) an osmoprotectant or (4) a source of potassium ions.

Date Recue/Date Received 2022-03-25

30. The method of claim 28 wherein the probiotic composition further comprises at least two of the following: (1) D-glucose, (2) D-fructose, (3) an osmoprotectant and (4) a source of potassium ions.

31. The method of claim 28 wherein the probiotic composition further comprises potassium chloride.

32. The method of claim 20 wherein the Bacillus species are one or more of Bacillus licheniformis, Bacillus subtillis, Bacillus amyloliquiefaciens, Bacillus polymyxa, Bacillus thuringiensis, Bacillus megaterium, Bacillus coagulans, Bacillus lentus, Bacillus clausii, Bacillus circulans, Bacillus firmus, Bacillus lactis, Bacillus laterosporus, Bacillus laevolacticus, Bacillus pumilus, Bacillus simplex, Bacillus sphaericus, Bacillus sonorensis, Bacillus, horneckiae, Bacillus axarquiensis, Bacillus mucilaginosus, Bacillus olivae.

33. The method of claim 20 wherein the Bacillus species are Bacillus licheniformis, or Bacillus subtilis, or a combination thereof.

34. The method according to claim 33 wherein the Bacillus species are two different strains of Bacillus licheniformis and at least one strain of Bacillus subtilis.

35. The method according to claim 34 wherein the Bacillus species comprises around 40% of a first strain of Bacillus licheniformis, around 40% of a second strain of Bacillus licheniformis, and around 20% of the at least one strain of Bacillus subtilis.

36. The method according to claim 28 wherein the Bacillus species are two different strains of Bacillus licheniformis and at least one strain of Bacillus subtilis.

37. The method according to claim 20 wherein the probiotic composition further comprises a preservative or germination inhibitor.

Date Recue/Date Received 2022-03-25

38. The method according to claim 37 wherein the preservative or germination inhibitor comprises propylparaben, methylparaben, NaCI, D-alanine, or a combination thereof.

39. The method according to claim 37 wherein the composition is heated to a temperature in a range of 44 C to 60 C.

40. The method of claim 20 wherein the food or beverage product wherein the food or beverage product is any variety of tea, hot chocolate, cereal, oatmeal, creamer, coffee, soup, gravy, or sauce and wherein the one or more buffers comprises a (1) phosphate buffer, (2) combination of a citrate and citric acid, (3) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, (4) Tris base, or (5) a combination thereof.

41. The method of claim 20 wherein the food or beverage product is any variety of tea, hot chocolate, coffee, soup, gravy, or sauce and wherein the one or more buffers comprises (1) phosphate buffer, (2) combination of a citrate and citric acid, (3) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, (4) Tris base, or (5) a combination thereof.

42. The probiotic composition of any one of claims 1 to 19, wherein all ingredients are GRAS (Generally Regarded as Safe) for human consumption under U.S. federal standards.

43. The method of any one of claims 20 to 41, wherein all ingredients in the probiotic composition are GRAS (Generally Regarded as Safe) for human consumption under U.S.
federal standards.

Date Recue/Date Received 2022-03-25