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

Abstract

The invention relates to the field of microorganisms, and provides a pediococcus pentosaceus VB341 with a lipid lowering effect, a culture device and application of the pediococcus pentosaceus VB341, the pediococcus pentosaceus VB341 is preserved in the China Center for Type Culture Collection on March 8, 2024, and the preservation number is CCTCC NO: M 2024433. The pediococcus pentosaceus VB341 disclosed by the invention has relatively strong ability of inhibiting lipase activity, has strong stability in intestinal juice and cholate, can be used for inhibiting fat hydrolysis, reducing the blood fat level, contributing to maintaining the healthy level of blood fat, contributing to controlling in-vivo fat and / or reducing weight, and is not easy to generate resistance.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a strain of Pediococcus pentosaceus VB341 with lipid-lowering effect, a culture device and an application thereof. Background Art

[0002] Fermented foods have always been a valuable source for probiotic development. Food-derived lactic acid bacteria not only offer high safety but also exhibit superior properties compared to other sample sources. Fermented foods have a long tradition in my country. Vegetables, meat, and dairy products found in daily human diets can be fermented with lactic acid bacteria to produce foods with unique flavors and probiotic properties. Although a variety of microorganisms participate in the food fermentation process, studies have shown that lactic acid bacteria play a primary role. Lactic acid bacteria can provide health benefits to the host by producing functionally active factors and regulating the intestinal flora.

[0003] Currently, the main methods for lowering blood lipid levels are medication and dietary intervention. Statins and fibrates are the most commonly used drugs in clinical practice. While these drugs are initially effective, they can produce varying degrees of side effects with long-term use. Currently, a low-fat or low-saturated fat diet is recommended. However, the taste and flavor of low-fat foods are relatively monotonous, resulting in low acceptance of this approach. People are eager to develop other dietary supplements that can lower cholesterol and blood lipids.

[0004] As early as the 1970s, people discovered that people who regularly consumed fermented dairy products (lactate bacteria) had lower cholesterol levels. Pediococcus pentosaceus, a member of the genus Pediococcus in the Streptococcus family, has been playing an increasingly important role in the application of lactic acid bacteria in recent years. In addition to its lactic acid-producing properties, Pediococcus pentosaceus can also enhance 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 provides a Pediococcus pentosaceus (Pediococcus Pentosaceus VB341. According to an embodiment of the present invention, 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: M 2024433.

[0007] The Pediococcus pentosaceus VB341 screened and obtained by the present invention has strong stability in intestinal fluid and bile salts, can secrete lipase inhibitors, and the hydrolysis 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 effects of lowering blood lipid levels and reducing weight. Furthermore, it can prevent, alleviate and / or treat diseases caused by hyperlipidemia and is not prone to resistance.

[0008] In a second aspect, the present invention provides a fermentation broth. According to an embodiment of the present invention, the fermentation broth is fermented from the Pediococcus pentosaceus VB341 described in the first aspect. The fermentation broth of the present invention can be used to inhibit fat hydrolysis, lower blood lipids, and / or reduce weight. Furthermore, it can prevent, alleviate, and / or treat diseases caused by hyperlipidemia and is not susceptible to developing resistance.

[0009] In a third aspect, the present invention provides a bacterial suspension. According to an embodiment of the present invention, the bacterial suspension comprises Pediococcus pentosaceus VB341 as described in the first aspect. The bacterial suspension of the present invention can be used to inhibit fat hydrolysis, lower blood lipids, and / or reduce weight. Furthermore, it can prevent, alleviate, and / or treat diseases caused by hyperlipidemia and is not susceptible to developing resistance.

[0010] In a fourth aspect, the present invention provides use of the 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 preparing a food, beverage, health product, feed, or additive that helps maintain healthy blood lipid levels and / or helps control body fat. The food, beverage, health product, feed, or additive of the present invention helps maintain healthy blood lipid levels and / or helps control body fat.

[0011] In a fifth aspect, the present invention provides the use of the 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 a food, beverage, medicine, health product, feed, or additive for inhibiting lipase activity, lowering blood lipid levels, and / or reducing weight. The food, beverage, health product, feed, or additive of the present invention can be used to inhibit lipase activity, and the medicine can be used to inhibit lipase activity, lower blood lipid levels, and / or reduce weight. Furthermore, the food, beverage, health product, feed, or additive of the present invention can prevent, alleviate, and / or treat diseases caused by hyperlipidemia and is not susceptible to the development of resistance.

[0012] In a sixth aspect, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises at least one of the 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 healthy blood lipid levels and / or helps control body fat.

[0013] In its seventh aspect, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises at least one of the 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, lower blood lipid levels, and / or reduce weight. Furthermore, it can prevent, alleviate, and / or treat diseases caused by hyperlipidemia and is less likely to develop resistance.

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

[0015] In its ninth aspect, the present invention provides a method for weight loss. According to an embodiment of the present invention, the method comprises administering to a subject the 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 previously mentioned, the Pediococcus pentosaceus VB341 of the present invention can effectively inhibit lipase activity, preventing excess fat from being hydrolyzed into absorbable small molecules in the intestines, thereby achieving a weight loss effect.

[0016] In a tenth aspect, the present invention provides a culture device or production device for the 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 an embodiment of the present invention is capable of mass-producing the Pediococcus pentosaceus VB341, fermentation broth, bacterial suspension, or composition described in the present invention.

[0017] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 This is a graph showing the test results of the choline tolerance of Pediococcus pentosaceus VB341 according to Example 3 of the present invention; Figure 2 This is a graph showing the test results of the ability of Pediococcus pentosaceus VB341 to tolerate artificial simulated gastric fluid according to Example 3 of the present invention; Figure 3 This is a graph showing the test results of the tolerance of Pediococcus pentosaceus VB341 to artificial simulated intestinal fluid according to Example 3 of the present invention; Figure 4 This is a graph showing the test results of cholesterol levels in the plasma of mice in the Control group, Model group, bacterial solution group, and drug (Xuezhikang) group according to Example 4 of the present invention; Figure 5 2 is a graph showing the detection results of triglyceride content in the plasma of mice in the Control group, Model group, bacterial solution group, and drug (Xuezhikang) group according to Example 4 of the present invention. DETAILED DESCRIPTION

[0020] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0021] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Thus, a feature specified as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "plurality" or "multiple" means at least two or two, for example, two, two, three, three, etc., unless otherwise specifically defined.

[0022] In this document, the terms “include”, “have” or “include” are open expressions, that is, they include the contents specified in the present invention but do not exclude other aspects.

[0023] As used herein, the term "optionally" generally means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0024] The endpoints of the ranges and any values ​​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 endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0025] In order to make the present invention more easily understood, 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 have the meaning commonly understood by those skilled in the art to which the present invention belongs.

[0026] As used herein, the terms "treat" and "alleviate" refer to agents used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have 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 alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a strain, fermentation broth, bacterial suspension or composition to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a strain, fermentation broth, bacterial suspension or composition described herein to an individual in need.

[0027] As used herein, the term "carrier" includes any solvent, pharmaceutical stabilizer, or combination thereof, which are known to those skilled in the art. Except where any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is encompassed.

[0028] strain In some embodiments, the present invention provides Pediococcus pentosaceus VB341, which was deposited in the China Center for Type Culture Collection on March 8, 2024, with a deposit number of CCTCC NO: M 2024433.

[0029] 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: 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.

[0030] The new strain of Pediococcus pentosaceus VB341 screened by the present invention has strong stability in intestinal fluid and bile salts, can secrete lipase inhibitors, and the hydrolysis 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, which helps to maintain healthy blood lipid (cholesterol and / or triglycerides) levels and achieve the effect of lowering blood lipids and / or reducing weight. Furthermore, it can prevent, alleviate and / or treat diseases caused by hyperlipidemia, such as metabolic syndrome, and is not easy to develop resistance, so it has high application value.

[0031] In this context, the term "Pediococcus pentosaceus" (Pediococcus pentosaceus) VB341", "Pediococcus pentosaceus VB341", "VB341 strain" and "VB341" are synonymous.

[0032] Lipases (glyceride hydrolases) belong to the class of carboxyl ester hydrolases and can gradually hydrolyze triglycerides into glycerol and fatty acids. Furthermore, lipases possess multiple catalytic capabilities, such as the hydrolysis, alcoholysis, esterification, transesterification, and reverse synthesis of triacylglycerols and other water-insoluble esters. They also exhibit other enzymatic activities, such as phospholipases, lysophospholipases, cholesterol esterases, and acylpeptide hydrolases. The different activities of lipases depend on the characteristics of the reaction system, such as promoting ester hydrolysis at the oil-water interface and enzymatic synthesis and transesterification in the organic phase. Pediococcus pentosaceus VB341 is described herein as being capable of producing lipase inhibitors that inhibit lipase activity, including but not limited to lipase hydrolysis. The lipases described herein include any natural or synthetic lipase, such as animal, plant, and microbial lipases.

[0033] Blood lipids, a general term for neutral fats (triglycerides) and lipids (phospholipids, glycolipids, sterols, and steroids) in plasma, are widely present in animals. They are essential for the basic metabolism of living cells. The main components of blood lipids are triglycerides and cholesterol. Triglycerides are involved in energy metabolism in the human body, while cholesterol is primarily used to synthesize cell membranes, steroid hormones, and bile acids. Furthermore, according to specific embodiments of the present invention, Pediococcus pentosaceus VB341 can effectively reduce blood lipid (cholesterol and / or triglyceride) levels in mouse plasma.

[0034] fermentation broth In some embodiments, the present invention provides a fermentation broth derived from the aforementioned Pediococcus pentosaceus VB341. The fermentation broth can be used to inhibit fat hydrolysis, lower blood lipids, and / or reduce weight. Furthermore, it can prevent, alleviate, and / or treat diseases caused by hyperlipidemia, and is less likely to develop resistance.

[0035] It should be noted that the "fermentation broth" of the present invention refers to the solution obtained after culturing Pediococcus pentosaceus VB341 for a period of time, which solution mainly contains Pediococcus pentosaceus VB341 and / or its metabolites; or the supernatant after further treatment by centrifugation, filtration, etc., which supernatant mainly contains metabolites of Pediococcus pentosaceus VB341.

[0036] bacterial suspension In some embodiments, the present invention provides a bacterial suspension comprising the aforementioned Pediococcus pentosaceus VB341. The bacterial suspension of the present invention can be used to inhibit fat hydrolysis, lower blood lipids, and / or reduce weight. Furthermore, the suspension can prevent, alleviate, and / or treat diseases caused by hyperlipidemia and is less likely to develop resistance.

[0037] It should be noted that the bacterial suspension can be obtained by treating the above fermentation broth by means of centrifugation, resuspension, etc.

[0038] use In some embodiments, the present invention provides the use of the aforementioned Pediococcus pentosaceus VB341, fermentation broth, or bacterial suspension in the preparation of a food, beverage, pharmaceutical, health product, feed, or additive that helps maintain healthy blood lipid levels (cholesterol and / or triglyceride) and / or helps control body fat. The food, beverage, health product, feed, or additive of the present invention can be used to maintain healthy blood lipid levels and / or help control body fat.

[0039] According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features: According to an embodiment of the present invention, the blood lipid includes at least one of cholesterol and triglycerides.

[0040] In some embodiments, the present invention provides the use of the aforementioned Pediococcus pentosaceus VB341, fermentation broth, or bacterial suspension in the preparation of a food, beverage, pharmaceutical, health product, feed, or additive for inhibiting lipase activity, lowering blood lipid levels, and / or reducing weight. The food, beverage, health product, feed, or additive of the present invention can be used to inhibit lipase activity, and the pharmaceutical can be used to inhibit lipase activity, lower blood lipid levels, and / or reduce weight. Furthermore, the present invention can prevent, alleviate, and / or treat diseases caused by hyperlipidemia and is less likely to develop resistance.

[0041] According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features: According to an embodiment of the present invention, the blood lipid includes at least one of cholesterol and triglycerides.

[0042] Food, beverages, medicines, health products, feed and / or additives The present invention provides a food, beverage, medicine, health product, feed, and / or additive comprising at least one of the aforementioned 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 healthy blood lipid levels and / or help control body fat. The medicine of the present invention can be used to inhibit lipase activity, lower blood lipid levels, and / or reduce weight. Furthermore, the medicine can prevent, alleviate, and / or treat diseases caused by hyperlipidemia and is less likely to develop resistance.

[0043] According to an embodiment of the present invention, the above-mentioned food, beverage, medicine, health product, feed or additive may further include at least one of the following additional technical features: According to an embodiment of the present invention, it further comprises pharmaceutically acceptable excipients or carriers, excipients or carriers acceptable in food or health products, or excipients or carriers acceptable in animal feed.

[0044] As used herein, "acceptable in food" refers to substances or compositions that can be consumed by humans, which may be adjusted according to food requirements in different countries.

[0045] As used herein, "acceptable in health products" refers to substances or compositions that can be consumed by humans, which may be adjusted according to the health product requirements of different countries.

[0046] As used herein, "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated therewith. Preferably, "pharmaceutically acceptable" as used herein means approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0047] As used herein, "adjuvant or carrier acceptable in animal feed" refers to a substance or composition that can be consumed by animals, which may be adjusted according to the animal feed requirements of different countries.

[0048] As used herein, the term "pharmaceutically acceptable carrier" includes any solvent, pharmaceutical stabilizer, or combination thereof, which are known to those skilled in the art. Except where any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is encompassed.

[0049] As used herein, the term "pharmaceutically acceptable excipient" includes any solvent suitable for the particular intended dosage form. Except to the extent that any conventional excipient is incompatible with the Pediococcus pentosaceus VB341 disclosed herein, such as by producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is contemplated by the present disclosure.

[0050] 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, beverages, health products, feed, and / or additives, or added to pharmaceuticals, thereby further obtaining food, beverages, health products, feed, and / or additives that can maintain healthy blood lipid levels and / or help control body fat, or obtaining pharmaceuticals that can inhibit lipase activity, lower blood lipid levels, and / or reduce weight, and prevent, alleviate, and / or treat diseases caused by hyperlipidemia. The diseases caused by hyperlipidemia include metabolic syndrome.

[0051] Illustratively, the aforementioned foods 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.

[0052] For example, the aforementioned medicines include but are not limited to: medicines for human use and medicines for veterinary use. The aforementioned medicines for veterinary use may be for pets, livestock, or wild animals.

[0053] Illustratively, the aforementioned health care products include but are not limited to: health care products for humans and health care products for animals.

[0054] Illustratively, the aforementioned additives include but are not limited to: food additives and feed additives.

[0055] It should be noted that the characteristics and advantages described above for Pediococcus pentosaceus VB341 are also applicable to the food, beverage, medicine, health product, feed or additive, and will not be repeated here.

[0056] Composition In some embodiments, the present invention provides a composition comprising at least one of the aforementioned Pediococcus pentosaceus VB341, a fermentation broth, and a bacterial suspension. The composition of the present invention can be used to inhibit lipase activity, lower blood lipids, and / or reduce weight. Furthermore, it can prevent, alleviate, and / or treat diseases caused by hyperlipidemia and is less likely to develop resistance.

[0057] According to an embodiment of the present invention, the above composition may further include at least one of the following additional technical features: According to an embodiment of the present invention, the composition further comprises an ingestible excipient and / or carrier.

[0058] According to an embodiment of the present invention, the excipient includes at least one selected from a binder, a disintegrant, a lubricant, a glidant, a stabilizer, a filler, a diluent and a sustained-release agent.

[0059] According to an embodiment of the present invention, the carrier includes at least one selected from sugars, calcium phosphates, alkaline earth metal stearates, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols and cereal hydrolyzed solids.

[0060] According to an embodiment of the present invention, the sugars include cellulose and its derivatives.

[0061] According to an embodiment of the present invention, the dosage form of the composition includes at least one selected from oral liquid, powder, granule, capsule, tablet, and pill.

[0062] method In some embodiments, the present invention provides an in vitro method for inhibiting lipase activity, comprising co-culturing at least one of the aforementioned Pediococcus pentosaceus VB341, fermentation broth, and bacterial suspension with a lipase. As previously described, the Pediococcus pentosaceus VB341 of the present invention can effectively inhibit lipase activity, preventing the hydrolysis of excess fat 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 in scientific research, to conduct in vitro experiments related to lipase activity.

[0063] In some embodiments, the present invention provides a method for weight loss, comprising administering to a subject at least one of the aforementioned Pediococcus pentosaceus VB341, fermentation broth, and bacterial suspension. As previously described, the Pediococcus pentosaceus VB341 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 intestines, thereby achieving a weight loss effect.

[0064] Device 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 embodiments of the present invention can achieve large-scale production of the Pediococcus pentosaceus VB341, fermentation broth, bacterial suspension, or composition described herein.

[0065] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0066] Example 1 Isolation and identification of strains The strain was isolated from homemade pickled vegetables stored in jars at a Harbin farm. A gradient dilution of the bacterial suspension was prepared using sterile saline solution. 100 μL of each dilution was spread onto MRS agar plates and incubated anaerobically at 37°C for 48 hours. Suspected lactic acid bacteria colonies were streaked and purified, and DNA was extracted. PCR amplification using specific primers was performed, followed by 16S rDNA sequencing. The corresponding strains were stored in glycerol tubes. Nine Pediococcus strains were selected based on 16S rDNA identification results and numbered B01-B09, including six strains of Pediococcus acidilactici and three strains of Pediococcus pentosaceus.

[0067] Example 2 Strain Screening Preparation of lipase (1) Prepare 0.02 mol / L anhydrous sodium hydrogen phosphate buffer solution (pH = 9.3); (2) Prepare 8% lipase solution with the above-mentioned anhydrous sodium hydrogen phosphate buffer, dissolve it in 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 refrigerator at -80°C. (3) Dilute the above 8% lipase solution to 1% concentration with 0.02 mol / L disodium hydrogen phosphate (pH = 10.3) and set aside.

[0068] 2.2 Preparation of fat emulsion (1) Preparation of 100 mmol / L tris-HCl buffer (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. (2) Take 0.3 mL of the emulsion stock solution, add 29.7 mL of the above-mentioned 100 mmol / L tris-HCl buffer (pH = 8.0), mix well, and obtain a 100-fold diluted emulsion. Mix the 100-fold diluted emulsion and 100 mmol / L tris-HCl buffer (pH = 8.0) in a 1:1 ratio to obtain a 200-fold diluted emulsion.

[0069] 2.3 Preparation of orlistat at different concentrations (1) Preparation of 100 μg / mL orlistat stock solution: Weigh 4 mg of orlistat standard and dissolve it in 40 mL of sodium hydrogen phosphate solution (pH = 10.3). Ultrasonicate until completely dissolved.

[0070] (2) Use 0.02 mol / L sodium hydrogen phosphate buffer (pH = 10.3) to 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.

[0071] The sources of the experimental reagents used in this example are shown in Table 1.

[0072] Table 1. Experimental materials

[0073] 2.4 Lipase inhibition experiment with orlistat (1) Orlistat and lipase inhibition experiments were performed using 96-well plates and divided into three groups; ①Blank 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; ② Negative control group with enzyme: 180 μL of 200-fold diluted emulsion + 10 μL of 1% lipase solution + 10 μL of sodium hydrogen phosphate buffer solution, pH = 10.3; ③ Orlistat positive control group: 180 μL of 200-fold diluted emulsion + 10 μL of 1% lipase solution + 10 μL of orlistat standard solution of different concentrations; Reagent solutions were added to a 96-well plate according to the aforementioned groups. For Group ③, the enzyme solution and orlistat solution were added first and reacted for 15 minutes before the emulsion was added. The emulsions in each group were added at the same time point to ensure consistent enzyme reaction times. The OD values ​​of the emulsions were measured at 490 nm using a microplate reader at 0, 45, and 2 hours of reaction time. The changes in the OD values ​​were used to calculate the enzyme's hydrolysis of the emulsion.

[0074] (2) Calculation method of lipase hydrolysis rate and orlistat lipase inhibition rate: Lipase hydrolysis rate % = (blank control group OD - enzyme solution negative control group OD) / blank control group OD * 100% The lipase inhibition rate of orlistat (%) = (hydrolysis rate of enzyme solution negative control group - hydrolysis rate of orlistat positive control group) / hydrolysis rate of enzyme solution negative control group * 100%.

[0075] 2.5 Screening of lipase inhibitor strains (1) MRS finished medium: Weigh 54 g of finished medium, add 1 L of pure water, stir to dissolve, and then divide into shake flasks. Sterilize at 121℃ for 15 min.

[0076] (2) Strain culture ① Bacterial source: glycerol tubes of the different strains described in Example 1 stored at -80°C.

[0077] ② Strain inoculation method: Use a glycerol tube directly connected to liquid culture medium for bacterial culture, and the inoculation volume range is 0.2% to 1%.

[0078] ③Liquid culture method: According to the growth characteristics of the strain, place it in an anaerobic workstation and culture at 37℃ for 18 to 24 hours.

[0079] (3) Sample preparation When performing lipase inhibitor testing, samples are divided into two types: supernatant and whole fluid, depending on the treatment method: Supernatant: Take 0.5 mL of the above bacterial solution, centrifuge at 14,000 rpm for 10 minutes, and collect the supernatant for analysis; Whole liquid: Take 0.5 mL of the above bacterial liquid and add it to a grinding tube. Grind at 4°C and 60 Hz for 2 minutes each time, for a total of 8 times. Centrifuge at 14,000 rpm for 10 minutes and collect the supernatant for analysis. (4) Sample testing The prepared supernatant and whole liquid samples were mixed with lipase for 15 minutes, and then the emulsion was added. The OD value of the emulsion was measured within a certain period of time. The change in the emulsion value (OD difference) was used to determine the inhibitory effect of the sample on the enzyme. The specific groups are as follows: Blank sample negative control group: 180 μL of 200-fold diluted emulsion + 10 μL of pH=8.0 tris-HCl buffer + 10 μL of MRS finished culture medium.

[0080] Sample test group 1: 180 μL of 200-fold diluted emulsion + 10 μL of 1% lipase solution + 10 μL of sample supernatant. Sample test group 2: 180 μL of 200-fold diluted emulsion + 10 μL of 1% lipase solution + 10 μL of sample solution. (5) Calculation formula for sample inhibition rate on enzyme Sample hydrolysis rate% = (blank sample negative control group OD-supernatant or whole liquid test group OD) / blank sample negative control group OD * 100% Lipase hydrolysis rate % = (blank control group OD - enzyme solution negative control group OD) / blank control group OD * 100% Sample inhibition rate % = (lipase hydrolysis rate - sample hydrolysis rate) / lipase hydrolysis rate * 100% (6) Screening results The experimental results are shown in Tables 2 and 3. This screening model was used to screen the lipase inhibitory abilities of nine strains (B01–B09) of different species. Only Pediococcus pentosaceus B08 showed an inhibitory effect on lipase activity. The in vitro lipase inhibitory ability of this strain was approximately equivalent to that of orlistat at a concentration of 5 μg / mL. This indicates that this strain has the ability to produce a lipase inhibitor, which can inhibit lipase activity and can be used to prepare weight-loss drugs, health foods, solid beverages, and other products.

[0081] Table 2 Inhibition rate of different concentrations of orlistat on 1% lipase

[0082] Table 3 Inhibition rate of active strains on lipase

[0083] 2.6 Strain identification The strain B08 obtained by screening in part 2.5 was sequenced and its molecular biological identification 16SrRNA sequence was compared with the strains in GenBank for homology. It was identified as Pediococcus pentosaceus. (Pediococcus pentosaceus) , named Pediococcus pentosaceus (Pediococcus pentosaceus) VB341 was deposited on March 8, 2024, at the China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number CCTCC NO: M 2024433. Its 16S rRNA sequence is shown in SEQ ID NO: 1:

[0084] Example 3 Stability of Pediococcus pentosaceus VB341 strain 3.1 Preparation of experimental materials The experimental materials used in this example are shown in Table 4: Table 4 Experimental materials

[0085] (1) Preparation of culture medium MRS medium: MRS broth 5.4% + 1.8% agar, pH = 6.5.

[0086] (2) Preparation of simulated gastric fluid: physiological saline (sodium chloride: 0.9%), first prepare a 1% concentration of sodium chloride solution, adjust the pH to different levels and then package, sterilize at 121℃ for 30 min, pepsin 0.3% (prepare 3% concentration, filter with sterile membrane), according to the ratio of 3% pepsin: 1% physiological saline = 1:9, mix well.

[0087] (3) Preparation of choline solution: Normal saline (sodium chloride: 0.9%), first prepare a 1% concentration of sodium chloride solution, add oxcholine at concentrations of 0%, 0.03%, 0.06%, 0.1%, 0.2%, and 0.3%, respectively, sterilize at 121℃ for 30 min, add 0.1% trypsin (prepare 1% concentration, filter with sterile membrane), and mix at a ratio of 1% trypsin: 1% normal saline of each choline concentration = 1:9.

[0088] (4) Preparation of intestinal fluid: Artificial simulated intestinal fluid was prepared according to the product instructions, and the pH of the intestinal fluid after preparation was 8.0.

[0089] 3.2 Experimental methods Take the frozen tube of Pediococcus pentosaceus VB341 strain, apply the stock solution to the blank plate, and culture it anaerobically in a 37℃ incubator for 1 day according to the growth characteristics. Scrape the bacterial lawn and make 20% glycerol tubes to store at -80℃ for later use. At the same time, dilute and count the viable bacteria. 9 ~10 10 CFU / mL is appropriate.

[0090] Take the counted bacterial suspension, thaw it and add it to pure water (as the control group) and the above-mentioned simulated choline solutions, gastric juice and intestinal juice. The addition amount is: 10 μL bacterial suspension + 990 μL pure water / simulated liquid. After mixing, count the number of viable bacteria in the 0 h group at time 0. Place the bacterial suspensions of the 2 h group and the 4 h group into anaerobic bags and culture in a 37°C incubator. Take them out and count the viable bacteria after 2 h and 4 h of culture, respectively.

[0091] Figure 1The results showed that the survival rate of Pediococcus pentosaceus VB341 in 0.03%-0.3% choline solution within 4 hours was as high as over 95%, indicating that Pediococcus pentosaceus VB341 can exist stably in pancreatic juice, that is, the strain can adapt to the intestinal environment. Figure 2 The results showed that Pediococcus pentosaceus VB341 died instantly in gastric juice at pH 1 and pH 2. In gastric juice at pH 3, the immediate mortality rate was 98%, 99.8% died within 2 hours, and all died within 4 hours. In gastric juice at pH 4, the bacteria were essentially stable within 2 hours, but approximately 50% died within 4 hours. Therefore, the instability of Pediococcus pentosaceus VB341 in gastric juice needs to be considered during subsequent dosage form development. Figure 3 The results showed that after being treated in intestinal fluid at pH 8.0 for 4 h, the survival rate of Pediococcus pentosaceus VB341 was 100%, indicating that the strain could exist stably in intestinal fluid.

[0092] Example 4 In vivo efficacy study of Pediococcus pentosaceus VB341 4.1 Experimental Design The experimental drug administration design is shown in Table 5: a total of 4 groups were set up, including the Control group, the Model group, the bacterial solution group, and the drug (Xuezhikang) group, with 10 mice in each group. The drinking water of the mice in each group was distilled water. The mice in the Control group were fed with ordinary feed (purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., 1010009), and the other three groups were fed with high-fat feed (purchased from Changzhou Shuyi Shuer Biotechnology Co., Ltd., PD6001, 60% kcal). After feeding for 17 weeks, the drugs were administered. Among them, the mice in the Control and Model groups were gavaged with 10 mL / kg body weight of distilled water; the mice in the bacterial solution group were gavaged with 1.0×10 10 CFU of Pediococcus pentosaceus VB341 per mouse; the drug (Xuezhikang) group was gavaged with 200 mg / kg body weight of Xuezhikang suspension. All mice in each group were gavaged once daily between 8:00 and 10:00 AM for 14 consecutive weeks.

[0093] Table 5 Animal experiment drug administration design

[0094] 4.2 Experimental Materials (1) Animals: Forty healthy male C57 BL / 6 mice aged 6 to 8 weeks were selected. Among them, 10 mice were fed with ordinary feed and the remaining 30 mice were fed with high-fat feed. After 17 weeks of feeding, these 30 mice were divided into three groups according to their body weight, with 10 mice in each group.

[0095] (2) Bacterial liquid: Inoculate the activated Pediococcus pentosaceus VB341 into MRS medium and culture at 37°C for 24 h. Centrifuge the fermentation liquid at 9000 rpm for 10 min. Discard the supernatant and add the protective agent (60 mg of maltodextrin as filler, 15 mg of sodium hyaluronate as suspending agent, and 20 mg of sodium glutamate as stabilizer) to the bacterial residue. Mix by pipetting and shaking. Adjust the viable bacterial count to about 1.0 × 10 10 CFU / vial, freeze-dried according to the conditions in Table 6.

[0096] Table 6 Freeze-drying process parameters

[0097] 4.3 Experimental testing After 14 weeks of continuous gavage, mice in each group were fasted with or without water the day before and anesthetized with isoflurane inhalation. Blood was collected from the orbital cavity of all animals and centrifuged at 3500 rpm for 15 minutes at 4°C to obtain plasma. Plasma cholesterol (TC) and triglyceride (TG) levels were measured according to the kit instructions (Nanjing Jiancheng Bioengineering Institute).

[0098] 4.4 Experimental Results The results are shown in Table 7 and Figure 4 As shown, the cholesterol content in the plasma of the Model group mice was significantly higher than that of the Control group mice ( p <0.0001), were 11.98±2.31 and 5.6±0.92 mM / L, respectively, indicating that the hypercholesterolemia model of mice was successfully established by feeding with a high-fat diet for 31 weeks; while feeding with high-fat diet, the bacterial solution and the positive control drug - Xuezhikang capsule were gavage-administered for 14 consecutive weeks. At the end of the experiment, the cholesterol levels in the plasma of the two groups of mice were reduced to 9.38±0.87 and 8.44±3.97 mM / L, respectively, which were significantly decreased compared with the Model group ( p <0.05); Table 7 and Figure 5 Results showed that after 28 weeks of high-fat diet feeding, the plasma triglyceride levels in the model group mice were slightly higher than those in the control group, reaching 0.6±0.13 and 0.75±0.09 mM / L, respectively. Triglyceride concentrations in the bacterial solution group and the positive control group (i.e., the drug (Xuezhikang) group) were lower than those in the model group, with comparable effects between the two groups. In summary, Pediococcus pentosaceus VB341 has the ability to lower plasma cholesterol in hypercholesterolemia model mice established by long-term high-fat diet feeding, and its effect is comparable to that of the positive control Xuezhikang capsule.

[0099] Table 7 Cholesterol and triglyceride concentrations in mouse plasma (mM / L)

[0100] In the description of this specification, reference to the terms "one embodiment," "some embodiments," "an embodiment," or "a specific embodiment" means that the specific features, structures, materials, or characteristics described in conjunction 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 suitable manner in any one or more embodiments. In addition, those skilled in the art may combine and combine different embodiments and features of different embodiments described in this specification without any contradiction.

[0101] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify 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, beverage, health product, feed or additive that helps maintain healthy blood lipid levels and / or helps control body fat.

5. 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.

6. 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.

7. The composition according to claim 6, characterized in that The dosage form of the composition includes at least one selected from oral liquid, powder, granule, capsule, tablet, and pill.

8. The composition according to claim 6, characterized in that The composition further comprises an excipient and / or a carrier.

9. The composition according to claim 8, 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.

10. 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.

11. A culture device or production device for the Pediococcus pentosaceus VB341 according to claim 1, the fermentation liquid according to claim 2, the bacterial suspension according to claim 3, or the composition according to any one of claims 6 to 9.

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