Pediococcus pentosaceus VB341 with lipid-lowering effect, culture device and its application

Abstract

The present invention relates to the field of microorganisms. The present invention provides a Pediococcus pentosaceus VB341 with lipid-lowering efficacy, a culture device and its application. The Pediococcus pentosaceus VB341 was deposited at the China Center for Type Culture Collection on March 8, 2024, with the deposit number CCTCC NO: M 2024433. The Pediococcus pentosaceus VB341 of the present invention has a strong ability to inhibit lipase activity, has strong stability in intestinal fluid and bile salts, can be used to inhibit fat hydrolysis, reduce blood lipid levels, help maintain a healthy blood lipid level, help control body fat and / or lose weight, and is not prone to generating resistance.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and specifically, to a Pediococcus pentosaceus VB341 with lipid-lowering efficacy, a culture device thereof, and applications thereof. Background Art

[0002] Fermented foods have always been a valuable source for the development of probiotics. Food-derived lactic acid bacteria not only have high safety, but also have better properties than other sample sources. In China, fermented foods have a long tradition of consumption. Vegetables, meats, dairy products, etc. in human daily foods can all be fermented by lactic acid bacteria to produce foods with special flavors and probiotic characteristics. Although multiple microorganisms are involved in the food fermentation process, existing research has shown that the main ones are lactic acid bacteria. Lactic acid bacteria can bring health benefits to the host by producing functional active factors and regulating the intestinal flora.

[0003] Currently, the methods for reducing blood lipid levels mainly include drug treatment and dietary intervention. Statins and fibrates are more commonly used clinically. The initial use of drugs has relatively significant efficacy, but long-term use will produce varying degrees of side effects. Currently, it is advocated to follow the dietary principle of low-fat or low-saturated-fat foods. However, the taste and flavor of low-fat foods are relatively monotonous. Therefore, the acceptance of the low-fat diet concept is low, and people expect to develop other dietary supplements that can reduce cholesterol and blood lipids.

[0004] As early as the 1970s, it was found that the cholesterol content in the bodies of people who often consumed fermented dairy products (lactic acid bacteria) was relatively low. Pediococcus pentosaceus belongs to the genus Pediococcus of the family Streptococcaceae. As a kind of lactic acid bacteria, it has gradually played an increasingly important role in the application of lactic acid bacteria in recent years. In addition to the function of lactic acid bacteria producing acid, Pediococcus pentosaceus can also improve the flavor, quality, and safety of fermented products. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0006] To this end, in the first aspect of the present invention, the present invention proposes a Pediococcus pentosaceus (Pediococcus pentosaceus) VB341. According to an embodiment of the present invention, the Pediococcus pentosaceus VB341 was deposited with the China Center for Type Culture Collection on March 8, 2024, and the deposit number is CCTCC NO: M 2024433.

[0007] Pediococcus pentosaceus VB341 screened by the present invention has strong stability in intestinal fluid and bile salts, and can secrete lipase inhibitors, reducing or inhibiting the hydrolysis activity of lipase on fat, so that excessive fat cannot be hydrolyzed into small molecule substances that can be absorbed by the body in the intestine, thereby achieving the effects of reducing blood lipid levels and weight loss. Further, it can prevent, relieve and / or treat diseases caused by hyperlipidemia and is not likely to produce resistance.

[0008] In the second aspect of the present invention, the present invention provides a fermentation broth. According to an embodiment of the present invention, the fermentation broth is fermented from Pediococcus pentosaceus VB341 described in the first aspect. The fermentation broth of the present invention can be used to inhibit fat hydrolysis, reduce blood lipid levels and / or lose weight. Further, it can prevent, relieve and / or treat diseases caused by hyperlipidemia and is not likely to produce resistance.

[0009] In the third aspect of the present invention, the present invention provides a bacterial suspension. According to an embodiment of the present invention, the bacterial suspension comprises Pediococcus pentosaceus VB341 described in the first aspect. The bacterial suspension of the present invention can be used to inhibit fat hydrolysis, reduce blood lipid levels and / or lose weight. Further, it can prevent, relieve and / or treat diseases caused by hyperlipidemia and is not likely to produce resistance.

[0010] In the fourth aspect of the present invention, the present invention provides the use of Pediococcus pentosaceus VB341 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, health products, feeds or additives that help maintain a healthy blood lipid level and / or help control body fat. The foods, beverages, health products, feeds or additives of the present invention help maintain a healthy blood lipid level and / or help control body fat.

[0011] In the fifth aspect of the present invention, the present invention provides the use of Pediococcus pentosaceus VB341 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, drugs, health products, feeds or additives that inhibit lipase activity, reduce blood lipid levels and / or lose weight. The foods, beverages, health products, feeds or additives of the present invention can be used to inhibit lipase activity, and the drugs can be used to inhibit lipase activity, reduce blood lipid levels and / or lose weight. Further, it can prevent, relieve and / or treat diseases caused by hyperlipidemia and is not likely to produce resistance.

[0012] In the sixth aspect of the present invention, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises at least one of Pediococcus pentosaceus VB341 described in the first aspect, the fermentation broth described in the second aspect and the bacterial suspension described in the third aspect. The composition of the present invention helps maintain a healthy blood lipid level and / or helps control body fat.

[0013] In the seventh aspect of the present invention, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises at least one of Pediococcus pentosaceus VB341 described in the first aspect, the fermentation broth described in the second aspect, and the bacterial suspension described in the third aspect. The composition of the present invention can be used to inhibit lipase activity, reduce blood lipid levels, and / or lose weight. Further, it can prevent, alleviate, and / or treat diseases caused by hyperlipidemia and is not prone to generating resistance.

[0014] In the eighth aspect of the present invention, 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 Pediococcus pentosaceus VB341 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 described above, Pediococcus pentosaceus VB341 of the present invention can effectively inhibit lipase activity.

[0015] In the ninth aspect of the present invention, the present invention provides a method for losing weight. According to an embodiment of the present invention, the method comprises: administering to a subject Pediococcus pentosaceus VB341 described in the first aspect, the fermentation broth described in the second aspect, or the bacterial suspension described in the third aspect. As described above, Pediococcus pentosaceus VB341 of the present invention can effectively inhibit lipase activity, such that excessive fat cannot be hydrolyzed into small molecules absorbable by the body in the intestine, thereby achieving the effect of weight loss.

[0016] In the tenth aspect of the present invention, the present invention provides a culture device or a production device for Pediococcus pentosaceus VB341 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 culture device or production device according to the embodiment of the present invention can mass-produce Pediococcus pentosaceus VB341, the fermentation broth, the bacterial suspension, or the composition of the present invention.

[0017] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. Description of the Drawings

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without creative efforts.

[0019] Figure 1 It is a detection result diagram of the choline tolerance ability of Pediococcus pentosaceus VB341 according to Example 3 of the present invention;

[0020] Figure 2 Detection result graph of the tolerance ability of Pediococcus pentosaceus VB341 to artificial simulated gastric juice according to Embodiment 3 of the present invention;

[0021] Figure 3 Detection result graph of the tolerance ability of Pediococcus pentosaceus VB341 to artificial simulated intestinal juice according to Embodiment 3 of the present invention;

[0022] Figure 4 Detection result graph of the cholesterol content in the plasma of mice in the Control group, Model group, bacterial liquid group, and drug (Xuezhikang) group according to Embodiment 4 of the present invention;

[0023] Figure 5 Detection result graph of the triglyceride content in the plasma of mice in the Control group, Model group, bacterial liquid group, and drug (Xuezhikang) group according to Embodiment 4 of the present invention. Detailed implementation manners

[0024] The embodiments of the present invention are described in detail below. The examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to explain the present invention, but should not be construed as limiting the present invention.

[0025] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meanings of "a plurality" and "a variety" are at least two, two kinds, for example, two, two kinds, three, three kinds, etc., unless otherwise clearly and specifically defined.

[0026] In this article, the term "comprising", "having" or "including" is an open expression, that is, it includes the content specified by the present invention, but does not exclude other aspects of the content.

[0027] In this article, the term "optionally" generally means that the subsequent event or condition may or may not occur, and this description includes the case where the event or condition occurs, and the case where the event or condition does not occur.

[0028] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0029] To facilitate a better understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein shall have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0030] As used herein, the terms "treat" and "alleviate" both refer to achieving a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein encompasses diseases in mammals, particularly humans, including: (a) preventing the occurrence of a disease or disorder in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as reducing the symptoms associated with the disease. "Treatment" as used herein encompasses any administration of a strain, fermentation broth, bacterial suspension, or composition to an individual to treat, cure, alleviate, improve, reduce, or inhibit the disease of the individual, including but not limited to administering the strain, fermentation broth, bacterial suspension, or composition described herein to an individual in need thereof.

[0031] As used herein, the term "carrier" includes any solvent, pharmaceutical stabilizer, or a composition thereof, which are known to those skilled in the art. Its use in a therapeutic or pharmaceutical composition is encompassed, except in cases where any conventional carrier is incompatible with the active ingredient.

[0032] Strain

[0033] In some embodiments, the present invention provides a Pediococcus pentosaceus VB341, which was deposited at the China Center for Type Culture Collection on March 8, 2024, with the deposit number CCTCC NO: M 2024433.

[0034] According to some specific embodiments of the present invention, the above-mentioned Pediococcus pentosaceus VB341 may further include at least one of the following additional technical features:

[0035] According to some specific embodiments of the present invention, the Pediococcus pentosaceus VB341 has a 16S rRNA sequence as shown in SEQ ID NO:1.

[0036] The newly obtained Pediococcus pentosaceus VB341 strain screened by the present invention has strong stability in intestinal fluid and bile salts, and can secrete lipase inhibitors, which reduce or inhibit the hydrolysis activity of lipase on fat, so that excessive fat cannot be hydrolyzed into small molecules that can be absorbed by the body in the intestine, helping to maintain a healthy level of blood lipids (cholesterol and / or triglycerides), and achieving the effect of reducing blood lipids and / or weight loss. Further, it can prevent, relieve and / or treat diseases caused by hyperlipidemia, such as metabolic syndrome, and is not prone to generating resistance, with high application value.

[0037] In this article, the term "Pediococcus pentosaceus" (Pediococcus pentosaceus) "VB341", "Pediococcus pentosaceus VB341", "VB341 strain" and "VB341" are synonymous.

[0038] Lipase (Lipase, glycerol ester hydrolase) belongs to the class of carboxyl ester hydrolases and can gradually hydrolyze triglycerides into glycerol and fatty acids. In addition, lipase has a variety of catalytic abilities. For example, it can catalyze the hydrolysis, alcoholysis, esterification, transesterification and reverse synthesis reaction of triacylglycerol esters and some other water-insoluble esters, and also exhibits the activities of some other enzymes, such as phospholipase, lysophospholipase, cholesterol esterase, acylpeptide hydrolase activity, etc. The exertion of different activities of lipase depends on the characteristics of the reaction system. For example, it promotes ester hydrolysis at the oil-water interface, while in the organic phase, it can carry out enzymatic synthesis and transesterification. In this article, Pediococcus pentosaceus VB341 can produce lipase inhibitors that inhibit lipase activity, and the lipase activity includes but is not limited to the hydrolysis activity of lipase. The lipase described in this article includes any natural or synthetic lipase, such as animal lipase, plant lipase and microbial lipase, etc.

[0039] Blood lipids are the general term for neutral fats (triglycerides) and lipids (phospholipids, glycolipids, sterols, steroids) in plasma, which are widely present in animals. They are essential substances for the basic metabolism of living cells. The main components of blood lipids are triglycerides and cholesterol, among which triglycerides participate in energy metabolism in the human body, while cholesterol is mainly used for synthesizing cell plasma membranes, steroid hormones and bile acids. In addition, according to the specific embodiments of the present invention, Pediococcus pentosaceus VB341 in this article can effectively reduce the blood lipid (cholesterol and / or triglyceride) level in the plasma of mice.

[0040] Fermentation broth

[0041] In some embodiments, the present invention provides a fermentation broth, which is fermented from the aforementioned Pediococcus pentosaceus VB341. The fermentation broth of the present invention can be used to inhibit fat hydrolysis, reduce blood lipids and / or lose weight. Further, it can prevent, relieve and / or treat diseases caused by hyperlipidemia, and is not prone to generating resistance.

[0042] It should be noted that the "fermentation broth" of the present invention refers to a solution obtained by culturing Pediococcus pentosaceus VB341 for a period of time, which mainly contains Pediococcus pentosaceus VB341 and / or its metabolites; or the supernatant obtained by further processing through means such as centrifugation and filtration, which mainly contains the metabolites of Pediococcus pentosaceus VB341.

[0043] Bacterial suspension

[0044] In some embodiments, the present invention provides a bacterial suspension, which includes the aforementioned Pediococcus pentosaceus VB341. The bacterial suspension of the present invention can be used to inhibit lipolysis, reduce blood lipid and / or lose weight. Further, it can prevent, alleviate and / or treat diseases caused by hyperlipidemia and is not prone to produce resistance.

[0045] It should be noted that the bacterial suspension can be obtained by processing the above-mentioned fermentation broth through means such as centrifugation and resuspension.

[0046] Use

[0047] In some embodiments, the present invention provides the use of the aforementioned Pediococcus pentosaceus VB341, fermentation broth or bacterial suspension in the preparation of foods, beverages, drugs, health products, feeds or additives that help maintain a healthy level of blood lipids (cholesterol and / or triglyceride) and / or help control body fat. The foods, beverages, health products, feeds or additives of the present invention can be used to maintain a healthy level of blood lipids and / or help control body fat.

[0048] According to the embodiments of the present invention, the above uses may further include at least one of the following additional technical features:

[0049] According to the embodiments of the present invention, the blood lipids include at least one of cholesterol and triglyceride.

[0050] In some embodiments, the present invention provides the use of the aforementioned Pediococcus pentosaceus VB341, fermentation broth or bacterial suspension in the preparation of foods, beverages, drugs, health products, feeds or additives that inhibit lipase activity, reduce blood lipid levels and / or lose weight. The foods, beverages, health products, feeds or additives of the present invention can be used to inhibit lipase activity, and the drugs can be used to inhibit lipase activity, reduce blood lipid levels and / or lose weight. Further, it can prevent, alleviate and / or treat diseases caused by hyperlipidemia and is not prone to produce resistance.

[0051] According to the embodiments of the present invention, the above uses may further include at least one of the following additional technical features:

[0052] According to the embodiments of the present invention, the blood lipids include at least one of cholesterol and triglyceride.

[0053] Food, beverage, medicine, health product, feed and / or additive

[0054] The present invention provides a food, beverage, medicine, health product, feed and / or additive, which comprises at least one of the foregoing Pediococcus pentosaceus VB341, fermentation broth or bacterial suspension. The food, beverage, health product, feed and / or additive of the present invention can be used to maintain a healthy blood lipid level and / or help control body fat. The medicine of the present invention can be used to inhibit lipase activity, reduce blood lipid level and / or lose weight. Further, it can prevent, relieve and / or treat diseases caused by hyperlipidemia and is not likely to produce resistance.

[0055] According to an embodiment of the present invention, the above-mentioned food, beverage, medicine, health product, feed or additive may further comprise at least one of the following additional technical features:

[0056] According to an embodiment of the present invention, it further comprises a pharmaceutically acceptable excipient or carrier, an excipient or carrier acceptable in food or health products, or an excipient or carrier acceptable in animal feed.

[0057] As used herein, "acceptable in food" refers to a substance or composition that can be consumed by humans and can be adjusted according to food requirements in different countries.

[0058] As used herein, "acceptable in health products" refers to a substance or composition that can be consumed by humans and can be adjusted according to health product requirements in different countries.

[0059] As used herein, "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammal being treated therewith. Preferably, "pharmaceutically acceptable" as used in the present invention refers to those approved by federal regulatory agencies or national governments or listed in the United States Pharmacopeia or other generally recognized pharmacopeias for use in animals, particularly in humans.

[0060] As used herein, "excipient or carrier acceptable in animal feed" refers to a substance or composition that can be consumed by animals and can be adjusted according to animal feed requirements in different countries.

[0061] As used herein, the term "pharmaceutically acceptable carrier" includes any solvent, drug stabilizer, or composition 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.

[0062] In this text, the term "pharmaceutically acceptable excipient" can include any solvent suitable for a specific target dosage form. Except for the scope where any conventional excipient is incompatible with Pediococcus pentosaceus VB341 disclosed in the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their uses are also within the scope contemplated by this disclosure.

[0063] According to an embodiment of the present invention, at least one of the aforementioned Pediococcus pentosaceus VB341, fermentation broth, and bacterial suspension is added or inoculated into food, beverage, health product, feed, and / or additive, or added to a drug, whereby food, beverage, health product, feed, and / or additive capable of maintaining a healthy blood lipid level and / or contributing to controlling body fat, or a drug capable of inhibiting lipase activity, reducing blood lipid level and / or losing weight, and capable of preventing, alleviating, and / or treating diseases caused by hyperlipidemia can be further obtained. Among them, the diseases caused by hyperlipidemia include metabolic syndrome.

[0064] Exemplarily, the aforementioned food includes but is 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.

[0065] Exemplarily, the aforementioned drugs include but are not limited to: human drugs, veterinary drugs. The aforementioned veterinary use can be for pets, livestock, and wild animals.

[0066] Exemplarily, the aforementioned health products include but are not limited to: human health products, veterinary health products.

[0067] Exemplarily, the aforementioned additives include but are not limited to: food additives, feed additives.

[0068] It should be noted that the features and advantages described above for Pediococcus pentosaceus VB341 also apply to the food, beverage, drug, health product, feed, or additive, and will not be elaborated here.

[0069] Composition

[0070] In some embodiments, the present invention provides a composition, which includes at least one of the aforementioned Pediococcus pentosaceus VB341, fermentation broth, and bacterial suspension. The composition of the present invention can be used to inhibit lipase activity, reduce blood lipid, and / or lose weight. Further, it can prevent, alleviate, and / or treat diseases caused by hyperlipidemia and is not prone to generating resistance.

[0071] According to an embodiment of the present invention, the above composition may further include at least one of the following additional technical features:

[0072] According to an embodiment of the present invention, the composition further comprises an ingestible excipient and / or carrier.

[0073] According to an embodiment of the present invention, the excipient comprises at least one selected from the group consisting of binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, and sustained-release agents.

[0074] According to an embodiment of the present invention, the carrier comprises at least one selected from the group consisting of saccharides, calcium phosphates, alkaline earth metal salts of stearic acid, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols, and hydrolyzed cereal solids.

[0075] According to an embodiment of the present invention, the saccharides comprise cellulose and its derivatives.

[0076] According to an embodiment of the present invention, the dosage form of the composition comprises at least one selected from the group consisting of oral liquids, powders, granules, capsules, tablets, and dripping pills.

[0077] Method

[0078] In some embodiments, the present invention provides a method for inhibiting lipase activity in vitro, the method comprising: co-culturing at least one of the aforementioned Pediococcus pentosaceus VB341, fermentation broth, and bacterial suspension with lipase. As described above, the Pediococcus pentosaceus VB341 of the present invention can effectively inhibit lipase activity, such that excessive fat cannot be hydrolyzed into small molecules absorbable by the body. This method can be used for non-diagnostic and / or non-therapeutic purposes, such as for scientific research and in vitro experiments related to lipase activity.

[0079] In some embodiments, the present invention provides a weight loss method, the method comprising: administering to a subject at least one of the aforementioned Pediococcus pentosaceus VB341, fermentation broth, and bacterial suspension. As described above, the Pediococcus pentosaceus VB341 of the present invention can effectively inhibit the activity of lipase, such that excessive fat cannot be hydrolyzed into small molecules absorbable by the body in the intestine, thereby achieving the effect of weight loss.

[0080] Device

[0081] In some embodiments, the present invention provides a culture device or production device for the aforementioned Pediococcus pentosaceus VB341, fermentation broth, bacterial suspension, or composition. The culture device or production device according to the embodiment of the present invention can achieve mass production of the Pediococcus pentosaceus VB341, fermentation broth, bacterial suspension, or composition of the present invention.

[0082] The solution of the present invention will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those without specific techniques or conditions noted in the embodiments, the techniques or conditions described in the literature in the art or according to the product specifications are followed. Those reagents or instruments without the manufacturer noted are all conventional products that can be obtained through commercial purchase.

[0083] Example 1 Isolation and Identification of Strains

[0084] The strains were collected and isolated from a self-made pickled vegetables in a jar from a rural household in Harbin. Different gradient dilution bacterial suspensions were prepared by gradient dilution with a sterile normal saline solution. 100 μL of each different gradient dilution was taken and spread on MRS agar plates, anaerobically cultured at 37 °C for 48 h. The suspected lactic acid bacteria colonies were picked and purified by streaking, and then DNA was extracted. After PCR amplification with specific primers, 16S rDNA was sequenced, and at the same time, the corresponding strains were stored in glycerol tubes. According to the 16S rDNA identification results, 9 strains of Pediococcus were selected, numbered B01 - B09, among which 6 strains were Pediococcus acidilactici and 3 strains were Pediococcus pentosaceus.

[0085] Example 2 Strain Screening

[0086] Preparation of lipase (1) Prepare a 0.02 mol / L disodium hydrogen phosphate buffer solution (pH = 9.3);

[0087] (2) Prepare an 8% lipase solution with the above disodium hydrogen phosphate buffer solution, dissolve it and put it into a grinding tube, grind 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 frozen at -80 °C in the refrigerator;

[0088] (3) Dilute the above 8% lipase solution to a 1% concentration with 0.02 mol / L disodium hydrogen phosphate with pH = 10.3 for standby.

[0089] 2.2 Preparation of fat emulsion

[0090] (1) Preparation of 100 mmol / L tris-HCl buffer solution (pH = 8.0): Weigh 1.21 g of tris reagent and dissolve it in 100 mL of sterile water. After dissolution, adjust the pH to 8.0 with dilute hydrochloric acid;

[0091] (2) Take 0.3 mL of the emulsion stock solution, add 29.7 mL of the above 100 mmol / L tris-HCl buffer solution (pH = 8.0), mix well to obtain an emulsion diluted 100 times. Mix the emulsion diluted 100 times and the 100 mmol / L tris-HCl buffer solution (pH = 8.0) evenly in a 1:1 ratio to obtain an emulsion diluted 200 times.

[0092] 2.3 Preparation of Orlistat at Different Concentrations

[0093] (1) Preparation of 100 μg / mL Orlistat Stock Solution: Weigh 4 mg of Orlistat standard and dissolve it in 40 mL of disodium hydrogen phosphate solution with pH = 10.3, and ultrasonicate until completely dissolved.

[0094] (2) Gradually dilute the 100 μg / mL Orlistat stock solution 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 solution with pH = 10.3.

[0095] The sources of the experimental reagents used in this example are shown in Table 1.

[0096] Table 1. Experimental Materials

[0097]

[0098] 2.4 Inhibition Experiment of Orlistat on Lipase

[0099] (1) Use a 96-well plate to conduct the inhibition experiment of Orlistat and lipase. The experiment is divided into 3 groups in total;

[0100] ① Blank control group: 180 μL of emulsion diluted 200 times + 10 μL of tris-Hcl buffer solution with pH = 8.0 + 10 μL of disodium hydrogen phosphate buffer solution with pH = 10.3;

[0101] ② Enzyme-added negative control group: 180 μL of emulsion diluted 200 times + 10 μL of 1% lipase solution + 10 μL of disodium hydrogen phosphate buffer solution with pH = 10.3;

[0102] ③ Orlistat positive control group: 180 μL of emulsion diluted 200 times + 10 μL of 1% lipase solution + 10 μL of Orlistat standard solution at different concentrations;

[0103] Add the respective reagent solutions to the 96-well plate according to the above groups. For the third group, first add the enzyme solution and Orlistat solution and react for 15 minutes, and then add the emulsion. The emulsions of each group are added at the same time point to ensure the same enzyme reaction time. Detect the OD value of the emulsion at 490 nm by a microplate reader at 0 h, 45 minutes, and 2 h of the reaction, and calculate the hydrolysis effect of the enzyme on the emulsion through the change of the OD value of the emulsion.

[0104] (2) Calculation methods for lipase hydrolysis rate and Orlistat inhibition rate on lipase:

[0105] Lipase hydrolysis rate % = (OD of blank control group - OD of enzyme solution negative control group) / OD of blank control group * 100%

[0106] Inhibitory rate of orlistat on 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%.

[0107] 2.5 Screening of lipase inhibitor strains

[0108] (1) MRS ready-made medium: Weigh 54 g of the ready-made medium, add 1 L of pure water, stir and dissolve, then dispense into shake flasks and sterilize at 121 °C for 15 min.

[0109] (2) Strain culture

[0110] ① Source of bacteria: Glycerol tubes of different strains described in Example 1 stored at -80 °C.

[0111] ② Strain inoculation method: The method of directly inoculating the glycerol tube into the liquid medium is used for culturing the bacterial solution, and the inoculation amount ranges from 0.2% to 1%.

[0112] ③ Liquid culture method: According to the growth characteristics of the strain, place it in an anaerobic workstation and culture at 37 °C for 18 - 24 h.

[0113] (3) Sample preparation

[0114] When detecting lipase inhibitors, according to different treatment methods, the samples are divided into two types: supernatant and whole liquid:

[0115] Supernatant: Take 0.5 mL of the above-mentioned bacterial solution, centrifuge at 14,000 rpm for 10 minutes, and take the supernatant for testing;

[0116] Whole liquid: Take 0.5 mL of the above-mentioned bacterial solution, add it to a grinding tube, grind at 4 °C and 60 Hz for 2 minutes each time, grind a total of 8 times, and then centrifuge at 14,000 rpm for 10 minutes, and take the supernatant for testing;

[0117] (4) Sample detection

[0118] Mix the prepared supernatant and whole liquid samples with lipase respectively for a 15-minute reaction, then add the emulsion, measure the OD value of the emulsion within a certain time, and judge the inhibitory effect of the sample on the enzyme through the change value of the emulsion (OD difference). The specific grouping is as follows:

[0119] 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 ready-made medium.

[0120] Sample detection group 1: 180 μL of emulsion diluted 200 times + 10 μL of 1% lipase solution + 10 μL of sample supernatant. Sample detection group 2: 180 μL of emulsion diluted 200 times + 10 μL of 1% lipase solution + 10 μL of sample whole liquid. (5) Calculation formula for the inhibition rate of the sample on the enzyme

[0121] 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%

[0122] Lipase hydrolysis rate % = (OD of blank control group - OD of enzyme solution negative control group) / OD of blank control group * 100%

[0123] Sample inhibition rate % = (lipase hydrolysis rate - sample hydrolysis rate) / lipase hydrolysis rate * 100%

[0124] (6) Screening results

[0125] The experimental results are shown in Table 2 and Table 3. Using this screening model, a total of 9 strains (B01 - B09) of different species were screened for their lipase inhibition ability. Only Pediococcus pentosaceus B08 had an inhibitory effect on the activity of lipase. The in vitro lipase inhibition ability of this strain was approximately equivalent to that of orlistat at a concentration of 5 μg / mL. That is, this strain has the ability to produce lipase inhibitors, can inhibit the activity of lipase, and can be used to prepare products such as weight loss drugs, health foods, or solid beverages.

[0126] Table 2 Inhibition rate of different concentrations of orlistat on 1% lipase

[0127]

[0128] Table 3 Inhibition rate of active strains on lipase

[0129]

[0130] 2.6 Strain identification

[0131] The Pediococcus pentosaceus B08 screened in part 2.5 was sequenced, and the 16S rRNA sequence of the molecular biological identification of the strain was compared with the strains in GenBank, and it was identified as Pediococcus pentosaceus (Pediococcus pentosaceus) , named Pediococcus pentosaceus (Pediococcus pentosaceus) VB341, was deposited in the China Center for Type Culture Collection on March 8, 2024. The deposit address is Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit number is CCTCC NO: M 2024433, and its 16S rRNA sequence is shown in SEQ ID NO:1

[0132]

[0133] Example 3 Stability of Pediococcus pentosaceus Strain VB341

[0134] 3.1 Preparation of experimental materials

[0135] The experimental materials used in this example are shown in Table 4 as follows:

[0136] Table 4 Experimental materials

[0137]

[0138] (1)Preparation of culture medium

[0139] MRS culture medium: MRS broth 5.4% + 1.8% agar, pH = 6.5.

[0140] (2)Preparation of simulated gastric juice: Normal saline (sodium chloride: 0.9%), first prepare a 1% concentration of sodium chloride solution, adjust different pH values and then aliquot, sterilize at 121 °C for 30 min, pepsin 0.3% (prepare a 3% concentration, filter with a sterile membrane), according to 3% pepsin: 1% normal saline = 1:9, mix well.

[0141] (3)Preparation of choline solution: Normal saline (sodium chloride: 0.9%), first prepare a 1% concentration of sodium chloride solution, add bovine choline at concentrations of 0%, 0.03%, 0.06%, 0.1%, 0.2%, and 0.3% respectively, sterilize at 121 °C for 30 min, trypsin 0.1% (prepare a 1% concentration, filter with a sterile membrane), according to 1% trypsin: 1% normal saline of each choline concentration = 1:9, mix well.

[0142] (4)Preparation of intestinal fluid: Artificial simulated intestinal fluid is prepared according to the product instruction manual, and after preparation, the pH of the intestinal fluid is 8.0.

[0143] 3.2 Experimental method

[0144] Take a cryotube of Pediococcus pentosaceus strain VB341, spread the original solution on a blank petri dish, anaerobically culture in a 37 °C incubator for 1 day according to the growth characteristics, scrape the bacterial lawn, prepare a 20% glycerol tube and store it at -80 °C for later use. At the same time, dilute and count, with the viable cell count preferably at 10 9 ~10 10 CFU / mL.

[0145] Take the counted bacterial suspension, thaw it and add pure water (as the control group) and the above-mentioned separately packed simulated choline solutions, gastric juice and intestinal juice. The addition amount is: 10 μL bacterial suspension + 990 μL pure water / simulated solution. After mixing, count the viable bacteria of the 0 h group immediately at 0 hour. Put the bacterial suspensions of the 2 h group and the 4 h group into an anaerobic bag and place them in an incubator at 37 °C. Take them out and count the viable bacteria at 2 h and 4 h of incubation respectively.

[0146] Figure 1 It shows that the survival rate of Pediococcus pentosaceus VB341 is as high as over 95% within 4 h in choline solutions with a concentration of 0.03% - 0.3%, indicating that Pediococcus pentosaceus VB341 can stably exist in pancreatic juice, that is, this strain can adapt to the intestinal environment. Figure 2 It shows that Pediococcus pentosaceus VB341 dies immediately in gastric juice with pH = 1 and pH = 2, the immediate mortality rate in gastric juice with pH = 3 is 98%, 99.8% die after 2 h, and all die after 4 h. In gastric juice with pH = 4, it is basically stable after 2 h and about 50% die after 4 h. Therefore, in the subsequent dosage form development process, the instability of Pediococcus pentosaceus VB341 in gastric juice needs to be considered. Figure 3 It shows that after being treated in intestinal juice with pH 8.0 for 4 h, the survival rate of Pediococcus pentosaceus VB341 is 100%, that is, this strain can stably exist in intestinal juice.

[0147] Example 4 In vivo efficacy study of Pediococcus pentosaceus VB341

[0148] 4.1 Experimental design

[0149] The experimental drug administration design is shown in Table 5: A total of 4 groups are set, namely the Control group, the Model group, the bacterial liquid group, and the drug (Xuezhikang) group. The number of mice in each group is 10. The drinking water for all mice in each group is distilled water. The mice in the Control group are fed with normal feed (purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., 1010009), and the other three groups are fed with high-fat feed (purchased from Changzhou Mouse One Mouse Two Biotechnology Co., Ltd., PD6001, 60% kcal). After feeding for 17 weeks, drug administration is carried out. Among them, the mice in the Control group and the Model group are intragastrically administered 10 mL / kg body weight of distilled water; the mice in the bacterial liquid group are intragastrically administered 1.0×10 10 CFU of Pediococcus pentosaceus VB341 bacterial liquid per mouse; the drug (Xuezhikang) group is intragastrically administered 200 mg / kg body weight of Xuezhikang suspension. Each group intragastrically administers all mice once within the time range of 8:00 - 10:00 every day for 14 consecutive weeks.

[0150] Table 5 Animal experiment drug administration design

[0151]

[0152] 4.2 Experimental materials

[0153] (1) Animals: Forty 6 - 8 - week - old healthy male C57BL / 6 mice were selected. Among them, 10 mice were fed with normal diet, and the remaining 30 mice were fed with high - fat diet. After 17 weeks of feeding, these 30 mice were evenly divided into 3 groups according to body weight, with 10 mice in each group.

[0154] (2) Bacterial suspension: The activated Pediococcus pentosaceus VB341 was inoculated into MRS medium and cultured at 37 °C for 24 h. The fermentation broth was centrifuged at 9000 rpm for 10 min. After discarding the supernatant, the bacterial residue was added with protective agents (60 mg of filler maltodextrin, 15 mg of suspending agent sodium hyaluronate, 20 mg of stabilizer sodium glutamate), and mixed well by pipetting and shaking. The viable count was adjusted to about 1.0×10 10 CFU / vial, and lyophilized according to the conditions in Table 6.

[0155] Table 6 Freeze - drying process parameters

[0156]

[0157] 4.3 Experimental detection

[0158] After continuous intragastric administration for 14 weeks, the mice in each group were fasted but allowed to drink water the day before. After anesthesia by inhaling isoflurane, orbital blood collection was performed on all experimental animals, and the blood was centrifuged at 4 °C and 3500 rpm for 15 min to obtain plasma. The cholesterol (TC) and triglyceride (TG) contents in the plasma were detected according to the kit instructions (Nanjing Jiancheng Bioengineering Institute).

[0159] 4.4 Experimental results

[0160] The results are shown in Table 7 and Figure 4 as follows. The cholesterol content in the plasma of the Model group mice was significantly higher than that of the Control group mice ( p < 0.0001), being 11.98 ± 2.31 and 5.6 ± 0.92 mM / L respectively, indicating that a hypercholesterolemia model in mice was successfully established by feeding with high - fat diet for 31 weeks; While continuously intragastrically administering the bacterial suspension and the positive control drug - Xuezhikang capsule for 14 weeks simultaneously during high - fat feeding, at the end of the experiment, the cholesterol contents in the plasma of the two groups of mice decreased to 9.38 ± 0.87 and 8.44 ± 3.97 mM / L respectively. Compared with the Model group, both were significantly decreased ( p < 0.05); Table 7 and Figure 5It was shown that after 28 weeks of high-fat diet feeding, the triglyceride content in the plasma of mice in the Model group was slightly higher than that in the Control group, being 0.6±0.13 and 0.75±0.09 mM / L respectively. The triglyceride concentrations in the mice in the bacterial liquid group and the positive control group (i.e., the drug (Xuezhikang) group) were decreased compared with the Model group, and the effects of the two groups were comparable. In summary, Pediococcus pentosaceus VB341 has the effect of reducing plasma cholesterol in hypercholesterolemia model mice established by long-term feeding with high-fat feed, and the effect is comparable to that of the positive control Xuezhikang capsule.

[0161] Table 7 Concentrations of cholesterol and triglyceride in mouse plasma (mM / L)

[0162]

[0163] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "embodiment" or "specific embodiment" means that the specific features, structures, materials or characteristics described in connection with the embodiment are included in at least one embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments in a suitable manner. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments and the features of different embodiments described in this specification.

[0164] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A Pediococcus pentosaceus (Pediococcus pentosaceus) VB341, characterized by: The Pediococcus pentosaceus VB341 was deposited in the China Center for Type Culture Collection on March 8, 2024, with a deposit number of CCTCC NO: M2024433.

2. A fermentation broth, characterized in that: The fermentation broth is obtained by fermenting the Pediococcus pentosaceus VB341 according to claim 1.

3. A bacterial suspension, characterized in that: The bacterial suspension contains the Pediococcus pentosaceus VB341 according to claim 1.

4. Use of the Pediococcus pentosaceus VB341 according to claim 1, the fermentation broth according to claim 2 or the bacterial suspension according to claim 3 in the preparation of food, feed or additives that help maintain healthy blood lipid levels and / or help control body fat.

5. The use according to claim 4, characterized in that The food includes beverages or health products.

6. Use of the Pediococcus pentosaceus VB341 according to claim 1, the fermentation broth according to claim 2 or the bacterial suspension according to claim 3 in the preparation of a medicine for inhibiting lipase activity, lowering blood lipid levels and / or reducing weight.

7. A composition, characterized in that The composition comprises at least one of the Pediococcus pentosaceus VB341 according to claim 1, the fermentation broth according to claim 2, and the bacterial suspension according to 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 liquid, powder, granule, capsule, tablet, and pill.

9. The composition according to claim 7, characterized in that The composition further comprises an excipient and / or a carrier.

10. The composition according to claim 9, characterized in that The excipient comprises at least one selected from a binder, a disintegrant, a lubricant, a glidant, a stabilizer, a filler, a diluent and a sustained-release agent; and / or The carrier comprises at least one selected from sugars, calcium phosphates, stearic acid alkaline earth metal salts, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols and cereal hydrolyzed solids.

11. A method for inhibiting lipase activity in vitro, characterized in that: include: At least one of the Pediococcus pentosaceus VB341 according to claim 1, the fermentation broth according to claim 2, and the bacterial suspension according to claim 3 is mixed with the lipase.

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