Probiotic strains selected by targeted in vivo enrichment to support healthy lactose digestion

Abstract

Methods and compositions are provided for the selection of probiotic strains by targeted in vivo (in the human gut) enrichment to aid in healthy lactose digestion. The selected probiotic strains may advantageously include Bifidobacterium adolescentis strain IVS-1 to reduce and / or prevent symptoms of lactose intolerance in subjects in need thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 306,547, filed February 4, 2022, and entitled “Probiotic Strain Selected by Targeted In Vivo (In the Human Gut) Enrichment to Aid with Healthy Lactose Digestion,” the entire contents of which are incorporated herein by reference.

[0002] Sequence table XML This application contains a Sequence Listing that has been submitted via electronic submission in XML file format and is incorporated herein by reference in its entirety. The XML file, created on Feb. 2, 2023, is named 93259.2.WOU1_Sequence Listing.xml and is 3305 bytes in size.

[0003] The present disclosure relates to compositions and methods for supporting lactose digestion that include the incorporation of a probiotic, for example a therapeutically effective amount of isolated Bifidobacterium adolescentis strain IVS-1. [Background technology]

[0004] Dairy foods are an important source of protein, riboflavin, and calcium, especially in the United States, Europe, Asia, Africa, and many other parts of the world. However, many individuals suffer from lactose intolerance. This condition results from the absence or insufficient production of the enzyme lactase, also known as beta-galactosidase. Acquired lactase deficiency is the most common disorder of complex carbohydrate absorption worldwide, affecting up to 75% of the world's population. In the United States, 15% of Caucasians, more than 50% of Hispanics, and more than 80% of African Americans suffer from lactose intolerance. The disorder is characterized by gastrointestinal symptoms of excessive gas production, abdominal pain, cramps, bloating, and diarrhea after excessive consumption of lactose-containing foods, such as dairy and dairy-based products.

[0005] During infancy, mammals have high levels of lactase activity in the lining of the upper digestive tract due to their reliance on lactose as the primary carbohydrate in their diet. However, in humans, lactase expression in the upper digestive tract decreases by approximately 90% between the ages of 2 and 5. This condition is called primary lactase deficiency. Many Northern Europeans, some Western Europeans, Mediterraneans, and their descendants have a mutation that prevents this natural decrease in lactase production. These individuals are able to consume milk and other dairy-based products without problems as adults. However, a large proportion of the world's population, such as Southern Europeans, East Asians, and sub-Saharan Africans, have primary lactase deficiency.

[0006] Alternatively, secondary lactase deficiency results from injury or disease of the small intestine. For example, celiac disease, inflammatory bowel syndrome (IBS) and Crohn's disease are often associated with lactase deficiency. These diseases occur in all ethnic groups.

[0007] Lactose is a disaccharide of glucose and galactose linked by a beta-D-glycosidic bond. The disaccharide is digested into its individual sugars by lactase (beta-D-galactosidase) produced in the small intestine by cells at the intestinal villus border. Since glucose and galactose are absorbed in the small intestine, the absence of lactase in the small intestine would allow undigested lactose to reach the large intestine. In the large intestine, resident bacteria metabolize lactose by fermentation, possibly producing gas in the large intestine depending on the particular bacterial species. The gas is responsible for symptoms such as pain, pressure, cramps and flatulence. In addition, undigested lactose increases the osmotic pressure in the intestine, causing increased water excretion and diarrhea.

[0008] One way to supplement lactase involves ingesting live or dead lactase-producing bacteria.For example, it is known that people with mild lactose intolerance can tolerate yogurt but not milk, even though yogurt and milk contain the same amount of lactose.This is because bacteria added to yogurt, such as Streptococcus, Lactobacillus and Bifidobacterium species, express functional lactase both during fermentation and digestion in the digestive tract.Therefore, yogurt products containing live and active cultures of these bacteria can be ingested to improve the ability to digest lactose.

[0009] Unfortunately, physicians have reported limited success treating lactose intolerance with many traditional probiotic bacterial cultures. A literature review conducted by a family physician at the University of Pittsburg (Non-Patent Document 1) concluded that, overall, the strategy was ineffective. The authors suggest that this is due to variation in the bacteria's survival rate and ability to produce lactase among different dairy products and supplements. According to the study, with a few exceptions, most of these products do not provide the human body with enough lactase activity at once to alleviate the symptoms of lactose intolerance. Other attempts to solve lactose intolerance with probiotics have used non-natural strains that 1) cannot survive the GI transit, 2) cannot colonize the intestine, or 3) cannot compete with lactose in the ecological context of the gut. Probiotic strains that cannot survive the transit may alleviate symptoms by releasing active lactase into the intestinal lumen upon dissolution, but are unlikely to provide long-term relief. Similarly, probiotics that cannot colonize will eventually be flushed through the intestine without providing long-term relief of symptoms. Strains that do not compete with lactose in the system may still allow undesirable species to access the substrate, leading to undesirable gas production, or may not metabolize the substrate fast enough to prevent osmosis from causing watery feces. Attempts to show a solution to lactose intolerance by supplementing with lactose-like prebiotic galactooligosaccharides (GOS) have ultimately been unsuccessful, and the Phase 3 trial data shown by Ritter Pharmaceuticals (Non-Patent Document 2) are likely due to a combination of 1) individual variations in the subject's microbiome and perhaps the lack of enrichable and competitive lactose-degrading strains, and 2) competition of dietary lactose with GOS for metabolism by resident lactose-degrading bacteria.

[0010] Symptoms of lactose intolerance were not alleviated when treatment with Lactobacillus acidophilus, a very common probiotic species, was used. Among the common yogurt-additive bacteria, L. acidophilus has one of the highest natural lactase levels and the ability to adhere to the intestinal wall. Nevertheless, a Tufts University study (Non-Patent Document 3) concluded that even this bacterium was ineffective against symptoms of lactose intolerance.

[0011] There remains a need in the art for improved methods and compositions for alleviating or preventing the symptoms of lactose intolerance. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] “Do probiotics reduce adult lactose intolerance?”J.Fam.Pract.,2005;v.54,No.7,p.613-620 [Non-Patent Document 2] https: / / www.globenewswire.com / news-release / 2019 / 09 / 12 / 1915140 / 0 / en / Ritter-Pharmaceuticals-Reports-Top-Line-Results-from-its-Liberatus-Phase-3-Trial-of-RP-G28-for-Lactose-Intolerance.html [Non-Patent Document 3] “A randomized trial of Lactobacillus acidophilus BG2F04 to treat lactose intolerance”, Am.J.Clin.Nutr.1999,69:140-146 Summary of the Invention

[0013] Provided herein are methods and compositions for probiotic strains selected by targeted in vivo (in the human gut) enrichment to aid in healthy lactose digestion. In Example 1, a method of treating lactose intolerance comprises administering to a subject in need thereof a therapeutically effective amount of isolated Bifidobacterium adolescentis strain IVS-1.

[0014] Example 2 is where the strain is at least 10 6 The method of Example 1 is directed to a method for administering the medicament according to the present invention in an amount of 0.1 CFU / day. Example 3 relates to the method according to example 1 or 2, wherein the strain is administered daily for one or more weeks.

[0015] Example 4 relates to a method according to any one of Examples 1-3, wherein the method improves lactose degradation and / or reduces lactose intolerance symptoms in a subject in need thereof. Example 5 relates to a method according to any one of Examples 1 to 4, wherein the Bifidobacterium adolescentis strain IVS-1 is pretreated with lactose to increase the initial lactase activity of the Bifidobacterium adolescentis strain IVS-1.

[0016] Example 6 relates to a method according to any one of Examples 1 to 5, wherein the method provides lactase to a subject in need thereof via administration of both live and inactive cultures of Bifidobacterium adolescentis strain IVS-1, and its actual and former cellular contents.

[0017] Example 7 relates to a method according to any one of Examples 1 to 6, wherein the method reduces gas production in the intestine. Example 8 relates to a method according to any one of Examples 1 to 7, wherein the method balances osmotic pressure in the intestine.

[0018] Example 9 relates to a method according to any one of Examples 1 to 8, wherein the method alters the gut microbiome to benefit the health and well-being of a subject in need thereof.

[0019] In Example 10, a food composition for treating lactose intolerance comprises Bifidobacterium adolescentis strain IVS-1, an inoculum of Bifidobacterium adolescentis strain IVS-1 exhibiting lactase activity.

[0020] Example 11 relates to the composition according to Example 10, wherein the food composition is selected from the group consisting of milk, curd, fermented milk based products, acidified milk, yogurt, desserts, snack foods, chocolates, candies, lozenges, frozen yogurt, milk powders, milk based powders, milk concentrates, cheeses, cheese spreads, dressings, beverages, ice creams, bars, additive powders, encapsulated and tableted supplements, fermented or non-fermented cereal based products, infant formulas, tablets, liquid bacterial suspensions, dry oral supplements, and wet oral supplements.

[0021] Example 12 relates to a composition according to Example 10 or 11, further comprising a pharma- ceutically acceptable carrier. Example 13 relates to a composition according to any one of Examples 10 to 12, further comprising a prebiotic selected from the group consisting of galactooligosaccharides (GOS), fructooligosaccharides (FOS), and inulin, mannan-oligosaccharides (MOS), arabinoxylan, or compositions obtained from bacterial cells, metabolites, enzyme activities, and polysaccharides of Bifidobacterium adolescentis strain IVS-1.

[0022] Example 14 relates to a composition according to any one of Examples 10 to 13, wherein the Bifidobacterium adolescentis strain IVS-1 is selected from a wild-type strain, a derivative strain, and / or a mutant strain.

[0023] Example 15 shows that Bifidobacterium adolescentis strain IVS-1 is 4 ~about 10 14 Concentration of CFU relates to the composition described in any one of Examples 10 to 14.

[0024] In Example 16, a method of treating lactose intolerance comprises administering to a subject in need thereof a therapeutically effective amount of Bifidobacterium adolescentis strain IVS-1 and increasing lactase activity in the gastrointestinal tract by Bifidobacterium adolescentis strain IVS-1 at least one day after administration.

[0025] Example 17 relates to the method according to Example 16, wherein the method further comprises increasing beta-galactosidase activity levels in the gastrointestinal tract via administration of Bifidobacterium adolescentis strain IVS-1.

[0026] Example 18 relates to the method according to Example 16 or 17, wherein the method further comprises reducing lactose levels in the digestive tract through administration of Bifidobacterium adolescentis strain IVS-1.

[0027] Example 19 relates to the method of any one of Examples 16-19, wherein the method further comprises delivering an effective amount of GOS together with Bifidobacterium adolescentis strain IVS-1.

[0028] Example 20 relates to the method of any one of Examples 16-19, wherein the method further comprises decreasing hydrogen production in the digestive tract and reducing symptoms associated with lactose intolerance in the subject.

[0029] While multiple embodiments are disclosed, still other embodiments of the present disclosure will become apparent to those skilled in the art from the following detailed description, which shows and describes illustrative embodiments of the present disclosure. As will be realized, the present disclosure can be modified in various obvious aspects without departing from the spirit and scope of the present disclosure. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not restrictive.

[0030] In the accompanying drawings, like elements are identified by like reference numerals among several preferred embodiments of the present disclosure. [Brief description of the drawings]

[0031] [Figure 1] FIG. 1 is a graph showing the growth of Bifidobacterium adolescentis strain IVS-1 compared to several other probiotic strains when grown in intestinal-like medium containing lactose. [Diagram 2] FIG. 1 is a graph showing Bifidobacterium adolescentis strain IVS-1 resulting in greater reduction in gas production from lactose spiked fecal samples compared to other probiotic strains. [Diagram 3] 1 is a graph showing expected results of treatment with Bifidobacterium adolescentis strain IVS-1 reducing hydrogen production and symptom scores. [Figure 4] FIG. 1 is a flow chart showing the clinical trial pathway for Example 4 visits, meal washout, baseline measurements and lactose challenge, treatment with Bifidobacterium adolescentis strain IVS-1 or placebo, and post-treatment measurements and lactose challenge. [Diagram 5] 1 is a graph showing predicted results of relative lactase activity for different probiotic strains and different growth conditions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The foregoing and other features and advantages of the present disclosure will be apparent from the following detailed description of exemplary embodiments, when read in conjunction with the accompanying drawings, which are merely illustrative of the disclosure rather than limiting, the scope of which is defined by the appended claims and their equivalents.

[0033] The embodiments of the present disclosure are described with reference to the drawings, in which like numerals refer to like elements throughout. The terms used in the description set forth herein are not intended to be interpreted in any limiting or restrictive manner, merely because they are utilized in conjunction with a detailed description of certain specific embodiments of the present disclosure. Furthermore, the embodiments of the present disclosure may include several novel features, none of which, taken alone, is solely responsible for its desirable attributes or is essential to the practice of the disclosure described herein.

[0034] Use of the terms "a," "an," "the," and similar referents in the context of describing the present disclosure should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. It will be further understood that the terms "comprises," "comprising," "includes," and / or "including," as used herein, specify the presence of stated features, elements, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, elements, steps, operations, elements, components, and / or groups thereof.

[0035] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each individual value falling within that range, unless otherwise indicated herein, and each individual value is incorporated into the specification as if it were set forth individually herein. The word "about," when accompanied by a numerical value, is to be interpreted as indicating a deviation of up to about 1 log, inclusive, from the recited numerical value. For example, the term "about" may be interpreted as indicating a deviation of 0.1 to 10 times the recited value. Any and all examples provided herein, or the use of exemplary language (such as "for example" or "such as"), are intended merely to better clarify the disclosure and do not limit the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0036] References to "one embodiment," "an embodiment," "exemplary embodiment," "various embodiments," etc. may indicate that the embodiment(s) of the present disclosure so described may include a particular feature, structure, or characteristic, but not all embodiments necessarily include a particular feature, structure, or characteristic. Further, repeated use of the phrase "in one embodiment" or "in an exemplary embodiment" does not necessarily refer to the same embodiment, but may.

[0037] As used herein, the term "method" refers to manners, means, techniques, and procedures for accomplishing a given task, including, but not limited to, manners, means, techniques, and procedures known or readily developed from known manners, means, techniques, and procedures by practitioners in the fields of chemistry, pharmacology, biology, biochemistry, and medicine. Unless otherwise expressly stated, it is never intended that any method or aspect described herein be interpreted as requiring that its steps be performed in a particular order. Thus, method claims are never intended to imply an order in any respect unless specifically recited in the claims or description that the steps are limited to a particular order. This is to be maintained for any possible non-express basis for interpretation, including the obvious meaning derived from the arrangement of steps or operational flow, grammatical construction or punctuation, or logical matters regarding the number or type of aspects described in the specification.

[0038] As used herein, "isolated" refers to obtaining a population of microbial cells in which at least about 80% (e.g., about 85%, 90%, 95%, 99% or 100%) of the cells are cells of a particular strain, e.g., Bifidobacterium adolescentis strain IVS-1 as described herein.

[0039] The term "probiotic" is used herein to refer to compositions comprising probiotic microorganisms. Probiotic bacteria are defined as live bacteria that, when administered in sufficient amounts, confer a health benefit on the host. Probiotic microorganisms are defined as "Live microorganisms which when administered in adequate amounts confer a health benefit on the host" (FAO / WHO 2002).

[0040] The expression "prebiotic" refers to a composition or a component of a composition that is selectively utilized by the host microorganisms to confer a health benefit. Prebiotics are generally non-viable food ingredients that are specifically fermented in the colon by bacteria that are considered to have a positive value, such as bifidobacteria, lactobacilli, and other short-chain fatty acid producing microorganisms. Prebiotics are also known as ingredients that allow certain changes in both the composition and / or activity of the gastrointestinal microflora that benefit the health and well-being of the host. The combined administration of probiotic strains with one or more prebiotic compounds, which, if optimally designed, can enhance the growth of the administered probiotics in vivo and provide additional or more pronounced health benefits, is called a synbiotic. A synbiotic is formally defined as "a mixture containing live microorganisms and substrate(s) that are selectively utilized by the host microorganisms to confer a health benefit to the host". Well-characterized prebiotics include, for example, galactooligosaccharides (GOS), fructooligosaccharides (FOS), and inulin. GOS and FOS refer to a group of oligomeric non-digestible carbohydrates that are often produced from monomers that use glycosidases to catalyze transgalactosylation reactions.These carbohydrates are often resistant to digestion by host secreted enzymes in the small intestine, and reach the colon intact and are available to the colonic microflora.Those skilled in the art will understand that other compounds that fall within the definition of prebiotics can also be used in the methods described herein.

[0041] As used herein, a "subject" can refer to a human or a non-human. Exemplary non-human subjects include, but are not limited to, livestock (e.g., pigs, cows, horses, goats, and sheep), poultry (e.g., large birds such as chickens and turkeys), and companion animals (e.g., pets such as dogs and cats).

[0042] In a further embodiment, the composition further comprises a pharma- ceutically acceptable carrier. As used herein, the term "pharma- ceutically acceptable carrier" refers to one or more solid or liquid filler diluents or encapsulating substances suitable for administration to humans or animals and compatible with active probiotic organisms. The term "compatible" refers to the components of the pharmaceutical composition that can be mixed with the Bifidobacterium adolescentis strain IVS-1, or its variants, further described herein, in a manner that allows for an interaction that substantially reduces the probiotic effectiveness of the organism selected for this disclosure under normal use conditions. A pharma- ceutically acceptable carrier must be of sufficiently high purity and sufficiently low toxicity to be suitable for administration to humans and animals to be treated.

[0043] A bacterial "strain" as used herein refers to a bacterium that remains genetically unchanged when grown or multiplied. It includes a large number of identical bacteria. A "wild-type strain" refers to the unmutated form of a bacterium found in nature. In the present context, the term "derived strain" should be understood as a strain derived from a mother strain, for example, by genetic engineering, conventional laboratory and commercial production culture, radiation and / or chemical treatment, and / or selection, adaptation, screening, etc. In certain embodiments, the derived strain is a functionally equivalent mutant, for example, a mutant having substantially the same or improved properties (e.g., with respect to probiotic properties) as the mother strain. Such derived strains are part of the present disclosure. The term "derived strain" includes strains obtained by subjecting the strains of the present disclosure to any conventionally used mutagenesis treatment, including treatment with chemical mutagens such as ethane methanesulfonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV light, or spontaneously occurring mutations.

[0044] "Mutant" or "Mutant Strain" refers to a natural (spontaneous, naturally occurring) mutant or induced mutant bacteria that contains one or more mutations in its genome (DNA) that are not present in the wild-type DNA. An "induced mutant" is a bacterium that has been mutated by human treatment, including any conventionally used mutagenic treatment, including treatment with (i) a mutagen that associates with or incorporates into DNA, such as a base analogue, e.g., an interchelator such as 2-aminopurine or ICR-191, or (ii) a chemical mutagen, including an alkylating agent such as nitrosoguanidine or hydroxylamine, or a mutagen that reacts with DNA, including an alkylating agent such as ethane methylsulfonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV radiation, or gamma radiation. In contrast, a "spontaneous mutant" or "naturally occurring mutant" has not been mutated by humans. A derivative such as a mutant may have undergone several mutagenesis treatments (a single treatment should be understood as one mutagenesis step followed by a screening / selection step), but typically no more than 20, no more than 10, or no more than 5 treatments are performed. In certain embodiments of a derivative such as a mutant, less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, or even less than 0.0001% of the nucleotides in the bacterial genome are altered (such as by substitution, insertion, deletion, or combination thereof) compared to the parent strain. The mutant bacteria described above are non-GMO, i.e., not modified by recombinant DNA techniques. As an alternative to the above-mentioned preferred method of providing mutants by random mutagenesis, it is also possible to provide such mutants by site-directed mutagenesis, for example by using appropriately designed PCR techniques or by using transposable elements that can be integrated into a bacterial replicon. When the mutants are provided as spontaneously occurring mutants, the wild-type strain above is subjected to a selection step without a preceding mutagenesis treatment.

[0045] composition The present disclosure provides compositions and methods that utilize probiotics to address negative symptoms associated with gas-producing and undesirable species in the digestive tract of a subject. In some embodiments, the probiotic comprises Bifidobacterium adolescentis. In a preferred embodiment, the probiotic comprises Bifidobacterium adolescentis strain IVS-1. Bifidobacterium adolescentis strain IVS-1 is an ecologically adapted probiotic strain that has the genetic ability to compete for lactose and lactose-like carbohydrates in the human intestine. Bifidobacterium adolescentis strain IVS-1 was identified by a process of in vivo selection (IVS) after enrichment in the human intestine by ingestion of probiotic GOS (chemically similar to lactose) by individuals that naturally host the strain. Thus, the Bifidobacterium adolescentis strain IVS-1 disclosed herein is provided at a concentration level that is much higher than that found naturally in an individual subject. This strain can outcompete gas-producing and other undesirable species in the human intestine for target carbohydrates when administered at sufficient levels for colonization.In some embodiments, Bifidobacterium adolescentis strain IVS-1 compensates for the lack of endogenous human lactase activity in the intestinal environment, thus reducing and / or eliminating the gastrointestinal symptoms of lactose intolerance.Reducing and / or eliminating the gastrointestinal symptoms of lactose intolerance allows people to consume a more diverse and dairy-rich diet.

[0046] The present disclosure provides for the use of the Bifidobacterium adolescentis strain designated IVS-1, an isolate of which is deposited with the American Type Culture Collection under accession number PTA-120614. In some embodiments, the present disclosure may include a substantially similar strain having more than 95% similarity to the isolate deposited with the American Type Culture Collection under accession number PTA-120614. The use of the Bifidobacterium adolescentis strain IVS-1, or a substantially similar strain thereof, can reduce and / or eliminate the undesirable intestinal effects of lactose for individuals with low or absent human lactase expression in the intestine. A full description of the Bifidobacterium adolescentis strain IVS-1 is further available in U.S. Patent No. 9,125,935, which is incorporated herein by reference.

[0047] Preferably, the compositions of the invention contain Bifidobacterium adolescentis strain IVS-1 having at least 95% identity to the 16S rRNA gene sequence when compared to the type strain identified below in SEQ ID NO: 1. In embodiments, the compositions of the invention contain Bifidobacterium adolescentis strain IVS-1 having at least 97% identity, at least 98% identity, at least 98.5% identity, at least 99% identity, at least 99.5% identity, or at least 99.9% identity to the 16S rRNA gene sequence when compared to the type strain identified below in SEQ ID NO: 1, and are further provided in the XML format sequence listing, which is incorporated herein by reference.

[0048] Sequence of the 16S rRNA gene from the microbial strain Bifidobacterium adolescentis strain IVS-1 (SEQ ID NO:1)

[0049] [ka]

[0050] [ka]

[0051] In some embodiments, this is measured by 16S rRNA gene sequencing. B. adolescentis (such as strain IVS-1) was identified as a taxon of interest that appears to increase upon lactose ingestion, as shown in Table 1. Operational taxonomic units (OTUs) construct "mathematically" defined taxa that are widely accepted and applied to describe bacterial communities using amplicon sequencing of the 16S rRNA gene.

[0052] Table 1: Proportion of bacterial taxa significantly affected by dietary treatment within treatment groups (<0.1% in at least one treatment) (FDR-adjusted P-value <0.1). Significant values ​​are shown in italics.

[0053] [Table 1]

[0054] a : If a strain could not be assigned to a type of strain (<97% homology), the RDP classifier was used to determine the most likely genus (80% cutoff). *There is a significant difference between the two treatment groups for OTU_1.

[0055] In one embodiment, the composition of the present invention includes any mutant of the B. adolescentis strain having accession number PTA-120614, which can be obtained by subjecting the strain to a mutagenesis treatment as described for obtaining the mutant. In some aspects, the mutant strain is selected for having desired properties such as activity of a particular enzyme, resistance to bile and acid, increased fermentation yield, and good storage stability. Alternatively, the selection is performed for spontaneously occurring mutants. Also, or alternatively, embodiments including derivatization of strains resulting from natural genetic drift occurring over the passage of strains during normal routine laboratory and commercial production activities may be utilized in the compositions or methods of the present disclosure.

[0056] Advantageously, Bifidobacterium adolescentis strain IVS-1 colonizes the human intestine or digestive tract at least 10-fold better than non-adapted strains of the same genus. In some embodiments, Bifidobacterium adolescentis strain IVS-1 outperforms other strains due to its ability to utilize and compete with lactose-like carbohydrates.

[0057] Genomic and in vitro data characterize the ability of the strain to consume lactose and lactose-like carbohydrates or produce beta-galactosidase (beta-gal or B-gal).Furthermore, as shown in the examples provided herein, Bifidobacterium adolescentis strain IVS-1 provides the ability to metabolize lactose, reduce gas production in vitro, and reduce lactose intolerance symptoms.

[0058] In some aspects, B. adolescentis strain IVS-1 provides superior B-gal activity to break down lactose. In some embodiments, colorimetric indicators of lactose degradation, such as, but not limited to, X-gal, ONPG, or similar, may be used in liquid form to quantify enzyme activity via spectrophotometric measurements. Absorption may be normalized to CFU levels (or proxies thereof) and presented as relative levels normalized to other B-gal producing microorganisms. In some aspects, B. adolescentis strain IVS-1 may provide greater B-gal activity to break down lactose compared to other probiotic options and enterobacteria.

[0059] In further embodiments, B-gal gene expression can be measured by RT-qPCR with primers specific for the B-gal gene. Expression can be normalized to standard housekeeping gene levels and CFU enumeration (or their proxies) and presented as relative levels normalized to the lowest B-gal gene expressing microorganism. In some embodiments, B. adolescentis strain IVS-1 can express greater B-gal activity to break down lactose compared to other probiotic options and enterobacteria.

[0060] As an additional advantage, B. adolescentis strain IVS-1 can deplete lactose from the environment more quickly than other probiotic options. In some aspects, lactose depletion rates can be measured by incubating test microorganisms with a pre-defined set of lactose, generating data normalized to CFU levels (or proxies thereof) and displaying it as a normalized relative level to other lactose-degrading organisms. In some aspects, B. adolescentis strain IVS-1 can eliminate lactose more quickly than other probiotic options and enterobacteria. In other embodiments, B. adolescentis strain IVS-1 even outperforms other organisms when performed in a competitive lactose co-incubator in liquid culture.

[0061] In some embodiments, the compositions may be provided as solid, liquid, or semi-solid compositions. The solid compositions described herein may be provided in the form of tablets, capsules, powders, or granules (including multiple granules). In further embodiments, the liquid and semi-solid compositions may be provided as liquid suspensions, pastes, or syrups. A review of conventional formulation techniques can be found, for example, in Lachman et al. The Theory and Practice of Industrial Pharmacy. India: CBS Publishers & Distributors Pvt. Limited, 2009 (incorporated herein by reference). In some aspects, the compositions may be prepared by methods known in the art and may be compressed, enteric coated, sugar coated, film coated, or multi-compressed, containing suitable binders, lubricants, diluents, disintegrants, colorants, grinding agents, flow inducers, and melting agents. Capsules, both soft and hard, with liquid or solid contents, may be prepared according to conventional techniques well known in the pharmaceutical industry. As an example, the active probiotic organisms may be filled into gelatin capsules using a suitable filling machine. In a further embodiment, the solid compositions described herein may be pellets.

[0062] In some embodiments, the composition is preferably given to the subject by oral administration. In some aspects, oral delivery can be via low water activity (preferably less than 0.2), non-hot food (preferably less than 60°C), and supplements. In some embodiments, the food composition can be any ingestible material, including but not limited to milk, curd, milk-based fermented products, acid milk, yogurt, desserts, snack foods, candy, lozenges, frozen yogurt, milk powder, milk-based powder, milk concentrate, cheese, cheese spread, dressings, beverages, ice cream, bars, additive powders, encapsulated and tableted supplements, fermented cereal-based products, infant formula, pet food, tablets, liquid bacterial suspension, dry oral supplement, wet oral supplement, dry or wet tube feeding, produced by using Bifidobacterium adolescentis strain IVS-1, or their derivatives or variants. In some embodiments, acceptable carriers can be used to standardize the strain concentration for proper administration. For example, in one embodiment, the composition may be a chocolate truffle, which may contain lactose (milk chocolate) or other dairy-containing ingredients.

[0063] The composition or dosage form may contain at least Bifidobacterium adolescentis strain IVS-1, or a variant thereof, as described above, such that the amount of strain available to an individual is about 10 3 ~about 10 14 CFU / day, e.g., about 10 6 ~about 10 13 CFU / day (approx. 10 8 ~about 10 12 CFU / day, or even about 10 9 ~about 10 13 CFU / day). This amount may depend on the individual body weight of the subject, and is preferably about 10 9 ~about 10 12 CFU / day, approximately 10 for pets 7 ~about 10 12CFU / day amount. However, it will be understood that the specific dose level of any particular subject will depend on various factors, including the activity of the specific compound used, the microbiota community structure and content, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, formulation combination, and the severity of the subject's specific target condition.

[0064] In a further embodiment, the composition further comprises one or more prebiotic substances.Suitable examples of prebiotic substances include, but are not limited to, lactose, galactooligosaccharides (GOS), inulin, and other fructans.However, other prebiotic substances are also contemplated, such as fructooligosaccharides (FOS), mannan-oligosaccharides (MOS), arabinoxylan, and even the composition obtained from bacterial cells, metabolites, enzyme activity, and polysaccharides of Bifidobacterium adolescentis strain IVS-1.

[0065] Microorganisms are involved in the production of food and feed products, including most dairy products. Bacterial cultures, especially those of bacteria commonly classified as lactic acid bacteria, are essential for the production of all fermented dairy products, cheese, and butter. These microbial cultures are often called starter cultures and perform a number of functions to impart specific characteristics to various dairy products. In order for a starter culture to perform its functions, it is essential that it contains a sufficient amount of viable cells. Thus, some embodiments of the present disclosure provide a starter culture composition comprising viable Bifidobacterium adolescentis strain IVS-1 or a variant thereof, the starter culture composition preferably comprising about 10 viable cells per gram of composition. 4 ~about 10 12 A starter culture composition is provided having a concentration of viable cells in the range of CFU.

[0066] The starter culture is typically used in the manufacture of a food or feed product by adding the starter culture composition according to the food or feed product starting material and maintaining the starting material thus inoculated under conditions in which the lactic acid bacteria are metabolically active. In a preferred embodiment, the food product is a milk-based product such as cheese, yogurt, butter, or a liquid fermented dairy product, for example buttermilk or drinking yogurt. The use of the present disclosure for the manufacture of products containing milk or other dairy products is particularly preferred.

[0067] Elimination and / or reduction of lactose intolerance symptoms The present disclosure further provides methods of reducing symptoms of lactose intolerance during treatment with IVS-1 strains and / or mutant derivatives thereof, and for extended periods of time after treatment has ceased. Thus, the methods of the present disclosure include partially, substantially, or completely reducing symptoms of lactose intolerance for days, weeks, months, years, or permanently. Such reduction is achieved by the methods and compositions described herein.

[0068] Individuals who may benefit from the methods and compositions of the present disclosure include those who suffer from symptoms of lactose intolerance, as described above. Any degree of lactose intolerance may be treated by the methods of the present disclosure. Symptoms of lactose intolerance include gas, bloating, abdominal discomfort, diarrhea, vomiting, and / or cramps. The effectiveness of treatment may be measured in several ways. Conventional measurements such as hydrogen gas production, fecal acidity, symptom surveys, dietary recall surveys, microbiome measurements, and / or blood glucose levels may be used before and after treatment. Alternatively, or in addition, the amount of lactose that can be consumed before the onset of one or more symptoms may be measured or evaluated before and after treatment, and thus, for example, if less hydrogen is produced on average by a given dose of lactose after treatment, the treatment is considered to be at least partially effective.

[0069] Alternatively, the individual does not precisely test the amount of hydrogen or measure effectiveness using, for example, a blood glucose test, but instead has a general sense of how much lactose the individual can ingest, as well as the type and degree of symptoms experienced following such ingestion.

[0070] "Partial" elimination of symptoms of lactose intolerance is statistically significant, statistically significant increase in the amount of lactose that can be ingested before the onset of symptoms, statistically significant decrease in the amount of breath hydrogen produced, or statistically significant (P<0.05) reduction in reporting of some symptoms in digestive studies. As used herein, "substantial" elimination of symptoms of lactose intolerance encompasses the above effects where the results are both statistically and clinically significant as measured by appropriate statistical evaluation of effect size. As used herein, "complete" or "substantially complete" elimination of symptoms of lactose intolerance indicates that normal amounts of lactose (i.e., the amount of lactose in the typical diet of the region and / or culture in which the individual normally lives) can be ingested after treatment without symptoms or with only infrequent occurrence of symptoms. Thus, for example, an individual may know that if the individual ingests a half cup (4 ounces) of milk, there will be no or minimal symptoms, but if the individual ingests more than one cup of milk, symptoms such as gas or diarrhea will occur. The individual may find that after treatment, one and a half cups of milk can be consumed, but three or more cups cause symptoms, indicating that the symptoms of lactose intolerance have substantially disappeared. Alternatively, the individual may find that after treatment, the normal diet of the geographic or cultural area can be consumed with little or no symptoms of lactose intolerance. The symptoms of lactose intolerance have then disappeared completely.

[0071] More specifically, efficacy may be measured by the reduction in the rate of symptoms of lactose intolerance. In this measurement, the severity of a given symptom or set of symptoms is measured before and after treatment, for example, using a Likert scale before and after. Exemplary symptoms include gas, bloating, diarrhea, cramps, abdominal pain, and vomiting. Any one or more of the symptoms may be measured. For example, an individual may be asked to rank one or more symptoms on a scale of increasing severity from 1 to 5. In one embodiment, a set of symptoms is ranked and the rankings are added. For example, gas, bloating, diarrhea, abdominal pain, and cramps may be ranked. The percentage reduction of symptoms from before to after treatment may be calculated, and the symptoms of lactose intolerance may be considered to have disappeared by that percentage reduction (e.g., if the symptoms are reduced by 50%, the symptoms of lactose intolerance are eliminated by 50%).

[0072] In some embodiments, B. adolescentis strain IVS-1 can be maintained in a subject after about 4 weeks of washout. This is important in addressing lactose intolerance because IVS-1 treatment can provide subjects with excess metabolic capacity in their gut microbiome to metabolize lactose for extended periods of time, even if not taken daily. This is different from traditional lactase treatment (e.g., Lactaid®), which must be administered immediately before or at the same time as a lactose-containing meal. The IVS-1 strain of the present disclosure can help precondition the microbiome by increasing its duration in the gut of a subject. In some embodiments, B. adolescentis strain IVS-1 is further different from other probiotic strains, which generally pass through the gut quickly and do not colonize.

[0073] In some embodiments, quantification of absolute cell numbers of IVS-1 in fecal samples may be performed by qPCR using strain-specific primers for B. adolescentis IVS-1. In such embodiments, IVS-1 has been shown to be maintained in some subjects by both added lactose and prebiotic GOS. In further embodiments, 16S rRNA gene bacterial community sequencing of clinical trial participants showed that OTUs consistent with B. adolescentis (OTU_1) increased during treatment with IVS-1 and remained higher than baseline after 4 weeks of washout. In some embodiments, for subjects receiving added lactose and additional prebiotic GOS, the percent B. adolescentis OTU_1 mean abundance was statistically significantly higher for the washout period than baseline.

[0074] In some embodiments, the disclosure provides a method of reducing symptoms of lactose intolerance in an individual exhibiting symptoms of lactose intolerance by administering to the individual increasing amounts of lactose for a period of time, where one or more symptoms of lactose intolerance are partially, substantially, or completely eliminated. In some embodiments, the symptom(s) of lactose intolerance remain partially, substantially, or completely eliminated at least about 1 day, 1 week, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, or more than 5 years after the end of treatment, or remain permanently eliminated after the end of treatment. In some embodiments, the disclosure provides a method of reducing symptoms of lactose intolerance in an individual exhibiting symptoms of lactose intolerance by administering to the individual increasing amounts of lactose for a period of time, where one or more symptoms of lactose intolerance are substantially eliminated for at least about 1 month after the end of treatment.

[0075] In some embodiments, the disclosure provides a method of reducing symptoms of lactose intolerance in an individual exhibiting symptoms of lactose intolerance by administering to the individual increasing amounts of lactose over a period of time, where the symptoms of lactose intolerance are reduced by an average of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, as measured as described herein. An "average" reduction is a reduction as measured in a population of individuals exhibiting symptoms of lactose intolerance, such as greater than about 2, 3, 4, 5, 10, 25, or 50 individuals. In some embodiments, the reduction in symptoms of lactose intolerance persists or is even greater (e.g., 50% reduction to 55% reduction) at least about 1 day, 1 week, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, or more than 5 years after termination of treatment. In some embodiments, the reduction in symptoms is permanent. In some embodiments, the disclosure provides a method for reducing symptoms of lactose intolerance in an individual exhibiting symptoms of lactose intolerance by administering to the individual increasing amounts of lactose for a period of time, where the symptoms of lactose intolerance, as measured as described herein, are reduced on average by at least about 20% and remain reduced by at least about 20% for at least about one month after the end of treatment. In some embodiments, the disclosure provides a method for reducing symptoms of lactose intolerance in an individual exhibiting symptoms of lactose intolerance by administering to the individual increasing amounts of lactose for a period of time, where the symptoms of lactose intolerance, as measured as described herein, are reduced on average by at least about 50% and remain reduced by at least about 50% for at least about one month after the end of treatment.

[0076] The total duration of treatment can be about 2 weeks to about 4 weeks, or about 1 week to about 10 weeks, or about 4 weeks to about 8 weeks, or about 3 weeks. During this period, the subject begins a treatment program and is then challenged with a lactose-containing product after the intervention. In some embodiments, treatment with an IVS-1 strain is combined with one or more additional substances, such as prebiotics, as described herein. In some embodiments, the total duration of treatment is about 1 day to about 30 days, or about 5 days to about 10 days, or about 2 days to about 15 days, or about 1 day to about 4 days, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In some embodiments, the total duration of treatment is about 3 days. In some embodiments, the total duration of treatment is about 14 days. It will be understood that these durations are averages and that individuals using the treatment may differ from the averages based on the severity of the individual's symptoms, existing microbiome content, number of days without treatment, etc. In some embodiments, the duration of treatment is based on the individual's symptoms or existing microbiome content. Thus, an individual may experience a recurrence of symptoms at a given lactose intake and may require adjustment of the treatment dose and / or time, or a reduction in lactose intake, until symptoms subside. Thus, in some embodiments, the duration of treatment is not definitively established at the outset, but continues until a maximum dose of lactose is achieved or a desired level of lactose tolerance is achieved.

[0077] Increasing the dosage of Bifidobacterium adolescentis strain IVS-1 can be accomplished by increasing the amount of Bifidobacterium adolescentis strain IVS-1 administered, either by increasing the amount of Bifidobacterium adolescentis strain IVS-1 per administration, the number of administrations, or both. In one embodiment, both strategies are used. Thus, in some embodiments of the present disclosure, Bifidobacterium adolescentis strain IVS-1 is initially administered with every meal, and then reduced to a single administration per day. A single administration per meal can last for a period of about 1 to about 30 days, about 1 to about 10 days, about 2 to about 20 days, about 6 to about 20 days, or about 20 days, and a single administration per day can last for about 1 to about 8 days, about 2 to about 10 days, about 2 to about 12 days, about 4 to about 14 days, about 16 days, or indefinitely if needed to maintain symptomatic remission. In some embodiments, lactose intake is minimized during the one-serving administration period, hi some embodiments, lactose intake may be increased after 1, 3, 5, or 7 days, or after 2, 3, or 4 weeks, or any combination thereof.

[0078] After treatment has ended, individual subjects are encouraged to enjoy dairy products or other lactose-containing foods regularly, such as every other day, twice a week, or once a week, to maintain colonization with the IVS-1 strain and thus maintain a reduction in symptoms of lactose intolerance.

[0079] Although an individual typically does not require more than one series of treatments, in some embodiments, an individual may have a series of treatments repeated. A series of treatments may be repeated when symptoms of lactose intolerance appear or increase to undesirable levels. In one embodiment, a "booster pack" of IVS treatments may be recommended to reestablish an individual who has lapsed lactose intake and is therefore prone to experiencing symptoms again. Alternatively, a series of treatments may be repeated at regular or predetermined intervals. Thus, a treatment may be repeated after about 1 month, 2 months, 3 months, 4 months, 6 months, 8 months, 10 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, or more than 5 years, or any combination thereof (e.g., a treatment may be repeated after 1 year, and then every 2-5 years). A treatment may be repeated in the same form (e.g., duration, dosage, timing of dosing, additional substances, etc.) used in the first treatment, or may be modified. For example, the duration of treatment may be shortened or extended, the dosage may be increased more quickly or more slowly, and / or a higher or lower dose of lactose may be used, a different lactose-containing product may be used (e.g., containing more or less, or less or more, of other substances in addition to lactose), etc. In another embodiment, a "maintenance" dosage may be given that comprises a lower than usual dosage administered daily to prevent the onset of symptoms.

[0080] In vivo selection (IVS) The present disclosure further provides a method for selecting and isolating one or more microbial strains that naturally occur in the gastrointestinal microbiota and exhibit improved characteristics in the presence of lactose. After isolation and characterization, the in vivo selected microbial strains may be used as therapeutics, and can be administered to animals as a synbiotic composition, optionally with prebiotics. For the purposes of this specification, gastrointestinal microbiota refers to the microbial population present in the gastrointestinal tract of a subject. The gastrointestinal tract typically includes the mouth, esophagus, stomach, small intestine, large intestine, rectum and anus.

[0081] In one embodiment, a method is provided in which the microbial strains present in one or more subjects' gastrointestinal microbiota can be specifically selected based on their positive response to the presence of prebiotics.Such a method typically begins with administering a candidate prebiotic to the subject.

[0082] Typically, a subject is administered at least one dose of a prebiotic (e.g., lactose or GOS), but more often several doses over a period of time (e.g., one or more doses per day for multiple days (e.g., about or at least one week, about or at least two weeks)). In some cases, to aid or further validate the correlation between the increase in microbial strains and the presence of lactose, the subject can be administered successively higher doses of the prebiotic over time.

[0083] The methods described herein can be used to identify additional microbial strains based on in vivo selection where certain strains preferentially increase in number or otherwise elicit a positive response due to the presence of a prebiotic, such as lactose or GOS. In addition to an increase in number of one or more microbial strains in the presence of a prebiotic compared to a baseline sample, a "positive response" can refer, for example, to an increase in metabolic activity by the microbial strain (i.e., in the absence of an increase in number), or both an increase in number and an increase in metabolism of lactose.

[0084] As described herein, this method has been used to identify a microbial strain that responds particularly well to prebiotics (i.e., lactose or GOS). As described herein, this microbial strain was identified as Bifidobacterium adolescentis strain IVS-1. This strain was deposited with the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Va. 20110) on October 8, 2013, and assigned the accession number PTA-120614.

[0085] Bifidobacterium adolescentis strain IVS-1 or other strains identified using the methods herein can be provided as a substantially pure population. As used herein, a "substantially pure population" of cells means that at least about 50% (e.g., about 55%, 60%, 65%, 70%, 75%, 80%, 90%, 99% or more) of the cells present are Bifidobacterium adolescentis strain IVS-1 as described herein. Methods for culturing Bifidobacterium adolescentis strain IVS-1 will be known to those skilled in the art. See, for example, Handbook of Culture Media for Food Microbiology, 2nd Ed., Vol 37, Corry et al., eds., 2003, Elsevier Science. In addition, there are commercially available selective media (e.g., MRS medium from Difco) that are specifically designed for culturing Bifidobacterium.

[0086] After the microbial strain is selected, identified, and isolated using the methods disclosed herein, and after the affinity of the microbial strain to a prebiotic (e.g., GOS or lactose) is confirmed, the microbial strain can be administered to a subject (e.g., as a probiotic). Considering the method by which the microbial strain was obtained, it is preferable that the microbial strain is administered to the subject in combination with a corresponding prebiotic (e.g., GOS lactose). In some embodiments, the microbial strain and the prebiotic are combined prior to administration to generate a synbiotic. Typically, the animal to which the rationally designed synbiotic is administered is an animal of the same species as the subject in which the microbial strain was originally identified. As described above with respect to the subject, the subject may be an animal, including a human or any number of non-human animals.

[0087] In some embodiments, the microbial strain and prebiotics (e.g., synbiotics) can be contained in food. Food includes any number of foods suitable for human consumption, such as, but not limited to, milk, yogurt, juice, water, cereal, chewing gum, crackers, candy, cookies, vitamin supplements, meat, and fruits or vegetables (i.e., blended fruits or vegetables, such as, for example, baby food). Food also includes feed products (e.g., suitable for consumption by livestock or companion animals), including dry animal feed. In certain embodiments, the Bifidobacterium adolescentis strain IVS-1 described herein can be mixed with GOS into liquid feed or drinking water, or can be combined with a carrier and applied to solid feed.

[0088] Compositions or foods comprising the microbial strains described herein (e.g., Bifidobacterium adolescentis strain IVS-1) and prebiotics (e.g., lactose or GOS) may further comprise a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers should be non-toxic to bacteria and animals and may also contain components that promote survival of the microorganism during storage. Liquid or gel-based carriers are well known in the art, such as water, fruit juice, glucose or fructose solutions, physiological electrolyte solutions, and glycols such as methanol, ethanol, propanol, butanol, ethylene glycol, and propylene glycol. Carriers also include oily carriers such as, for example, white petrolatum, isopropyl myristate, lanolin or lanolin alcohol, mineral oil, perfume oil or essential oil, nasturtium extract oil, sorbitan monooleate, cetylstearyl alcohol, hydroxypropylcellulose (MW=100,000-1,000,000), or detergents (e.g., polyoxyl stearates or sodium lauryl sulfate).Other suitable carriers include water-in-oil or oil-in-water emulsions, where a mixture of emulsifiers and emollients is provided.

[0089] The composition or food product comprising the microbial strain (e.g., Bifidobacterium adolescentis strain IVS-1) and prebiotic (e.g., lactose or GOS) described herein may also include natural or synthetic flavors and food-quality colorants, thickeners such as corn starch, guar gum, xanthan gum, binders, disintegrants, coatings, lubricants, stabilizers, solubilizers, suspending agents, excipients, and diluents. For example, additional ingredients may be included to improve mouthfeel, improve shelf life, and provide nutritional benefits. It will be understood by those skilled in the art that any additional ingredients in the composition must be compatible with maintaining the viability of the microbial strain.

[0090] Administration of a composition or food comprising a microbial strain (e.g., Bifidobacterium adolescentis strain IVS-1) and a prebiotic (e.g., lactose or GOS) as described herein can be accomplished by any method that delivers at least a portion of the microorganism and prebiotic to the animal's digestive tract. Enteral administration (e.g., oral, sublingual, or rectal) is therefore preferred, although other routes are not excluded. In general, the formulation of a composition depends on its intended delivery route. For example, a composition comprising Bifidobacterium adolescentis strain IVS-1 (with or without lactose or GOS) as described herein can be formulated as a powder, granules, tablet, capsule, liquid suspension, paste, or syrup.

[0091] Probiotics have been reported to provide health benefits including: (1) relief of intestinal disorders such as constipation and diarrhea caused by infections due to pathogenic organisms, antibiotics, or chemotherapy, (2) stimulation and regulation of the immune system, (3) antitumor effects due to inactivation or inhibition of carcinogenic compounds in the digestive tract by reducing the activity of gut bacterial enzymes such as beta-glucuronidase, azoreductase, and nitroreductase, (4) reduction in the production of toxic end products such as ammonia, phenols, and other metabolites of proteins known to affect liver cirrhosis, (5) reduction in serum cholesterol and blood pressure, (6) maintenance of mucosal integrity, (7) relief of symptoms of lactose intolerance, and (8) prevention of vaginitis. Thus, the beneficial effects attributed to probiotics include increased resistance to infections, a healthier immune system, reduced irritable bowel syndrome, reduced blood pressure, reduced serum cholesterol, milder allergies, and tumor regression. For example, in animals, probiotics can enhance weight gain or weight loss and improve meat quality and milk production. Importantly, probiotics can be used to establish and maintain a healthy (e.g., balanced) gastrointestinal flora in animals and reduce the effects of gastrointestinal diseases.Gastrointestinal diseases include, but are not limited to, diarrhea, constipation, loose stools, abdominal distension, ulcerative colitis, Crohn's disease, irritable bowel syndrome, extremely irritable bowel syndrome, food toxicity, food allergies, pseudomembranous colitis, hemorrhagic colitis, gastritis, gastroduodenal ulcer, dental caries, and periodontitis.In some embodiments, bifidobacteria can prevent or reduce the effects of metabolic disorders such as obesity and type 2 diabetes by reducing intestinal permeability.Reducing intestinal permeability can improve metabolic endotoxemia and metabolic inflammation, both of which are involved in obesity and related metabolic disorders. EXAMPLES

[0092] The following examples are set forth to provide those of skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and / or methods claimed herein are made and evaluated, are intended to be purely exemplary of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. However, those of skill in the art should, in light of this disclosure, understand that many changes can be made in the specific embodiments disclosed and still obtain a similar or similar result without departing from the spirit and scope of the disclosure.

[0093] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.

[0094] Example 1 Genomic characterization The genome of Bifidobacterium adolescentis IVS-1 strain was sequenced via 454 Pyrosequencing to determine whether the strain contained the B-galactosidase gene for lactose metabolism. The genome was submitted to NCBI and analyzed by the NCBI Prokaryotic Genome Annotation Automated Computational Analysis Pipeline. Based on the results, it was confirmed that the IVS-1 strain contained the B-galactosidase gene for lactose metabolism (see https: / / www.ncbi.nlm.nih.gov / protein / KIM01121.1?report=genpept).

[0095] Example 2 In vivo characterization The growth of Bifidobacterium adolescentis strain IVS-1 was evaluated in comparison with a number of other probiotic strains in a simulated gastrointestinal environment containing lactose. The optical density (OD600) of each strain was measured at approximately 10 4An inoculum of CFU was used and measured during growth on 1% lactose. Growth was completed in wells of a 96-well plate incubated in a plate reader in an anaerobic chamber. Data showing the growth of each probiotic strain are provided in Figure 1.

[0096] The data given in Figure 1 give the average of four replicates for each strain. For clarity, standard deviations are shown only for B. adolescentis strain IVS-1, but the replicate variation was similar for the other strains shown in Figure 1. Probiotic strains were previously prepared by growing to late logarithmic phase in liquid MRS or RCM (both rich media), centrifuging, and resuspending the pellet in enteroid medium containing 7.5% DMSO as a cryopreservative. Aliquots were then frozen.

[0097] As shown in Figure 1, the IVS-1 strain was confirmed to grow much faster than the other strains in a simulated gastrointestinal environment containing lactose. Without being limited to a particular mechanism or theory, the increased growth rate of the IVS-1 strain may contribute to its superior properties in reducing gas.

[0098] Example 3 Gas production during fecal fermentation The amount of gas produced and the relative change in gas production were evaluated by B. adolescentis strain IVS-1 and a number of additional probiotics. Gas production measurements were performed after inoculation of diluted fecal samples with an intestinal-like medium containing 1% lactose. 7A range of different probiotics, ranging in CFU, were utilized in the analysis. Changes in gas production were compared to a control containing no probiotics, as shown in Figure 2. Probiotic strains were pre-prepared by growing to late logarithmic phase in liquid MRS or RCM (both rich media), centrifuging, resuspending the pellet in enteroid medium containing 7.5% DMSO as a cryopreservative, and then freezing an aliquot of each strain. Heat-killed IVS-1 controls were incubated at 70°C for 5 minutes before being added. The experiment was performed in three replicates, with each of four different fecal donors. Individual measurements for each of the four fecal donors are shown as circles, triangles, squares, or crosses in Figure 2.

[0099] As shown in Figure 2, B. adolescentis IVS-1 significantly reduced gas from lactose more than the other strains. The results showed that the IVS-1 strain gave superior results in this preclinical model of gas produced from gastrointestinal lactose metabolism. Because excess gas production is a major cause of lactose intolerance symptoms, the results indicate that B. adolescentis strain IVS-1 may be used to treat lactose intolerance by reducing the amount of gas produced.

[0100] Example 4 Hydrogen production and reduction in symptom scores A clinical trial with B. adolescentis strain IVS-1 treatment is being conducted to analyze hydrogen production and reduction in symptom scores for individuals administered lactose. IVS-1 treatment is expected to be superior to placebo in terms of resulting gas levels and lactose intolerance symptom scores, as shown in Figure 3. Breath hydrogen is measured using a handheld respirometer, and symptom scores are based on subjective severity and / or frequency surveys rated by the study subjects themselves.

[0101] Breath hydrogen test: This is an 8-hour breath hydrogen test. Participants who pass a pre-qualification screening indicating they are lactose intolerant will fast for 12 hours before the test and will then be given commercial milk to drink. Each subject will be provided with milk containing 0.5g of lactose per kg of body weight, assuming a 5% lactose content (12g per 240ml cup) with a maximum of 50g of lactose. This dose of lactose is within the physiological range of typical dietary intake and has historically caused some elevation of symptoms. Breath samples to measure hydrogen levels are taken at the following time points: 0 (before milk administration), 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, and 8 hours, for a total of 10 breath samples. Participants are asked to record any symptoms related to lactose intolerance experienced by the participant at these same time intervals using a provided form. A further flow chart of the procedure can be found in FIG. 4.

[0102] Example 5 Relative lactase activity of different probiotics / growth conditions In this test, a colorimetric indicator of lactose degradation (X-gal, ONPG, or similar) is used in liquid format and mixed in standardized amounts with various strains grown in broth medium to quantify lactase (B-gal) enzyme activity through spectrophotometric measurement of color change. Various strains will be as shown in Figure 5. Absorption is standardized to CFU levels (or their proxies) and shown as relative levels normalized to the minimum lactase activity producing microorganism in this test. The expected results shown in Figure 5 indicate that IVS-1 is more likely to generate more B-gal activity to break down lactose than other probiotic options. Furthermore, the amount of lactase activity is expected to be higher when IVS-1 is provided with lactose during growth in preparing the strains for this test. Pretreatment of IVS-1 with lactose provides a synergistic effect on IVS-1 growth and lactase activity.

[0103] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0104] While the present disclosure has been described in connection with various embodiments, it will be understood that the disclosure is capable of further modifications. This application is intended to cover in general any variations, uses, or adaptations of the present disclosure in accordance with the principles of the present disclosure, including departures from the present disclosure within known practice in the art to which the disclosure pertains.

Claims

1. A method of treating lactose intolerance, comprising administering to a subject in need thereof a therapeutically effective amount of isolated Bifidobacterium adolescentis strain IVS-1.

2. The strain is at least 10 ６ 13. The method of claim 1, wherein the amount is administered in CFU / day.

3. 3. The method of claim 1 or 2, wherein the strain is administered daily for one week or more.

4. The method according to any one of claims 1 to 3, wherein said method improves lactose degradation and / or reduces lactose intolerance symptoms in a subject in need thereof.

5. 5. The method of any one of claims 1 to 4, wherein the Bifidobacterium adolescentis strain IVS-1 is pretreated with lactose to increase the initial lactase activity of the Bifidobacterium adolescentis strain IVS-1.

6. 6. The method of any one of claims 1 to 5, wherein the method provides lactase to a subject in need thereof through administration of both live and inactive cultures of Bifidobacterium adolescentis strain IVS-1, and its actual and former cellular contents.

7. The method of any one of claims 1 to 6, wherein the method reduces gas production in the intestine.

8. The method of any one of claims 1 to 7, wherein the method balances osmotic pressure in the intestine.

9. 9. The method of any one of claims 1 to 8, wherein the method alters the gut microbiome to benefit the health and well-being of a subject in need thereof.

10. A food composition for treating lactose intolerance comprising Bifidobacterium adolescentis strain IVS-1, wherein an inoculum of Bifidobacterium adolescentis strain IVS-1 exhibits lactase activity.

11. 11. The food composition of claim 10, wherein the food composition is selected from the group consisting of milk, curd, milk based fermented products, acidified milk, yogurt, desserts, snack foods, chocolates, candies, lozenges, frozen yogurt, milk powders, milk based powders, milk concentrates, cheese, cheese spreads, dressings, beverages, ice cream, bars, additive powders, encapsulated and tableted supplements, fermented or non-fermented cereal based products, infant formulas, tablets, liquid bacterial suspensions, dry oral supplements, and wet oral supplements.

12. The composition of claim 10 or 11, further comprising a pharma- ceutically acceptable carrier.

13. 13. The composition of any one of claims 10 to 12, further comprising a prebiotic selected from the group consisting of galactooligosaccharides (GOS), fructooligosaccharides (FOS), and inulin, mannan-oligosaccharides (MOS), arabinoxylan, or compositions obtained from bacterial cells, metabolites, enzyme activities, and polysaccharides of Bifidobacterium adolescentis strain IVS-1.

14. The composition according to any one of claims 10 to 13, wherein the Bifidobacterium adolescentis strain IVS-1 is selected from a wild-type strain, a derivative strain and / or a mutant strain.

15. Bifidobacterium adolescentis strain IVS-1 was found to be approximately 10 ４ ~about 10 １４ The composition according to any one of claims 10 to 14, wherein the concentration is CFU.

16. 1. A method of treating lactose intolerance, comprising administering to a subject in need thereof a therapeutically effective amount of Bifidobacterium adolescentis strain IVS-1 and increasing lactase activity by the Bifidobacterium adolescentis strain IVS-1 in the gastrointestinal tract at least one day after administration.

17. 17. The method of claim 16, further comprising increasing beta-galactosidase activity levels in the gastrointestinal tract through administration of Bifidobacterium adolescentis strain IVS-1.

18. 18. The method of claim 16 or 17, further comprising reducing lactose levels in the gastrointestinal tract through administration of Bifidobacterium adolescentis strain IVS-1.

19. 19. The method of any one of claims 16 to 18, further comprising delivering an effective amount of GOS with said Bifidobacterium adolescentis strain IVS-1.

20. 20. The method of any one of claims 16-19, further comprising decreasing hydrogen production in the gastrointestinal tract and reducing symptoms associated with lactose intolerance in the subject.

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