Acidic spore-based probiotic compositions

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of spore-based probiotic compositions. A heat-treated, acidic, spore-based probiotic composition is provided, comprising a plurality of viable probiotic microorganisms with biological or therapeutic activity in the gastrointestinal tract. A method for producing the heat-treated, acidic, spore-based probiotic composition is also provided. Background Technology

[0002] Probiotics are increasingly recognized for their important and beneficial role in human health, particularly in restoring or improving the composition of the gut microbiota.

[0003] Probiotics are typically provided as dietary supplements, often in capsules and powders. However, liquid is the preferred method of delivery because it allows for better absorption and distribution of the probiotics in the gastrointestinal tract. Furthermore, liquid media are convenient and eliminate the need to swallow pills or tablets. Probiotics can be found in beverages; however, these beverages typically require refrigerated delivery, storage, and use to maintain the activity / viability of the probiotics, and have a limited shelf life.

[0004] Acidic beverages (including most fruit juices, teas, sodas, and sports drinks) are enjoyed by a large number of consumers worldwide. While some acids are naturally occurring, such as citric acid in citrus drinks, it is also known that adding acid to beverages can provide a tangy and robust flavor, thus balancing the sweetness of the sugars present in the drink, and also acting as a preservative. Many acidic beverages do not require refrigeration and have a shelf life of several months.

[0005] With increasing interest in probiotics and consumers seeking convenient ways to regularly incorporate them into their diets, probiotic-containing, shelf-stable ready-to-drink acidic beverages seem like an obvious choice. However, such beverages face challenges. To be considered safe and shelf-stable, acidic beverages need to undergo sterilization or pasteurization, typically provided as a heat treatment process. However, most probiotic strains, even those available as spores, have low viability in the low pH, high-temperature environment required for sterilizing acidic beverages. Consequently, many commercially available probiotic acidic beverages fail to meet labeling claims regarding the number and type of viable microorganisms present in the product and do not deliver the amount needed for the health benefits.

[0006] US 10 494 684 B2 involves Bacillus subtilis identified as MB40. Bacillus subtilisThe strain (deposited with ATCC accession number PTA-122264) was used. In Example 2, the viability of MB40 in a hot tea preparation made from commercially available Orange Pekoe and Broken Pekoe tea bags was evaluated, with 84% viability reported by total colony count (TPC) after steeping the tea bags containing MB40 in boiling water (100°C) for 4 minutes. However, Example 2 did not mention the pH of the tea preparation, which could vary considerably depending on factors such as steeping time, tea brand / tea blend, and the tea-to-water ratio used to brew the tea preparation. In another example (identified as Example 12 in US 10 494 684 B2), MB40 viability was tested over a 10-month period at various pH levels (pH 2, 4, and 6) and solid percentage levels (solids composed of varying amounts of sugar and pea protein), concluding that the results indicated good viability of MB40 across a wide range of solid percentage levels at pH 4 to 6.

[0007] WO 2023 / 196003 A1 relates to a Bacillus subtilis strain identified as BS50 (deposited under ATCC accession number PTA-127287). In Example 4, the stability of BS50 in a hot beverage matrix was tested using commercially available broken white tea bags, concluding that increasing the steeping temperature reduced BS50 spore survival, while at least 50% of the spores survived at steeping temperatures up to 100°C. In a similar experiment, BS50 was added to ground coffee and brewed, reporting 100% BS50 survival in the resulting grounds. However, this example does not mention the actual pH of the tea preparation and the brewed coffee, which can vary considerably. Summary of the Invention

[0008] This invention is based on the unexpected and inventive discovery that Bacillus subtilis strains, known as probiotic microorganisms, can survive heat treatment in an acidic environment. Such probiotic Bacillus subtilis strains can be provided to acidic beverages, which are then heat-sterilized or pasteurized to improve shelf life. The resulting shelf-stable acidic probiotic beverages do not require refrigeration, resulting in significantly reduced energy consumption in the manufacture, packaging, delivery, and storage of the beverages.

[0009] The present invention provides a heat-treated acidic beverage containing viable Bacillus subtilis spores, and a method for producing such a heat-treated acidic beverage. Attached Figure Description

[0010] Figure 1The survival of endospores of Bacillus probiotic strains in acidic solution is shown at 0 and 7 days after heat treatment. CTRL = No pasteurization, i.e., no heating or pH change; HU58 = Bacillus subtilis HU58™; DE111 = Bacillus subtilis DE111®; BC30 = Bacillus coagulans BC30™.

[0011] Definition : As used herein, the verb “comprising” and its variations as used in this specification and claims are used in their non-limiting sense to mean including the item following the word, but not excluding items not specifically mentioned. Furthermore, the indefinite article “a” or “an” refers to an element and does not exclude the possibility of more than one element unless the context explicitly requires the presence of one and only one element. Therefore, the indefinite article “a” or “an” generally means “at least one.”

[0012] Unless otherwise defined or explicitly indicated by the context, all percentages are weight percentages (percentage w / w or "% (w / w)"). The term "% (w / v)" refers to the concentration of a substance in a solution, expressed as a weight percentage per unit volume.

[0013] This document uses the term "about" to refer to a value or parameter, including aspects of that value or parameter itself. For example, a description of "about X" includes aspect "X". When used in conjunction with a measurement, "about" includes at least a range covering the uncertainty associated with the method of measuring that particular value, and may include a range of two standard deviations positive or negative around the said value.

[0014] The accompanying drawings are for illustrating certain convenient embodiments of the invention and should not be construed as limiting the invention.

[0015] Furthermore, it will be understood that, unless otherwise stated, the embodiments described in connection with one of the aspects described herein can also be applied to the other aspects.

[0016] Unless otherwise defined or explicitly indicated by the 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 pertains.

[0017] Bacterial Count: The terms “bacterial count” and “bacterial load” are used interchangeably and have their general meanings well known and understood by those skilled in the art. In the context of this invention, bacterial count is expressed as viable bacterial colony-forming units per gram or per milliliter (abbreviated as CFU / g and CFU / ml, respectively). Bacterial count can be determined by any method known to those skilled in the art, such as, but not limited to, viable bacterial colony-forming units (CFU), quantitative polymerase chain reaction (qPCR), flow cytometry, live / dead staining, propidium azide bromide qPCR (PMA-qPCR), microscopy, metabolic assays, spectrophotometry, or any combination thereof. Methods used to determine bacterial count (such as those disclosed herein) are common and will be apparent to those skilled in the art.

[0018] Colony: The term "colony," when used in conjunction with "clone," "isolated strain," or "cell," refers to a visible aggregate or group of microbial cells. Colonies can be grown and obtained from solid culture media, such as agar.

[0019] Composition: In the context of this invention, the term "composition" covers any type of composition comprising Bacillus subtilis spores of the present invention. In the context of this invention, the composition may be a liquid composition or a dry composition.

[0020] Origin: In the context of this invention, "originate" means that the Bacillus subtilis spores used according to the invention are derived from an origin sample. As used herein, the term "originate" is interchangeable with the terms "derived" and "obtained," and refers to the source from which the spores were obtained. In some embodiments, the origin sample contains Bacillus subtilis strains found, constituted, or known to reside in an environmental and / or microbiome niche. In the context of this invention, the microbiome is the genetic material of all microorganisms (bacteria, fungi, protozoa, and viruses) living on or within the human body.

[0021] Effective amount: As used herein, “effective amount” is defined as the amount that effectively achieves the desired biological outcome (such as reducing, preventing, or treating a disease or condition and / or inducing a specific beneficial effect). In some embodiments, the effective amount is the amount provided in a single serving of probiotic beverage, where multiple servings need to be consumed over time to observe the beneficial effect.

[0022] Isolated: The term “isolated” means that one or more microorganisms (such as bacterial strains) described herein are in a form or environment that does not exist in nature, that is, the one or more microorganisms are at least partially isolated from one or more or all naturally occurring components associated with them in nature.

[0023] Probiotics: In the context of this invention, the term "probiotics" refers to microorganisms that, upon consumption, may have a beneficial effect on the health of a subject (e.g., a human subject). Typically, probiotics are generally defined as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host," such as restoring or improving the composition of the gut microbiota. See, for example, FAO / WHO, Guidelines for the evaluation of probiotics in food, London, Ontario, Canada (2002), which is incorporated herein by reference in its entirety. Therefore, the acidic probiotic beverage obtained according to the method of the invention may have a beneficial effect on the health of a subject (e.g., a human) upon ingestion of the acidic probiotic beverage.

[0024] Probiotic Beverage: In the context of this invention, a "probiotic beverage" comprises at least a probiotic composition and water. The probiotic composition contains at least one viable probiotic microorganism. The probiotic composition may contain one or more colonizing probiotic bacterial strains, which may be spore-based probiotic bacterial strains. In some embodiments, the probiotic composition contains a Bacillus subtilis species. In another embodiment, the probiotic composition contains Bacillus subtilis HU58™ (Bacillus subtilis with accession number EF101709). Bacillus species may exist in the form of spores (also called endospores) or in a vegetative state.

[0025] Acidic Probiotic Beverages: In the context of this 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 juices, fruit juice drinks, flavored waters, sodas, sports drinks, yogurt-based beverages (which may be based on dairy or dairy substitutes), kefir-based beverages, kombucha-based beverages, tea-based beverages, or coffee-based beverages. The fruit juice may be a fermented fruit juice. Acidic probiotic beverages can be obtained through a fermentation process that increases acidity, thereby creating an environment conducive to the growth and activity of probiotics. Additionally, certain milk-based and plant-based fermented beverages, such as some types of yogurt drinks and fermented 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 and 4.0, such as between 2.0 and 3.7, such as between 2.0 and 3.5, such as between 2.0 and 3.0.

[0026] Prebiotics and postbiotics: In the context of this invention, the terms "prebiotic" and "postbiotic" have their general meanings well known and understood by those skilled in the art. As used herein, the term "prebiotic" refers to any compound, nutrient, or other microorganism used to support or enhance the desired probiotic health benefits or to assist the growth and / or activity of probiotic microorganisms. The term "postbiotic" refers to probiotic cells killed by heating or other means and delivered to consumers as dead cells.

[0027] Spore: The terms “spore” and “endospore” are used interchangeably and have their general meanings well known and understood by those skilled in the art. As used herein, the term spore refers to a microorganism in its dormant, protected state.

[0028] Spore-forming microorganisms: The term "spore-forming microorganism" refers to a microorganism capable of forming spores. In the method of the present invention, viable Bacillus subtilis spores are derived from spore-forming microbial cells, which may be wild-type cells or mutants thereof. In embodiments, the spore-forming microbial cells are obtained from the environment or a microbiome niche.

[0029] Stable: The term “stable” is a term known in the art, and in a preferred aspect, stable is intended to mean the ability of a microorganism to remain in spore form until it is applied to a subject (particularly a human subject) to improve the subject’s health.

[0030] Suspension: The term "suspension" refers to materials that are suspended in a fluid.

[0031] Viable: As used in this article with respect to Bacillus subtilis spores, the term “viable” or “viable” means that a cell or spore is capable of multiplying on or in a suitable growth medium or substrate when conditions (such as temperature, moisture, nutrient availability, pH, etc.) are favorable for germination and / or microbial growth over a given period of time. Detailed Implementation

[0032] This disclosure provides heat-treated acidic beverages comprising probiotic compositions and methods for producing such heat-treated acidic probiotic beverages.

[0033] It is well known in the art that lowering pH reduces the heat resistance and viability of bacterial strains, including the viability of spores from probiotic sporulating bacteria such as Bacillus species. Surprisingly, this paper shows that Bacillus subtilis strains exhibit better viability compared to other probiotic sporulating bacteria, including other Bacillus species such as Bacillus coagulans. The ability to survive in low pH and high temperature environments indicates that Bacillus subtilis strains can survive pasteurization and / or other heat treatments used to sterilize beverages. Therefore, Bacillus subtilis strains may be better suited for certain probiotic applications, such as shelf-stable acidic probiotic beverages requiring sterilization or pasteurization steps.

[0034] In a first aspect, the present invention relates to a method for producing a heat-treated acidic probiotic beverage containing viable Bacillus subtilis spores, wherein the method comprises the following steps: (a) Preparing an acidic beverage containing viable Bacillus subtilis spores, wherein the pH of the beverage is between 2.0 and 4.6; and (b) Heat treatment of the beverage; At least 70% of the initial Bacillus subtilis spores remained viable after heat treatment.

[0035] Acidic probiotic beverages include any acidic beverage to which probiotics can be added. In some embodiments, the pH of an acidic probiotic beverage is between 2.0 and 4.6. In other embodiments, the pH of an acidic probiotic beverage is between 3.7 and 4.3. Acidic beverages typically have a pH of 4.6 or lower. Fruit juices and sodas often have a pH below 4.0. Acids and acidifiers provide a tangy and robust flavor, helping to balance the sweetness of the sugars present in the beverage and are a key factor in the beverage's taste. Acids can also act as preservatives and can reduce the growth of bacteria and fungi in beverages, thereby improving shelf stability, also known as shelf life. Acidic beverages can be produced using weak acids and / or acids naturally present in fruits. Citric acid, naturally present in citrus fruits, and malic acid, naturally present 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 can also be used to produce acidic beverages.

[0036] In some embodiments, the acidic probiotic beverage may be fruit juice, fruit drink, flavored water, soda water, sports drink, yogurt-based beverage, kefir-based beverage, kombucha-based beverage, tea-based beverage, or coffee-based beverage.

[0037] In some embodiments, the acidic probiotic beverage may be fruit juice, fruit drink, flavored water, soda water, sports drink, yogurt-based beverage, kombucha-based beverage, tea-based beverage, or coffee-based beverage.

[0038] In some embodiments, the acidic probiotic beverage is selected from fruit juice, fruit drinks, flavored water, soda water, sports drinks, yogurt-based beverages, kefir-based beverages, and kombucha-based beverages.

[0039] In some embodiments, the acidic probiotic beverage is a fruit juice, a fruit juice cocktail, lemonade, cider, or a beverage flavored with fruit juice. 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, grapefruit, or any combination thereof.

[0040] In some embodiments, the acidic probiotic beverage is soda water. Soda water is a carbonated beverage that typically contains carbonated water and sweeteners, as well as natural and / or artificial flavorings. Soda water can be pure sparkling water (seltzer). Soda water can be sugar-free, meaning it contains no sweeteners or may contain only artificial sweeteners. Soda water can be "zero sugar," meaning it may contain no carbohydrates. Soda water may also contain caffeine, colorings, preservatives, and optional additional ingredients. Soda water may also be referred to as a soft drink, pure sparkling water, "pop," or "soda pop."

[0041] In some embodiments, the acidic probiotic beverage is flavored water. Flavored water can be carbonated flavored water, such as sparkling water.

[0042] In some embodiments, acidic probiotic beverages are sports drinks. Sports drinks contain electrolytes such as sodium, potassium, and chloride, and typically also contain carbohydrates, often in the form of sugars such as glucose, sucrose, or high-fructose corn syrup. Sports drinks can be sugar-free, meaning they contain no sweeteners or may contain only artificial sweeteners. Sports drinks can be “zero-sugar,” in which case they may contain no carbohydrates. Sports drinks may contain caffeine, vitamins, minerals, proteins, amino acids, natural or artificial flavorings, and optional additional ingredients. Sports drinks may also be referred to as vitamin water, fitness water, fortified water, energy drinks, or rehydration drinks.

[0043] In some embodiments, the acidic probiotic beverage is tea or a tea-based beverage. The tea can be green tea, black tea, oolong tea, yellow tea, white tea, decaffeinated tea, herbal tea, or any combination thereof. The herbal tea can be rosehip tea, chamomile tea, gynostemma pentaphyllum tea, peppermint tea, rooibos tea, ginger tea, ginseng tea, lemongrass tea, or any combination thereof. In some embodiments, the tea-based beverage is fermented tea. In some embodiments, the acidic probiotic beverage is tea or a tea-based beverage with a pH below 4.0.

[0044] In another embodiment, the fermented tea is kombucha or a kombucha-based beverage. Kombucha is produced by symbiotic fermentation of sugary tea using a symbiotic culture of bacteria and yeast. Kombucha or a kombucha-based beverage may further contain fruit juice and / or flavorings. In some embodiments, the kombucha or kombucha-based beverage may contain ethanol or be alcoholic. In other embodiments, the kombucha or kombucha-based beverage is non-alcoholic.

[0045] In some embodiments, the acidic probiotic beverage is milk-based. Milk-based beverages can be derived from fermented dairy products such as kefir, oysters, and yogurt.

[0046] In some embodiments, the acidic probiotic beverage is yogurt-based. Yogurt-based beverages can be based on dairy products and / or dairy alternatives. Examples of dairy-alternative yogurt-based 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 nut yogurt-based beverages; or legume-based yogurt-based beverages, such as chickpea, broad bean, lentil, pea, or soybean yogurt-based beverages.

[0047] In some embodiments, the acidic probiotic beverage is coffee or a 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 contain dairy components, such as milk or cream, and / or plant-based alternatives, such as oat, almond, soy, rice, or cashew milk, or any other non-dairy alternatives known in the art. The coffee or coffee-based beverage may further contain flavoring ingredients, such as cocoa, chocolate, artificial cocoa compositions, vanilla, artificial vanilla compositions, or any combination thereof.

[0048] Acidic probiotic beverages may further contain one or more additional ingredients to improve or alter the flavor of the beverage. These additional ingredients include acidifiers, thickeners, buffers or pH adjusters, chelating agents, colorants, emulsifiers, excipients, flavorings, penetrants, acceptable carriers, preservatives, stabilizers, sugars, sweeteners, conditioning agents, minerals, and / or vitamins. These additional ingredients may be added in any suitable amount.

[0049] Those skilled in the art will further recognize that, in addition to probiotics, acidic probiotic beverages also contain food ingredients and / or ingredients intended for nutritional benefits. Other ingredients include vitamins, minerals, amino acids, proteins, fiber, prebiotics (such as oligosaccharides, galacto-oligosaccharides, inulin, and polydextrose), and metabiotics.

[0050] The heat-treated acidic probiotic beverage of the present invention can be consumed by human or animal subjects, preferably by human subjects. In some embodiments, the heat-treated acidic probiotic beverage has beneficial effects on the subjects after consumption, such as beneficial effects on the subjects' intestinal health.

[0051] In the human body, the gut is the primary location of the human microbiome and is considered essential for lifelong health. The gut microbiome helps digest food, regulates the immune system, protects against other bacteria that cause disease, and produces vitamins, including B vitamins B12, thiamine, and riboflavin; as well as vitamin K, which is necessary for blood clotting.

[0052] The gut microbiome is a collection of a vast number of bacteria, viruses, fungi, and protozoa that colonize the gastrointestinal tract and outnumber human cells by more than 10 times. Early exposure (such as through delivery (maternal microbes), infant diet (selective substrates), antibiotics (selective killing), probiotics (selective enrichment), and physical environment (environmental microbes)) leads to the colonization of the gut microbiome, which contributes to the development of the immune system, gut homeostasis, and host metabolism.

[0053] Disruption of the gut microbiota is associated with a growing number of diseases. See, for example, MB Azad et al. Gut microbiota of healthy Canadian infants: profiles by mode of delivery and infant diet at 4 months [Gut Microbiota of Healthy Canadian Infants: An Overview by Delivery Method and Diet of 4-Month-Old Infants], 185 Can. Med. Ass'n J. [Journal of the Canadian Medical Association] 385 (2013), which is incorporated herein by reference in its full text. Recent advances in metagenomics have enhanced our understanding of the gut microbiome, suggesting that it may provide important immune and metabolic benefits in humans.

[0054] The heat-treated acidic probiotic beverage disclosed herein contains probiotics in the form of Bacillus subtilis spores. Preferably, in order for the Bacillus subtilis spores to induce a probiotic effect in subjects after ingestion of the heat-treated acidic probiotic beverage, the probiotics should remain substantially viable at the intestinal level. There is some evidence supporting the importance of viability in human studies, with viable bacteria exhibiting greater immunological activity than non-viable bacteria. See, for example, M. Kaila et al. Viable versus inactivated lactobacillus strain GG in acute rotavirus diarrhea[Live Lactobacillus strain GG versus inactivated Lactobacillus strain GG in acute rotavirus diarrhea], 72 Archives of Disease in Childhood 51 (1995); PV Kirjavainen et al., Probiotic bacteria in the management of atopic disease: underscoring the importance of viability [Probiotics in the management of atopic diseases: Emphasizing the importance of vitality], 36 J. Pediatric Gastroenterology & Nutrition 223 (2003), each of these references is incorporated herein by reference in its full text.

[0055] In a preferred embodiment, the probiotics are resistant to gastric acid digestion and bile salts to reach the intestine intact and are non-pathogenic to subjects consuming the probiotic ready-to-eat food product.

[0056] Many commercially available probiotics are Gram-positive sporogenic bacteria, supplied in the form of their spores (also known as endospores). As spores, bacteria can survive harsh environments, making it possible to process them in a dry form, allowing them to be supplied, for example, in capsules and powders. Ingesting these capsules or powders then introduces the probiotics into the intestines. However, ingesting probiotics in capsules or powders may reduce their effectiveness. Liquid media allow for better absorption of probiotics and better distribution in the gastrointestinal tract.

[0057] The appropriate liquid medium for delivering probiotics to the gut needs to be a beverage that consumers want to drink. Acidic beverages (including most fruit juices, tea-based drinks, coffee-based drinks, sodas, and sports drinks) are enjoyed by a large number of consumers worldwide. To be considered safe and shelf-stable, acidic beverages need to undergo sterilization or pasteurization, which is typically provided as a heat treatment. However, while probiotics can survive under harsher conditions in spore form, most sporulating bacteria cannot survive under all harsh conditions, especially in the presence of multiple environmental stressors. Most probiotic strains, even those that can be supplied as spores, have low viability in the low pH, high temperature environment required for sterilizing acidic beverages. One solution is to sterilize the acidic beverage and the sporulating probiotics under separate conditions and then combine them by packaging them together under aseptic conditions (see, for example, WO 2019 / 231910, which is incorporated herein by reference in its entirety). However, such a manufacturing method is cumbersome, requires several additional processing steps, and increases production costs.

[0058] This invention includes a method for producing a heat-treated acidic probiotic beverage, wherein probiotics containing viable Bacillus subtilis spores are added to an acidic beverage, and the beverage is subsequently heat-treated, during which an effective amount of Bacillus subtilis spores survive. Because the probiotics are not separately sterilized and then combined with the beverage under aseptic conditions, the beverage processing requires fewer steps and is more cost-effective and resource-efficient. This invention also includes heat-treated acidic probiotic beverages produced by the method of this invention. Preferably, the heat treatment is pasteurization or cooking sterilization.

[0059] Probiotics are measured in colony-forming units (“CFU”), and can be measured in CFU / g or CFU / vol. Alternatively, a given dose of probiotics can be delivered in total CFU. The effective dose (also known as the effective amount) is typically measured in hundreds of millions of CFU per gram or milliliter, and in some cases, it can be higher. In some embodiments, probiotics are taken with a meal. In other embodiments, probiotics are taken between meals. Probiotics may survive better if taken with liquids, as liquids can dilute stomach acid and allow probiotics to enter the digestive tract more quickly. Probiotics can be taken for short periods or long periods.

[0060] This invention is based on the unexpected discovery that various Bacillus subtilis strains are highly resistant to sterilization heat treatment under acidic liquid conditions.

[0061] In some embodiments, the acidic probiotic beverage of the present invention contains spores of Bacillus subtilis HU58™ (also known as Bacillus subtilis HU58), which is deposited at the National Center for Biotechnology Research, USA, accession number EF101709. (Bacillus Genetic Depository Center, USA) Bacillus The Genetic Stock Center (BGSC) designates Bacillus HU58 as 3A34, while NCIMB Ltd designates it as 30283. HU58™ is commercially available as a dried spore composition, manufactured and marketed, for example, by Microbiome Labs UK Ltd. Commercially available spore compositions of Bacillus subtilis HU58 are also marketed by Novozymes (formerly Novozymes A / S, Bagsvaerd, Denmark) under the trade name ProSilience™ HU58™.

[0062] In some embodiments, the acidic probiotic beverage of the present invention contains Bacillus subtilis DE111® (also known as Bacillus subtilis DE111 or Bacillus subtilis desert subsp.) Bacillus subtilis subspecies inaquosorum The spores of the fungus have been deposited at the Agricultural Research Service Culture Collection (NRRL) in the United States, accession number NRRL B-67989.

[0063] In some embodiments, the acidic probiotic beverage of the present invention contains more than one strain of Bacillus subtilis. In other embodiments, the beverage of the present invention contains only one strain of Bacillus subtilis.

[0064] In some embodiments, in addition to Bacillus subtilis spores, the heat-treated acidic probiotic beverage may also contain additional probiotics. These additional probiotics may be probiotic microorganisms in spore form or in a nutrient state. The additional probiotics will also be resistant to the low pH, high-temperature heat treatment required for sterilization of the beverage. Examples of additional probiotic microorganisms may include, but are not specifically limited to, strains of Lactobacillus, Bacillus, Bifidobacterium, Lactococcus, Propionibacterium, Enterococcus, Escherichia, Streptococcus, Pediococcus, and Saccharomyces. For example, additional probiotic microorganisms may be selected from the following list: Lactobacillus acidophilus (… Lactobacillus acidophilus Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), Lactobacillus fermentum ( Lactobacillus fermentum Lactobacillus casei ( Lactobacillus Lactobacillus bulgaricus ( casei Lactobacillus gasseri ( Lactobacillus bulgaricus Lactobacillus Lactobacillus helveticus ( gasseri Lactobacillus johnsonii ( Lactobacillus helveticus Lactobacillus Lactobacillus ( ) johnsonii Lactobacillus plantarum ( Lactobacillus lactis Lactobacillus Lactobacillus reuteri ( plantarum ), Lactobacillus salivarius ( Lactobacillus reuteri Lactobacillus Lactobacillus paracasei ( salivarius ), species of the genus Bifidobacterium ( Lactobacillus paracasei Bifidobacterium longum ( Bifidobacterium sp. ), Bifidobacterium infantis ( Bifidobacterium longum Bifidobacterium animalis ( Bifidobacterium infantis Bifidobacterium bifidum ( Bifidobacterium animalis Bifidobacterium adolescentis ( )Bifidobacterium bifidum ), Bifidobacterium lactis ( Bifidobacterium adelocentis Bacillus coagulans ( Bifidobacterium lactis ), Bacillus licheniformis ( Bacillus coagulans ), Enterococcus faecalis ( Bacillus licheniformis ), Enterococcus faecalis ( Enterococcus faecalis ), Lactococcus lactis ( Enterococcus faecium ), Streptococcus salivarius ( Lactococcus lactis ), brewer's yeast ( Streptococcus salivarius ), Bula yeast ( Saccharomyces cerevisiae ) and their combinations.

[0065] In the method of this invention, an acidic probiotic beverage containing viable Bacillus subtilis spores is heat-treated. After heat treatment, the number of viable Bacillus subtilis spores 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 100% of the spores present before heat treatment.

[0066] In some embodiments, the acidic probiotic beverage contains a sufficient amount of spores to ensure that an effective quantity of spores remains viable after heat treatment. In some embodiments, the effective quantity of Bacillus subtilis spores is at least 1 x 10⁻⁶. 4 CFU / ml. In some embodiments, the concentration of Bacillus subtilis strain in the acidic probiotic beverage prior to heat treatment may be at least 1 x 10⁻⁶ CFU / ml. 4 1 colony forming unit (CFU) / ml, at least 3 x 10 4 CFU / ml, at least 5 x 10 4 CFU / ml, at least 7 x 10 4 CFU / ml, at least 9x10 4 CFU / ml, at least 1x10 5 1 colony forming unit (CFU) / ml, at least 5 x 10 5 CFU / ml, at least 7 x 10 5 CFU / ml, at least 9x10 5 CFU / ml, at least 1x10 6 CFU / ml, at least 2 x 10 6 CFU / ml, at least 3 x 10 6 CFU / ml, at least 4 x 10 6 CFU / ml, at least 5 x 10 6 CFU / ml, at least 6 x 10 6 CFU / ml, at least 7 x 10 6 CFU / ml, at least 8 x 106 CFU / ml, at least 9x10 6 CFU / ml, at least 1x10 7 CFU / ml, at least 2 x 10 7 CFU / ml, at least 3 x 10 7 CFU / ml, at least 4 x 10 7 CFU / ml, at least 5 x 10 7 CFU / ml, at least 6 x 10 7 CFU / ml, at least 7 x 10 7 CFU / ml, at least 8 x 10 7 CFU / ml, at least 9x10 7 CFU / ml, at least 1x10 8 CFU / ml, at least 2 x 10 8 CFU / ml, at least 3 x 10 8 CFU / ml, at least 4 x 10 8 CFU / ml, at least 5 x 10 8 CFU / ml, at least 6 x 10 8 CFU / ml, at least 7 x 10 8 CFU / ml, at least 8 x 10 8 CFU / ml, at least 9x10 8 CFU / ml, at least 1x10 9 CFU / ml, 5x10 9 CFU / ml, at least 1x10 10 CFU / ml, at least 5 x 10 10 CFU / ml, at least 1x10 11 CFU / ml, at least 5 x 10 11 CFU / ml or at least 1x10 12 CFU / ml.

[0067] After heat treatment, the probiotic beverage of the present invention contains an effective amount of Bacillus subtilis spores. In some embodiments, the effective amount of Bacillus subtilis spores is at least 1 x 10⁻⁶. 4 CFU / ml. In some embodiments, the heat-treated probiotic beverage contains at least 1 x 10 CFU / ml. 4 CFU / ml, at least 2 x 10 4 CFU / ml, at least 3 x 10 4 CFU / ml, at least 4 x 10 4 CFU / ml, at least 5 x 10 4 CFU / ml, at least 6 x 10 4 CFU / ml, at least 7 x 10 4CFU / ml, at least 8 x 10 4 CFU / ml, at least 9x10 4 CFU / ml, at least 1x10 5 CFU / ml, at least 2 x 10 5 CFU / ml, at least 3 x 10 5 CFU / ml, at least 4 x 10 5 CFU / ml, at least 5 x 10 5 CFU / ml, at least 6 x 10 5 CFU / ml, at least 7 x 10 5 CFU / ml, at least 8 x 10 5 CFU / ml, at least 9x10 5 CFU / ml, at least 1x10 6 CFU / ml, at least 2 x 10 6 CFU / ml, at least 3 x 10 6 CFU / ml, at least 4 x 10 6 CFU / ml, at least 5 x 10 6 CFU / ml, at least 6 x 10 6 CFU / ml, at least 7 x 10 6 CFU / ml, at least 8 x 10 6 CFU / ml, at least 9x10 6 CFU / ml, at least 1x10 7 CFU / ml, at least 2 x 10 7 CFU / ml, at least 3 x 10 7 CFU / ml, at least 4 x 10 7 CFU / ml, at least 5 x 10 7 CFU / ml, at least 6 x 10 7 CFU / ml, at least 7 x 10 7 CFU / ml, at least 8 x 10 7 CFU / ml, at least 9x10 7 CFU / ml, at least 1x10 8 CFU / ml, at least 2 x 10 8 CFU / ml, at least 3 x 10 8 CFU / ml, at least 4 x 10 8 CFU / ml, at least 5 x 10 8 CFU / ml, at least 6 x 10 8 CFU / ml, at least 7 x 10 8 CFU / ml, at least 8 x 10 8 CFU / ml, at least 9x10 8 CFU / ml, at least 1x109 CFU / ml, 5x10 9 CFU / ml, at least 1x10 10 CFU / ml, at least 5 x 10 10 CFU / ml, at least 1x10 11 CFU / ml, at least 5 x 10 11 CFU / ml or at least 1x10 12 CFU / ml.

[0068] In some embodiments, the probiotic acidic beverage is canned or packaged prior to heat treatment for in-container pasteurization. In other embodiments, the beverage is heat-treated and then canned or packaged via aseptic filling or hot filling using methods known in the art.

[0069] In the method of this invention, the acidic probiotic beverage is heat-treated to sterilize it. Heat treatment can also be referred to as pasteurization. Heat treatment aims to make the beverage safe to consume by killing harmful microorganisms, thereby reducing the risk of food poisoning. Heat treatment also increases the shelf life or shelf stability of the beverage because it eliminates microorganisms in the beverage that could degrade it by spoiling it or reducing its quality. In some embodiments, the heat treatment is performed at 85°C-95°C for 5-30 minutes. In other embodiments, the heat treatment is performed at 85°C-95°C for 10-15 minutes. In some embodiments, the heat treatment is performed at 95°C for 5, 10, 15, 20, 25, or 30 minutes. In some embodiments, the heat treatment is performed at 85°C-95°C for one minute or less. In some embodiments, the heat treatment is performed at 85°C-95°C for 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 seconds.

[0070] In some embodiments, the acidic probiotic beverage is heat-treated by high-temperature short-time (HTST) pasteurization. For this purpose, the beverage is heated to a temperature between 71.5°C and 74°C for 15-30 seconds, or to a temperature between 74°C and 76°C for 15-20 seconds. After heat treatment, the beverage can then be rapidly cooled to 4°C-5.5°C.

[0071] In some embodiments, the acidic probiotic beverage is heat-treated by ultrapasteurization. For this purpose, the beverage is heated to a temperature between 70°C and 75°C for 20-30 minutes. In some embodiments, the beverage is heated to a temperature between 80°C and 85°C for 20-30 seconds.

[0072] In some embodiments, the acidic probiotic beverage is heat-treated by ultra-high temperature (UHT) treatment. UHT treatment can be direct or indirect. In some embodiments, UHT treatment is performed at 135°C-154°C for 1-10 seconds. In other embodiments, UHT treatment is performed at 140°C-150°C for 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In still other embodiments, UHT treatment is performed at 140°C-145°C for 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In some embodiments, UHT treatment is performed at 143°C for 4, 5, 6, 7, or 8 seconds.

[0073] In some embodiments, the probiotics in the heat-treated acidic probiotic beverage are determined to have good viability for several days after heat treatment. In the methods and compositions of the present invention, the number of viable Bacillus 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 100% of the spores present before heat treatment.

[0074] Preferred embodiments

[0075] 1. A method for producing a heat-treated acidic probiotic beverage containing viable Bacillus subtilis spores, wherein the method comprises the following steps: (a) Preparing an acidic beverage containing viable Bacillus subtilis spores, wherein the pH of the beverage is between 2.0 and 4.6; and (b) Heat treatment of the beverage; At least 70% of the initial Bacillus subtilis spores remained viable after heat treatment.

[0076] 2. The method as described in Example 1, wherein at least 1x10 4 5x10 4 1x10 5 2x10 5 3x10 5 4x10 5 5x10 5 6x10 5 7x10 5 8x10 5 9x10 5 1x10 6 2x10 6 3x10 6 4x10 6 5x10 6 6x106 7x10 6 8x10 6 9x10 6 Or at least 1x10 7 These Bacillus subtilis spores at CFU / ml are viable after heat treatment.

[0077] 3. The method as described in any of the preceding embodiments, wherein the number of viable Bacillus 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 100% of the spores present prior to the heat treatment.

[0078] 4. The method as described in any of the foregoing embodiments, wherein at least 70% of the spores present prior to the heat treatment are viable 7 days after the heat treatment.

[0079] 5. The method as described in any of the foregoing embodiments, wherein at least 1x10 4 5x10 4 1x10 5 2x10 5 3x10 5 4x10 5 5x10 5 6x10 5 7x10 5 8x10 5 9x10 5 1x10 6 2x10 6 3x10 6 4x10 6 5x10 6 6x10 6 7x10 6 8x10 6 9x10 6 Or at least 1x10 7 These Bacillus subtilis spores at CFU / ml were viable 7 days after the heat treatment.

[0080] 6. The method as described in any 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 100% of the spores present prior to the heat treatment are viable 7 days after the heat treatment.

[0081] 7. The method as described in any of the foregoing embodiments, wherein the heat treatment is an ultra-high temperature (UHT) treatment, a high temperature short time (HTST) treatment, or a low temperature long time (LTLT) treatment.

[0082] 8. The method as described in Example 7, wherein the UHT treatment is performed at 135°C-154°C for 1-10 seconds.

[0083] 9. The method as described in Example 7, wherein the HTST treatment is performed at 70°C-85°C for 10-30 seconds.

[0084] 10. The method as described in Example 7, wherein the LTLT treatment is performed at 55°C-70°C for 15-30 minutes.

[0085] 11. The method as described in any of the foregoing embodiments, wherein the beverage has a pH below 4.0.

[0086] 12. The method as described in any of the preceding embodiments, wherein the beverage has a pH of 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.

[0087] 13. The method as described in any of the foregoing embodiments, wherein the beverage has a pH of 2.0 to 4.0, 2.5 to 4.5, or 2.5 to 3.5.

[0088] 14. The method as described in any of the foregoing embodiments, wherein the beverage is fruit juice, fruit juice drink, flavored water, soda water, sports drink, kombucha-based beverage, yogurt-based beverage, tea-based beverage, or coffee-based beverage.

[0089] 15. The method as described in any of the foregoing embodiments, wherein the beverage is fruit juice, fruit juice drink, flavored water, soda water, sports drink, kombucha-based beverage, or yogurt-based beverage.

[0090] 16. The method as described in any of the foregoing embodiments, wherein the beverage is fruit juice, fruit juice drink, flavored water, soda water, or sports drink.

[0091] 17. The method as described in any one of Examples 1-15, wherein the beverage is a kombucha-based beverage.

[0092] 18. The method as described in any one of Examples 1-15 or 17, wherein the beverage is a non-alcoholic kombucha-based beverage.

[0093] 19. The method as described in any of the foregoing embodiments, wherein the beverage is a carbonated beverage.

[0094] 20. The method as described in any of the foregoing embodiments, wherein the Bacillus subtilis spores are derived from Bacillus subtilis HU58™ (Bacillus subtilis with accession number EF101709) and / or Bacillus subtilis DE111® (Bacillus subtilis with accession number NRRL B-67989).

[0095] 21. The method as described in any of the foregoing embodiments, wherein the Bacillus subtilis spores are derived from Bacillus subtilis HU58™ (Bacillus subtilis with accession number EF101709).

[0096] 22. The method as described in any of the preceding embodiments, wherein the beverage contains viable Bacillus subtilis spores from more than one strain.

[0097] 23. The method as described in any of the preceding embodiments, wherein the beverage further comprises viable spores derived from at least one probiotic species that is not Bacillus subtilis.

[0098] 24. The method as described in any of the preceding embodiments, wherein the beverage further comprises viable spores derived from at least one probiotic species selected from the genera *Lactobacillus*, *Bacillus*, *Bifidobacterium*, *Lactococcus*, *Propionibacterium*, *Enterococcus*, *Escherichia*, *Streptococcus*, *Pediococcus*, and / or *Saccharomyces*.

[0099] 25. A method for producing a heat-treated acidic probiotic beverage containing viable Bacillus subtilis HU58™ (accession number EF101709) spores, wherein the method comprises the following steps: (a) Preparing an acidic beverage containing viable Bacillus subtilis HU58™ spores, wherein the pH of the beverage is between 2.0 and 4.6; and (b) Heat treatment of the beverage; At least 70% of the initial Bacillus subtilis HU58™ spores were viable after heat treatment.

[0100] 26. A heat-treated acidic probiotic beverage containing viable Bacillus subtilis spores, wherein at least 1 x 10⁻⁶ spores are present. 4 These Bacillus subtilis spores at CFU / ml are viable.

[0101] 27. The heat-treated acidic probiotic beverage as described in Example 26, wherein the pH of the beverage is between pH 2.0 and 4.6.

[0102] 28. The heat-treated acidic probiotic beverage as described in any one of Examples 26 or 27, wherein the pH of the beverage is below pH 4.0.

[0103] 29. The heat-treated acidic probiotic beverage as described in any one of Examples 26-28, wherein the beverage has a pH of 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.

[0104] 30. The method of any one of Examples 26-29, wherein the beverage has a pH of 2.0 to 4.0, 2.5 to 4.5, or 2.5 to 3.5.

[0105] 31. A heat-treated acidic probiotic beverage as described in any one of Examples 26-30, wherein at least 1 x 10 4 2x10 4 3x10 4 4x10 4 5x10 4 6x10 4 7x10 4 8x10 4 9x10 4 1x10 5 2x10 5 3x10 5 4x10 5 5x10 5 6x10 5 7x10 5 8x10 5 9x10 5 1x10 6 2x10 6 3x10 6 4x10 6 5x10 6 6x10 6 7x10 6 8x10 6 9x10 6 1x10 7 2x10 7 3x10 7 4x10 7 5x10 7 6x10 7 7x10 7 8x10 7 9x10 7 Or at least 1x10 8 These Bacillus subtilis spores at CFU / ml are viable.

[0106] 32. The heat-treated acidic probiotic beverage as described in any one of Examples 26-31, wherein at least 1 x 10 5 2x10 5 3x10 5 4x10 5 5x10 5 6x10 5 7x10 5 8x10 5 9x10 5 1x10 6 2x10 6 3x10 6 4x10 6 5x10 6 6x10 6 7x10 6 8x10 6 9x10 6 Or at least 1x10 7 These Bacillus subtilis spores at CFU / ml remained viable after 7 days of storage.

[0107] 33. The heat-treated acidic probiotic beverage as described in any one of Examples 26-32, wherein the beverage is a fruit juice, fruit juice drink, flavored water, soda water, sports drink, kombucha-based beverage, yogurt-based beverage, tea-based beverage, or coffee-based beverage.

[0108] 34. The heat-treated acidic probiotic beverage as described in any one of Examples 26-33, wherein the beverage is fruit juice, fruit juice drink, flavored water, soda water, sports drink, kombucha-based beverage or yogurt-based beverage.

[0109] 35. The heat-treated acidic probiotic beverage as described in any one of Examples 26-34, wherein the beverage is fruit juice, fruit juice drink, flavored water, soda water or sports drink.

[0110] 36. The heat-treated acidic probiotic beverage as described in any one of Examples 26-35, wherein the beverage is a kombucha-based beverage.

[0111] 37. The heat-treated acidic probiotic beverage as described in any one of Examples 26-34 or 36, wherein the beverage is a non-alcoholic kombucha-based beverage.

[0112] 38. The heat-treated acidic probiotic beverage as described in any one of Examples 26-37, wherein the beverage is a carbonated beverage.

[0113] 39. The heat-treated acidic probiotic beverage as described in any one of Examples 26-38, wherein the Bacillus subtilis spores are derived from Bacillus subtilis HU58™ (Bacillus subtilis with accession number EF101709) and / or Bacillus subtilis DE111® (Bacillus subtilis with accession number NRRL B-67989).

[0114] 40. The heat-treated acidic probiotic beverage as described in any one of Examples 26-39, wherein the Bacillus subtilis spores are derived from Bacillus subtilis HU58™ (Bacillus subtilis with accession number EF101709).

[0115] 41. The heat-treated acidic probiotic beverage as described in any one of Examples 26-40, wherein the beverage contains viable Bacillus subtilis spores from more than one strain.

[0116] 42. The heat-treated acidic probiotic beverage as described in any one of Examples 26-41, wherein the beverage contains viable spores derived from at least one probiotic species that is not Bacillus subtilis.

[0117] 43. The heat-treated acidic probiotic beverage as described in any one of Examples 26-42, wherein the beverage contains viable spores derived from at least one probiotic species selected from the genera *Lactobacillus*, *Bacillus*, *Bifidobacterium*, *Lactococcus*, *Propionibacterium*, *Enterococcus*, *Escherichia*, *Streptococcus*, *Pediococcus*, and / or *Saccharomyces*.

[0118] 44. A heat-treated acidic probiotic beverage comprising viable Bacillus subtilis spores produced by any one of Examples 1-25.

[0119] Example Example 1: Spore survival during pasteurization of acidic beverages In this example, the survival of endospores from Bacillus probiotic strains was evaluated to test the performance of probiotics in pasteurized acidic beverages. The study included endospores from three commercially available Bacillus strains: Bacillus subtilis HU58™ (Novison, Bageswede, Denmark), Bacillus subtilis DE111® (Deerland Probiotics and Enzymes, Kennesaw, Georgia, USA), and Bacillus coagulans BC30™ (Kerry Group, Tralee, Ireland).

[0120] To prepare spore suspensions, endospores of each strain were diluted in three different acidic solutions at pH 2.5, pH 3.5, and pH 4.5. The spore dosage was 1 x 10⁻⁶. 6 CFU / mL was used to simulate the standard dose in beverages, and 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 incubated at room temperature for 7 days. After 7 days, the samples were serially diluted and then spread onto agar plates suitable for the growth of Bacillus strains. For all three test strains, the tested dilutions and agar plates used were identical. The resulting colony-forming units per milliliter (CFU / mL) were recorded by counting the number of colonies formed on the agar plates after incubation at 37°C.

[0121] To evaluate the immediate effects of heat treatment at low pH, the study also included serial dilutions of samples obtained on day 0 (i.e., immediately after heat shock). Finally, control samples that were not subjected to heat shock were included to determine the effect of heat treatment on the activation of endospores.

[0122] The results of the study show Saccharomyces boulardii In the diagram. From Figure 1 Figure 1 Clearly, the two commercially available Bacillus subtilis strains (HU58™ and DE111®) were superior to the commercially available Bacillus coagulans strain (BC30™). This is evident from the higher CFU / mL observed immediately after pasteurization ('0 days') and after incubation ('7 days') for both HU58 and DE111. The differences in stability were particularly significant at lower pH levels (pH 2.5 and pH 3.5). Acidic beverages with a pH range of 2.5–3.5 include fruit juices, fruit drinks (including lemonade and cider), sodas, sports drinks, and tea-based beverages (including kombucha-based beverages).

[0123] As illustrated herein with reference to two commercially relevant strains, the stability observed for Bacillus subtilis endospores thus allows for the production of heat-treated acidic probiotic beverages in which Bacillus subtilis spores are added to the beverage prior to pasteurization or sterilization by heat treatment. Therefore, using the method of the present invention, more stable probiotic-based acidic beverages with improved storage stability and reduced risk of contamination can be obtained.

Claims

1. A method for producing a heat-treated acidic probiotic beverage containing viable Bacillus subtilis spores, wherein the method comprises the following steps: (a) Preparing an acidic beverage containing viable Bacillus subtilis spores, wherein the pH of the beverage is between 2.0 and 4.6; and (b) Heat treatment of the beverage; At least 70% of the initial Bacillus subtilis spores were viable after the heat treatment.

2. The method of claim 1, wherein the number of viable Bacillus 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 100% of the spores present prior to the heat treatment.

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

4. The method as described in any 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 100% of the spores present prior to the heat treatment are viable 7 days after the heat treatment.

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

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

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

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

9. The method of any of the preceding claims, wherein the beverage has a pH of less than 4.0, such as 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 containing viable Bacillus subtilis spores, wherein at least 1 x 10⁻⁶ spores are present. 4 The Bacillus subtilis spores at CFU / ml are viable, and the pH of the beverage is between pH 2.0 and 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 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 as described in any one of claims 10 or 11, wherein at least 1 x 10 5 2x10 5 3x10 5 4x10 5 5x10 5 6x10 5 7x10 5 8x10 5 9x10 5 1x10 6 2x10 6 3x10 6 4x10 6 5x10 6 6x10 6 7x10 6 8x10 6 9x10 6 Or at least 1x10 7 The Bacillus subtilis spores at CFU / ml were viable after 7 days of storage.

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

14. The heat-treated acidic probiotic beverage according to any one of claims 10-13, wherein the Bacillus subtilis spores are derived from Bacillus subtilis HU58™ (Bacillus subtilis with accession number EF101709) and / or Bacillus subtilis DE111® (Bacillus subtilis with accession number NRRL B-67989).

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

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