CN117143769A - Lactobacillus plantarum CCFM1275 capable of converting glycyrrhizic acid and rutin and soyabean glycoside - Google Patents

Lactobacillus plantarum CCFM1275 capable of converting glycyrrhizic acid and rutin and soyabean glycoside Download PDF

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Publication number
CN117143769A
CN117143769A CN202311073347.4A CN202311073347A CN117143769A CN 117143769 A CN117143769 A CN 117143769A CN 202311073347 A CN202311073347 A CN 202311073347A CN 117143769 A CN117143769 A CN 117143769A
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lactobacillus plantarum
ccfm1275
food
product
rutin
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崔树茂
唐鑫
刘飞
毛丙永
张秋香
赵建新
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Jiangnan University
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Jiangnan University
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Abstract

The invention discloses a lactobacillus plantarum CCFM1275 capable of converting glycyrrhizic acid and rutin and daidzin, belonging to the technical field of microorganisms. The screened lactobacillus plantarum CCFM1275 is preserved in the microorganism strain preservation center of Guangdong province at the year 2022, 09 and 15, and the preservation number is GDMCC No:62799; the lactobacillus plantarum CCFM1275 can generate micromolecular phenols through converting glycyrrhizic acid, can generate micromolecular active substances through converting rutin, and can also convert daidzin into daidzein. Therefore, the application of the lactobacillus plantarum CCFM1275 to products containing glycyrrhizic acid, rutin and daidzin has great application prospect.

Description

Lactobacillus plantarum CCFM1275 capable of converting glycyrrhizic acid and rutin and soyabean glycoside
Technical Field
The invention relates to a lactobacillus plantarum CCFM1275 capable of converting glycyrrhizic acid and rutin and soyabean glycoside, belonging to the technical field of microorganisms.
Background
Licorice is widely accepted by asian countries as a medicinal and traditional edible plant. In recent years, chemical components and pharmacological actions of licorice are increasingly paid attention to. The identified liquorice is mainly divided into compounds such as saponin, flavone, coumarin, polysaccharide and the like, and the active ingredients of the liquorice have various pharmacological effects such as liver protection, anti-tumor, anti-inflammatory, antiviral and the like. Triterpenoid saponins in Glycyrrhrizae radix are regarded as main drug effect substances with liver protecting activity, and mainly comprise glycyrrhizic acid (GL), mono-glucuronic acid Glycyrrhetinic Acid (GAMG) and Glycyrrhetinic Acid (GA), especially high in glycyrrhizic acid content. However, research shows that glycyrrhizic acid has activity less than that of active small molecules such as glycyrrhetinic acid, glycyrrhetinic acid and the like.
Rutin is a flavonoid compound widely existing in the roots, stems, leaves, flowers, fruits and seeds of edible or medicinal plants. Some common traditional Chinese medicines, such as flos Sophorae Immaturus, herba Rutaceae, fructus Hordei vulgaris, fructus Hippophae, semen Ginkgo, fructus Lycii, herba Leonuri, bupleuri radix, prunellae Spica, aloe, folium Eucalypti Globueli and tobacco leaf, etc., contain rutin. As a safe and low-toxicity natural active ingredient, rutin has various effects of resisting oxidation, reducing blood fat, resisting cancer, resisting inflammation and the like, and plays an important role in improving and preventing various metabolic diseases such as obesity, diabetes, cancer and the like. However, a great deal of human body studies have shown that rutin is absorbed and utilized with a large individual difference.
The radix puerariae is firstly carried in Shen nong Ben Cao Jing (Shen nong's herbal), and has a long history of dual purposes of medicine and food. The main components of the composition comprise isoflavone, triterpene, saponin, polysaccharide, etc. Wherein the radix Puerariae isoflavone is the main pharmacologically active ingredient, and the existing forms are generally free aglycone and combined glycoside, the former is mainly daidzein, and the latter is mainly puerarin (C) 21 H 20 O 9 ) Soyabean glycosides, and the like. Researches show that the pueraria isoflavone has wide antioxidant activity and great potential in the aspects of inhibiting alcohol absorption, accelerating alcohol metabolism, excreting and the like. However, the absorption of daidzin (glycosidic isoflavones) in humans is very low and the actual effect is that of daidzein produced by the deslycosidation of other metabolites of isoflavones, such as daidzein.
Thus, the complex metabolic genes through the intestinal flora are key to the conversion of glycyrrhizic acid, rutin and daidzin, but the following problems still remain: some people lack of flora capable of converting glycyrrhizic acid, rutin and daidzin, so that the effects are poor when eating foods containing glycyrrhizic acid, rutin and daidzin; the food and pharmaceutical fields lack strains capable of producing active metabolites by converting glycyrrhizic acid, rutin and daidzin.
Lactobacillus plantarum (Lactobacillus plantarum) is one of lactobacillus, and has positive gram staining, anaerobic or facultative anaerobic, no spores, acid resistance and bile salt resistance, the optimal growth temperature is 30-37 ℃, and the lactobacillus commonly found in dairy products, meat, vegetables and fruit juice can play a beneficial role through intestines and stomach and colonization in intestinal tracts. The lactobacillus plantarum has wide application in the fields of food fermentation, industrial lactic acid fermentation, medical care and the like.
Therefore, the lactobacillus plantarum strain capable of converting glycyrrhizic acid, rutin and soyabean glycoside has strong specificity and high conversion rate, and can have great application value in industry.
Disclosure of Invention
The invention provides a lactobacillus plantarum strain capable of converting glycyrrhizic acid, rutin and daidzin.
The technical scheme of the invention is as follows:
the invention provides a lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1275 which is deposited in the microorganism strain collection of Guangdong province at 2022, 09 and 15 days, and the deposited number is GDMCC No:62799, the preservation address is 5 buildings of Guangzhou Md.A. No. 100 college, no. 59.
The lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1275 strain is circular, convex or lens-shaped after being cultured on MRS culture medium for 48 hours, is slightly white, is opaque and has a soft surface which is smooth to be sticky.
As a specific technical scheme, the colony is obtained by the following method:
(1) Taking the feces of healthy people from Jiangsu tin-free as a sample, properly diluting with sterile water, coating a certain amount of the feces on an MRS solid culture medium, inversely culturing for 48 hours at 37 ℃, and selecting colonies with different forms for streaking separation after colonies grow out until pure single colonies with consistent forms are obtained;
(2) Inoculating the single colonies screened in the step (1) into 5mL MRS liquid culture medium containing glycyrrhizic acid or rutin respectively, and culturing at 37 ℃ for 48 hours to obtain test tube bacterial liquid;
(3) Taking 1mL of the test tube bacterial liquid obtained in the step (2), centrifuging for 3min at 8000r/min in a sterile centrifuge tube, discarding the upper layer culture medium, freeze-drying the obtained bacterial mud, and selecting a strain with stronger converted glycyrrhizic acid and rutin;
(4) And (3) carrying out PCR amplification on the strain obtained in the step (3) to obtain 16S rDNA, sending the PCR product to Suzhou gold intellectual biosciences, sequencing, and comparing the sequencing result with a nucleic acid sequence in NCBI to finally obtain 1 lactobacillus plantarum, namely the lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1275.
The lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1275 provided by the invention can convert glycyrrhizic acid into other small molecular substances, can also convert rutin into other small molecular active substances, and can also convert daidzein into daidzein.
The invention provides a microbial agent, which comprises lactobacillus plantarum CCFM1275 or fermentation liquor thereof, or freeze-dried powder containing lactobacillus plantarum CCFM1275, or thallus inactivated by lactobacillus plantarum CCFM1275, or lysate containing lactobacillus plantarum CCFM1275, or extract of lactobacillus plantarum CCFM1275, and the fermentation liquor contains lactobacillus plantarum CCFM1275.
In one embodiment of the present invention, the microbial agent comprises lactobacillus plantarum CCFM1275 in an amount of at least: 1X 10 8 CFU/ml or 1X 10 8 CFU/g。
The invention also provides a culture medium which contains the lactobacillus plantarum CCFM1275 or the microbial agent.
In one embodiment of the invention, lactobacillus plantarum CCFM1275 is added in an amount of at least: 1X 10 8 CFU/ml or 1X 10 8 CFU/g。
The invention provides a product, which contains the lactobacillus plantarum CCFM1275 or the microbial agent or the culture medium.
In one embodiment of the invention, the product comprises a food product, a health product, a pharmaceutical, a special medical use food product, a cosmetic product, a feed or a feed additive.
In one embodiment of the present invention, the lactobacillus plantarum CCFM1275 is added in an amount of not less than 1X 10 in the product 8 CFU/ml。
In one embodiment of the invention, the medicament is a medicament containing lactobacillus plantarum CCFM1275, a medicament carrier and/or a pharmaceutical excipient.
In one embodiment of the invention, the food product comprises a solid food product, a semi-solid food product, a liquid food product.
In one embodiment of the invention, the food is a health food; or the food is dairy products, bean products or fruit and vegetable products produced by using a starter containing the lactobacillus plantarum CCFM 1275; or the food is a beverage or snack containing the lactobacillus plantarum CCFM 1275.
In one embodiment of the invention, the fermented food product comprises a dairy product, a soy product or a fruit and vegetable product; the dairy product comprises milk, sour cream or cheese; the fruit and vegetable product comprises cucumber, carrot, beet, celery or cabbage product.
In one embodiment of the invention, the health product contains the lactobacillus plantarum CCFM1275 and a carrier and/or auxiliary materials.
In one embodiment of the invention, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.
In one embodiment of the invention, the pharmaceutical excipients comprise excipients and/or additives.
In one embodiment of the invention, the excipient comprises a binder, filler, disintegrant, and/or lubricant.
In one embodiment of the invention, the additive comprises a solubilizer, a co-solvent and/or a preservative.
In one embodiment of the invention, the dosage form of the medicine comprises powder, granules, capsules, tablets, pills or oral liquid.
In one embodiment of the invention, the dosage form of the pharmaceutical product comprises a pill, tablet, lozenge, lyophilized powder, granule, capsule, aqueous solution, alcoholic solution, oily solution, syrup, emulsion, suspension, suppository, solution for injection or infusion, ointment, gel, tincture, cream, patch, lotion, spray, aerosol, powder mist, effervescent tablet, transdermal therapeutic system, microcapsule, implant or stick.
The invention provides a product, which contains the lactobacillus plantarum CCFM1275 or the microbial agent or the culture medium and glycyrrhizic acid.
In one embodiment of the invention, the product comprises a food product, a health product, a pharmaceutical, a special medical use food product, a cosmetic product, a feed or a feed additive.
In one embodiment of the present invention, the lactobacillus plantarum CCFM1275 is added in an amount of not less than 1X 10 in the product 8 CFU/ml or 1X 10 8 CFU/g。
The invention provides a product, which contains the lactobacillus plantarum CCFM1275 or the microbial agent or the culture medium, and also contains one or more of rutin, soyabean glycoside and glycyrrhizic acid.
In one embodiment of the invention, the product comprises a food product, a health product, a pharmaceutical product, a special medical use food product, a cosmetic product, a feed or a feed additive.
In one embodiment of the present invention, the lactobacillus plantarum CCFM1275 is added in an amount of not less than 1X 10 in the product 8 CFU/ml or 1X 10 8 CFU/g。
The invention also provides a product for promoting the activity of the soybean glycoside, the glycyrrhizic acid and the rutin to be exerted, and the product contains the lactobacillus plantarum CCFM1275 or the microbial agent or the culture medium.
In one embodiment of the invention, the product comprises a food product, a health product, a pharmaceutical product, a special medical use food product, a cosmetic product, a feed or a feed additive.
In one embodiment of the present invention, the lactobacillus plantarum CCFM1275 is added in an amount of not less than 1X 10 in the product 8 CFU/ml or 1X 10 8 CFU/g。
In one embodiment of the invention, the medicine contains lactobacillus plantarum CCFM1275, a medicine carrier and/or a pharmaceutical adjuvant.
The invention provides application of lactobacillus plantarum CCFM1275 or the microbial agent or the culture medium in preparing products capable of promoting rutin, daidzin and glycyrrhizic acid to be active.
In one embodiment of the invention, the product comprises a food product, a health product, a pharmaceutical product, a special medical use food product, a cosmetic product, a feed or a feed additive.
In one embodiment of the present invention, the lactobacillus plantarum CCFM1275 is added in an amount of not less than 1X 10 in the product 8 CFU/ml or 1X 10 8 CFU/g。
In one embodiment of the invention, the pharmaceutical product contains lactobacillus plantarum CCFM1275 or the microbial agent, the pharmaceutical carrier and/or the pharmaceutical excipients.
The invention also provides a fermentation method for converting rutin, soyabean glycoside or glycyrrhizic acid by using the lactobacillus plantarum, which comprises the steps of adding the lactobacillus plantarum CCFM1275 into a reaction system containing rutin, soyabean glycoside or glycyrrhizic acid for fermentation.
In one embodiment of the invention, the fermentation process comprises the steps of:
(1) The lactobacillus plantarum is streaked on MRS solid culture medium, and the plate is inversely cultured for 48 hours at 37 ℃. And (3) picking single bacterial colony, inoculating the single bacterial colony into 5mL of MRS liquid culture medium, and culturing for 48 hours at 37 ℃ to prepare bacterial liquid.
(2) Adding 5% (v/v) of lactobacillus plantarum bacterial liquid into fermentation base material rich in glycyrrhizic acid, soyabean glycoside or rutin, and fermenting at 37 ℃ for 48h.
(3) After fermentation, 8000r/min, centrifuging for 15min, collecting fermentation supernatant, and cold preserving at 4deg.C.
In one embodiment of the invention, in the step (2), the concentration of glycyrrhizic acid in the fermentation base material rich in glycyrrhizic acid, rutin and daidzin is 10g/L, the concentration of rutin is 0.2g/L, and the concentration of daidzin is 0.8g/L.
The food in the invention comprises solid food, semi-solid food and liquid food.
The food in the invention is a health food; or the food is dairy products, bean products or fruit and vegetable products produced by using a starter containing the lactobacillus plantarum CCFM 1275; or the food is a beverage or snack containing the lactobacillus plantarum CCFM 1275.
The fermented food in the invention comprises dairy products, bean products or fruit and vegetable products; the dairy product comprises milk, sour cream or cheese; the fruit and vegetable product comprises cucumber, carrot, beet, celery or cabbage product.
The health product contains the lactobacillus plantarum CCFM1275 and a carrier and/or auxiliary materials.
The medicine or the drug in the invention contains lactobacillus plantarum CCFM1275, a drug carrier and/or a pharmaceutic adjuvant.
The drug carrier in the invention comprises microcapsules, microspheres, nanoparticles and/or liposomes.
The pharmaceutical excipients in the invention comprise excipients and/or additives.
The excipient in the present invention comprises a binder, a filler, a disintegrant and/or a lubricant.
The additives of the present invention comprise solubilizers, co-solvents and/or preservatives.
The dosage forms of the medicine comprise powder, granules, capsules, tablets, pills or oral liquid.
The dosage forms of the medicine in the present invention include pills, tablets, troches, freeze-dried powders, granules, capsules, aqueous solutions, alcoholic solutions, oily solutions, syrups, emulsions, suspensions, suppositories, solutions for injection or infusion, ointments, gels, tinctures, creams, patches, lotions, sprays, aerosols, powder mists, effervescent tablets, transdermal therapeutic systems, microcapsules, implants or sticks.
Advantageous effects
The invention provides lactobacillus plantarum CCFM1275 which has the following characteristics:
(1) The invention discloses a lactobacillus plantarum CCFM1275 capable of converting glycyrrhizic acid, which can convert the glycyrrhizic acid into other small-molecule active substances, so that the function of the glycyrrhizic acid can be effectively improved, the problem that part of people have poor liquorice utilization effect due to the problem of intestinal flora of the people is solved, the efficacy is better exerted, and the invention has the characteristics of simplicity in operation, low energy consumption and the like.
(2) The lactobacillus is a widely used probiotic bacteria, the lactobacillus plantarum is a main starter for fermenting plant food, the lactobacillus plantarum with rutin conversion capability is safe and has good probiotic potential, and the lactobacillus plantarum is simultaneously used with rutin and has biological effects of probiotics and flavonoid natural products, so that the lactobacillus plantarum has good application prospect in functional foods such as fermented foods, novel dietary supplements and the like.
(3) The invention discloses a lactobacillus plantarum CCFM1275 capable of converting soybean glycoside, which can convert soybean glycoside into daidzein and other active molecules and enhance the efficacy of the soybean glycoside.
Preservation of biological materials
Lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1275, taxonomic designation Lactiplantibacillus plantarum, was deposited at the Cantonese microorganism strain collection at month 09 and 15 of 2022 under the accession number GDMCC No:62799 the preservation address is building 5 of Guangzhou Miao 100 # college of first-vogue 59, and the university of Guangdong province institute of microorganisms.
Drawings
FIG. 1 is a rutin liquid chromatogram.
FIG. 2 is a diagram showing the rutin conversion effect of Lactobacillus plantarum.
FIG. 3 is a liquid phase diagram of each component of glycyrrhizic acid before fermentation.
FIG. 4 is a liquid phase diagram of glycyrrhizic acid components after fermentation.
FIG. 5 is a total ion flow diagram of glycyrrhizic acid before and after fermentation.
FIG. 6 is a total ion flow diagram of rutin extract before and after fermentation.
FIG. 7 is a graph showing the effect of the fermentation of soybean glycoside of Lactobacillus plantarum.
FIG. 8 is a diagram showing the estimation of rutin metabolic pathway.
FIG. 9 is a diagram showing the estimation of the metabolic pathway of glycyrrhizic acid.
Detailed Description
The present invention now provides the following explanations of terms and methods to better illustrate the present invention and to guide those skilled in the art to practice the present invention.
The terms "comprising," "including," "having," "containing," and "containing" as used herein mean "including but not limited to," "having but not limited to," "containing but not limited to," and are used interchangeably with the corresponding phrases.
The term "or" as used herein is used herein to mean, and is used interchangeably with, the term "and/or" unless the context clearly indicates otherwise.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the suitable methods, examples of materials, are set forth below for purposes of illustration only and are not intended to be limiting.
Definition of technical terms:
the term "strain" as used herein refers to a microorganism of a particular species having common characteristics. The terms "strain" and "cell" are used interchangeably herein unless indicated to the contrary.
The term "plate" as used herein refers to a plate culture medium, which is the most commonly used form of solid medium used to obtain pure culture of microorganisms, and which is a solid plane of the medium formed by cooling solidified solid medium in a sterile petri dish, often referred to simply as a culture plate, or plate.
The term "medium" as used herein refers to a medium comprising the chemical elements necessary for the growth of the microorganism together with at least one carbon source and one nitrogen source.
The term "culturing" as used herein means culturing the microorganism for a period of time until a desired target is reached.
The term "licorice" as used herein: licorice is a plant of the herb perennial Leguminosae (Leguminosae), native to asia, europe and parts of africa. Several licorice has been recorded in pharmacopoeias of countries including china, japan, uk and united states as one of the famous and old herbs, and licorice is one of materials commonly used in the feed industry and the food industry, and has important medicinal and economic values. Among many liquorice, glabra liquorice and licorice which are main streams in the multinational formulary and market are selected as research objects for research.
The term "glycyrrhizic acid" as used herein:
glycyrrhizic acid is the main medicinal component in licorice, and is a triterpene compound which is more studied in licorice. Triterpenes are usually produced by the mevalonate pathway (MVA pathway) in the cytoplasm and the methylerythrose phosphate pathway (MEP pathway) in the plastid, through the action of a series of enzymes to produce isopentenyl pyrophosphate (isopentenyl diphosphate, IPP), with the possible plastid membrane exchange of isopentenyl pyrophosphate. The isomerization of IPP to dimethylallyl pyrophosphate (dimethylallyl diphosphate, DMAPP) is carried out by isopentenyl pyrophosphate isomerase (isopentenyl diphosphate isomerase, IPPI), followed by the stepwise condensation of two molecules of IPP and one molecule of DMAPP under the catalysis of farnesyl pyrophosphate synthase (farnesyl pyrophosphate synthase, FPPS) to give farnesyl pyrophosphate (farnesyl pyrophosphate, FPP). Then, two molecules of FPP are subjected to squalene synthase (squalene synthase, SQS) catalytic condensation to obtain squalene (squalene), and are subjected to squalene cyclooxygenase (Squalene epoxidase, SQE) catalytic condensation to obtain 2, 3-oxasqualene (2, 3-oxaqualene), and triterpenes and sterols with different configurations and functions are obtained under the action of different oxasqualene cyclases (oxidosqualene cyclases, OSCs). 2, 3-oxidation squalene is used AS an important branch point in the glycyrrhizic acid synthesis path, beta-Amyrin alcohol (beta-Amyrin) is obtained under the action of beta-Amyrin synthase (beta-AS), and recent researches find that miRNA-n244 is potentially targeted in the process.
In licorice, beta-amyrin alcohol has three directions, namely glycyrrhetinic acid, soyasapogenol B and oleanolic acid. Cytochrome P450 monooxygenase CYP88D6 and CYP72A154 respectively catalyze two-step oxidation reaction at the C-11 position and three-step oxidation reaction at the C-30 position of beta-amyrin to obtain glycyrrhetinic acid (Glycyrrhetinic acid), and miR2119 can potentially target the first-step oxidation reaction of the C-11 of beta-amyrin. The glycyrrhizic acid (Glycyrrhizic acid) is obtained by transferring two glucuronic acids to C-3 position of glycyrrhetinic acid sequentially by glycyrrhizic acid UDP-glycosyltransferase (GuUGT 3).
Glycyrrhrizae radix has antiinflammatory, antibacterial, enzyme inhibiting, and other pharmacological activities. The Glycyrrhrizae radix contains glycyrrhizic acid, liquiritin, isoliquiritigenin and liquiritigenin. Mainly flavonoids and triterpenes. Glycyrrhizic acid formula C 42 H 62 O 16 Molecular weight 822.92. Glycyrrhizic acid is the most important pharmaceutical active ingredient reflecting the quality of licorice.
The term "flavonoid" as used herein: flavonoids are a large class of compounds that are widely found in nature. The natural flavonoids can be divided into six classes: flavonoids (flavones), flavanones (flavanones), flavonols (flavonols), isoflavones (isoflavones), anthocyanidins (anthocyanides), flavanols (flavanols) [ or catechins ]. Flavonoids have a wide variety of biological activities, but cannot be synthesized in humans and can only be obtained from foods.
The term "rutin" as used herein: rutin, also known as rutin, vitamin P, has the molecular formula C27H30O16, is a natural flavonoid glycoside, and belongs to flavonol glycoside widely existing in plants, and the two glycoside are glucose and rhamnose. The appearance is light yellow or light green crystalline powder. Is soluble in pyridine, formyl and alkali liquor, slightly soluble in ethanol, acetone and ethyl acetate, and almost insoluble in chloroform, ether, benzene, carbon disulfide and petroleum ether. Has antiinflammatory, antioxidant, antiallergic, and antiviral effects
The term "fermentation broth" as used herein refers to a liquid culture medium into which a microorganism strain is introduced, after a period of time, the microorganism utilizes the nutrients in the culture medium to synthesize bacterial cells and secretion products, and the liquid after metabolism of the microorganism is called a fermentation broth.
The term "strain extract" as used herein refers to an extract obtained by fermentation of a strain of lactobacillus salivarius, which is inoculated into a suitable medium, fermented under conventional fermentation conditions to synthesize and secrete the product into the medium, and then purified.
The term "lysate of a strain" as used herein means a lysate of a strain obtained by inoculating cultured Lactobacillus salivarius into a cell lysate, lysing under conventional conditions, centrifuging, and purifying.
The invention provides a lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1275 which is deposited in the microorganism strain collection of Guangdong province at 2022, 09 and 15 days, and the deposited number is GDMCC No:62799, the preservation address is 5 buildings of Guangzhou Md.A. No. 100 college, no. 59.
The lactobacillus plantarum strain of the invention also comprises mutants, variants and/or progeny of the lactobacillus plantarum strain.
By "mutant" is meant any microorganism produced by modification of a parent lactobacillus plantarum strain. For example, the mutant may be a microorganism produced by genetic modification of a lactobacillus plantarum strain. By "variant" is meant a naturally occurring microorganism derived from a parent lactobacillus plantarum strain. For example, the variant may be a microorganism produced by lactobacillus plantarum adapted to specific cell culture conditions. By "progeny" is meant any microorganism produced by propagation or multiplication of a parent lactobacillus plantarum strain or mutant, variant thereof, which itself may be identified as the same or substantially the same strain as the parent strain. It will be appreciated that, in view of the asexual propagation process, the progeny strain is almost identical in gene to the parent lactobacillus plantarum strain. Thus, the progeny strain is identical in gene to the parent strain and can be considered a "clone" of the parent strain. Or the progeny strain is substantially identical in gene to the parent strain.
The "culture" in the present invention refers to a product obtained by culturing a strain in a medium, and the product may include the strain itself. The lysate refers to a product obtained by treating a strain with enzymes, ultrasound, homogenization and the like. The term "extract" refers to a product obtained by subjecting a strain to a solvent extraction or the like. The term "inactivated product" refers to a product obtained by treating a strain with heat, pressure, or a drug.
The invention is further illustrated below in connection with specific examples, but the invention is not limited by the regulations of the examples.
The brain heart extract Broth (BHI) in the following examples was purchased from the ocean biotechnology company, high tech industrial park, peninsula; rutin standard (HPLC more than or equal to 98%) is purchased from Shanghai Charcup technology Co., ltd; dimethyl sulfoxide was purchased from ala Ding Huaxue reagent limited; acetic acid, chromatographic grade methanol was purchased from Sigma-Aldrich Sigma Aldrich (Shanghai) trade limited; glycyrrhizic acid (product number: G810519, G810520, CAS: 1405-86-3), glycyrrhetinic acid (product number: G810497, CAS: 471-53-4), and monoglucuronic acid glycyrrhetinic acid (product number: G939067, CAS: 34096-83-8) were purchased from Michelin corporation; anhydrous ethanol was purchased from national pharmaceutical group chemical reagent limited; acetonitrile is chromatographic purity and is purchased from Soy glycoside of national pharmaceutical systems chemical reagent Limited company and is purchased from Allatin (purity 98%, shanghai, china); daidzein is purchased from Ala ding (purity not less than 98%, shanghai, china).
The following examples relate to the following media:
MRS liquid Medium (g/L): 10g/L peptone, 5g/L yeast extract, 10g/L beef extract, 20g/L glucose, 2g/L anhydrous sodium acetate, 2g/L, K hydrogen diamine citrate 2 HPO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.58g/L、MnSO 4 ·7H 2 O0.25 g/L, tween-80 1g/L and distilled water 1000g/L.
MRS solid cultureBase (g/L): 10g/L peptone, 5g/L yeast extract, 10g/L beef extract, 20g/L glucose, 2g/L anhydrous sodium acetate, 2g/L, K hydrogen diamine citrate 2 HPO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.58g/L、MnSO 4 ·7H 2 O0.25 g/L, tween-80 1g/L, agar 20g/L, and distilled water 1000g/L.
Fermentation base material (g/L) containing glycyrrhizic acid: 10g/L glycyrrhizic acid, 10g/L peptone, 5g/L yeast extract, 10g/L beef extract, 20g/L glucose, 2g/L anhydrous sodium acetate, 2g/L, K hydrogen diamine citrate 2 HPO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.58g/L、MnSO 4 ·7H 2 0.25g/L of O, 1g/L of Tween-80 and 1000g/L of distilled water.
BHI liquid medium: 10g/L tryptone, 17.5g/L beef heart infusion powder, 5.0g/L sodium chloride, 2.0g/L glucose and 2.5g/L disodium hydrogen phosphate.
Fermentation base containing soyabean glycoside (g/L): 0.08g/L of soyasaponin, 10g/L of peptone, 5g/L of yeast extract, 10g/L of beef extract, 20g/L of glucose, 2g/L of anhydrous sodium acetate, 2g/L, K of diamine hydrogen citrate 2 HPO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.58g/L、MnSO 4 ·7H 2 0.25g/L of O, 1g/L of Tween-80 and 1000g/L of distilled water.
The detection method involved in the following examples is as follows:
and (3) detecting the rutin content after fermentation by HPLC:
1. preparation of standard solution
Rutin 1mg is accurately weighed by using an analytical balance (0.0001 g), and 5mL of methanol solution is added for full dissolution (ultrasonic dissolution assisting) to prepare 200 mug/mL of single standard substance mother liquor for standby. The standard mother liquor is preserved at-20deg.C in dark place, and has a shelf life of 6 months.
And (3) absorbing a certain amount of methanol, diluting and uniformly mixing rutin single standard substance mother liquor to prepare serial mixed standard working solutions with single standard concentration of 2.5 mug/mL, 5 mug/mL, 25 mug/mL, 50 mug/mL, 100 mug/mL and 200 mug/mL respectively for preparing standard curves. And (5) refrigerating at 4 ℃ and keeping in dark, wherein the effective period is 1 week.
All the sample solutions prepared were filtered through a 0.22 μm microporous organic filter.
2. Chromatographic conditions
The apparatus was carried out using a Waters e2695, using an XBiridge C18 (5 μm, 4.6X1250 mm) liquid chromatography column, with a sample temperature set at 25℃and a gradient elution at a flow rate of 1.00mL/min, with a column temperature of 30℃and a sample loading of 20. Mu.L. Mobile phase: 0.1% acetic acid aqueous solution (A), 100% methanol solution (B). Gradient elution procedure: 0-5 min,20% B; 5-20 min, 20-70% of B; 20-25 min, 70-50% of B; 25-26 min, 50-20% of B; 26-30 min,20% B. Ultraviolet detector wavelength: 283nm.
3. Standard curve making
And (3) measuring the standard substance by using a high performance liquid chromatography, recording peak areas, and respectively measuring rutin standard curves by taking the concentration of each substance as an abscissa and taking the measured peak areas as an ordinate. The liquid chromatogram of rutin standard substance is shown in figure 1, and the retention time of rutin is 17.3min.
4. Sample detection
Filtering the obtained fermentation liquor by a 0.22 mu m microporous organic filter membrane, adding the filtered fermentation liquor into a sample injection bottle, measuring a sample by adopting a high performance liquid chromatography, recording the peak area of each substance in the sample, substituting a standard curve to calculate the substance content in the sample, and calculating the rutin consumption according to the substrate content before and after fermentation.
Consumption (%) = [ post-conversion molar amount (mol)/initial substrate molar amount (mol) ]. Times.100
HPLC (high performance liquid chromatography) detection of glycyrrhizic acid content after glycyrrhizic acid fermentation:
1. the standard preparation treatment method is established:
10mg of GL (glycyrrhizic acid), GAMG (glycyrrhetinic acid monoglucuronide) and GA (glycyrrhetinic acid) standard substances dried to constant weight at 105 ℃ are respectively weighed, prepared into standard substance mother liquor solutions of 1.0g/L by using methanol in 3 volumetric flasks, diluted into standard substance solutions of 20 g/L, 40 g/L, 80 g/L, 160 g/mL and 320g/mL respectively, filtered by a 0.22 mu m filter membrane, sampled to 10 mu L and subjected to HPLC detection, and the concentrations (g/mL) of GL, GAMG and GA are plotted as an abscissa to obtain 3 standard curves.
2. Establishment of liquid chromatography detection method
Chromatographic column: x Bridge RC18 (250X 4.6mm,5 μm); mobile phase: water (pH 2.85+0.6% (v/v) acetic acid): methanol=19: 81 (v/v); a detector: an ultraviolet detector (UV) 254nm; sample injection amount: 10. Mu.L; column temperature: 35 ℃; elution conditions: the flow rate was 1.0mL/min.
The retention times of glycyrrhizic acid (GL), mono glucuronic acid Glycyrrhetinic Acid (GAMG) and Glycyrrhetinic Acid (GA) were 7.5min, 15.9min and 22.7min, respectively.
HPLC (high performance liquid chromatography) detection of the content of the soybean glycoside and the soybean aglycone after fermentation:
HPLC detection: preliminary analysis of the lactobacillus plantarum CCFM1275 fermented soybean glycoside was performed using a high performance liquid chromatograph (Waters 2695). Chromatographic column: atlantis T3 (250×4.6mm,5 um), column temperature: 35 ℃. Mobile phase: 0.1% acetic acid water (phase a), methanol (phase B); a detector: ultraviolet detector (UA) 280nm. Elution conditions: gradient elution, elution gradient profile: 0min,100% B;0-10min,0% -30% A;10-30min, 30-70% of A;30-35min, 70-0% of A; flow rate: 1mL/min, sample injection volume: 5. Mu.L.
Example 1: screening, strain identification and preservation of lactobacillus plantarum
1. Screening
Taking feces of healthy adult as sample, diluting with sterile physiological saline solution to 10 times -6 Then respectively taking 100 mu L of dilution factors of 10 -4 、10 -5 、10 -6 Plating on MRS solid culture medium, culturing at 37deg.C for 48 hr, observing and recording colony morphology; selecting colonies with different forms on an MRS solid culture medium for streaking separation, culturing at 37 ℃ for 48 hours, and then selecting single colonies with different forms on the MRS solid culture medium again for streaking separation until pure single colonies with consistent forms are obtained; pure bacterial colony on MRS solid culture medium is selected and inoculated into 5mL MRS liquid culture medium, and the culture is carried out for 24 hours at 37 ℃; taking 1mL of bacterial liquid in a sterile centrifuge tube, centrifuging at 8000r/min for 3min, discarding an upper layer culture medium, and freeze-drying the obtained bacterial mud.
2. Authentication
The isolated strain is subjected to PCR amplification of 16S rDNA (shown as SEQ ID NO. 1), the PCR product is sent to Suzhou gold only biotechnology limited company for sequencing, the result obtained by sequencing is subjected to nucleic acid sequence comparison in NCBI, and the result shows that 1 lactobacillus plantarum is finally obtained and named as lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1275.
3. Preservation of
Lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1275 is inoculated in 5mL MRS liquid culture medium and cultured for 24 hours at 37 ℃; taking 1mL of bacterial liquid in a sterile centrifuge tube, centrifuging at 8000r/min for 3min, discarding an upper layer culture medium, re-suspending bacterial mud in 30% glycerol solution, and preserving at-80 ℃.
Example 2: rutin conversion from lactobacillus plantarum
The method comprises the following specific steps:
(1) The lactobacillus plantarum CCFM1275 of example 1 was streaked on MRS solid medium and the plates were incubated upside down at 37 ℃ for 48h; and (3) picking single colony, inoculating into 5mL of MRS liquid culture medium, and culturing at 37 ℃ for 48 hours to prepare seed liquid.
(2) Lactobacillus plantarum fermented rutin
Preparation of BHI liquid culture medium:
10g/L tryptone, 17.5g/L beef heart infusion powder, 5.0g/L sodium chloride, 2.0g/L glucose and 2.5g/L disodium hydrogen phosphate.
Preparing a rutin solution:
weighing rutin standard substance, preparing into concentrated dimethyl sulfoxide (DMSO) solution of rutin at concentration of 100g/L, placing into an ultra-clean bench, continuously irradiating with ultraviolet for 30min, pouring into sterilized BHI culture medium, shaking thoroughly, and packaging to obtain fermentation culture medium for subsequent experiment.
Fermenting rutin by lactobacillus plantarum:
inoculating the seed solution prepared in the step (1) into a BHI liquid culture medium with an inoculum size of 4% (v/v), adding a rutin solution into the culture medium according to a final concentration of 200 mug/mL, and culturing at 37 ℃ for 24 hours to prepare a fermentation broth.
The fermentation broths were taken in 0h and 24h respectively in 1mL to 2mL EP tubes, centrifuged at 8000rpm for 5min, the cells were removed, and the supernatants were transferred to 5mL EP tubes.
(3) Detecting rutin content in fermentation liquid
Adding ethyl acetate with the same volume into an EP pipe filled with fermentation liquor for extraction respectively, carrying out ultrasonic treatment for 30min, fully mixing, centrifuging at 8000rpm for 5min, taking 1ml of supernatant, respectively filling the supernatant into 2ml of EP pipe, and carrying out freeze concentration on the fermentation liquor by using a freeze dryer under the condition of 45 ℃ and 2000 rpm; adding 200 mu L of methanol for redissolution, and shaking and mixing uniformly.
Sucking rutin control solution and the sample solution to be tested, determining according to chromatographic conditions, determining a liquid phase diagram of the control as shown in figure 1, calculating the rutin content in the fermentation product by an external standard method, and obtaining the rutin concentration in the lactobacillus plantarum fermentation liquid as a result, wherein the specific content of each component is shown in table 1, and the consumption is as follows: consumption of rutin added to the fermentation medium (FIG. 2).
Table 1: content of rutin before and after fermentation of Lactobacillus plantarum CCFM1275 in 200 μg/L rutin
The result shows that after the fermentation is finished, the rutin content in the fermentation medium is reduced to 14.65 mug/L compared with that before the fermentation. Therefore, after the lactobacillus plantarum CCFM1275 is adopted to ferment rutin, the rutin is greatly converted into other active micromolecular substances.
(4) Analysis of metabolites of rutin before and after fermentation and conversion of lactobacillus plantarum CCFM1275
Taking 1mL of fermentation liquor respectively, centrifuging and collecting the supernatant at 4 ℃ and 10000g for 5min;
1) Remove 100. Mu.L supernatant in a 1.5mL centrifuge tube;
2) 400 μl methanol was added: acetonitrile= (1:1, v/v) (pre-cooling at-20 ℃ in advance) to precipitate proteins;
3) Swirling for 30s;
4) Ice bath ultrasound for 10min;
5) Placing the sample in a refrigerator at-20 ℃ for incubation for 1h, and improving the protein precipitation rate (removing protein by secondary precipitation);
6) High-speed centrifugation at 15000rpm for 15min at 4 ℃;
7) Collecting supernatant, and vacuum concentrating;
8) Redissolved, add 200 μl acetonitrile: water (1:1) post vortex for 30s;
9) Centrifuging at 15000rpm for 15min at 4deg.C, filtering the supernatant with 0.22 μm filter membrane, transferring appropriate volume, and loading into sample injection vial for machine detection;
10 QC preparation: transferring the same volume from the sample to be measured to a new sample injection bottle, and uniformly mixing to obtain QC (Quality Control);
11 Blanc preparation:
1mL acetonitrile: mixing water (1:1, v/v) to a sample injection bottle uniformly;
LC-MS/MS detection:
the rutin fermentation broth was further analyzed using a high resolution liquid chromatography-mass spectrometry (Q exact LC-MS/MS). Chromatographic column: hypersil GOLD C18 (1.9 μm×2.1mm×100 mm), sheath air flow rate: 35arb, flow rate of assist gas: 15arb, ion transport capillary temperature: 320℃the autosampler temperature and the column temperature were 4℃and 30℃respectively. Mobile phase: 0.1% formic acid water (phase a), acetonitrile (phase B). Elution conditions: gradient elution, elution gradient profile: 0-3min,5% B;3-9min,5-30% B;9-15min,30-100% B;15-16min,100% B;16-16.5min,100-5% B;16.5-20min,5% B; flow rate: sample volume of 0.3 mL/min: 2. Mu.L.
Screening active ingredients in rutin samples:
the whole phenolic spectrum, including small molecular organic acid and phenolic acid substances, in the rutin extract after fermentation for 0h and 48h through lactobacillus plantarum CCFM1275 fermentation, namely before and after non-fermentation, is studied by a non-targeted metabonomics method, and the rutin extract is subjected to ESI mode collection based on an LC-MS system sensitivity mode, and the total ion flow diagram is shown in figure 6. Finally, after the rutin extract was fermented for 48 hours by CCFM1275, 19 active ingredients (only the ingredients related to the metabolic pathway are shown in Table 2 as shown in Table 2, and FIG. 8) were identified in total, mainly organic acid compounds (the lower part of the table corresponds to the primary mass spectrum).
Table 2: active small molecular substances detected in rutin fermentation liquid and change of substances before and after fermentation
Note that: "+" represents an increase in post-fermentation material compared to pre-fermentation material and "-" represents a decrease in post-fermentation material compared to pre-fermentation.
Example 3: conversion of glycyrrhizic acid by Lactobacillus plantarum
The method comprises the following specific steps:
(1) The lactobacillus plantarum CCFM1275 of example 1 was streaked on MRS solid medium and the plates were incubated upside down at 37 ℃ for 48h; and (3) picking single colony, inoculating into 5mL of MRS liquid culture medium, and culturing at 37 ℃ for 48 hours to prepare seed liquid.
(2) Lactobacillus plantarum fermented glycyrrhizic acid
Preparation of a fermentation base material containing glycyrrhizic acid:
10g/L glycyrrhizic acid, 10g/L peptone, 5g/L yeast extract, 10g/L beef extract, 20g/L glucose, 2g/L anhydrous sodium acetate, 2g/L, K hydrogen diamine citrate 2 HPO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.58g/L、MnSO 4 ·7H 2 0.25g/L of O, 1g/L of Tween-80 and distilled water.
Lactobacillus plantarum fermented glycyrrhizic acid:
and (2) adding 5% (v/v) of seed liquid of the lactobacillus plantarum CCFM1275 prepared in the step (1) into a fermentation base material containing glycyrrhizic acid, and fermenting at a constant temperature of 37 ℃ for 48 hours.
(3) After fermentation is completed, 8000r/min, centrifuging for 15min, collecting fermentation supernatant to obtain fermented glycyrrhizic acid fermented product, and refrigerating at 4deg.C.
(4) Sucking the fermented product (sample to be tested) obtained in the step (3), and filtering with 0.45 μm membrane. Respectively sucking the reference substance solution and the sample solution to be tested, and carrying out sample injection, wherein the specific content of each component is shown in a table 3 (fig. 3-4) according to the chromatographic conditions:
table 3: the content of each component before and after fermentation of the lactobacillus plantarum CCFM1275 in 10g/L glycyrrhizic acid
The results show that: the results showed that after the fermentation was completed, the glycyrrhizic acid content in the fermentation medium was reduced to 3.39g/L. Therefore, after the lactobacillus plantarum CCFM1275 is adopted to ferment glycyrrhizic acid, the glycyrrhizic acid is greatly converted into other active micromolecular substances.
(5) Analysis of metabolites of glycyrrhizic acid before and after fermentation and conversion by lactobacillus plantarum CCFM1275
Taking 1mL of fermentation liquor respectively, centrifuging and collecting supernatant at 4 ℃ and 10000g for 5min
1) Remove 100. Mu.L supernatant in a 1.5mL centrifuge tube;
2) 400 μl methanol was added: acetonitrile= (1:1, v/v) (pre-cooling at-20 ℃ in advance) to precipitate proteins;
3) Swirling for 30s;
4) Ice bath ultrasound for 10min;
5) Placing the sample in a refrigerator at-20 ℃ for incubation for 1h, and improving the protein precipitation rate (removing protein by secondary precipitation);
6) High-speed centrifugation at 15000rpm at 4℃for 15min,
7) Collecting supernatant, and vacuum concentrating;
8) Redissolved, add 200 μl acetonitrile: water (1:1) post vortex for 30s;
9) Centrifuging at 15000rpm for 15min at 4deg.C, filtering the supernatant with 0.22 μm filter membrane, transferring appropriate volume, and loading into sample injection vial for machine detection;
10 QC preparation: transferring the same volume from the sample to be measured to a new sample injection bottle, and uniformly mixing to obtain QC (Quality Control);
11 Blanc preparation:
1mL acetonitrile: mixing water (1:1, v/v) to a sample injection bottle uniformly;
LC-MS/MS detection:
the glycyrrhizin fermentation broth was further analyzed using a high resolution liquid chromatography-mass spectrometry (QExactive LC-MS/MS). Chromatographic column: hypersil GOLD C18 (1.9 μm×2.1mm×100 mm), sheath air flow rate: 35arb, flow rate of assist gas: 15arb, ion transport capillary temperature: 320℃the autosampler temperature and the column temperature were 4℃and 30℃respectively. Mobile phase: 0.1% formic acid water (phase a), acetonitrile (phase B). Elution conditions: gradient elution, elution gradient profile: 0-3min,5% B;3-9min,5-30% B;9-15min,30-100% B;15-16min,100% B;16-16.5min,100-5% B;16.5-20min,5% B; flow rate: sample volume of 0.3 mL/min: 2. Mu.L.
Screening active ingredients in a licorice sample:
the whole phenol spectrum including free and bound phenol in glycyrrhizic acid before and after fermentation for 0h and 48h by Lactobacillus plantarum CCFM1275 is studied by a non-targeted metabonomics method, and ESI mode collection is performed on the licorice extract based on the sensitivity mode of an LC-MS system, and the total ion flow diagram is shown in FIG. 5.
Referring to the data, combining with an on-line Chinese medicine database, and collecting information such as compound name, molecular formula, molecular weight, retention time and the like. The data base is imported into Compound Discoverer 3.3 software, the Compound Discoverer 3.3 software is utilized to analyze the acquired data, and the possible structure of the compound is deduced by researching fragment ions, lost groups, mass spectrum cracking rules and the like of each molecular ion of the high collision energy channel. Finally, 22 active ingredients (only the ingredients related to the metabolic pathway are shown in table 4, fig. 9, as shown in table 4) were identified in total after fermentation of glycyrrhizic acid for 48h by CCFM1275, mainly organic acids and amino acid compounds (the lower part of the table corresponds to the primary mass spectrum).
Table 4: active micromolecular substances detected in glycyrrhizic acid fermentation liquid and change of substances before and after fermentation
And (3) injection: "+" represents an increase in post-fermentation material compared to pre-fermentation material and "-" represents a decrease in post-fermentation material compared to pre-fermentation.
Example 4: transformation of soybean glycoside by lactobacillus plantarum
The method comprises the following specific steps:
(1) The lactobacillus plantarum CCFM1275 of example 1 was streaked on MRS solid medium and the plates were incubated upside down at 37 ℃ for 48h; and (3) picking single colony, inoculating into 5mL of MRS liquid culture medium, and culturing at 37 ℃ for 48 hours to prepare seed liquid.
(2) Preparation of a fermentation base containing daidzin:
0.08g/L of soyasaponin, 10g/L of peptone, 5g/L of yeast extract, 10g/L of beef extract, 20g/L of glucose, 2g/L of anhydrous sodium acetate, 2g/L, K of diamine hydrogen citrate 2 HPO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.58g/L、MnSO 4 ·7H 2 O0.25 g/L, tween-80 1g/L and distilled water 1000g.
And (2) adding 5% (v/v) of the bacterial liquid of the lactobacillus plantarum CCFM1275 prepared in the step (1) into a fermentation base material containing the soyabean glycoside, and fermenting at the constant temperature of 37 ℃ for 24 hours.
(3) After fermentation is completed, 8000r/min, centrifuging for 15min, collecting fermentation supernatant to obtain fermented soybean glycoside, and refrigerating at 4deg.C.
(4) Sucking the fermented product (sample to be tested) obtained in the step (3), and filtering with 0.45 μm membrane. Respectively sucking the reference substance solution and the sample solution to be tested, and carrying out sample injection, and determining according to the chromatographic conditions, wherein the specific content of each component is shown in table 5:
Table 5: content of each component before and after fermentation of Lactobacillus plantarum CCFM1275 in 80mg/L soybean glycoside
The results show that: the results showed that after the fermentation was completed, the daidzin content in the fermentation medium was reduced to 13mg/L and converted to daidzein 4.4mg/L.
Example 5: application of lactobacillus plantarum CCFM1275
The lactobacillus plantarum CCFM1275 can be used for preparing a starter, and the specific preparation process is as follows:
MRS medium: 10g of tryptone, 10g of beef extract, 5g of yeast powder, 20g of glucose, 5g of sodium acetate, 2g of diammonium hydrogen citrate, 2g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate heptahydrate, 1mL of Tween 80, 0.25g of manganese sulfate monohydrate, fixing the volume of water to 1000mL, adjusting the pH value to 6.5, and sterilizing for 15-25min at 119-123 ℃.
And (3) a protective agent: 100g/L-150g/L skimmed milk powder, 100g/L-150g/L maltodextrin and 140g/L-160g/L trehalose.
Inoculating Lactobacillus plantarum CCFM1275 into MRS culture medium, culturing at 37deg.C under constant temperature and humidity for 18-20 hr, collecting thallus, and re-suspending thallus cells with protectant to reach thallus concentration of 10 10 CFU/mL, then the suspension is cultured for 50-70min under the condition of constant temperature and constant humidity at 37 ℃ and dried. The drying is carried out by vacuum freeze drying after pre-freezing for 8-14h at the temperature of minus 15-minus 20 ℃.
Example 6: application of lactobacillus plantarum CCFM1275
The lactobacillus plantarum CCFM1275 can be used for manufacturing lactobacillus milk beverage, and the specific preparation process is as follows:
sterilizing raw milk skim milk at 95deg.C for 20min, cooling to 4deg.C, adding lactobacillus plantarum CCFM1275 obtained by screening in example 1 or the starter prepared in example 5 to obtain thallus concentration of 10 6 And (3) refrigerating and preserving at the temperature of 4 ℃ to obtain the milk beverage containing the lactobacillus plantarum CCFM1275 viable bacteria.
Example 7: application of lactobacillus plantarum CCFM1275
Lactobacillus plantarum CCFM1275 can be used for manufacturing soymilk, and the preparation process is as follows:
soft water is adopted to soak soybeans, the water content is three times the volume of the original soybeans, the soybeans are soaked for 1 to 2 hours at the temperature of 80 ℃, and then soybean hulls are removed. Then, leaching the soaking water, adding boiling water to grind pulp, and preserving the temperature for 10-15 min under the condition that the temperature is higher than 80 ℃. Filtering the slurry with 150 mesh filter membrane, centrifuging to obtain centrifugate, and adding into coarse soybean milkAnd (3) heating to 140-150 ℃, and then rapidly introducing the hot crude soymilk into a vacuum cooling chamber for vacuumizing, wherein the peculiar smell substances in the crude soymilk are rapidly discharged along with water vapor. Vacuum degassing, cooling to 37deg.C, inoculating lactobacillus plantarum CCFM1275 selected in example 1 or the starter prepared in example 5 to make lactobacillus plantarum CCFM1275 concentration reach 10 6 And (3) refrigerating and preserving at 4 ℃ to obtain the soybean milk containing lactobacillus plantarum CCFM1275 viable bacteria.
Example 8: application of lactobacillus plantarum CCFM1275
The lactobacillus plantarum CCFM1275 can be used for manufacturing fruit and vegetable beverages, and the specific preparation process is as follows:
cleaning fresh vegetables (such as one or more of cucumber, carrot, beet, celery or cabbage), squeezing, instant sterilizing at high temperature for 2s, immediately cooling to about 37deg.C, and inoculating lactobacillus plantarum CCFM1275 ferment to a concentration of 10 6 And (3) refrigerating and preserving at the temperature of 4 ℃ to obtain the fruit and vegetable beverage containing the lactobacillus plantarum CCFM1275 viable bacteria.
Example 9: application of lactobacillus plantarum CCFM1275
The lactobacillus plantarum CCFM1275 can be used for manufacturing capsule products, and the specific preparation process is as follows:
the lactobacillus plantarum CCFM1275 is cultured for 24 hours on an MRS culture medium, centrifuged for 20 minutes at the temperature of 4 ℃ and 4000 Xg, washed twice by PBS, added with 4% skim milk powder and 6% lactose which are mixed for 10 minutes by the weight of the powder containing the lactobacillus plantarum CCFM1275 finally obtained, added with sterile 2% calcium chloride and 3% sodium alginate, stirred for 10 minutes at 150 Xg, then left to stand for 30 minutes, finally washed and filtered, and the obtained filtrate is frozen and dried for 20 hours to obtain powder containing the lactobacillus plantarum CCFM1275, and the powder is filled into commercial medicinal microcapsules to obtain the capsule product.
Comparative example 1: influence of microbial strains on rutin conversion
The specific embodiment is the same as example 2, except that the modified lactobacillus plantarum CCFM1275 is replaced by lactobacillus reuteri (Lactobacillus reuteri) CCFM8631 (accession number GDMCC No.14394, described in the patent application text with publication number CN 107523526A); bifidobacterium adolescentis (Bifidobacterium adoltestencis) CCFM8630 (accession number GDMCC No. 14395, described in patent text publication number CN 107699517A); lactobacillus plantarum (Lacticaseibacillus plantarum) CCFM8724 has a preservation number of CGMCC No.5492 (described in patent application publication No. CN 102533618A).
And detecting the rutin change rate before and after fermentation. The specific contents of the components are shown in Table 6:
table 6: rutin content before and after fermentation of each strain
The results show that: the other strains have no strong capacity of converting rutin by lactobacillus plantarum CCFM 1275.
Comparative example 2: effect of microbial species on glycyrrhizic acid conversion
The specific embodiment is the same as in example 3, except that lactobacillus plantarum CCFM1275 is respectively adjusted to: bifidobacterium breve (Bifidobacterium breve) CCFM1025 deposit No.60386 is described in the patent application publication No. CN 114533765 a; lactobacillus plantarum (Lacticaseibacillus plantarum) CCFM8724 has a preservation number of CGMCC No.5492 and is described in patent application text with a publication number of CN 102533618A; bifidobacterium animalis (Bifidobacterium animalis) CCFM1155 accession number GDMCC No.61495 is described in the patent document with publication number CN 113088473A. Detecting glycyrrhizic acid content of each component before and after fermentation. The specific contents of the components are shown in Table 7:
Table 7: the content of each component before and after fermentation of each strain
The results show that: none of the other strains had the ability to convert glycyrrhizic acid to other active small molecules.
Comparative example 3: effect of microbial species on conversion of daidzin
The specific embodiment is the same as in example 4, except that lactobacillus plantarum CCFM1275 is respectively adjusted to: bifidobacterium breve (Bifidobacterium breve) CCFM1025 deposit No.60386 is described in the patent application publication No. CN 114533765 a; lactobacillus plantarum (Lacticaseibacillus plantarum) CCFM8724 has a preservation number of CGMCC No.5492 and is described in patent application text with a publication number of CN 102533618A; bifidobacterium animalis (Bifidobacterium animalis) CCFM1155 accession number GDMCC No.61495 is described in the patent document with publication number CN 113088473A. The content of daidzin and daidzein before and after fermentation was measured, and the specific content of each component is shown in table 8:
table 8: the content of each component before and after fermentation of each strain
The results show that: none of the remaining strains had the ability to convert soyaglycosides to daidzein.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (15)

1. Lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1275 was deposited with the Cantonese microorganism strain collection at month 15 of 2022 under the accession number GDMCC No:62799.
2. a microbial agent, which is characterized by comprising the lactobacillus plantarum CCFM1275 or a fermentation liquid thereof according to claim 1, or a freeze-dried powder of the lactobacillus plantarum CCFM1275 according to claim 1, or a thallus inactivated by the lactobacillus plantarum CCFM1275, or a lysate of the lactobacillus plantarum CCFM1275, or an extract of the lactobacillus plantarum CCFM1275, and the fermentation liquid contains the lactobacillus plantarum CCFM1275.
3. The microbial agent according to claim 2, wherein the lactobacillus plantarum CCFM1275 is added in an amount of at least: 1X 10 8 CFU/ml or 1X 10 8 CFU/g。
4. A culture medium comprising the lactobacillus plantarum CCFM1275 of claim 1 or the microbial agent of claim 2 or 3.
5. A product comprising the lactobacillus plantarum CCFM1275 of claim 1 or the microbial agent of claim 3 or 4 or the culture medium of claim 5.
6. The product according to claim 5, wherein the product comprises a food, a health product, a medicament, a special medical use food, a cosmetic, a feed or a feed additive; preferably, the food comprises solid food, semi-solid food, liquid food; preferably, the food is a health food; or the food is dairy products, bean products or fruit and vegetable products produced by using a starter containing the lactobacillus plantarum CCFM 1275; or the food is a beverage or snack containing the lactobacillus plantarum CCFM 1275.
7. The product according to claim 5 or 6, wherein lactobacillus plantarum CCFM1275 is added in an amount of at least: 1X 10 8 CFU/ml or 1X 10 8 CFU/g。
8. A product, which is characterized in that the product contains the lactobacillus plantarum CCFM1275 of claim 1 or the microbial agent of claim 2 or 3 or the culture medium of claim 4, and also contains one or more of rutin, daidzin and glycyrrhizic acid.
9. The product according to claim 8, characterized in that the product comprises a food, a health product, a medicament, a special medical use food, a cosmetic, a feed or a feed additive; preferably, the food comprises solid food, semi-solid food, liquid food; preferably, the food is a health food; or the food is dairy products, bean products or fruit and vegetable products produced by using a starter containing the lactobacillus plantarum CCFM 1275; or the food is a beverage or snack containing the lactobacillus plantarum CCFM 1275.
10. Product according to claim 8 or 9, characterized in that in the product lactobacillus plantarum CCFM1275 is added in an amount of at least: 1X 10 8 CFU/ml or 1X 10 8 CFU/g。
11. Use of lactobacillus plantarum CCFM1275 according to claim 1 or the microbial agent according to claim 2 or 3 or the culture medium according to claim 4 for the preparation of a product that promotes the absorption of rutin, daidzin, glycyrrhizic acid simultaneously.
12. Use according to claim 11, characterized in that the product contains the lactobacillus plantarum CCFM1275 according to claim 1 or the microbial agent according to claim 2 or 3 or the culture medium according to claim 4.
13. The use according to claim 12, wherein the product comprises a food product, a health product, a medicament, a special medical use food product, a cosmetic product, a feed or a feed additive; preferably, the food comprises solid food, semi-solid food, liquid food; preferably, the food is a health food; or the food is dairy products, bean products or fruit and vegetable products produced by using a starter containing the lactobacillus plantarum CCFM 1275; or the food is a beverage or snack containing the lactobacillus plantarum CCFM 1275.
14. The use according to claim 13, wherein the lactobacillus plantarum CCFM1275 is added in an amount of not less than 1 x 10 in said product 8 CFU/ml or 1X 10 8 CFU/g。
15. A fermentation method for converting rutin, soyaoside and/or glycyrrhizic acid by using lactobacillus plantarum is characterized in that the method is characterized in that lactobacillus plantarum CCFM1275 according to claim 1 is added into a reaction system containing rutin, soyaoside and/or glycyrrhizic acid for fermentation.
CN202311073347.4A 2023-08-23 2023-08-23 Lactobacillus plantarum CCFM1275 capable of converting glycyrrhizic acid and rutin and soyabean glycoside Pending CN117143769A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117598958A (en) * 2024-01-23 2024-02-27 佛山市汇腾生物技术有限公司 Sophora japonica fermentation composition, cosmetic with anti-wrinkle effect, and preparation method and application thereof

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