Lipase inhibitor-producing lactobacillus rhamnosus strain VB336, use thereof, and culture apparatus thereof

Abstract

A lipase inhibitor-producing Lactobacillus rhamnosus strain VB336, use thereof, and a culture apparatus thereof. The Lactobacillus rhamnosus strain VB336 was deposited in China Center for Type Culture Collection on March 8, 2024 under an accession number of CCTCC M 204432. The Lactobacillus rhamnosus strain VB336 has a relatively strong ability to inhibit lipase activity and inhibit common pathogenic bacteria, is resistant to gastric acid and bile salts, and has strong stability in the intestinal fluid. The strain can be used for inhibiting lipolysis and / or for weight loss, regulating gut microbiota, and preventing, alleviating, and / or treating diseases caused by infection with pathogenic bacteria.

Description

A strain of Lactobacillus rhamnosus VB336 producing lipase inhibitors and its application and culture device

[0001] Priority information

[0002] This application claims priority to Chinese Patent Application No. 202410825955.4, filed on June 25, 2024, entitled "A strain of Lactobacillus rhamnosus VB336 producing a lipase inhibitor and its application and culture device", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of microbiology, specifically to a strain of Lactobacillus rhamnosus VB336 that produces a lipase inhibitor, its application, and a cultivation device thereof. Background Technology

[0004] The human gut is home to a vast array of microorganisms, forming a complex ecosystem that is vital to human health. Lactobacillus is an important probiotic in the human gut, playing a crucial role in maintaining the gut microecological balance and enhancing the function of the immune system.

[0005] Researching the causes of obesity and developing safe and effective weight-loss drugs has become a global research focus. Many currently developed and used weight-loss drugs act on the central nervous system, resulting in significant side effects, such as central nervous system excitation (including insomnia, tension, anxiety, nightmares, irritability, etc.), increased or decreased blood pressure, and valvular heart disease. Pancreatic lipase inhibitors, however, are a relatively safe new type of weight-loss drug. They selectively inhibit the hydrolytic activity of lipase, preventing excess fat from being hydrolyzed into smaller hydrolysates in the intestines, thus preventing absorption and storage by the body. This does not affect the absorption of other nutrients, thereby achieving weight loss. Therefore, developing suitable lipase inhibitors could significantly improve the current unfavorable situation in the weight-loss drug market.

[0006] Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent.

[0008] Therefore, in a first aspect, the present invention provides a *Lactobacillus rhamnosus* VB336. According to an embodiment of the present invention, the *Lactobacillus rhamnosus* VB336 was deposited at the China Center for Type Culture Collection on March 8, 2024, with accession number CCTCC M 2024432.

[0009] The *Lactobacillus rhamnosus* VB336 obtained by screening in this invention can secrete lipase inhibitors. The hydrolytic activity of lipase on fat is reduced or inhibited, so that excess fat cannot be hydrolyzed into small molecules that can be absorbed by the body in the intestine, thereby achieving the effect of weight loss. In addition, this bacterium can inhibit common pathogens and can be used to regulate the intestinal flora and prevent, alleviate and / or treat diseases caused by pathogen infection.

[0010] In a second aspect, the present invention provides a fermentation broth. According to an embodiment of the present invention, the fermentation broth is derived from the fermentation of *Lactobacillus rhamnosus* VB336 described in the first aspect. The fermentation broth of the present invention can be used to inhibit lipolysis and / or weight loss, regulate intestinal flora, and prevent, alleviate, and / or treat diseases caused by pathogenic bacterial infections.

[0011] In a third aspect, the present invention provides a bacterial suspension. According to an embodiment of the present invention, the bacterial suspension comprises *Lactobacillus rhamnosus* VB336 as described in the first aspect. The bacterial suspension of the present invention can be used to inhibit lipolysis and / or weight loss, regulate intestinal flora, and prevent, alleviate, and / or treat diseases caused by pathogenic bacterial infections.

[0012] In a fourth aspect, the present invention discloses the use of *Lactobacillus rhamnosus* VB336 described in the first aspect, the fermentation broth described in the second aspect, or the bacterial suspension described in the third aspect in the preparation of foods, beverages, pharmaceuticals, health products, feeds, or additives that inhibit lipase activity and / or promote weight loss. The foods, beverages, health products, feeds, or additives of the present invention can be used to inhibit lipase activity and / or promote weight loss, as well as to regulate intestinal flora. The pharmaceuticals of the present invention can be used to inhibit lipase activity and / or promote weight loss, as well as to regulate intestinal flora.

[0013] In a fifth aspect, the present invention provides the use of the *Lactobacillus rhamnosus* VB336 described in the first aspect, the fermentation broth described in the second aspect, or the bacterial suspension described in the third aspect in the preparation of foods, beverages, pharmaceuticals, health products, feeds, or additives that inhibit the activity of pathogenic bacteria. The foods, beverages, health products, feeds, or additives of the present invention can be used to regulate intestinal flora, and the pharmaceuticals of the present invention can be used to regulate intestinal flora and prevent, alleviate, and / or treat diseases caused by pathogenic bacterial infections.

[0014] In a sixth aspect, the present invention provides a composition. According to an embodiment of the invention, the composition comprises at least one of the following: *Lactobacillus rhamnosus* VB336 as described in the first aspect, the fermentation broth as described in the second aspect, and the bacterial suspension as described in the third aspect. The composition of the present invention can be used to inhibit lipase activity and / or achieve weight loss, regulate intestinal flora, and prevent, alleviate, and / or treat diseases caused by pathogenic bacterial infections.

[0015] In a seventh aspect, the present invention provides a method for inhibiting lipase activity in vitro. According to an embodiment of the present invention, the method comprises: mixing at least one of the *Lactobacillus rhamnosus* VB336 described in the first aspect, the fermentation broth described in the second aspect, and the bacterial suspension described in the third aspect with the lipase. As previously stated, the *Lactobacillus rhamnosus* VB336 of the present invention can effectively inhibit lipase activity.

[0016] In an eighth aspect, the present invention provides a weight loss method. According to an embodiment of the present invention, the method includes administering to a subject the *Lactobacillus rhamnosus* VB336 described in the first aspect, the fermentation broth described in the second aspect, or the bacterial suspension described in the third aspect. As previously mentioned, the *Lactobacillus rhamnosus* VB336 of the present invention can effectively inhibit the activity of lipase, preventing excess fat from being hydrolyzed into small molecules that can be absorbed by the body in the intestine, thereby achieving a weight loss effect.

[0017] In a ninth aspect, the present invention provides a method for inhibiting the growth of pathogenic bacteria in vitro. According to an embodiment of the present invention, the method includes: co-culturing at least one of the *Lactobacillus rhamnosus* VB336 described in the first aspect, the fermentation broth described in the second aspect, and the bacterial suspension described in the third aspect with a pathogenic bacterium. As previously stated, the *Lactobacillus rhamnosus* VB336 of the present invention can effectively inhibit the growth of pathogenic bacteria.

[0018] In a tenth aspect, the present invention provides a cultivation or production apparatus for the *Lactobacillus rhamnosus* VB336 described in the first aspect, the fermentation broth described in the second aspect, the bacterial suspension described in the third aspect, or the composition described in the sixth aspect. The cultivation or production apparatus according to embodiments of the present invention is capable of mass-producing the *Lactobacillus rhamnosus* VB336, fermentation broth, bacterial suspension, or composition described in the present invention.

[0019] In an eleventh aspect, the present invention provides the use of *Lactobacillus rhamnosus* VB336 described in the first aspect, the fermentation broth described in the second aspect, the bacterial suspension described in the third aspect, or the composition described in the sixth aspect in inhibiting lipase activity and / or weight loss in an individual. As previously stated, *Lactobacillus rhamnosus* VB336 of the present invention can effectively inhibit lipase activity, preventing excess fat from being hydrolyzed into small molecules that can be absorbed by the body in the intestine, thereby achieving a weight loss effect. Therefore, it can be used to inhibit lipase activity in an individual, and further, can effectively reduce weight and prevent, alleviate, and / or treat related diseases caused by lipase.

[0020] In a twelfth aspect of the invention, a method for inhibiting lipase activity and / or losing weight is provided. According to an embodiment of the invention, the method includes administering to the individual the *Lactobacillus rhamnosus* VB336 described in the first aspect, the fermentation broth described in the second aspect, the bacterial suspension described in the third aspect, or the composition described in the sixth aspect. As previously mentioned, the *Lactobacillus rhamnosus* VB336 of the present invention can effectively inhibit lipase activity, preventing excess fat from being hydrolyzed into small molecules that can be absorbed by the body in the intestine, thereby achieving a weight loss effect. Therefore, it can be used to inhibit lipase activity in an individual, and further, can effectively reduce weight and prevent, alleviate, and / or treat related diseases caused by lipase.

[0021] In a thirteenth aspect, the present invention provides the use of the *Lactobacillus rhamnosus* VB336 described in the first aspect, the fermentation broth described in the second aspect, the bacterial suspension described in the third aspect, or the composition described in the sixth aspect in the prevention, relief, and / or treatment of individual obesity. As previously stated, the *Lactobacillus rhamnosus* VB336 of the present invention can effectively inhibit lipase activity, preventing excess fat from being hydrolyzed into small molecules that the body can absorb in the intestines, thereby achieving a weight loss effect. Therefore, it can be used to prevent, relieve, and / or treat individual obesity, and further, can effectively prevent, relieve, and / or treat obesity-related diseases.

[0022] In a fourteenth aspect, the present invention provides a method for preventing, alleviating, and / or treating obesity in an individual, comprising administering to the individual the *Lactobacillus rhamnosus* VB336 described in the first aspect, the fermentation broth described in the second aspect, the bacterial suspension described in the third aspect, or the composition described in the sixth aspect. As previously stated, the *Lactobacillus rhamnosus* VB336 of the present invention can effectively inhibit lipase activity, preventing excess fat from being hydrolyzed into small molecules that can be absorbed by the body in the intestine, thereby achieving a weight loss effect. Therefore, it can be used to prevent, alleviate, and / or treat obesity in an individual, and further, can effectively prevent, alleviate, and / or treat obesity-related diseases.

[0023] In a fifteenth aspect, the present invention provides for the use of *Lactobacillus rhamnosus* VB336 as described in the first aspect, the fermentation broth as described in the second aspect, the bacterial suspension as described in the third aspect, or the composition as described in the sixth aspect in the prevention, alleviation, and / or treatment of diseases related to pathogenic bacterial infections. As previously stated, *Lactobacillus rhamnosus* VB336 can inhibit the activity of common pathogenic bacteria and has good tolerance to gastric acid and bile salts, as well as strong stability in intestinal fluid. Therefore, this strain and its related products can effectively prevent, alleviate, and / or treat diseases related to common pathogenic bacterial infections.

[0024] In a sixteenth aspect, the present invention provides a method for preventing, alleviating, and / or treating diseases related to individual pathogenic bacterial infections. According to an embodiment of the invention, the method comprises administering to the individual the *Lactobacillus rhamnosus* VB336 described in the first aspect, the fermentation broth described in the second aspect, the bacterial suspension described in the third aspect, or the composition described in the sixth aspect. As previously mentioned, *Lactobacillus rhamnosus* VB336 can inhibit the activity of common pathogenic bacteria and has good tolerance to gastric acid and bile salts, as well as strong stability in intestinal fluid. Therefore, this strain and its related products can effectively prevent, alleviate, and / or treat diseases related to common pathogenic bacterial infections.

[0025] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 is a scanning electron microscope image of Lactobacillus rhamnosus VB336 according to Example 2 of the present invention;

[0028] Figure 2 shows the test results of choline tolerance of Lactobacillus rhamnosus VB336 according to Example 4 of the present invention;

[0029] Figure 3 shows the test results of the artificial simulated gastric juice tolerance of Lactobacillus rhamnosus VB336 according to Example 4 of the present invention;

[0030] Figure 4 shows the test results of the artificial intestinal fluid tolerance of Lactobacillus rhamnosus VB336 according to Example 4 of the present invention. Detailed Implementation

[0031] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0032] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "multiple" or "a plurality of" means at least two, two types, such as two, two, three, three, etc., unless otherwise explicitly specified.

[0033] In this document, the terms “comprising,” “having,” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0034] In this document, the term “optionally” generally means that an event or condition described below may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0035] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0036] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] In this document, the terms “treatment” and “relief” refer to the attainment of a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, “treatment” encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in susceptible individuals who have not yet been diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing disease-related symptoms. As used herein, “treatment” encompasses any medication that administers a strain, fermentation broth, bacterial suspension, or composition to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual’s disease, including but not limited to administering to an individual in need a strain, fermentation broth, bacterial suspension, or composition described herein.

[0038] In this document, the term "carrier" includes any solvent, pharmaceutical stabilizer, or combination thereof, which are known to those skilled in the art. Except in cases where any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is covered.

[0039] strains

[0040] In some embodiments, the present invention provides a Lactaseibacillus rhamnosus VB336, which was deposited at the China Center for Type Culture Collection on March 8, 2024, with accession number CCTCC M 2024432.

[0041] According to some specific embodiments of the present invention, the above-mentioned Lactobacillus rhamnosus VB336 may further include at least one of the following additional technical features:

[0042] According to some specific embodiments of the present invention, the Lactobacillus rhamnosus VB336 has a 16S rRNA sequence as shown in SEQ ID NO:1.

[0043] The novel *Lactobacillus rhamnosus* VB336 strain obtained by screening in this invention can secrete lipase inhibitors. The hydrolytic activity of lipase on fat is reduced or inhibited, so that excess fat cannot be hydrolyzed into small molecules that can be absorbed by the body in the intestine, thereby achieving the effect of weight loss. In addition, this bacterium can inhibit common pathogens and can be used to regulate the intestinal flora, prevent, alleviate and / or treat diseases caused by pathogen infection, and has high application value.

[0044] In this article, the terms “Lactobacillus rhamnosus VB336”, “Lactobacillus rhamnosus VB336”, “Lactobacillus rhamnosus VB336”, “Lactobacillus rhamnosus VB336”, “VB336 strain”, and “VB336” are synonymous.

[0045] Lipases (glycerol ester hydrolases) belong to the class of carboxyl ester hydrolases and can progressively hydrolyze triglycerides into glycerol and fatty acids. Furthermore, lipases possess diverse catalytic abilities; for example, they can catalyze the hydrolysis, alcoholysis, esterification, transesterification, and reverse synthesis of triglycerides and other water-insoluble esters. They also exhibit activities similar to other enzymes, such as phospholipase, lysophospholipase, cholesterol esterase, and acylpeptide hydrolase. The different activities of lipases depend on the characteristics of the reaction system; for example, they promote ester hydrolysis at the oil-water interface, while in organic phases they can catalyze synthesis and transesterification. In this study, *Lactobacillus rhamnosus* VB336 can produce lipase inhibitors that inhibit lipase activity, including but not limited to lipase hydrolytic activity. The lipases described herein include any natural or synthetic lipases, such as animal lipases, plant lipases, and microbial lipases.

[0046] Fermentation liquid

[0047] In some embodiments, the present invention provides a fermentation broth derived from *Lactobacillus rhamnosus* VB336 as described above. The fermentation broth of the present invention can be used to inhibit lipolysis and / or weight loss, regulate intestinal flora, and prevent, alleviate, and / or treat diseases caused by pathogenic bacterial infections.

[0048] It should be noted that the "fermentation broth" of the present invention refers to the solution obtained after culturing Lactobacillus rhamnosus VB336 for a period of time, which mainly contains Lactobacillus rhamnosus VB336 and its metabolites; or the supernatant after further treatment by centrifugation, filtration and other means, which mainly contains the metabolites of Lactobacillus rhamnosus VB336.

[0049] bacterial suspension

[0050] In some embodiments, the present invention provides a bacterial suspension comprising the aforementioned *Lactobacillus rhamnosus* VB336. The bacterial suspension of the present invention can be used to inhibit lipolysis and / or weight loss, regulate intestinal flora, and prevent, alleviate, and / or treat diseases caused by pathogenic bacterial infections.

[0051] It should be noted that the bacterial suspension can be obtained by processing the above-mentioned fermentation broth through centrifugation, resuspension and other means.

[0052] use

[0053] In some embodiments, the present invention proposes the use of the aforementioned *Lactobacillus rhamnosus* VB336, fermentation broth, or bacterial suspension in the preparation of foods, beverages, pharmaceuticals, health products, feeds, or additives that inhibit lipase activity and / or promote weight loss. The foods, beverages, health products, feeds, or additives of the present invention can be used to inhibit lipase activity and / or promote weight loss, as well as to regulate intestinal flora. The pharmaceuticals of the present invention can be used to inhibit lipase activity and / or promote weight loss, as well as to regulate intestinal flora.

[0054] In some embodiments, the present invention proposes the use of the aforementioned *Lactobacillus rhamnosus* VB336, fermentation broth, or bacterial suspension in the preparation of foods, beverages, pharmaceuticals, health products, feeds, or additives that inhibit the activity of pathogenic bacteria. The foods, beverages, health products, feeds, or additives of the present invention can be used to regulate intestinal flora, and the pharmaceuticals of the present invention can be used to regulate intestinal flora and prevent, alleviate, and / or treat diseases caused by pathogenic bacterial infections.

[0055] According to embodiments of the present invention, the above-mentioned uses may further include at least one of the following additional technical features:

[0056] According to an embodiment of the present invention, the pathogenic bacteria include at least one of Escherichia coli, Staphylococcus aureus, Salmonella, Listeria, Shigella, Enterococcus faecalis, Clostridium perfringens, Malassezia, Candida albicans, Porphyromonas gingivalis, and Streptococcus mutans.

[0057] Food, beverages, medicines, health products, feed or additives

[0058] This invention provides a food, beverage, pharmaceutical, health product, feed, or additive, wherein the food, beverage, pharmaceutical, health product, feed, or additive includes at least one of the aforementioned *Lactobacillus rhamnosus* VB336, fermentation broth, or bacterial suspension. The food, beverage, health product, feed, or additive of this invention can be used to inhibit lipase activity and / or achieve weight loss, as well as regulate intestinal flora. The pharmaceutical product of this invention can be used to inhibit lipase activity and / or achieve weight loss, as well as regulate intestinal flora, and prevent, alleviate, and / or treat diseases caused by pathogenic bacterial infections.

[0059] According to embodiments of the present invention, the aforementioned food, beverage, pharmaceutical, health product, feed, or additive may further include at least one of the following additional technical features:

[0060] According to embodiments of the present invention, it further includes pharmaceutically acceptable excipients or carriers, food or health product acceptable excipients or carriers, or animal feed acceptable excipients or carriers.

[0061] In this article, "acceptable in food" refers to substances or compositions that are edible for human consumption, which may be adjusted according to the food requirements of different countries.

[0062] In this article, "acceptable in health supplements" refers to substances or compositions that can be consumed by humans, which may be adjusted according to the health supplement requirements of different countries.

[0063] In this document, "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used herein means approved by a federal regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

[0064] In this article, “acceptable adjuvants or carriers in animal feed” refers to substances or compositions that can be consumed by animals, and these can be adjusted according to the animal feed requirements of different countries.

[0065] In this document, the term "pharmaceutically acceptable carrier" includes any solvent, drug stabilizer, or combination thereof known to those skilled in the art. It covers the use of any conventional carrier in therapeutic or pharmaceutical compositions, except in cases where any conventional carrier is incompatible with the active ingredient.

[0066] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent suitable for the specific target dosage form. The use of any conventional excipients that are incompatible with the Lactobacillus rhamnosus VB336 disclosed herein, such as any adverse biological effects produced or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this disclosure.

[0067] According to embodiments of the present invention, at least one of the aforementioned Lactobacillus rhamnosus VB336, fermentation broth, and bacterial suspension is added to or inoculated into food, beverage, health product, feed, or additive, or added to a drug, thereby further obtaining food, beverage, health product, feed, or additive that inhibits lipase activity and / or causes weight loss, and regulates intestinal flora function, or obtaining a drug that can inhibit lipase activity and / or cause weight loss, regulate intestinal flora function, and prevent, alleviate, and / or treat pathogen infection or diseases caused by pathogen infection.

[0068] For example, the aforementioned food products include, but are not limited to: probiotic tablets, fermented dairy products (such as probiotic yogurt), probiotic solid beverages, probiotic milk powder, probiotic cheese, probiotic soy products, probiotic candies, probiotic fermented vegetables, etc.

[0069] For example, the aforementioned drugs include, but are not limited to, human drugs and veterinary drugs. The aforementioned veterinary drugs can be for pets, livestock, or wild animals.

[0070] For example, the aforementioned health products include, but are not limited to: health products for human use and health products for veterinary use.

[0071] It should be noted that the characteristics and advantages described above for Lactobacillus rhamnosus VB336 also apply to this food, beverage, pharmaceutical, health product, feed, or additive, and will not be repeated here.

[0072] Composition

[0073] In some embodiments, the present invention provides a composition comprising at least one of the aforementioned *Lactobacillus rhamnosus* VB336, fermentation broth, and bacterial suspension. The composition of the present invention can be used to inhibit lipase activity and / or achieve weight loss, regulate intestinal flora, and prevent, alleviate, and / or treat diseases caused by pathogenic bacterial infections.

[0074] According to embodiments of the present invention, the above composition may further include at least one of the following additional technical features:

[0075] According to embodiments of the present invention, the composition further includes an ingestible excipient and / or a carrier.

[0076] According to an embodiment of the present invention, the excipient includes at least one selected from adhesives, disintegrants, lubricants, flow aids, stabilizers, fillers, diluents, and sustained-release agents.

[0077] According to an embodiment of the present invention, the carrier comprises at least one selected from sugars, cellulose and its derivatives, calcium phosphates, alkaline earth metal stearates, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols, and hydrolyzed cereal solids.

[0078] According to embodiments of the present invention, the dosage form of the composition includes at least one selected from oral liquids, powders, granules, capsules, tablets, and pills.

[0079] method

[0080] In some embodiments, the present invention provides a method for in vitro inhibition of lipase activity, the method comprising: co-culturing at least one of the aforementioned *Lactobacillus rhamnosus* VB336, fermentation broth, and bacterial suspension with lipase. As previously stated, the *Lactobacillus rhamnosus* VB336 of the present invention can effectively inhibit lipase activity, preventing excess fat from being hydrolyzed into small molecules that can be absorbed by the body. This method can be used for non-diagnostic and / or non-therapeutic purposes, such as for scientific research.

[0081] In some embodiments, the present invention proposes a weight loss method comprising administering to a subject at least one of the aforementioned *Lactobacillus rhamnosus* VB336, fermentation broth, and bacterial suspension. As previously stated, the *Lactobacillus rhamnosus* VB336 of the present invention can effectively inhibit lipase activity, preventing excess fat from being hydrolyzed into small molecules that can be absorbed by the body in the intestine, thereby achieving a weight loss effect.

[0082] In some embodiments, the present invention provides a method for inhibiting the growth of pathogenic bacteria in vitro, the method comprising: co-culturing at least one of the aforementioned *Lactobacillus rhamnosus* VB336, fermentation broth, and bacterial suspension with pathogenic bacteria. As previously stated, the *Lactobacillus rhamnosus* VB336 of the present invention can effectively inhibit the growth of pathogenic bacteria.

[0083] According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

[0084] According to an embodiment of the present invention, the pathogenic bacteria include at least one of Escherichia coli, Staphylococcus aureus, Salmonella, Listeria, Shigella, Enterococcus faecalis, Clostridium perfringens, Malassezia, Candida albicans, Porphyromonas gingivalis, and Streptococcus mutans.

[0085] Device

[0086] In some embodiments, the present invention provides a culture or production apparatus for the aforementioned *Lactobacillus rhamnosus* VB336, fermentation broth, bacterial suspension, or composition. The culture or production apparatus according to embodiments of the present invention enables large-scale production of the *Lactobacillus rhamnosus* VB336, fermentation broth, bacterial suspension, or composition described herein.

[0087] Therapeutic uses and treatment methods

[0088] In some embodiments, the present invention proposes the use of *Lactobacillus rhamnosus* VB336 as described in the first aspect, the fermentation broth as described in the second aspect, the bacterial suspension as described in the third aspect, or the composition as described in the sixth aspect in inhibiting lipase activity and / or weight loss in an individual. As previously stated, *Lactobacillus rhamnosus* VB336 of the present invention can effectively inhibit lipase activity, preventing excess fat from being hydrolyzed into small molecules that can be absorbed by the body in the intestine, thereby achieving a weight loss effect. Therefore, it can be used to inhibit lipase activity in an individual, and further, can effectively reduce weight and prevent, alleviate, and / or treat related diseases caused by lipase.

[0089] In some embodiments, the present invention provides a method for inhibiting lipase activity and / or losing weight in an individual. According to embodiments of the present invention, the method includes administering to the individual the *Lactobacillus rhamnosus* VB336 described in the first aspect, the fermentation broth described in the second aspect, the bacterial suspension described in the third aspect, or the composition described in the sixth aspect. As previously mentioned, the *Lactobacillus rhamnosus* VB336 of the present invention can effectively inhibit lipase activity, preventing excess fat from being hydrolyzed into small molecules that the body can absorb in the intestines, thereby achieving a weight loss effect. Therefore, it can be used to inhibit lipase activity in an individual, and further, can effectively reduce weight and prevent, alleviate, and / or treat related diseases caused by lipase.

[0090] In some embodiments, the present invention proposes the use of *Lactobacillus rhamnosus* VB336 as described in the first aspect, the fermentation broth as described in the second aspect, the bacterial suspension as described in the third aspect, or the composition as described in the sixth aspect in the prevention, alleviation, and / or treatment of individual obesity. As previously stated, *Lactobacillus rhamnosus* VB336 of the present invention can effectively inhibit lipase activity, preventing excess fat from being hydrolyzed into small molecules that can be absorbed by the body in the intestine, thereby achieving a weight loss effect. Therefore, it can be used to prevent, alleviate, and / or treat individual obesity, and further, can effectively prevent, alleviate, and / or treat obesity-related diseases.

[0091] In some embodiments, the present invention provides a method for preventing, alleviating, and / or treating obesity in an individual, comprising administering to the individual the *Lactobacillus rhamnosus* VB336 described in the first aspect, the fermentation broth described in the second aspect, the bacterial suspension described in the third aspect, or the composition described in the sixth aspect. As previously stated, the *Lactobacillus rhamnosus* VB336 of the present invention can effectively inhibit lipase activity, preventing excess fat from being hydrolyzed into small molecules that the body can absorb in the intestines, thereby achieving a weight loss effect. Therefore, it can be used to prevent, alleviate, and / or treat obesity in an individual, and further, can effectively prevent, alleviate, and / or treat obesity-related diseases.

[0092] In some embodiments, the present invention provides for the use of *Lactobacillus rhamnosus* VB336 as described in the first aspect, the fermentation broth as described in the second aspect, the bacterial suspension as described in the third aspect, or the composition as described in the sixth aspect in the prevention, alleviation, and / or treatment of diseases related to pathogenic bacterial infections. As previously mentioned, *Lactobacillus rhamnosus* VB336 can inhibit the activity of common pathogenic bacteria and has good tolerance to gastric acid and bile salts, as well as strong stability in intestinal fluid. Therefore, this strain and its related products can effectively prevent, alleviate, and / or treat diseases related to common pathogenic bacterial infections.

[0093] According to embodiments of the present invention, the above-mentioned uses may further include at least one of the following additional technical features:

[0094] According to an embodiment of the present invention, the pathogenic bacteria include at least one of Escherichia coli, Staphylococcus aureus, Salmonella, Listeria, Shigella, Enterococcus faecalis, Clostridium perfringens, Malassezia, Candida albicans, Porphyromonas gingivalis, and Streptococcus mutans.

[0095] In some embodiments, the present invention provides a method for preventing, alleviating, and / or treating diseases related to individual pathogenic bacterial infections. According to embodiments of the invention, the method includes administering to the individual the *Lactobacillus rhamnosus* VB336 described in the first aspect, the fermentation broth described in the second aspect, the bacterial suspension described in the third aspect, or the composition described in the sixth aspect. As previously mentioned, *Lactobacillus rhamnosus* VB336 can inhibit the activity of common pathogenic bacteria and has good tolerance to gastric acid and bile salts, as well as strong stability in intestinal fluid. Therefore, this strain and its related products can effectively prevent, alleviate, and / or treat diseases related to common pathogenic bacterial infections.

[0096] According to embodiments of the present invention, the above-mentioned uses may further include at least one of the following additional technical features:

[0097] According to an embodiment of the present invention, the pathogenic bacteria include at least one of Escherichia coli, Staphylococcus aureus, Salmonella, Listeria, Shigella, Enterococcus faecalis, Clostridium perfringens, Malassezia, Candida albicans, Porphyromonas gingivalis, and Streptococcus mutans.

[0098] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0099] Example 1: Isolation and Identification of Strains

[0100] Samples were collected from different sources, including 20 fresh fecal samples from healthy infants and 20 fresh fecal samples from healthy adults. 0.5g of each fecal sample was added to a 15mL sterile collection tube containing 5mL PBS solution and vortexed to mix. Then, 1mL of each solution was serially diluted to prepare bacterial suspensions of different gradients. 100μL of each gradient of bacterial suspension was plated on MRS agar plates and anaerobically cultured at 37℃ for 48h. After incubation, colonies with different morphologies were picked, streaked, purified, and DNA extracted. PCR amplification was performed using specific primers, followed by sequencing of 16S rRNA. Glycerol tubes containing the corresponding strains were also preserved. Based on the 16S rRNA identification results, 148 Lactobacillus strains were selected, numbered A01-A148. Among them, there were 10 strains of Lactobacillus casei, 24 strains of Lactobacillus paracasei, 32 strains of Lactobacillus rhamnosus, 49 strains of Lactobacillus plantarum, 8 strains of Lactobacillus fermentum, 9 strains of Lactobacillus reuteri, and 16 strains of Lactobacillus acidophilus.

[0101] Example 2: Strain Screening

[0102] 1.1 Preparation of lipase

[0103] (1) Prepare a 0.02 mol / L anhydrous disodium hydrogen phosphate buffer solution (pH = 9.3);

[0104] (2) Prepare an 8% lipase solution using the above anhydrous disodium hydrogen phosphate buffer, dissolve it and put it into a grinding tube, grind it at 60 Hz for 2 minutes at 4°C, centrifuge and take the supernatant, adjust the pH to 8.0 with NaOH solution, and store it in a freezer at -80°C.

[0105] (3) Dilute the above 8% lipase solution with 0.02mol / L disodium hydrogen phosphate at pH=10.3 to a concentration of 1% and set aside.

[0106] 2.2 Preparation of Fat Emulsions

[0107] (1) Preparation of 100 mmol / L tris-HCl buffer (pH=8.0): Weigh 1.21 g tris reagent and dissolve it in 100 mL of sterile water. After dissolving, adjust the pH to 8.0 with dilute hydrochloric acid.

[0108] (2) Take 0.3 mL of the stock emulsion and add 29.7 mL of the above 100 mmol / L tris-HCl buffer (pH = 8.0), mix well, and obtain an emulsion diluted 100 times. Mix the 100-fold diluted emulsion and 100 mmol / L tris-HCl buffer (pH = 8.0) at a ratio of 1:1 to obtain an emulsion diluted 200 times.

[0109] 2.3 Preparation of Orlistat at Different Concentrations

[0110] (1) Preparation of 100 μg / mL orlistat stock solution: Weigh 4 mg of orlistat standard and dissolve it in 40 mL of pH 10.3 disodium hydrogen phosphate solution, and sonicate until completely dissolved.

[0111] (2) The 100 μg / mL orlistat stock solution was gradually diluted to 50 μg / mL, 25 μg / mL, 10 μg / mL, 8 μg / mL, 5 μg / mL, 2.5 μg / mL and 1 μg / mL with 0.02 mol / L disodium hydrogen phosphate buffer at pH 10.3.

[0112] The sources of the experimental reagents used in this embodiment are shown in Table 1.

[0113] Table 1. Experimental Materials

[0114] 2.4 Orlistat inhibition experiment on lipase

[0115] (1) An inhibition experiment of orlistat and lipase was conducted using 96-well plates. The experiment was divided into 3 groups.

[0116] ① Blank control group: 180 μL of 200-fold diluted emulsion + 10 μL of pH 8.0 Tris-HCl buffer + 10 μL of pH 10.3 disodium hydrogen phosphate buffer;

[0117] ② Enzyme-added negative control group: 180 μL of 200-fold diluted emulsion + 10 μL of 1% lipase solution + 10 μL of pH=10.3 disodium hydrogen phosphate buffer;

[0118] ③ Orlistat positive control group: 180 μL of emulsion diluted 200 times + 10 μL of 1% lipase solution + 10 μL of orlistat standard solution of different concentrations;

[0119] In 96-well plates, reagent solutions were added according to the groups described above. For group ③, the enzyme solution and orlistat solution were added first, and the reaction was allowed to proceed for 15 minutes before the emulsion was added. The emulsion was added at the same time point in all groups to ensure that the enzyme reaction time was the same. The OD value of the emulsion was measured at 490 nm using a microplate reader at 0 h, 45 min, and 2 h. The hydrolytic effect of the enzyme on the emulsion was calculated by measuring the changes in the OD value.

[0120] (2) Calculation methods for lipase hydrolysis rate and orlistat inhibition rate of lipase:

[0121] Lipase hydrolysis rate % = (OD of blank control group - OD of enzyme solution negative control group) / OD of blank control group * 100%

[0122] Orlistat inhibition rate of lipase % = (hydrolysis rate of enzyme solution negative control group - hydrolysis rate of orlistat positive control group) / hydrolysis rate of enzyme solution negative control group * 100%.

[0123] 2.5 Screening of lipase inhibitor strains

[0124] (1) MRS finished culture medium: Weigh 54g of finished culture medium, add 1L of pure water, stir to dissolve, and then dispense into shake flasks and sterilize at 121℃ for 15min.

[0125] (2) Strain culture

[0126] ①Inoculum source: Glycerol tubes of the different strains described in Example 1, stored at -80℃.

[0127] ② Inoculation method: The bacterial culture was carried out by directly inoculating the liquid culture medium with glycerol tubes, and the inoculation amount ranged from 0.2% to 1%.

[0128] ③ Liquid culture method: According to the growth characteristics of the strain, place it in an anaerobic workstation at 37℃ and culture for 18-24 hours.

[0129] (3) Sample preparation

[0130] When performing lipase inhibitor detection, the processing method varies, and the sample is divided into two types: supernatant and whole solution.

[0131] Supernatant: Take 0.5 mL of the above bacterial culture, centrifuge at 14,000 rpm for 10 minutes, and take the supernatant for testing;

[0132] Whole liquid: Take 0.5 mL of the above bacterial solution, add it to a grinding tube, and grind for 2 minutes at 4℃ and 60 Hz for a total of 8 times. After grinding, centrifuge at 14000 rpm for 10 minutes and take the supernatant for testing.

[0133] (4) Sample testing

[0134] The prepared supernatant and whole liquid samples were mixed with lipase and reacted for 15 minutes, then an emulsion was added. The OD value of the emulsion was measured over a certain period of time. The inhibitory effect of the sample on the enzyme was judged by the change in the emulsion value (OD difference). The specific groupings are as follows:

[0135] Blank sample negative control group: 180 μL of emulsion diluted 200 times + 10 μL of pH=8.0 Tris-HCl buffer + 10 μL of MRS finished culture medium.

[0136] A. Sample detection group 1: 180 μL of emulsion diluted 200 times + 10 μL of 1% lipase solution + 10 μL of sample supernatant.

[0137] B. Sample detection group 2: 180 μL of emulsion diluted 200 times + 10 μL of 1% lipase solution + 10 μL of whole sample solution.

[0138] (5) Formula for calculating the inhibition rate of enzyme by the sample

[0139] Sample hydrolysis rate % = (OD of blank sample negative control group - OD of supernatant or whole liquid detection group) / OD of blank sample negative control group * 100%

[0140] Lipase hydrolysis rate % = (OD of blank control group - OD of enzyme solution negative control group) / OD of blank control group * 100%

[0141] Sample inhibition rate % = (Lipase hydrolysis rate - Sample hydrolysis rate) / Lipase hydrolysis rate * 100%

[0142] (6) Screening Results

[0143] The experimental results are shown in Tables 2 and 3. Using this screening model, a total of 148 strains from different species were screened for their lipase inhibitory abilities. Five strains with lipase inhibitory activity were selected, among which *Lactobacillus rhamnosus* A36 showed the best inhibitory effect on lipase. Specific data are shown in Table 2. The in vitro inhibitory ability of this strain on lipase is equivalent to a concentration of 5–8 μg / mL orlistat. That is, this strain has the ability to produce lipase inhibitors and can inhibit lipase activity, making it suitable for the preparation of weight-loss drugs, health foods, or solid beverages.

[0144] Table 2. Results of Lipase Inhibitor Screening

[0145] Table 3. Lipase Inhibition Ability of Active Strains

[0146] 2.6 Strain Identification

[0147] The *Lactobacillus rhamnosus* A36 strain obtained from the screening in section 2.5 was sequenced, and the 16S rRNA sequence of the strain was compared with the homology of strains in GenBank. It was identified as *Lactobacillus rhamnosus* and named *Lactobacillus rhamnosus* VB336. It was deposited on March 8, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC M 2024432. The scanning electron microscope image of *Lactobacillus rhamnosus* VB336 is shown in Figure 1, and the 16S rRNA sequence is shown in SEQ ID NO:1.

[0148] Example 3: Physiological and biochemical characteristics of Lactobacillus rhamnosus strain VB336

[0149] The culture medium required for detecting the physiological and biochemical characteristics of Lactobacillus rhamnosus VB336, namely MRS agar medium, was prepared as follows: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, agar 20 g / L, Tween 80 1.0 g / L, pH = 6.7, and autoclaved at 121℃ for 15 min.

[0150] The physiological and biochemical characteristics of this strain were analyzed according to Bergey's Manual of Bacteriological Identification, and the specific test results are as follows:

[0151] (1) Contact enzyme: Take a small loop of Lactobacillus rhamnosus VB336 that has been cultured at 37℃ under anaerobic conditions for 24 hours and apply it to a glass slide that has been dotted with 3% hydrogen peroxide. Observe immediately. If bubbles are produced, it is positive; if no bubbles are produced, it is negative.

[0152] (2) Oxidase: Wet the oxidase test paper with distilled water, pick up a single Lactobacillus rhamnosus VB336 colony with a fine glass rod, and smear it on the test paper. If it turns blue or blue-purple within 30 seconds, it is a strong positive result; if it does not change color within 2 minutes, it is a negative result.

[0153] (3) Milk decomposition: Take a test tube containing 2 mL of sterile water, and use an inoculation needle to pick 1-3 Lactobacillus rhamnosus VB336 colonies from the plate and grind them carefully in sterile water to prepare a bacterial suspension. Add 100 μL of the bacterial suspension to the milk tube. Incubate at 37℃ for 24-48 h. After the incubation, observe the results. If the layers turn pink and coagulate, it is a positive result; if there is no color change, it is a negative result.

[0154] (4) Hydrolysis of hippuric acid: Add 100 μL of the bacterial suspension from step (3) to a biochemical tube and incubate at 37°C for 4 hours. After incubation, slowly add 0.2 mL of ninhydrin solution along the tube wall without shaking. Observe after incubation at 37°C for 10 minutes. A dark purple color indicates a positive result, while a light purple color or no color change indicates a negative result.

[0155] (5) Indole test: Add 100 μL of the bacterial suspension from step (3) to peptone water, incubate at 37°C for 24 h, and then add 0.2 mL to 0.3 mL of Kovacs indole reagent. If red appears on the upper layer, it is a positive reaction of indole; otherwise, it is negative.

[0156] (6) Urease: Add 100 μL of the bacterial suspension from step (3) into a biochemical tube and incubate at 37°C for 24 h. If the upper layer turns rose red, it is positive; otherwise, it is negative.

[0157] (7) Hydrogen sulfide production: Add 100 μL of the bacterial suspension from step (3) into a biochemical tube and incubate at 37°C for 18-24 h. If black appears, it is positive; otherwise, it is negative.

[0158] (8) Glucose semi-solid: Add 100 μL of the bacterial suspension from step (3) to a biochemical tube and incubate at 37°C for 18-24 h. If yellow appears, it is positive; if grayish-purple, purple, or purplish-red appears, it is negative.

[0159] (9) Arginine double hydrolase: Add 100 μL of the bacterial suspension from step (3) to a biochemical tube and incubate at 37°C for 18–24 h. After inoculation, cover the culture medium surface with sterile liquid paraffin. A purple or purplish-red color indicates a positive result, while a yellow color indicates a negative result.

[0160] (10) Amygdalin / rhamnose: Add 100 μL of the bacterial suspension from step (3) to a biochemical tube and incubate at 37°C for 18–24 h. A yellow color indicates a positive result, while a light red or red color indicates a negative result.

[0161] (11) Glucose / xylose / melbiose / minobiose / trehalose / galactose / arabinose / sorbitol: Add 100 μL of the bacterial suspension from step (3) to a biochemical tube and incubate at 37°C for 18–24 h. A yellow color indicates a positive result, while a blue or blue-green color indicates a negative result.

[0162] (12) D-ribose / melanotriose / mannose / fructose: Add 100 μL of the bacterial suspension from step (3) into a biochemical tube and incubate at 37°C for 24–48 h. A yellow color indicates a positive result, while a grayish-purple, purple, or purplish-red color indicates a negative result.

[0163] (13) Cellobiose / maltose / mannitol / salicylic acid / sucrose / raffinose / inulin / lactose: Add 100 μL of the bacterial suspension from step (3) into a biochemical tube and incubate at 37°C for 24–48 h. A yellow color indicates a positive result, while a purple or purplish-gray color indicates a negative result.

[0164] (14) Nitrate reduction: Add 100 μL of the bacterial suspension from step (3) to a biochemical tube and incubate at 37°C for 18–24 h. After incubation, add 2–3 drops each of nitrate reduction reagent A and solution B and observe immediately. If it turns red, it is positive; if it does not change color, it is negative.

[0165] (15) Gluconate: Add 100 μL of the bacterial suspension from step (3) to a biochemical tube and incubate at 37°C for 24–48 h. After incubation, add Benedict's reagent at a volume ratio of 1:1, mix well, boil for 10 min, and observe the results after cooling. A yellow, orange, or orange-red precipitate indicates a positive result, while a blue or green precipitate indicates a negative result.

[0166] (16) VP assay: Add 100 μL of the bacterial suspension from step (3) to a biochemistry tube and incubate at 37°C for 2–4 days. Add the VP kit and shake the tube thoroughly, then observe the results. A positive reaction will appear immediately or within minutes. If negative, incubate at 37°C for another 4 hours before observing again.

[0167] (17) Methyl red (MR): Add 100 μL of the bacterial suspension from step (3) into a biochemical tube, incubate at 37°C for 2–4 days, add one drop of methyl red reagent, and observe immediately. A bright red color indicates a positive result, while a yellow color indicates a negative result.

[0168] (18) Gelatin liquefaction: Add 100 μL of the bacterial suspension from step (3) into a biochemical tube and incubate at 37°C for 24–72 h. After removing the tube, place it in a refrigerator at 2–8°C for 30 min and observe. If it is still liquid, the experimental result is positive; otherwise, it is negative.

[0169] (19) Starch hydrolysis: Add 100 μL of the bacterial suspension from step (3) into a biochemical tube and incubate at 37°C for 48–96 h. After incubation, add iodine solution. If it does not turn blue, the result is positive; if it turns blue, the result is negative.

[0170] (20) Aesculin: Add 100 μL of the bacterial suspension from step (3) into a biochemical tube and incubate at 37°C for 24–48 h. After inoculation, cover the culture medium surface with sterile liquid paraffin. If black appears, it is positive; if purple or purplish-gray, it is negative.

[0171] The physiological and biochemical test results of this strain are shown in Table 4.

[0172] Table 4. Physiological and biochemical results of Lactobacillus rhamnosus VB336 Note: + indicates positive; - indicates negative.

[0173] Example 4: Stability of Lactobacillus rhamnosus strain VB336

[0174] 4.1 Preparation of Experimental Materials

[0175] (1) Preparation of culture medium

[0176] MRS medium: 5.4% MRS broth + 1.8% agar, pH = 6.5

[0177] (2) Preparation of simulated gastric juice: Physiological saline (sodium chloride: 0.9%), first prepare a 1% sodium chloride solution, adjust to different pH and dispense, sterilize at 121℃ for 30 min, pepsin 0.3% (prepare a 3% concentration, filter with sterile membrane), mix 3% pepsin: 1% physiological saline = 1:9.

[0178] (3) Preparation of choline solution: Physiological saline (sodium chloride: 0.9%), first prepare a 1% sodium chloride solution, add bovine choline at concentrations of 0%, 0.033%, 0.066%, 0.11%, 0.22%, and 0.33% respectively, sterilize at 121℃ for 30 min, trypsin 0.1% (prepared to 1% concentration, filtered through a sterile membrane), mix 1% trypsin: 1% physiological saline of each choline concentration = 1:9, and mix well.

[0179] (4) Preparation of intestinal fluid: Artificial simulated intestinal fluid is prepared according to the product instructions.

[0180] 4.2 Experimental Methods

[0181] Take cryopreservation tubes of Lactobacillus rhamnosus VB336 strain, spread the stock solution onto blank Petri dishes, and anaerobically incubate at 37°C for 1 day according to growth characteristics. Scrape off the bacterial colony and prepare 20% glycerol tubes for storage at -80°C for later use. Simultaneously, dilute and count the viable cells to achieve a viable count of 10-1. 9 -10 10 The recommended concentration is CFU / mL.

[0182] Take the counted bacterial suspension, thaw it, and add it to the pre-packaged simulated choline solution, gastric juice, and intestinal juice. The addition volume is: 10 μL of bacterial suspension + 990 μL of simulated solution. After mixing, count the viable bacteria in the 0h group at time 0. Place the bacterial suspensions of the 2h and 4h groups into anaerobic bags and incubate them in a 37℃ incubator. Take them out and count the viable bacteria after 2h and 4h of incubation, respectively.

[0183] Table 5. Experimental Materials

[0184] As shown in Figure 2, *Lactobacillus rhamnosus* VB336 maintained a survival rate of over 95% within 4 hours in a 0.03%-0.3% choline solution, indicating that *Lactobacillus rhamnosus* VB336 can stably exist in pancreatic juice, meaning that this strain can adapt to the intestinal environment. As shown in Figure 3, *Lactobacillus rhamnosus* VB336 died immediately in gastric juice at pH=1 and pH=2, with a 100% mortality rate after 2 hours in gastric juice at pH=3, and over 80% mortality after 2 hours and approximately 99% mortality after 4 hours in gastric juice at pH=4. Therefore, the instability of *Lactobacillus rhamnosus* VB336 in gastric juice needs to be considered in subsequent dosage form development. As shown in Figure 4, *Lactobacillus rhamnosus* VB336 maintained a 100% survival rate after 4 hours of treatment in intestinal fluid at pH 8.0, indicating that this strain can stably exist in intestinal fluid.

[0185] Example 5: Antibacterial activity of Lactobacillus rhamnosus strain VB336

[0186] (1) Prepare MRS agar medium for Lactobacillus rhamnosus strain VB336:

[0187] The following ingredients were added: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, agar 20 g / L, Tween 80 1.0 g / L, pH 6.7. The mixture was autoclaved at 121°C for 15 min. The culture conditions were anaerobic incubation at 37°C for 1 day.

[0188] (2) Antibacterial model

[0189] The antibacterial experiment used the double-layer plate culture method:

[0190] Preparation of the lower experimental bacterial layer: After thawing the activated Lactobacillus rhamnosus VB336 cryovials, take 2 μL (viable bacterial count of 10⁻⁶). 6 The strains were inoculated on MRS solid plates, with two spots for each strain, and cultured overnight at 37°C under anaerobic conditions.

[0191] Preparation of the upper pathogenic bacteria layer: After high-temperature sterilization, the upper culture medium is cooled to 40℃-50℃, and different pathogenic bacteria are added as needed, with a final pathogenic bacteria concentration of 10. 8 Add 7 mL of culture medium containing pathogenic bacteria to the pre-grown Lactobacillus rhamnosus VB336 plate. After the plate solidifies, place it under appropriate culture conditions according to the growth conditions of different pathogenic bacteria. After the upper plate has grown well, observe whether an inhibition zone forms around the Lactobacillus rhamnosus VB336 colony and measure the size of the inhibition zone to determine the antibacterial ability of Lactobacillus rhamnosus VB336.

[0192] The results are shown in Table 6. Lactobacillus rhamnosus VB336 showed good inhibitory effects on 11 pathogenic bacteria, including Escherichia coli, Staphylococcus aureus, Salmonella, Listeria, Shigella, Enterococcus faecalis, and Clostridium perfringens, which are all enteric pathogens; Malassezia, which is a dermatopathogenic bacterium; Candida albicans, which is a vaginal pathogen; and Porphyromonas gingivalis and Streptococcus mutans, which are oral pathogens.

[0193] Table 6. Antibacterial activity of Lactobacillus rhamnosus VB336

[0194] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "embodiment," or "specific embodiment," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment. Furthermore, the specific features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments and features described in this specification.

[0195] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A Lactobacillus rhamnosus VB336, characterized in that, The Lactobacillus rhamnosus VB336 was deposited at the China Center for Type Culture Collection on March 8, 2024, with accession number CCTCC M 2024432.

2. A fermentation broth, characterized in that, The fermentation broth is obtained by fermentation of Lactobacillus rhamnosus VB336 as described in claim 1.

3. A bacterial suspension, characterized in that, The bacterial suspension comprises Lactobacillus rhamnosus VB336 as described in claim 1.

4. The use of Lactobacillus rhamnosus VB336 as described in claim 1, the fermentation broth as described in claim 2, or the bacterial suspension as described in claim 3 in the preparation of food, beverage, pharmaceutical, health product, feed, or additive that inhibits lipase activity and / or reduces weight.

5. The use of Lactobacillus rhamnosus VB336 as described in claim 1, the fermentation broth as described in claim 2, or the bacterial suspension as described in claim 3 in the preparation of food, beverages, pharmaceuticals, health products, feed, or additives that inhibit the activity of pathogenic bacteria.

6. The use according to claim 5, characterized in that, The pathogens include at least one of the following: Escherichia coli, Staphylococcus aureus, Salmonella, Listeria, Shigella, Enterococcus faecalis, Clostridium perfringens, Malassezia, Candida albicans, Porphyromonas gingivalis, and Streptococcus mutans.

7. A composition, characterized in that, The composition comprises at least one of Lactobacillus rhamnosus VB336 as described in claim 1, the fermentation broth as described in claim 2, and the bacterial suspension as described in claim 3.

8. The composition according to claim 7, characterized in that, The dosage form of the composition includes at least one selected from oral liquids, powders, granules, capsules, tablets, and pills.

9. The composition according to claim 7, characterized in that, The composition further includes excipients and / or carriers.

10. The composition according to claim 9, characterized in that, The excipients include at least one selected from adhesives, disintegrants, lubricants, flow aids, stabilizers, fillers, diluents, and sustained-release agents.

11. The composition according to claim 10, characterized in that, The carrier includes at least one selected from sugars, cellulose and its derivatives, calcium phosphates, alkaline earth metal stearates, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols, and hydrolyzed cereal solids.

12. A method for inhibiting lipase activity in vitro, characterized in that, include: At least one of the Lactobacillus rhamnosus VB336 of claim 1, the fermentation broth of claim 2, and the bacterial suspension of claim 3 is mixed with the lipase.

13. A method for inhibiting the growth of pathogenic bacteria in vitro, characterized in that, include: At least one of the Lactobacillus rhamnosus VB336 of claim 1, the fermentation broth of claim 2, and the bacterial suspension of claim 3 is co-cultured with the pathogen.

14. The method according to claim 13, characterized in that, The pathogens include at least one of the following: Escherichia coli, Staphylococcus aureus, Salmonella, Listeria, Shigella, Enterococcus faecalis, Clostridium perfringens, Malassezia, Candida albicans, Porphyromonas gingivalis, and Streptococcus mutans.

15. A culture or production apparatus for Lactobacillus rhamnosus VB336 as described in claim 1, the fermentation broth as described in claim 2, the bacterial suspension as described in claim 3, or the composition as described in claim 7.

16. Use of the Lactobacillus rhamnosus VB336 of claim 1, the fermentation broth of claim 2, the bacterial suspension of claim 3, or the composition of any one of claims 7 to 11 in inhibiting lipase activity and / or weight loss in an individual.

17. A method for inhibiting individual lipase activity and / or losing weight, characterized in that, This includes administering to the individual the Lactobacillus rhamnosus VB336 of claim 1, the fermentation broth of claim 2, the bacterial suspension of claim 3, or the composition of any one of claims 7 to 11.

18. Use of the Lactobacillus rhamnosus VB336 of claim 1, the fermentation broth of claim 2, the bacterial suspension of claim 3, or the composition of any one of claims 7 to 11 in the prevention, relief, and / or treatment of individual obesity.

19. A method for preventing, alleviating, and / or treating obesity in an individual, characterized in that, This includes administering to the individual the Lactobacillus rhamnosus VB336 of claim 1, the fermentation broth of claim 2, the bacterial suspension of claim 3, or the composition of any one of claims 7 to 11.

20. Use of the Lactobacillus rhamnosus VB336 of claim 1, the fermentation broth of claim 2, the bacterial suspension of claim 3, or the composition of any one of claims 7 to 11 in the prevention, relief, and / or treatment of diseases related to pathogenic bacterial infections.

21. A method for preventing, alleviating, and / or treating diseases related to an individual's pathogenic infection, characterized in that, This includes administering to the individual the Lactobacillus rhamnosus VB336 of claim 1, the fermentation broth of claim 2, the bacterial suspension of claim 3, or the composition of any one of claims 10 to 17.

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