An acidic spore-based probiotic composition

Abstract

The present invention relates to methods for producing a heat-treated acidic probiotic beverage comprising viable Bacillus subtilis spores, and the heat-treated acidic probiotic beverage produced by the methods.

Description

[0001] AN ACIDIC SPORE-BASED PROBIOTIC COMPOSITION

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of spore-based probiotic compositions. A heat- treated acidic spore-based probiotic composition is provided that comprises a plurality of viable probiotic microorganisms having a biological or therapeutic activity in the gastrointestinal tract. Also provided are methods of producing a heat-treated acidic spore-based probiotic compositions.

[0004] BACKGROUND OF THE INVENTION

[0005] Probiotics are increasingly recognized as a playing an important, beneficial role in human health, particularly in restoring or improving the composition of intestinal microflora.

[0006] Probiotics are typically provided as dietary supplements, frequently as capsules and powders. However, liquid is a preferred method of delivering probiotics, as a liquid medium allows for better absorption of the probiotics and better distribution across the gastrointestinal tract. Further, a liquid medium is convenient and does not require swallowing of pills or tablets. Probiotics may be found in beverages, however, these beverages typically require refrigerated delivery, storage, and use to maintain the probiotic activity / viability, and have a limited shelf-life.

[0007] Acidic beverages, which include most juices, teas, sodas, and sports drinks, are enjoyed by a large number of consumers world-wide. Although some of the acid is naturally occurring, such as citric acid in citrus drinks, it is also known that the addition of acids to a beverage can provide a tart and tangy taste that can balance the sweetness of the sugar present in the beverage, and also function as a preservative. Many acidic beverages do not require refrigeration and have a shelf-life of several months.

[0008] As the interest in probiotics increases and consumers look for convenient ways to introduce probiotics into their diets on a regular basis, shelf-stable, ready-to-drink acidic beverages comprising probiotics seems to be an obvious choice. However, such a beverage faces challenges. To be considered safe and shelf-stable, the acidic beverage needs to undergo sterilization or pasteurization, which typically is provided as a heat treatment. However, most probiotic strains, even those which can be supplied as spores, have low survivability in the low pH, high temperature environment required to sterilize an acidic beverage. Consequently, many commercially available probiotic acidic beverages do not meet label claims with regards to the numbers and types of viable microbes present in the product, and do not deliver the quantity that needs to be consumed for a health benefit.

[0009] US 10 494684 B2 relates to a Bacillus subtilis strain identified as MB40 (deposited under ATCC Accession No. PTA-122264). In Example 2, the survivability of MB40 steeped in a hot tea preparation made from store-bought orange pekoe and pekoe cut black tea teabags was assessed, reporting 84% survivability via Total Place Count (TPC) after MB40-containing teabag was steeped in boiling water (100 °C) for 4 minutes. Example 2 is, however, silent on the pH of the tea preparation, which may vary greatly depending on, e.g., steeping time, the tea brand / tea mixture, and tea-to-water ratio used to brew the tea preparation. In another example, identified as Example 12 in US 10 494 684 B2, the survivability of MB40 is tested at various pH (pH 2, 4 and 6) and percent-solid levels (solids made up of varying amounts of sugar and pea protein) over a 10-month period, concluding that the results illustrate that MB40 displays good survivability within the range of pH 4 to 6 across a broad range of percent-solid levels.

[0010] WO 2023 / 196003 A1 relates to a Bacillus subtilis strain identified as BS50 (deposited under ATCC Accession No. PTA-127287). In Example 4, BS50 stability in hot beverage matrices is tested using store bought pekoe cut black tea teabags, concluding that increased steeping temperatures decreased the BS50 spore survival, while at least 50% of the spores survived steeping temperatures up to 100 °C. In a similar experiment, adding BS50 to ground coffee and brewing the resulting grounds, reporting 100% survival of BS50. The Example is, however, silent on the actual pH of the tea preparation and brewed coffee, which can vary greatly..

[0011] SUMMARY OF THE INVENTION

[0012] The present invention is based on the surprising and inventive finding that strains of Bacillus subtilis which are known to be probiotic microorganisms can survive a heat-treatment in an acidic environment. Such probiotic B. subtilis strains may be provided to an acidic beverage which is subsequently heat-sterilized or pasteurized to improve shelf-life. The resulting shelfstable acidic probiotic beverage does not require refrigeration, so that manufacturing, packaging, delivery, and storage of the beverage are significantly less energy intensive.

[0013] The invention provides a heat-treated acidic beverage which comprises viable spores from Bacillus subtilis, and methods of producing such a heat-treated acidic beverage.

[0014] BRIEF DESCRIPTION OF DRAWINGS

[0015] Fig. 1 shows the survival of endospores of Bacillus probiotic strains in an acidic solution at 0 days and 7 days following a heat treatment. CTRL = no pasteurization, i.e., no heat or pH change; HU58 = Bacillus subtilis HU58™; DE111 = B. subtilis DE111®; BC30 = B. coagulans BC30™.

[0016] Definitions:

[0017] As used herein, the verb “comprise” as is used in this description and in the claims and its conjugations are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements are present, unless the context clearly requires that there is one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”. Unless defined otherwise or clearly indicated by context, all percentages are percentage by weight (percent w / w or “% (w / w)”). The term "% (w / v)" refers to the concentration of a substance in a solution, expressed as a percentage by weight per unit volume.

[0018] Reference to "about" a value or parameter herein includes aspects that are directed to that value or parameter per se. For example, description referring to "about X" includes the aspect "X". When used in combination with measured values, "about" includes a range that encompasses at least the uncertainty associated with the method of measuring the particular value and can include a range of plus or minus two standard deviations around the stated value.

[0019] The drawings featured in the figures are for the purpose of illustrating certain convenient embodiments of the invention and are not to be considered as limitation thereto.

[0020] Furthermore, it will be appreciated that embodiments described in connection with one of the aspects described herein may equally be applied to the other aspects, unless otherwise stated.

[0021] Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0022] Bacterial count: The term "bacterial count" and "bacterial load" are used interchangeably and have their normal meaning which is well known and understood by those of skill in the art. In the context of the invention, the bacterial count is given in the measures viable bacterial colony forming units per grams or milliliters, abbreviated CFU / g and CFU / ml, respectively. The bacterial count may be determined by any method known to the skilled person such as, but not limited to, by viable bacterial colony forming unit (CFU), quantitative polymerase chain reaction (qPCR), flow cytometry, live-dead staining, Propidium Monoazide qPCR (PMA-qPCR), microscopy, a metabolic assay, spectrophotometry, or any combination thereof. Methods for determining bacterial count, such as those disclosed herein, are common and would be apparent to a person of skill in the art.

[0023] Colony: The term “colony”, referred to in connections with a “clone” or “isolate” or “cell”, means a visible cluster or grouping of microbial cells. The colony may be grown and obtained from a solid medium, such as agar.

[0024] Composition: In the context of the invention, the term "composition" encompasses any kind of compositions comprising a Bacillus subtilis spore of the invention. In the context of the invention, the composition may be a liquid composition or a dry composition.

[0025] Derived from: In the context of the invention, “derived from” means the Bacillus subtilis spores used in accordance with the invention are being derived from an origin sample. As used herein, the term "being derived" is interchangeable with the term "originated" and "obtained" and refers to the source from which the spores are obtained. In some embodiments, the origin sample comprises a Bacillus subtilis strain found, constituted in, or known to reside in an environmental and / or microbiome niche. In the context of the invention, the microbiome is the genetic material of all microbes (bacteria, fungi, protozoa, and viruses) that live on or in the human body.

[0026] Effective amount: As used herein, an "effective amount" is defined as an amount effective to achieve a desired biological result, such as reducing, preventing, or treating a disease or condition and / or inducing a particular beneficial effect. In some embodiments, the effective amount is the amount provided in a single serving probiotic beverage where consumption of multiple servings is required over time for the beneficial effect to be observed.

[0027] Isolated: The term “isolated” means that the one or more microorganisms, such as bacterial strains, described herein are in a form or environment which does not occur in nature, that is, the one or more microorganisms are at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature.

[0028] Probiotic: In the context of the invention, the term “probiotic” refers to microorganisms that may have a beneficial effect on the health of a subject, such as on the health of a human subject, upon consumption. Typically, probiotics are commonly defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host,” such as restoring or improving the composition of intestinal microflora. See, e.g., KQ)N,'J\-\Q), Guidelines for the evaluation of probiotics in food, London, Ontario, Canada (2002), incorporated by reference herein in its entirety. The acidic probiotic beverage obtained according to the methods of the invention, may, thus, have a beneficial effect on the health of a subject, such as a human, upon ingestion of the acidic probiotic beverage.

[0029] Probiotic beverage: In the context of the invention, a “probiotic beverage” comprises at least a probiotic composition and water. The probiotic composition comprises at least one viable probiotic microorganism. The probiotic composition may comprise one or more colonizing probiotic bacterial strains which may be spore-based probiotic bacterial strains. In some embodiments, the probiotic composition comprises species of Bacillus subtilis. In further embodiments, the probiotic composition comprises Bacillus subtilis HLI58™ (B. subtilis having accession number EF101709). The Bacillus species may be present in the form of spores, also referred to as endospores, or in a vegetative state.

[0030] Acidic probiotic beverage: In the context of the invention, an “ acidic probiotic beverage” comprises at least a probiotic composition and water, and has a low pH level, typically below pH 4.6. Examples of acidic probiotic beverages include fruit juice, fruit drink, flavored water, soda, sports drink, yogurt-based beverage (which may be based on dairy or dairy alternatives), kefirbased beverages, kombucha-based beverage, tea-based beverage, or coffee-based beverage. The fruit juices may be fermented fruit juices. Acidic probiotic beverages may be obtained by fermentation processes that result in increased acidity, creating an environment conducive to probiotic growth and activity. Additionally, certain dairy-based and plant-based fermented beverages, such as some types of yogurt drinks and cultured buttermilk, also fall into this category. In a preferred embodiment, the acidic probiotic beverage according to the invention has a pH below 4.0. In another preferred embodiment, the acidic probiotic beverage according to the invention has a pH between 2.0-4.0, such as between pH 2.0-3.7, such as between pH 2.0-3.5, such as between pH 2.0-3.0.

[0031] Prebiotic and Postbiotic: In the context of the invention, the terms “prebiotic” and “postbiotic” have their normal meaning which is well known and understood by those of skill in the art. The term "prebiotic" as used herein refers to any compound, nutrient or additional microorganism used to support or enhance a desired probiotic health effect or to assist the growth and / or activity of probiotic microorganism. The term "postbiotic" refers to probiotic cells that have been killed by heat or other means and are delivered to the consumer as dead cells.

[0032] Spore: The terms "spore" and "endospore" are used interchangeable and have their normal meaning which is well known and understood by a person of skill in the art. As used herein, the term spore refers to a microorganism in its dormant, protected state.

[0033] Sporulating microorganism: The term "sporulating microorganism" means a microorganism that is able to sporulate. In the methods of the invention, the viable Bacillus subtilis spores are derived from sporulating microbial cell which may be a wild-type cell or a mutant thereof. In an embodiment, the sporulating microbial cell is obtained from an environmental or microbiome niche.

[0034] Stable: The term “stable” is a term that it known in the art, and in a preferred aspect, stable is intended to mean the ability of a microorganism to remain in a spore form until it is administered to a subject, in particular a human subject, to improve the health of the subject.

[0035] Suspension: The term “suspension” refers to material suspended in a fluid.

[0036] Viable: As used herein with respect to Bacillus subtilis spores, the terms “viable” or “viability” means the cells or spores are capable of propagating on or in a suitable growth medium or substrate when conditions, such as, e.g., temperature, moisture, nutrient availability, pH, etc., are favorable for germination and / or microbial growth at a given period of time.

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0038] The present disclosure provides heat-treated acidic beverages comprising probiotic compositions and methods of producing these heat-treated acidic probiotic beverages.

[0039] It is well-known in the art that lowering of pH decreases the heat tolerance and survivability of bacterial strains, including the survivability of spores of probiotic spore-forming bacteria such as Bacillus spp. Surprisingly, it is shown here that Bacillus subtilis strains have better survivability compared to other probiotic spore-forming bacteria, including other species of Bacillus such as Bacillus coagulans. The ability to survive in an environment of low pH and high temperature indicates the B. subtilis strains may be able to survive pasteurization and / or other heat treatments used to sterilize beverages. Therefore, Bacillus subtilis strains can be better suited for certain probiotic applications, such as shelf-stable acidic probiotic beverages which require a sterilization or pasteurization step. In a first aspect the present invention relates to a method of producing a heat-treated acidic probiotic beverage comprising viable Bacillus subtilis spores, wherein the method comprises the following steps:

[0040] (a) preparing an acidic beverage comprising viable B. subtilis spores, wherein the pH of the beverage is between 2.0 to 4.6; and

[0041] (b) heat-treating said beverage; wherein at least 70% of the initial B. subtilis spores are viable after the heat-treatment.

[0042] An acidic probiotic beverage includes any acidic beverage into which probiotics may be added. In some embodiments, the pH of the acidic probiotic beverage is between 2.0 to 4.6. In further embodiments, the pH of the acidic probiotic beverage is between 3.7 to 4.3. An acidic beverage typically has a pH of 4.6 or lower. Juices and sodas frequently have a pH below 4.0. Acids and acidulants provide a tartness and tangy taste that helps balance the sweetness of sugar present in the beverage, and are key factors in the taste of the beverage. Acids may also act as a preservative and can reduce the growth of bacteria and fungi in a beverage, thereby improving shelf-stability, also referred to as shelf-life. Weak acids and / or acids that naturally occur in fruits may be used to produce an acidic beverage. Citric acid, which occurs naturally in citrus, and malic acid, which occurs naturally occurs in apples, pears, and cherries, are added to many beverages including fruit drinks, sports drinks, and tea-based beverages. Phosphoric acid is added to cola drinks. Acetic acid, adipic acid, ascorbic acid, fumaric acid, butyric acid, gluconic acid, lactic acid, tartaric acid, and sorbic acid may also be used to produce an acidic beverage.

[0043] In some embodiments, the acidic probiotic beverage may be a fruit juice, fruit drink, flavored water, soda, sports drink, yogurt-based beverage, kefir-based beverage, kombucha- based beverage, tea-based beverage, or coffee-based beverage.

[0044] In some embodiments, the acidic probiotic beverage may be a fruit juice, fruit drink, flavored water, soda, sports drink, yogurt-based beverage, kombucha-based beverage, teabased beverage, or coffee-based beverage.

[0045] In some embodiments, the acidic probiotic beverage is selected from a fruit juice, fruit drink, flavored water, soda, sports drink, yogurt-based beverage, kefir-based beverage, and kombucha-based beverage.

[0046] In some embodiments, the acidic probiotic beverage is a fruit juice, fruit juice cocktail, lemonade, cider, or a beverage flavored with fruit juices. The fruit component may be orange, grapefruit, lemon, lime, apple, pear, peach, apricot, prune, plum, strawberry, raspberry, cranberry, blueberry, pineapple, banana, mango, grape, tomato, pomegranate, papaya, coconut, kiwi, lychee, yuzu, or any combination thereof.

[0047] In some embodiments, the acidic probiotic beverage is a soda. A soda is a carbonated beverage, typically comprising carbonated water and sweeteners and natural and / or artificial flavorings. A soda may be a seltzer. A soda may be sugar-free, so that it comprises no sweeteners or may only comprise artificial sweeteners. A soda may be “zero sugar”, where it may not comprise carbohydrates. A soda may also comprise caffeine, colorings, preservatives, and optionally additional ingredients. A soda may also be referred to as a soft drink, seltzer, “pop”, or “soda pop”.

[0048] In some embodiments, the acidic probiotic beverage is a flavored water. The flavored water may be carbonated, such as for example a sparkling water.

[0049] In some embodiments, the acidic probiotic beverage is a sports drink. A sports drink comprises electrolytes, such as sodium, potassium, and chloride, and typically also comprises carbohydrates, frequently in the form of sugars such as glucose, sucrose, or high-fructose corn syrup. A sports drink may be sugar-free, so that it comprises no sweeteners or may only comprise artificial sweeteners. A sports drink may be “zero sugar”, where it may not comprise carbohydrates. A sports drink may comprise caffeine, vitamins, minerals, protein, amino acids, natural or artificial flavorings, and optionally additional ingredients. A sports drink may also be referred to as a vitamin water, a fitness water, an enhanced water, an energy drink, or a hydration beverage.

[0050] In some embodiments, the acidic probiotic beverage is a tea or a tea-based beverage. The tea may be green tea, black tea, oolong tea, yellow tea, white tea, decaffeinated tea, herbal tea, or any combination thereof. The herbal tea may be rosehip tea, chamomile tea, jiaogulan tea, peppermint tea, rooibos tea, ginger tea, ginseng tea, lemongrass tea, or any combination thereof. In some embodiments, the tea-based beverage is a fermented tea. In some embodiments, the acidic probiotic beverage is a tea or a tea-based beverage having pH below 4.0.

[0051] In further embodiments, the fermented tea is kombucha, or is a kombucha-based beverage. Kombucha is produced by symbiotic fermentation of sugared tea using a symbiotic culture of bacteria and yeast. Kombucha or kombucha-based beverages may further comprise fruit juices and / or spices. In some embodiments, kombucha or kombucha-based beverages may comprise ethanol, or be alcoholic. In other embodiments, the kombucha or kombucha-based beverage is non-alcoholic.

[0052] In some embodiments, the acidic probiotic beverage is milk-based. A milk-based beverage may be derived from fermented dairy products, such as kefir, Ayran, and yogurt.

[0053] In some embodiments, the acidic probiotic beverage is yogurt-based. The yogurt-based beverage may be based on dairy and / or a dairy alternative. Examples of dairy alternative yogurtbased beverages include grain-based yogurt-based beverages, such as oat- or corn-based yogurt-based beverages, nut-based yogurt-based beverages, such as peanut-, almond-, coconut- , cashew-, hazelnut-, or macadamia-based yogurt-based beverages, or legume-based yogurtbased beverages, such as chickpea-, faba-, lentil-, pea-, or soy-based yogurt-based beverages.

[0054] In some embodiments, the acidic probiotic beverage is a coffee or coffee-based beverage. The coffee may be roasted coffee, unroasted green coffee, decaffeinated coffee, or any combination thereof. The coffee or coffee-based beverage may further comprise a dairy component, such as milk or cream, and / or a plant-based substitute, such as oat, almond, soy, rice, or cashew milk, or any other non-dairy substitute known in the art. The coffee or coffeebased beverage may further comprise flavoring ingredients, such as cocoa, chocolate, artificial cocoa compositions, vanilla, artificial vanilla compositions, or any combination thereof.

[0055] An acidic probiotic beverage may further comprise one or more additional ingredients to improve or alter the taste of the beverage. Additional ingredients include acidulants, thickeners, buffers or agents for pH adjustment, chelating agents, colorants, emulsifiers, excipient, flavorants, osmotic agents, acceptable carriers, preservatives, stabilizers, sugars, sweeteners, texturizers, minerals, and / or vitamins. The additional ingredients can be added in any suitable amount.

[0056] It will be further recognized by one of skill in the art that the acidic probiotic beverage comprises food ingredients and / or ingredients for nutritional benefit in addition to the probiotics. Additional ingredients include vitamins, minerals, amino acids, protein, fiber, prebiotics (such as fructo-oligosaccharides, galacto-oligosaccharides, inulin, and polydextrose) and postbiotics.

[0057] The heat-treated acidic probiotic beverage of the invention can be ingested by human or animal subjects, preferably by human subjects. In some embodiments, the heat-treated acidic probiotic beverage has a beneficial effect on the subject upon consumption, such as a beneficial effect on the gut health of the subject.

[0058] In humans, the gut is the main location of the human microbiome and is believed to be essential to human health throughout life. The gut microbiome helps digest food, regulate the immune system, protect against other bacteria that cause disease, and produce vitamins (including the B vitamins B12, thiamine, and riboflavin; and Vitamin K, which is required for blood coagulation).

[0059] The gut microbiome is a vast collection of bacteria, viruses, fungi, and protozoa that colonize the gastrointestinal tract and outnumber human cells 10-fold. Exposures in early life, such as through mode of delivery (maternal microbes), infant diet (selective substrates), antibiotics (selective killing), probiotics (selective enrichment), and physical environment (environmental microbes), results in colonization of gut microbiota which contributes to the development of the immune system, intestinal homeostasis and host metabolism.

[0060] Disruption of the gut microbiota is associated with a growing number of diseases. See, e.g., M.B. Azad, et al., Gut microbiota of healthy Canadian infants: profiles by mode of delivery and infant diet at 4 months, 185 CAN. MED. ASS’N J. 385 (2013), incorporated by reference herein in its entirety. Recent advances in metagenomics have enhanced our understanding of the gut microbiome, suggesting that it can provide important immune and metabolic benefits to humans.

[0061] The heat-treated acidic probiotic beverage disclosed herein comprises a probiotic in the form of Bacillus subtilis spores. Preferably, for the Bacillus subtilis spores to elicit a probiotic effect on a subject following ingestion of the heat-treated acidic probiotic beverage, the probiotic should remain substantially viable at the level of the intestine. There is some evidence to support the importance of viability in human studies, with viable bacteria having greater immunological effects than nonviable bacteria. See, e.g., M. Kaila, et al., Viable versus inactivated lactobacillus strain GG in acute rotavirus diarrhea, 72 ARCHIVES OF DISEASE IN CHILDHOOD 51 (1995); P.V. Kirjavainen, et al., Probiotic bacteria in the management of atopic disease: underscoring the importance of viability, 36 J. PEDIATRIC GASTROENTEROLOGY & NUTRITION 223 (2003); each of which is incorporated by reference herein in its entirety.

[0062] In preferred embodiments, the probiotic is resistant to gastric acid digestion and to bile salts to reach the intestine intact and is non-pathogenic to the subject consuming the probiotic instant food product.

[0063] Many commercially available probiotics are gram positive spore-forming bacteria, which are provided in their spore form, also referred to as the endospore. As spores, the bacteria can survive harsh environments which enable processing of the bacteria in a dry form, so that it can be supplied for example in capsules and powders. Ingestion of the capsules or powders then introduces the probiotics to the gut. However, ingestion of probiotics in a capsule or powder can reduce the effectiveness of the probiotics. A liquid medium allows for better absorption of the probiotics and better distribution across the gastrointestinal tract.

[0064] A suitable liquid medium for delivery of probiotics to the gut needs to be a beverage that a consumer wants to drink. Acidic beverages, which include most juices, tea-based beverages, coffee-based beverages, sodas, and sports drinks, are enjoyed by a large number of consumers world-wide. To be considered safe and shelf-stable, the acidic beverage needs to undergo sterilization or pasteurization, which typically is provided as a heat treatment. However, although in spore form probiotics can survive in harsher conditions, most spore-forming bacteria cannot survive in all harsh conditions, especially if there are multiple environmental stressors. Most probiotic strains, even those which can be supplied as spores, have low survivability in the low pH, high temperature environment required to sterilize an acidic beverage. One solution is to sterilize the acidic beverage and the spore-forming probiotic under separate conditions, and then combine them by packaging them together under sterile conditions (see for example WO 2019 / 231910, herein incorporated by reference in its entirety). However, such a method of manufacturing is cumbersome, requires multiple additional processing steps, and raises the cost of production.

[0065] The present invention includes a method of producing a heat-treated acidic probiotic beverage where the probiotic, comprising viable B. subtilis spores, is added to the acidic beverage, and the beverage is subsequently heat-treated, with an effective amount of the B. subtilis spores surviving the heat treatment. Because the probiotics are not sterilized separately and then combined with the beverage under sterile conditions, the processing of the beverage requires fewer steps and more cost- and resource- efficient. The present invention also includes the heat-treated acidic probiotic beverage produced by the methods of the invention. Preferably, the heat-treatment is a pasteurization or a retort sterilization.

[0066] Probiotics are measured by colony forming units (“CFUs”) and can be measured as CFUs / g or CFUs / vol. Alternatively, a given probiotic dosage can be delivered as a total in CFUs. Effective dosages, also referred to as effective amounts, are usually in the hundreds of millions of CFUs per gram or per milliliter, and in some cases are higher. In some embodiments, probiotics are taken with meals. In other embodiments, probiotics are taken between meals. Probiotics may survive better if taken with liquids, which can dilute stomach acid and move the probiotics more quickly into the digestive tract. Probiotics may be taken short-term or long-term.

[0067] The present invention is based on the surprising finding that multiple strains of B. subtilis are highly tolerant of a sterilizing heat treatment in liquid acidic conditions.

[0068] In some embodiments, the acidic probiotic beverage of the invention comprises spores of Bacillus subtilis HU58™, also referred to as B. subtilis HU58, has been deposited with the National Center for Biotechnology Research under the accession number EF101709. The Bacillus Genetic Stock Center (“BGSC”) assigned number for Bacillus HU58 is 3A34, and the NCIMB Ltd. assigned strain number is 30283. HU58™ is available commercially as a dried spore composition, manufactured and sold, e.g., by Microbiome Labs UK Ltd. A commercially available spore composition of Bacillus subtilis HU58 is also sold by Novonesis (previously (Novozymes A / S, Bagsvaerd, Denmark) under the tradename ProSilience™ HU58™.

[0069] In some embodiments, the acidic probiotic beverage of the invention comprises spores of Bacillus subtilis DE111®, also referred to as B. subtilis DE111 or Bacillus subtilis subspecies inaquosorum, has been deposited with the Agricultural Research Service Culture Collection (NRRL), under the accession number NRRL B-67989.

[0070] In some embodiments, the acidic probiotic beverage of the invention comprises more than one strain of B. subtilis. In other embodiments, the beverage of the invention comprises only one strain of B. subtilis.

[0071] In some embodiments, the heat-treated acidic probiotic beverage may comprise in addition to the Bacillus subtilis spores, further probiotics. The further probiotics may be probiotic microorganisms in the form of spores or in a vegetative state. The further probiotics will also be tolerant of the low pH, high temperature heat treatment required for sterilization of the beverage. Examples of further probiotics microorganisms may include, but are not particularly limited to, strains of the genera Lactobacillus, Bacillus, Bifidobacterium, Lactococcus, Propionibacterium, Enterococcus, Escherichia, Streptococcus, Pediococcus, and Saccharomyces. For example, the further probiotic microorganism may be selected from list of Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus paracasei, Bifidobacterium sp., Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium adelocentis, Bifidobacterium lactis, Bacillus coagulans, Bacillus licheniformis, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Streptococcus salivarius, Saccharomyces cerevisiae, Saccharomyces boulardii, and combinations thereof.

[0072] In the methods of the invention, an acidic probiotic beverage comprising viable B. subtilis spores is heat-treated. Following heat treatment, the number of viable B. subtilis spores is at least

[0073] 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least

[0074] 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least

[0075] 98%, at least 99%, or is 100% of the spores present before the heat treatment.

[0076] In some embodiments, an acidic probiotic beverage comprises a sufficient amount of spores so that an effective amount of spores remain viable after heat-treatment. In some embodiments, the effective amount of B. subtilis spores is at least 1x104CFU / ml. In some embodiments, the concentration of a B. subtilis strain in the acidic probiotic beverage prior to heat treatment may be at least 1x104colony forming units (CFU) / ml, at least 3x104CFU / ml, at least 5x104CFU / ml, at least 7x104CFU / ml, at least 9x104CFU / ml, at least 1x105colony forming units (CFU) / ml, at least 5x105CFU / ml, at least 7x105CFU / ml, at least 9x105CFU / ml, at least 1x106CFU / ml, at least 2x106CFU / ml, at least 3x106CFU / ml, at least 4x106CFU / ml, at least 5x106

[0077] CFU / ml, at least 6x106CFU / ml, at least 7x106CFU / ml, at least 8x106CFU / ml, at least 9x106

[0078] CFU / ml, at least 1x107CFU / ml, at least 2x107CFU / ml, at least 3x107CFU / ml, at least 4x107

[0079] CFU / ml, at least 5x107CFU / ml, at least 6x107CFU / ml, at least 7x107CFU / ml, at least 8x107

[0080] CFU / ml, at least 9x107CFU / ml, at least 1x108CFU / ml, at least 2x108CFU / ml, at least 3x108CFU / ml, at least 4x108CFU / ml, at least 5x108CFU / ml, at least 6x108CFU / ml, at least 7x108CFU / ml, at least 8x108CFU / ml, at least 9x108CFU / ml, at least 1x109CFU / ml, 5x109CFU / ml, at least 1x1010CFU / ml, at least 5x1O10CFU / ml, at least 1x1011CFU / ml, at least 5x1011CFU / ml, or at least 1x1012CFU / ml.

[0081] Following heat treatment, the probiotic beverage of the invention comprises an effective amount of B. subtilis spores. In some embodiments, the effective amount of B. subtilis spores is at least 1x104CFU / ml. In some embodiments, the probiotic beverage after heat treatment comprises at least 1x104CFU / ml, at least 2x104CFU / ml, at least 3x104CFU / ml, at least 4x104CFU / ml, at least 5x104CFU / ml, at least 6x104CFU / ml, at least 7x104CFU / ml, at least 8x104CFU / ml, at least 9x104CFU / ml, at least 1x105CFU / ml, at least 2x105CFU / ml, at least 3x105CFU / ml, at least 4x105CFU / ml, at least 5x105CFU / ml, at least 6x105CFU / ml, at least 7x105

[0082] CFU / ml, at least 8x105CFU / ml, at least 9x105CFU / ml, at least 1x106CFU / ml, at least 2x106

[0083] CFU / ml, at least 3x106CFU / ml, at least 4x106CFU / ml, at least 5x106CFU / ml, at least 6x106

[0084] CFU / ml, at least 7x106CFU / ml, at least 8x106CFU / ml, at least 9x106CFU / ml, at least 1x107

[0085] CFU / ml, at least 2x107CFU / ml, at least 3x107CFU / ml, at least 4x107CFU / ml, at least 5x107

[0086] CFU / ml, at least 6x107CFU / ml, at least 7x107CFU / ml, at least 8x107CFU / ml, at least 9x107CFU / ml, at least 1x108CFU / ml, at least 2x108CFU / ml, at least 3x108CFU / ml, at least 4x108CFU / ml, at least 5x108CFU / ml, at least 6x108CFU / ml, at least 7x108CFU / ml, at least 8x108CFU / ml, at least 9x108CFU / ml, at least 1x109CFU / ml, 5x109CFU / ml, at least 1x1O10CFU / ml, at least 5x1010CFU / ml, at least 1x1O11CFU / ml, at least 5x1011CFU / ml, or at least 1x1012CFU / ml.

[0087] In some embodiments, the probiotic acidic beverage is canned or packaged before heat treatment, so that in-container pasteurization is performed. In other embodiments, the beverage is heat treated and then canned or packaged via sterile filling or hot filling using methods known in the art.

[0088] In methods of the invention, the acidic probiotic beverage is heat-treated to sterilize the beverage. The heat-treatment may also be referred to as pasteurization. The heat-treatment is intended to make the beverage safe for consumption by killing harmful microorganisms, thereby reducing the risk of food poisoning. The heat-treatment also increases shelf-life or shelf-stability of the beverage, as it eliminates microorganisms in the beverage which may degrade the beverage, either by causing it to spoil or by decreasing the quality of the beverage. In some embodiments, the heat treatment is 85-95°C for 5-30 minutes. In further embodiments, the heat treatment is 85-95°C for 10-15 minutes. In some embodiments, the heat treatment is 95°C for 5, 10, 15, 20, 25, or 30 minutes. In some embodiments, the heat treatment is 85-95°C for one minute or less. In some embodiments, the heat treatment is 85-95°C for 10,15, 20, 25, 30, 35, 40, 45, 50,55, or 60 seconds.

[0089] In some embodiments, the acidic probiotic beverage is heat-treated by High Temperature Short Time (HTST) pasteurization. For this, the beverage is heated to a temperature between 71.5-74°C for 15-30 seconds, or is heated to a temperature between 74-76°C for 15-20 seconds. Following heat treatment, the beverage may then be rapidly cooled to 4-5.5°C.

[0090] In some embodiments, the acidic probiotic beverage is heat-treated by ultra pasteurization. For this, the beverage is heated to a temperature between 70-75°C for 20-30 minutes. In some embodiments, the beverage is heated to a temperature between 80-85°C for 20-30 seconds.

[0091] In some embodiments, the acidic probiotic beverage is heat-treated by an Ultra High Temperature (UHT) treatment. The UHT treatment may be direct or indirect. In some embodiments, the UHT treatment is 135-154°C for 1-10 seconds. In further embodiments, the UHT treatment is 140-150°C for 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In further embodiments, the UHT treatment is 140-145°C for 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In some embodiments, the UHT treatment is 143°C for 4, 5, 6, 7, or 8 seconds.

[0092] In some embodiments, the probiotics of the heat-treated acidic probiotic beverage are determined to have good survivability several days after the heat treatment. In the methods and compositions of the invention, the number of viable B. subtilis spores after 7 days of storage is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% of the spores present before the heat treatment.

[0093] PREFERRED EMBODIMENTS

[0094] 1. A method of producing a heat-treated acidic probiotic beverage comprising viable Bacillus subtilis spores, wherein the method comprises the following steps:

[0095] (a) preparing an acidic beverage comprising viable B. subtilis spores, wherein the pH of the beverage is between 2.0 to 4.6; and

[0096] (b) heat-treating said beverage; wherein at least 70% of the initial B. subtilis spores are viable after the heat-treatment.

[0097] 2. The method of embodiment 1 , wherein at least 1x104, 5x104, 1x105, 2x105, 3x105, 4x105, 5x105, 6x105, 7x105, 8x105, 9x105, 1x106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, or at least 1x107CFU / ml of the B. subtilis spores are viable after heat treatment.

[0098] 3. The method of any one of the preceding embodiments, wherein the number of viable B. subtilis spores is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% of the spores present before the heat treatment.

[0099] 4. The method of any one of the preceding embodiments, wherein at least 70% of the spores present before the heat treatment are viable 7 days after the heat-treatment.

[0100] 5. The method of any one of the preceding embodiments, wherein at least 1x104, 5x104, 1x105, 2x105, 3x105, 4x105, 5x105, 6x105, 7x105, 8x105, 9x105, 1x106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, or at least 1x107CFU / ml of the B. subtilis spores are viable 7 days after the heat-treatment.

[0101] 6. The method of any one of the preceding embodiments, wherein at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% of the spores present before the heat treatment are viable 7 days after heat treatment.

[0102] 7. The method of any one of the preceding embodiments, wherein the heat treatment is an Ultra High Temperature (UHT) treatment, high temperature short-time (HTST) treatment, or low temperature long-time (LTLT) treatment. 8. The method of embodiment 7, wherein the UHT treatment is 135-154°C for 1-10 seconds.

[0103] 9. The method of embodiment 7, wherein the HTST treatment is 70-85°C for 10-30 seconds.

[0104] 10. The method embodiment 7, wherein the LTLT treatment is 55-70°C for 15-30 minutes.

[0105] 11. The method of any one of the preceding embodiments, wherein the beverage has a pH below 4.0.

[0106] 12. The method of any one of the preceding embodiments, wherein the beverage has a pH from 2.0 to 4.0, 2.0 to 3.7, 2.0 to 3.5, 2.0 to 3.0, 2.5 to 4.5, or 2.5 to 3.5.

[0107] 13. The method of any one of the preceding embodiments, wherein the beverage has a pH from 2.0 to 4.0, 2.5 to 4.5, or 2.5 to 3.5.

[0108] 14. The method of any one of the preceding embodiments, wherein the beverage is a fruit juice, fruit drink, flavored water, soda, sports drink, kombucha-based beverage, yogurt-based beverage, tea-based beverage, or coffee-based beverage.

[0109] 15. The method of any one of the preceding embodiments, wherein the beverage is a fruit juice, fruit drink, flavored water, soda, sports drink, kombucha-based beverage, or yogurt-based beverage.

[0110] 16. The method of any one of the preceding embodiments, wherein the beverage is a fruit juice, fruit drink, flavored water, soda, or sports drink.

[0111] 17. The method of any one of embodiments 1-15, wherein the beverage is a kombucha-based beverage.

[0112] 18. The method of any one of embodiments 1-15 or 17, wherein the beverage is a kombucha- based beverage that is not alcoholic.

[0113] 19. The method of any one of the preceding embodiments, wherein the beverage is carbonated.

[0114] 20. The method of any one of the preceding embodiments, wherein the B. subtilis spores are derived from B. subtilis HLI58™ (8. subtilis having accession number EF101709) and / or B. subtilis DE111® (8. subtilis having accession number NRRL B-67989). 21. The method of any one of the preceding embodiments, wherein the B. subtilis spores are derived from B. subtilis HLI58™ (B. subtilis having accession number EF101709).

[0115] 22. The method of any one of the preceding embodiments, wherein the beverage comprises viable B. subtilis spores from more than one strain.

[0116] 23. The method of any one of the preceding embodiments, wherein the beverage additionally comprises viable spores from at least one probiotic species that is not Bacillus subtilis.

[0117] 24. The method of any one of the preceding embodiments, wherein the beverage additionally comprises viable spores from at least one probiotic species of Lactobacillus, Bacillus, Bifidobacterium, Lactococcus, Propionibacterium, Enterococcus, Escherichia, Streptococcus, Pediococcus, and / or Saccharomyces.

[0118] 25. A method of producing a heat-treated acidic probiotic beverage comprising viable spores of Bacillus subtilis HLI58™ (having accession number EF101709), wherein the method comprises the following steps:

[0119] (a) preparing an acidic beverage comprising viable 8. subtilis HLI58™ spores, wherein the pH of the beverage is between 2.0 to 4.6; and

[0120] (b) heat-treating said beverage; wherein at least 70% of the initial 8. subtilis HLI58™ spores are viable after the heat-treatment.

[0121] 26. A heat-treated acidic probiotic beverage comprising viable Bacillus subtilis spores, wherein at least 1x104CFU / ml of the 8. subtilis spores are viable.

[0122] 27. The heat-treated acidic probiotic beverage of embodiment 26, wherein the pH of the beverage is between pH 2.0 to 4.6.

[0123] 28. The heat-treated acidic probiotic beverage of any one of embodiments 26 or 27, wherein the pH of the beverage is below pH 4.0.

[0124] 29. The heat-treated acidic probiotic beverage of any one of embodiments 26-28, wherein the beverage has a pH from 2.0 to 4.0, 2.0 to 3.7, 2.0 to 3.5, 2.0 to 3.0, 2.5 to 4.5, or 2.5 to 3.5.

[0125] 30. The method of any one of embodiments 26-29, wherein the beverage has a pH from 2.0 to 4.0, 2.5 to 4.5, or 2.5 to 3.5. 31. The heat-treated acidic probiotic beverage of any one of embodiments 26-30, wherein at least 1x104, 2x104, 3x104, 4x104, 5x104, 6x104, 7x104, 8x104, 9x104, 1x105, 2x105, 3x105, 4x105, 5x105, 6x105, 7x105, 8x105, 9x105, 1x106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, 1x107, 2x107, 3x107, 4x107, 5x107, 6x107, 7x107, 8x107, 9x107or at least 1x108CFU / ml of the B. subtilis spores are viable.

[0126] 32. The heat-treated acidic probiotic beverage of any one of embodiments 26-31 , wherein at least 1x105, 2x105, 3x105, 4x105, 5x105, 6x105, 7x105, 8x105, 9x105, 1x106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, or at least 1x107CFU / ml of the B. subtilis spores are viable after 7 days of storage.

[0127] 33. The heat-treated acidic probiotic beverage of any one of embodiments 26-32, wherein the beverage is a fruit juice, fruit drink, flavored water, soda, sports drink, kombucha-based beverage, yogurt-based beverage, tea-based beverage, or coffee-based beverage.

[0128] 34. The heat-treated acidic probiotic beverage of any one of embodiments 26-33, wherein the beverage is a fruit juice, fruit drink, flavored water, soda, sports drink, kombucha-based beverage, or yogurt-based beverage.

[0129] 35. The heat-treated acidic probiotic beverage of any one of embodiments 26-34, wherein the beverage is a fruit juice, fruit drink, flavored water, soda, or sports drink.

[0130] 36. The heat-treated acidic probiotic beverage of any one of embodiments 26-35, wherein the beverage is a kombucha-based beverage.

[0131] 37. The heat-treated acidic probiotic beverage of any one of embodiments 26-34 or 36, wherein the beverage is a kombucha-based beverage that is not alcoholic.

[0132] 38. The heat-treated acidic probiotic beverage of any one of embodiments 26-37, wherein the beverage is carbonated.

[0133] 39. The heat-treated acidic probiotic beverage of any one of embodiments 26-38, wherein the B. subtilis spores are derived from B. subtilis HLI58™ (B. subtilis having accession number EF101709) and / or B. subtilis DE111® (B. subtilis having accession number NRRL B-67989).

[0134] 40. The heat-treated acidic probiotic beverage of any one of embodiments 26-39, wherein the 8. subtilis spores are derived from 8. subtilis HLI58™ (8. subtilis having accession number EF101709). 41. The heat-treated acidic probiotic beverage of any one of embodiments 26-40, wherein the beverage comprises viable B. subtilis spores from more than one strain.

[0135] 42. The heat-treated acidic probiotic beverage of any one of embodiments 26-41 , wherein the beverage comprises viable spores from at least one probiotic species that is not Bacillus subtilis.

[0136] 43. The heat-treated acidic probiotic beverage of any one of embodiments 26-42, wherein the beverage comprises viable spores from at least one probiotic species of Lactobacillus, Bacillus, Bifidobacterium, Lactococcus, Propionibacterium, Enterococcus, Escherichia, Streptococcus, Pediococcus, and / or Saccharomyces.

[0137] 44. A heat-treated acidic probiotic beverage comprising viable B. subtilis spores produced by the method of any of embodiments 1-25.

[0138] EXAMPLES

[0139] Example 1 : Survival of spores in pasteurization of acidic beverages

[0140] In this example, the survival of endospores of Bacillus probiotic strains were evaluated to test the performance of the probiotics in pasteurized acidic beverages. Endospores of three commercially available Bacillus strains were included in the study: Bacillus subtilis HU58™ (Novozymes A / S, Bagsvaerd, Denmark), B. subtilis DE111® (Deerland Probiotics and Enzymes, Kennesaw, Georgia, USA) and B. coagulans BC30™ (Kerry Group, Tralee, Ireland).

[0141] To prepare the spore suspensions, endospores of each strain were diluted in three different acidic solutions of pH 2.5, pH 3.5 and pH 4.5. Dosing of spores were 1x106CFU / mL to mimic standard dosing in beverages and the different pH solutions were obtained based on citric acid and disodium hydrogen phosphate, with a target buffer strength of 20 mM. The samples were then heat shocked at 72°C for 30 seconds and then incubated at room temperature for 7 days. After 7 days, the samples were subjected to serial dilutions, which were then spread on agar plates suitable for growth of the Bacillus strains. The dilutions tested and agar plates used were the same for all of the three tested strains. The resulting colony forming units per millilitre (CFU / mL) were recorded by counting the number of colonies formed on the agar plates following incubation at 37°C.

[0142] To evaluate the immediate effect of the heat treatment at low pH, a serial dilution based on samples taken at day 0 (i.e. immediately after heat shock) were also included in the study. Finally, control samples, which were not subjected to the heat shock, were also included to determine effect of the heat treatment on activation of the endospores.

[0143] The results from the study are shown in the graph in Figure 1. From Figure 1 , it is clear that the two commercially available Bacillus subtilis strains (HU58™ and DE111®) outperformed the commercially available B. coagulans strain (BC30™). This is seen from the higher CFU / mL observed for HLI58 and DE111 both immediately after pasteurization (‘0 days’) and following incubation (7 days’). The difference in stability is particularly prominent at lower pH (pH 2.5 and pH 3.5). Acidic beverages with a 2.5-3.5 pH range include fruit juices, fruit drinks including lemonades and ciders, sodas, sports drinks, and tea-based beverages including kombucha- based beverages.

[0144] The stability observed for the endospores of B. subtilis, as exemplified herein by two commercially relevant strains, will thus allow for the production of heat-treated acidic probiotic beverages, where the probiotic B. subtilis spores are added to a beverage prior to pasteurization or sterilization by heat treatment. Thus, using the methods of the present invention, more stable probiotic-based acidic beverages can be obtained having improved storage stability and reduced risk of contamination.

Claims

CLAIMS1. A method of producing a heat-treated acidic probiotic beverage comprising viable Bacillus subtilis spores, wherein the method comprises the following steps:(a) preparing an acidic beverage comprising viable B. subtilis spores, wherein the pH of the beverage is between 2.0 to 4.6; and(b) heat-treating said beverage; wherein at least 70% of the initial B. subtilis spores are viable after the heat-treatment.

2. The method of claim 1 , wherein the number of viable B. subtilis spores is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% of the spores present before the heat treatment.

3. The method of claim 1 or 2, wherein at least 70% of the spores present before the heat treatment are viable 7 days after the heat-treatment.

4. The method of any one of the preceding claims, wherein at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% of the spores present before the heat treatment are viable 7 days after heat treatment.

5. The method of any one of the preceding claims, wherein the heat treatment is an Ultra High Temperature (UHT) treatment, high temperature short-time (HTST) treatment, or low temperature long-time (LTLT) treatment.

6. The method of any one of the preceding claims, wherein the beverage is a fruit juice, fruit drink, flavored water, soda, sports drink, kombucha-based beverage, yogurt-based beverage, teabased beverage, or coffee-based beverage.

7. The method of any one of the preceding claims, wherein the B. subtilis spores are derived from B. subtilis HU58™ (8. subtilis having accession number EF101709) and / or B. subtilis DE111® (8. subtilis having accession number NRRL B-67989).

8. The method of any one of the preceding claims, wherein the beverage additionally comprises viable spores from at least one probiotic species that is not Bacillus subtilis.

9. The method of any one of the preceding claims, wherein the beverage has a pH of less than 4.0, such as a pH from 2.0 to 4.0, 2.0 to 3.7, 2.0 to 3.5, 2.0 to 3.0, or 2.5 to 3.5.

10. A heat-treated acidic probiotic beverage comprising viable Bacillus subtilis spores, wherein at least 1x104CFU / ml of the B. subtilis spores are viable and wherein the pH of the beverage is between pH 2.0 to 4.6.

11. The heat-treated acidic probiotic beverage of claim 10, wherein the beverage has a pH of less than 4.0, such as a pH from 2.0 to 4.0, 2.0 to 3.7, 2.0 to 3.5, 2.0 to 3.0, or 2.5 to 3.5.

12. The heat-treated acidic probiotic beverage of any one of claims 10 or 11 , wherein at least 1x105, 2x105, 3x105, 4x105, 5x105, 6x105, 7x105, 8x105, 9x105, 1x106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, or at least 1x107CFU / ml of the B. subtilis spores are viable after 7 days of storage.

13. The heat-treated acidic probiotic beverage of any one of claims 10-12, wherein the beverage is a fruit juice, fruit drink, flavored water, soda, sports drink, kombucha-based beverage, yogurtbased beverage, tea-based beverage, or coffee-based beverage.

14. The heat-treated acidic probiotic beverage of any one of claims 10-13, wherein the B. subtilis spores are derived from B. subtilis HLI58™ (8. subtilis having accession number EF101709) and / or 8. subtilis DE111® (8. subtilis having accession number NRRL B-67989).

15. The heat-treated acidic probiotic beverage of any one of claims 10-14, wherein the beverage comprises viable spores from at least one probiotic species that is not Bacillus subtilis.

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