Fermentation of Traditional Chinese Medicine by Using a Novel Lactobacillus paracasei Strain and Its Application

Abstract

The present invention relates to the field of microbial technology, and particularly to the use of a novel Lactobacillus paracasei strain for traditional Chinese medicine fermentation and its applications. The present invention provides Lactobacillus paracasei LP-K2102, which has a preservation number of CGMCC No. 28936. The Lactobacillus paracasei LP-K2102 of the present invention has functions such as high biological activity, acid resistance, bile salt resistance, strong antioxidant ability, regulation of flora balance, prevention and treatment of diseases, and health care, and has strong plant fermentation ability and can improve the functions of the products after fermentation.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to the fermentation of traditional Chinese medicine using a novel Lactobacillus paracasei strain and its application. Background Art

[0002] Lactobacillus paracasei is a Gram-positive probiotic that does not produce spores. In August 2022, the National Health Commission of China issued a notice changing its genus from Lactobacillus to Paracasei. It is mainly used in probiotic products and the fermentation of traditional Chinese medicine. The higher the biological activity of Lactobacillus paracasei, the higher the yield in industrial production, and the stronger the efficacy, which can further reduce production costs and improve production efficiency. Therefore, screening for Lactobacillus paracasei with high activity has become the goal of the industry.

[0003] When developing probiotic products with Lactobacillus paracasei, it is necessary to have strong resistance to gastric acid and bile salts to ensure effective passage through the stomach and small intestine to reach the large intestine and play a role. The increase of harmful bacteria such as Escherichia coli will lead to dysbiosis and cause various diseases. If Lactobacillus paracasei has a good inhibitory effect on harmful bacteria, it will accelerate the restoration of the microbial balance. Superoxide anion is a harmful compound produced in the oxidative reaction of the body, which can promote the aging and diseases of the body and cause health problems. Clearing superoxide anion is beneficial to maintaining the health of the body.

[0004] Traditional Chinese medicines that are both edible and medicinal can be applied to food due to their safety, but their efficacy is relatively not ideal. How to improve the efficacy of traditional Chinese medicines that are both edible and medicinal has become a difficult problem in the industry. The problem of low efficacy of plants such as traditional Chinese medicines that are both edible and medicinal can be solved through traditional Chinese medicine fermentation technology, but finding an excellent strain for fermenting traditional Chinese medicine is the key to traditional Chinese medicine fermentation.

[0005] Existing Lactobacillus paracasei has low biological activity, weak resistance to gastric acid and bile salts, weak functions of inhibiting harmful bacteria and regulating microbial balance, weak ability to scavenge superoxide anion and antioxidant, and low plant fermentation ability and product function after fermentation. There is no report on Lactobacillus paracasei strains that have significant performance in both probiotics and the fermentation of herbs such as traditional Chinese medicine. Summary of the Invention

[0006] In view of this, the present invention provides the fermentation of traditional Chinese medicine using a novel Lactobacillus paracasei strain and its application. The present invention provides Lactobacillus paracasei LP-K2102, with a preservation number of CGMCC No. 28936. The Lactobacillus paracasei LP-K2102 of the present invention has high biological activity, acid resistance, bile salt resistance, strong antioxidant ability, functions of preventing and treating diseases, health care, and regulating microbial balance, has strong plant fermentation ability and improves the function of the product after fermentation, and its comprehensive performance is superior to other Lactobacillus paracasei strains independently screened by the applicant and commercially available Lactobacillus paracasei strains.

[0007] To achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0008] The present invention provides Lacticaseibacillus paracasei LP-K2102, with the preservation number of CGMCC No. 28936.

[0009] In some specific embodiments of the present invention, the Lacticaseibacillus paracasei LP-K2102 includes one or more of its viable bacteria, inactivated bacteria, fermentation broth, exosomes or metabolites.

[0010] In some specific embodiments of the present invention, the Lacticaseibacillus paracasei LP-K2102 has high biological activity, acid tolerance and / or bile salt tolerance.

[0011] In some specific embodiments of the present invention, the pH value of acid tolerance includes 2.0 - 3.0.

[0012] In some specific embodiments of the present invention, the bile salt tolerance includes tolerance to 0.3% bile salt.

[0013] The present invention also provides the application of the Lacticaseibacillus paracasei LP-K2102 in the preparation of any of the following:

[0014] (I), microecological products, probiotic products, synbiotics and / or postbiotics; and / or

[0015] (II), products for plant fermentation;

[0016] The microecological products, probiotic products, synbiotic products, postbiotic products and / or the products for plant fermentation include but are not limited to one or more of drugs, health foods, foods for special medical purposes, functional foods, foods, feeds, feed additives or pet foods; and / or

[0017] The dosage forms of the microecological products, probiotic products, synbiotic products, postbiotic products and / or the products for plant fermentation include but are not limited to one or more of powder, granule, capsule, tablet, pill, extract, liquid preparation, sugar or jelly; and / or

[0018] The sugar includes but is not limited to gummy candy and / or compressed tablets; and / or

[0019] The plant fermentation includes, but is not limited to, one or more of traditional Chinese medicine fermentation, fruit and vegetable fermentation, or new food ingredient fermentation;

[0020] The traditional Chinese medicine fermentation includes, but is not limited to, the fermentation of traditional Chinese medicines that are both food and medicine;

[0021] The traditional Chinese medicines include, but are not limited to, one or more of American ginseng, wolfberry, or Chinese yam.

[0022] The present invention also provides a product, including the Lacticaseibacillus paracasei LP-K2102;

[0023] The product includes any of the following:

[0024] (I), a microecological product, a probiotic product, a synbiotic product, and / or a postbiotic product; and / or

[0025] (II), a product for plant fermentation;

[0026] The microecological product, probiotic product, synbiotic product, postbiotic product, and / or the product for plant fermentation include, but are not limited to, one or more of medicines, health foods, foods for special medical purposes, functional foods, foods, feeds, feed additives, or pet foods; and / or

[0027] The dosage forms of the microecological product, probiotic product, synbiotic product, postbiotic product, and / or the product for plant fermentation include, but are not limited to, one or more of powders, granules, capsules, tablets, pills, extracts, liquid preparations, sugars, or jellies; and / or

[0028] The sugars include, but are not limited to, gummy candies and / or compressed tablets; and / or

[0029] The plant fermentation includes, but is not limited to, one or more of traditional Chinese medicine fermentation, fruit and vegetable fermentation, or new food ingredient fermentation;

[0030] The traditional Chinese medicine fermentation includes, but is not limited to, the fermentation of traditional Chinese medicines that are both food and medicine;

[0031] The traditional Chinese medicines include, but are not limited to, one or more of American ginseng, wolfberry, or Chinese yam.

[0032] The present invention also provides a product prepared by fermenting with the Lacticaseibacillus paracasei LP-K2102.

[0033] In some specific embodiments of the present invention, the product includes, but is not limited to, a product of plant fermentation; and / or

[0034] The product includes, but is not limited to, one or more of pharmaceuticals, health foods, foods for special medical purposes, functional foods, foods, feeds, feed additives, or pet foods; and / or

[0035] The dosage forms of the product include, but are not limited to, one or more of powders, granules, capsules, tablets, pills, extracts, liquid preparations, sugars, or jellies; and / or

[0036] The sugars include, but are not limited to, gummy candies and / or compressed tablets; and / or

[0037] The plant fermentation includes, but is not limited to, one or more of traditional Chinese medicine fermentation, fruit and vegetable fermentation, or novel food ingredient fermentation;

[0038] The traditional Chinese medicine fermentation includes, but is not limited to, the fermentation of traditional Chinese medicines that are both medicine and food;

[0039] The traditional Chinese medicines include, but are not limited to, one or more of American ginseng, wolfberry, or Chinese yam.

[0040] The present invention also provides the Lactiplantibacillus paracasei LP-K2102; and / or

[0041] The product; and / or

[0042] The application of the product in any of the following:

[0043] (1) Preparing a product for inhibiting the growth of harmful bacteria; and / or

[0044] (2) Preparing a product for promoting the growth of beneficial bacteria; and / or

[0045] (3) Preparing a product for regulating the balance of the flora; and / or

[0046] (4) Preparing an antioxidant product; and / or

[0047] (5) Improving the performance of the product in plant fermentation; and / or

[0048] (6) Preparing a drug or preparation for preventing and / or treating diseases; and / or

[0049] (7) Preparing a health care product.

[0050] In some specific embodiments of the present invention, the harmful bacteria include, but are not limited to, Staphylococcus aureus and / or Escherichia coli; and / or

[0051] The beneficial bacteria include, but are not limited to, Bifidobacterium and / or Lactobacillus; and / or

[0052] The described microbial balance includes, but is not limited to, one or more of intestinal microbiota, vaginal microbiota, skin microbiota, oral microbiota, gastric microbiota, or urinary tract microbiota; and / or

[0053] The described antioxidant includes, but is not limited to, increasing the activity of superoxide dismutase; and / or

[0054] The described improvement of the product performance of plant fermentation includes, but is not limited to, improving safety and / or enhancing immune function; and / or

[0055] The described prevention and / or treatment of diseases includes, but is not limited to, one or more of digestive system diseases, locomotor system diseases, respiratory system diseases, urinary system diseases, reproductive system diseases, circulatory system diseases, endocrine system diseases, nervous system diseases, or immune system diseases; and / or

[0056] The described health care includes, but is not limited to, one or more of nutritional care, maintaining physical health, relieving hangover and protecting the liver, nourishing the stomach, nourishing the lungs, beauty care, ovarian maintenance, promoting the intestinal development and maturation of children, digestion and absorption, nutritional care, or growth and development;

[0057] The described digestive system diseases include, but are not limited to, one or more of spleen deficiency, diarrhea, irritable bowel syndrome, ulcerative colitis, Crohn's disease, antibiotic-associated diarrhea, pseudomembranous colitis, secondary diarrhea of pediatric pneumonia, intestinal cancer, adjuvant treatment for tumor radiotherapy and chemotherapy, abdominal distension and indigestion, constipation, foul-smelling stools, hepatobiliary diseases, viral diarrhea, feeding intolerance, necrotizing enterocolitis, jaundice, oral ulcers, thrush, bad breath, or nutritional absorption disorder diseases;

[0058] The described hepatobiliary diseases include, but are not limited to, liver diseases;

[0059] Preferably, the described hepatobiliary diseases include, but are not limited to, one or more of fatty liver, hepatitis, cirrhosis, or liver cancer;

[0060] Preferably, the described hepatobiliary diseases include, but are not limited to, alcoholic liver disease and / or non-alcoholic liver disease;

[0061] Preferably, the described hepatobiliary diseases include, but are not limited to, one or more of alcoholic fatty liver, alcoholic hepatitis, non-alcoholic fatty liver, or non-alcoholic steatohepatitis; and / or

[0062] The described locomotor system diseases include, but are not limited to, one or more of arthritis, osteoporosis, or rickets;

[0063] Preferably, the described locomotor system diseases include, but are not limited to, one or more of degenerative arthritis, rheumatoid arthritis, or ankylosing spondylitis; and / or

[0064] The respiratory diseases include, but are not limited to, one or more of moistening the lungs to relieve cough, lung nodules, pneumonia, coronavirus disease 2019, coronavirus infection, recurrent respiratory tract infection, or antiviral infection; and / or

[0065] The urinary system diseases include, but are not limited to, kidney deficiency;

[0066] Preferably, the urinary system diseases include, but are not limited to, kidney yin deficiency and / or kidney yang deficiency; and / or

[0067] The reproductive system diseases include, but are not limited to, one or more of vaginitis, uterine fibroids, dysmenorrhea, breast nodules, cold uterus, breast cancer, or menopausal syndrome; and / or

[0068] The circulatory system diseases include, but are not limited to, one or more of cardiovascular and cerebrovascular system diseases, hyperlipidemia, lipid-lowering, or hypertension; and / or

[0069] The endocrine system diseases include, but are not limited to, one or more of hyperglycemia, abnormal blood glucose, diabetes, hyperuricemia, gout, or thyroid nodules; and / or

[0070] The nervous system diseases include, but are not limited to, one or more of insomnia, relieving emotions, abnormal sleep, headache, regulating snoring, refreshing, depression, or anxiety; and / or

[0071] The immune system diseases include, but are not limited to, one or more of allergic diseases, allergy, skin diseases, enhancing immunity, or allergic rhinitis; and / or

[0072] Maintaining good health includes, but is not limited to, one or more of removing dampness, supplementing qi, supplementing yang, supplementing blood, supplementing yin, antioxidant, detoxification, anti-aging, weight loss, eye care, relieving eye fatigue, nourishing, weight management, brain strengthening, maintaining brain health, oral health, removing oil after meals, brain tonifying, hair care, hair nourishing, or blackening hair; and / or

[0073] Beauty care includes, but is not limited to, one or more of anti-glycation, moisturizing, anti-aging, or whitening.

[0074] The present invention also provides a drug or drug combination, comprising any of the following, any other active ingredient, and an acceptable excipient:

[0075] (a), the Lacticaseibacillus paracasei LP-K2102; and / or

[0076] (b), the product; and / or

[0077] (c), the product.

[0078] The present invention also provides a method for preventing and / or treating a disease, including administering to a subject any of the following:

[0079] (a), the Lacticaseibacillus paracasei LP-K2102; and / or

[0080] (b), the said product; and / or

[0081] (c), the said product; and / or

[0082] (d), the said drug or drug combination.

[0083] The present invention includes but is not limited to providing the following beneficial effects:

[0084] Aiming at the problems of unsatisfactory functions of existing probiotic strains such as biological activity, acid resistance, bile salt resistance, antioxidant ability, plant fermentation, and regulation of flora, the present invention provides a strain of Lacticaseibacillus paracasei LP-K2102 with comprehensive performance superior to that of Lacticaseibacillus paracasei in the market, and its preservation number is CGMCC No. 28936. The Lacticaseibacillus paracasei LP-K2102 of the present invention has high biological activity, acid resistance, bile salt resistance, strong antioxidant ability, strong plant fermentation ability, and the function of regulating the balance of flora, can improve the plant fermentation ability and the function of the product after fermentation, and its comprehensive performance is superior to other Lacticaseibacillus paracasei screened independently by the applicant and Lacticaseibacillus paracasei sold in the market.

[0085] Biological preservation description

[0086] Biological material: LP-K2102, classified and named: Lacticaseibacillus paracasei, was preserved in the General Microbiology Center of the China Microbial Culture Collection Center on November 10, 2023. The address is: No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the preservation number is CGMCC No. 28936.

[0087] The LP-K2102 described in the present invention is the strain with the preservation number of CGMCC No. 28936 as above. Detailed implementation manners

[0088] The present invention discloses the use of a novel Lactobacillus paracasei strain for traditional Chinese medicine fermentation and its applications. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve the same. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes, or appropriate alterations and combinations, to the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0089] Based on the above deficiencies and requirements existing in the prior art in this field, the object of the present invention is to provide a novel Lactobacillus paracasei LP-K2102, which is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, deposit number: CGMCC No. 28936. This strain of Lactobacillus paracasei is superior to the Lactobacillus paracasei strains already on the market and other Lactobacillus paracasei strains screened by the inventors in terms of biological activity, the ability to tolerate gastric acid and bile salts, inhibiting harmful bacteria, promoting beneficial bacteria and regulating the balance of the flora, antioxidant activity, plant fermentation ability, and the functions of the products after fermentation.

[0090] The regulation of the balance of the flora includes the flora of the human body such as the intestinal flora, vaginal flora, skin flora, oral flora, gastric flora, and urinary tract flora.

[0091] The inhibition of bacteria includes inhibiting Staphylococcus aureus, Escherichia coli, etc.

[0092] The antioxidant activity includes increasing the activity of superoxide dismutase (SOD);

[0093] The plant fermentation ability includes traditional Chinese medicine fermentation, fruit and vegetable fermentation, new food ingredient fermentation, etc. Traditional Chinese medicine fermentation further includes the fermentation of traditional Chinese medicines that are both medicine and food.

[0094] The improvement of the functions of plant fermentation products refers to improving the functions of the above plants through the above-mentioned plant fermentation, and further includes improving safety and enhancing immune functions.

[0095] A microecological product, probiotic product, or postbiotic product includes an active ingredient; the active ingredient includes Lactobacillus paracasei LP-K2102, deposit number: CGMCC No. 28936. The above-mentioned microecological product, probiotic product, or postbiotic product also includes: excipients; the excipients are selected from pharmaceutical excipients or food excipients; the products include drugs, health foods, foods for special medical purposes, foods, feeds, feed additives, pet foods, etc.; the dosage forms of the products include powders, granules, capsules, tablets, pills, extracts, oral liquids, candies, jellies, and candies further include soft candies and compressed tablets.

[0096] A plant fermentation product, the fermentation strains used at least include Lactobacillus paracasei LP-K2102, deposit number: CGMCC No. 28936. The plant fermentation product further includes: excipients; the excipients are selected from pharmaceutical excipients or food excipients; the products include drugs, health foods, foods for special medical purposes, foods, feeds, feed additives, pet foods, etc.; the dosage forms of the products include powders, granules, capsules, tablets, pills, extracts, oral liquids, sugars, jellies, and the sugar further includes soft candies and compressed tablets.

[0097] The Lactobacillus paracasei LP-K2102 described in the present invention includes live bacterial cells, inactivated bacterial cells, metabolites, etc.

[0098] Unless otherwise specified, the raw materials and reagents used in the fermentation of traditional Chinese medicine by the novel Lactobacillus paracasei strain and its application provided by the present invention can be purchased from the market.

[0099] The present invention will be further described below in conjunction with embodiments:

[0100] Preparation Example Preparation of Bacterial Powder and Powder

[0101] Preparation Example Explanation: Here, the powder of Lactobacillus paracasei LP-K2102 is taken as an example for specific description. Those skilled in the art can easily master the preparation methods of other Lactobacillus paracasei LP-K2102 preparations through this embodiment, and those skilled in the art can easily master the preparation methods of other dosage forms through this embodiment, and will not be described one by one here. The preparation method is not limited to that described in the embodiments of the present invention, and any well-known method that can achieve the preparation purpose can be used. The preparation description of the embodiments is only an explanation of the present invention, not a limitation on the protection scope of the present invention.

[0102] 1 Preparation of Bacterial Powder

[0103] The seed liquid of Lactobacillus paracasei LP-K2102 was cultured for 16 h, and the OD was measured at 600 nm to be 5.0. It was inoculated into MRS liquid medium at an inoculation amount of 1% by volume ratio. After culturing at 37 °C for 24 h, the obtained culture solution was centrifuged to separate the bacterial cells, and then the bacterial cells were freeze-dried under vacuum to prepare a dry bacterial powder.

[0104] 2 Preparation of Dosage Forms Such as Powder

[0105] According to the above steps, the Lactobacillus paracasei LP-K2102 was made into a bacterial powder, and then relevant excipients were added as needed to make various dosage forms. Preferably, according to the viable count of the Lactobacillus paracasei LP-K2102 bacterial powder, a powder was made so that the viable count was not less than 1×10 7 CFU / g, and then bagged.

[0106] Example 1 Biological Activity Experiment

[0107] Experimental method: Lactobacillus paracasei LP-K2102, Lactobacillus paracasei - self-screened 1, Lactobacillus paracasei - self-screened 2, Lactobacillus paracasei - commercially available 1, and Lactobacillus paracasei - commercially available 2 strains were separately activated in MRS liquid medium for 7 h, diluted with sterile water to an OD value of 1.0, inoculated into 100 mL of MRS liquid medium at an inoculation amount of 1%, and statically cultured at 37 °C for 16 - 24 hours (3 parallels), and samples were taken at the 24th hour to detect the OD value. The results are shown in Table 1.

[0108] Table 1 OD values of the biological activities of strains

[0109]

[0110] Conclusion: After statistical analysis, there was a highly significant difference between the Lactobacillus paracasei LP-K2102 group and other groups (P < 0.01). The OD value of Lactobacillus paracasei LP-K2102 after 24 h of culture was higher than that of other strains, indicating that the biological activity of Lactobacillus paracasei LP-K2102 was higher than that of other strains.

[0111] Example 2 Acid tolerance experiment

[0112] Experimental method: The pH values of MRS liquid medium were adjusted to 2.0 and 3.0 with 1 mol / L HCl respectively; sterilized at 121 °C for 15 min. Lactobacillus paracasei LP-K2102, Lactobacillus paracasei - self-screened 1, Lactobacillus paracasei - self-screened 2, Lactobacillus paracasei - commercially available 1, and Lactobacillus paracasei - commercially available 2 strains were separately activated in MRS liquid medium for 16 h, diluted with sterile water to an OD value of 5.0, and the inoculation amount was 2% to make the initial viable bacteria count in the medium the same. Incubated at a constant temperature of 37 °C, samples were taken at 0 h and 4 h to measure the OD value. Among them, the survival rate of the strain (%) = Nt / N0 × 100%, where N0 represents the OD value of the strain at 0 h and Nt represents the OD value of the viable bacteria count of the strain at 4 h. The results are shown in Table 2 and Table 3.

[0113] Table 2 Data table of strains under the condition of pH 2.0

[0114]

[0115]

[0116] Table 3 Data table of strains under the condition of pH 3.0

[0117]

[0118] Conclusion: Through statistical analysis, the difference between the LP-K2102 group of Lactobacillus paracasei and other groups was extremely significant (P<0.01). 1. Under the condition of pH2.0, the survival rates of the 5 strains of Lactobacillus paracasei all decreased to varying degrees with the extension of time. Among them, the survival rate of Lactobacillus paracasei LP-K2102 was the highest, indicating that it had the strongest acid tolerance under the condition of pH2.0. 2. Under the condition of pH3.0, the survival rate of Lactobacillus paracasei LP-K2102 was the highest, indicating that it had the strongest acid tolerance under the condition of pH3.0.

[0119] Example 3 Experiment on bile salt tolerance

[0120] Experimental method: After weighing the porcine bile salt, it was stirred and dissolved with pure water and added to MRS broth to prepare a bile salt liquid medium with a concentration of 0.3%. Lactobacillus paracasei LP-K2102, Lactobacillus paracasei - self-screened 1, Lactobacillus paracasei - self-screened 2, Lactobacillus paracasei - commercially available 1, and Lactobacillus paracasei - commercially available 2 strains were activated in MRS liquid medium for 16 h. The OD value of the seed liquid was diluted to 5.0, and inoculated into the bile salt liquid medium containing 0.3% at an inoculation amount of 2%, and cultured at 37°C for 4 h. Samples were taken at 0 h and 4 h to measure OD, and the survival rate was statistically analyzed. Among them, the survival rate of the strain (%) = Nt / N0×100%, where N0 represents the OD value of the strain at 0 h, and Nt represents the OD value of the viable bacteria of the strain at 4 h. The results are shown in Table 4.

[0121] Table 4 Survival rates of strains under the condition of 0.3% bile salt

[0122]

[0123] Conclusion: Through statistical analysis, the difference between the LP-K2102 group of Lactobacillus paracasei and other groups was extremely significant (P<0.01). Under the condition of 0.3% bile salt, the survival rates of the 5 strains of Lactobacillus paracasei all decreased. Among them, the survival rate of Lactobacillus paracasei LP-K2102 was the highest, indicating that this strain had the strongest tolerance to 0.3% bile salt and could quickly adapt to high bile salt concentrations.

[0124] Example 4 Experiment on the detection of superoxide dismutase (SOD) activity

[0125] Experimental method:

[0126] (I) Cell culture RAW264.7 cells were added to a sterile culture dish, and the number of cells was 5*10 5 cells / dish, and cultured in a cell incubator at 37°C and 5% CO2 for 24 h.

[0127] (2) Cell model establishment: Discard the old culture medium, add 0.8 mmol / L H2O2 solution to the culture dish to damage the cells. The control group is added with cell culture medium (DMEM high-glucose medium + 10% fetal bovine serum; Manufacturer of DMEM high-glucose medium: Bioss, product number C7076; Manufacturer of fetal bovine serum: Nanjing Senbeijia Biotechnology Co., Ltd., product number BC-SE-FBS07), 3 ml per culture dish, and culture in a 37 °C, 5% CO2 cell incubator for 24 h.

[0128] (3) Drug addition treatment: Discard the old culture medium. The control group and the H2O2 group are added with cell culture medium, and the experimental group is added with the supernatant of the fermentation broth diluted 25 times, 3 ml per culture dish, and culture in a 37 °C, 5% CO2 cell incubator for 24 h.

[0129] Among them:

[0130] The supernatant of the fermentation broth is obtained by fermenting Lactobacillus casei LC-K0313, Lactobacillus casei - self-screened, Lactobacillus casei - commercially available group 1, Lactobacillus casei - commercially available group 2, and Lactobacillus casei - commercially available group 3. The specific preparation method is as follows: Take the preservation tubes of Lactobacillus casei LC-K0313, Lactobacillus casei - self-screened, Lactobacillus casei - commercially available 1, Lactobacillus casei - commercially available 2, and Lactobacillus casei - commercially available 3 and melt them at room temperature, inoculate them into the MRS seed medium at an inoculation amount of 1% and activate for 16 h, dilute the OD value to 5 with sterile water, inoculate at an inoculation amount of 1% into 100 mL of MRS liquid medium, statically culture in a 37 °C incubator for 24 hours, place it in an autoclave and sterilize at 115 °C for 30 min, and after cooling, centrifuge at 8000 rpm for 10 minutes with a tabletop centrifuge to obtain the fermentation supernatant.

[0131] (4) Sample treatment: Discard the old culture medium, add 1 ml of trypsin (Manufacturer: EMD MilliporeCorp, product number: 3788142) to each culture dish, after enzymatic digestion for 2 minutes, collect the cells into a centrifuge tube with a cell scraper, add the extraction solution and ultrasonically crush (power 200 W, ultrasonic treatment for 20 minutes), centrifuge at 8000 rpm and 4 °C for 10 minutes to obtain the sample, and place it on ice for further measurement.

[0132] (V) Sample determination According to the instructions of the Superoxide Dismutase (SOD) Activity Detection Kit (Beijing Solarbio Science & Technology Co., Ltd., product number: BC0170), add 70 μL of the sample, 45 μL of Reagent 1, 20 μL of Reagent 2, 35 μL of Reagent 3, 20 μL of distilled water, and 10 μL of Reagent 4 into a 96-well plate in sequence, mix well, incubate in a water bath at 37 °C for 30 min, measure the absorbance A at a wavelength of 560 nm, and record it as A determination; in the control group, Reagent 2 is not added, and distilled water is used to make up the volume, and the remaining operations are the same as those of the experimental group, and the absorbance is recorded as A control; in Blank Group 1, the supernatant of the fermentation broth is replaced with distilled water, and the remaining operations are the same as those of the experimental group, and the absorbance is measured and recorded as A1 blank; in Blank Group 2, the supernatant of the fermentation broth is replaced with distilled water, Reagent 2 is not added, and distilled water is used to make up the volume, and the remaining operations are the same as those of the experimental group, and the absorbance is measured and recorded as A2 blank.

[0133] ΔA determination = A determination - A control, ΔA blank = A1 blank - A2 blank.

[0134] Inhibition percentage = (ΔA blank - ΔA determination) / ΔA blank × 100%.

[0135] SOD activity (U / mg prot) = 10 × inhibition percentage / (1 - inhibition percentage) / Cpr × F, where Cpr is the protein sample concentration, mg / ml; F is the sample dilution factor.

[0136] Improvement rate = (SOD of Lactobacillus paracasei LP-K2102 group - SOD of H2O2 group) / SOD of H2O2 group * 100%. The results are shown in Table 5.

[0137] Table 5 SOD Activity Detection

[0138]

[0139] Conclusion: After statistical analysis, there was a highly significant difference between the Lactobacillus paracasei LP-K2102 group and other groups (P < 0.01). The experimental data showed that the Superoxide Dismutase (SOD) activity of the fermentation broth of Lactobacillus paracasei LP-K2102 was the highest, indicating that this strain had the strongest antioxidant ability.

[0140] Example 5 Antibacterial Ability Experiment

[0141] Experimental method: Take the preservation tubes of Lactobacillus paracasei LP-K2102, Lactobacillus paracasei - self-screened 1, Lactobacillus paracasei - self-screened 2, Lactobacillus paracasei - commercially available 1, and Lactobacillus paracasei - commercially available 2, and inoculate them into MRS broth at an inoculation amount of 1% and activate them at 37 °C for 16 h to obtain the activated bacterial solution. Pour 20 mL of MRS agar medium into each plate, take 100 μL of the test bacterial solution (Escherichia coli, Staphylococcus aureus) for gradient dilution, and the concentration of the diluted bacterial solution is 105- 6 About cfu / ml is sufficient. Respectively, take 100 μL of the diluted bacterial solution and drop it on the solid plate, and spread it with a spreading rod, and set it aside upright. Gently place the sterilized Oxford cup on the surface of the plate medium (3 per plate). After standing for 5 min, suck 100 μL of the activated bacterial solution and add it to the Oxford cup. After the bacterial solution has fully diffused for 2 h, transfer the plate to a 37 °C incubator and culture for 24 - 72 h. Observe the phenomenon at regular intervals, measure the diameter of the inhibition zone with a ruler, and take pictures for record. The results are shown in Table 6.

[0142] Table 6 Data of the diameter of the inhibition zone of strains against Staphylococcus aureus and Escherichia coli

[0143]

[0144] Conclusion: Through statistical analysis, the difference between the Lactobacillus paracasei LP-K2102 group and other groups was extremely significant (P < 0.01). 1. By comparing the diameters of the inhibition zones of 5 strains of Lactobacillus paracasei against Staphylococcus aureus, it was shown that Lactobacillus paracasei LP-K2102 had the strongest ability to inhibit Staphylococcus aureus. 2. By comparing the diameters of the inhibition zones of 5 strains of Lactobacillus paracasei against Escherichia coli, it was shown that Lactobacillus paracasei LP-K2102 had the strongest ability to inhibit Escherichia coli.

[0145] Example 6 Fermentation of traditional Chinese medicine experiment

[0146] Experimental method: According to the material-liquid ratio of American ginseng to water being 8:100, wolfberry to water being 8:100, and Chinese yam to water being 15:100, add water and boil for extraction for 1 - 2 h respectively. Filter and collect the supernatant, add 4% (W / V) crystalline fructose, sterilize at 121 °C for 30 min after packaging. Respectively, take the strains Lactobacillus paracasei LP-K2102, Lactobacillus paracasei - self-screened 1, Lactobacillus paracasei - self-screened 2, Lactobacillus paracasei - commercially available 1, Lactobacillus paracasei - commercially available 2 and activate them in MRS broth at 37 °C for 16 h. Inoculate according to an inoculation amount of 2%, and culture at 37 °C for 48 h. Sample at 24 h to detect the OD value and pH value. The results are shown in Tables 7 - 9.

[0147] Table 7 Experimental data of fermented American ginseng

[0148]

[0149] Conclusion: Through statistical analysis, the difference between the Lactobacillus paracasei LP-K2102 group and other groups was extremely significant (P < 0.01). The experimental results showed that when fermenting American ginseng with Lactobacillus paracasei LP-K2102 for 24 h to 48 h, the OD value was the highest and the pH value was the lowest, indicating that this strain grew best and produced the most acid in the American ginseng fermentation broth.

[0150] Table 8 Experimental data of fermented wolfberry

[0151]

[0152]

[0153] Conclusion: Through statistical analysis, the difference between the Lactobacillus paracasei LP-K2102 group and other groups was extremely significant (P<0.01). The experimental results showed that when fermenting wolfberry with Lactobacillus paracasei LP-K2102 for 24h to 48h, the OD value was the highest and the pH value was the lowest, indicating that this bacterium grew best and produced the most acid in the wolfberry fermentation broth.

[0154] Table 9 Experimental data of fermented yam

[0155]

[0156] Conclusion: Through statistical analysis, the difference between the Lactobacillus paracasei LP-K2102 group and other groups was extremely significant (P<0.01). The experimental results showed that when fermenting yam with Lactobacillus paracasei LP-K2102 for 24h to 48h, the OD value was the highest and the pH value was the lowest, indicating that this bacterium grew best and produced the most acid in the yam fermentation broth.

[0157] Example 7 Immunity-enhancing experiment

[0158] Experimental method:

[0159] (1) Cell culture RAW264.7 cells were added to a 96-well plate, with 1×10 5 cells per well, and cultured at 37°C under 5% CO2 for 16h.

[0160] (2) Cell modeling 0.8 mmol / L H2O2 solution was added to the 96-well plate to damage the cells. The control group was added with cell culture medium (DMEM high-glucose medium + 10% fetal bovine serum; manufacturer of DMEM high-glucose medium: Bioss, product number C7076; manufacturer of fetal bovine serum: Nanjing Senbeijia Biotechnology Co., Ltd., product number BC-SE-FBS07), and cultured at 37°C under 5% CO2 for 24h.

[0161] (3) Drug addition treatment The supernatant of the fermented American ginseng solution was diluted 40 times and then added to the 96-well plate, 200 μl per well. The H2O2 group and the control group were added with cell culture medium, and cultured at 37°C under 5% CO2 for 24h.

[0162] (4) Neutral red staining, lysis, and detection: After discarding the culture medium, add neutral red staining solution at a concentration of 10% to each well and continue culturing for 4 h. Aspirate the neutral red staining solution, add 150 μL / well of neutral red lysis solution, mix well for 10 min, and detect at a wavelength of 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Phagocytosis index = OD value of the experimental group / OD value of the control group, and the improvement rate (%) = (phagocytosis index of the experimental group - phagocytosis index of the H2O2 group) / phagocytosis index of the H2O2 group × 100%. The results are shown in Table 10:

[0163] Among them:

[0164] The supernatant of the fermented American ginseng liquid was obtained by fermenting the American ginseng extract with Lactobacillus casei LC-K0313, Lactobacillus casei - self-screened, Lactobacillus casei - commercially available 1 group, Lactobacillus casei - commercially available 2 group, and Lactobacillus casei - commercially available 3 group. The specific preparation method is as follows: Extract according to the ratio of the material-liquid ratio of American ginseng to water of 8:100. Add 4% crystalline fructose to the extract by mass and sterilize at 115 °C for 30 minutes to obtain the American ginseng culture medium. Take the preservation tubes of Lactobacillus casei LC-K0313, Lactobacillus casei - self-screened, Lactobacillus casei - commercially available 1, Lactobacillus casei - commercially available 2, and Lactobacillus casei - commercially available 3 and melt them at room temperature. Inoculate them into the MRS seed medium at an inoculation amount of 1% and activate for 16 h. Inoculate the seed liquid into the American ginseng culture medium at an inoculation amount of 2% and statically culture in an incubator at 37 °C for 24 h. Place it in an autoclave and sterilize at 115 °C for 30 min. After cooling, centrifuge at 8000 rpm for 10 min using a tabletop centrifuge to obtain the supernatant of the fermented American ginseng liquid.

[0165] Table 10 Experimental data on the immune-enhancing effect of the fermented American ginseng liquid

[0166]

[0167] Conclusion: After statistical analysis, the difference between the Lactobacillus paracasei LP-K2102 group and other groups was significant (P < 0.05). Through the cell phagocytosis index experiment, it was proved that after fermenting American ginseng with Lactobacillus paracasei LP-K2102 for 24 h, the phagocytosis index of the fermented liquid was higher than that of the fermented liquids of other strains, indicating that after fermentation with this strain, the immunity of the fermented liquid was improved more than that of other strains.

[0168] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Lacticaseibacillus paracasei LP-K2102, characterized in that, Its preservation number is CGMCC No. 28936.

2. The fermentation broth of Lacticaseibacillus paracasei LP-K2102 as described in claim 1.

3. The application of Lacticaseibacillus paracasei LP-K2102 as described in claim 1 or the fermentation broth as described in claim 2 in the preparation of any of the following: (I), microecological products; (II), products for plant fermentation; The microecological products are probiotic products, synbiotics or postbiotic products; The products for plant fermentation are foods, feeds or feed additives; The foods are health foods, foods for special medical purposes, functional foods or pet foods; The dosage form of the microecological products or the products for plant fermentation is powder, granule, capsule, tablet, pill, extract or liquid preparation; The plant fermentation is traditional Chinese medicine fermentation, fruit and vegetable fermentation or new food raw material fermentation; The traditional Chinese medicine fermentation is the fermentation of traditional Chinese medicine with both edible and medicinal properties; The traditional Chinese medicine is American ginseng, wolfberry or Chinese yam.

4. A product, characterized in that, It includes Lacticaseibacillus paracasei LP-K2102 as described in claim 1 or the fermentation broth as described in claim 2; The products are any of the following: (I), microecological products; (II), products for plant fermentation; The microecological products are probiotic products, synbiotics or postbiotic products; The products for plant fermentation are foods, feeds or feed additives; The foods are health foods, foods for special medical purposes, functional foods or pet foods; The dosage form of the microecological products or the products for plant fermentation is powder, granule, capsule, tablet, pill, extract or liquid preparation; The plant fermentation is traditional Chinese medicine fermentation, fruit and vegetable fermentation or new food raw material fermentation; The traditional Chinese medicine fermentation is the fermentation of traditional Chinese medicine with both edible and medicinal properties; The traditional Chinese medicine is American ginseng, wolfberry or Chinese yam.

5. Lacticaseibacillus paracasei LP-K2102 as described in claim 1; The fermentation broth as described in claim 2; The application of the products as described in claim 4 in any of the following: (1), preparing products for inhibiting the growth of harmful bacteria; (2), preparing products for regulating the balance of flora; (3), preparing antioxidant products; (4), improving the performance of products for plant fermentation; The harmful bacteria are Staphylococcus aureus and / or Escherichia coli; The flora balance is the intestinal flora; The antioxidant is to increase the activity of superoxide dismutase; The improvement of the performance of products for plant fermentation is to enhance the immune function.

Images