Fermentation of traditional Chinese medicine using new strain of Bacillus casei and its application

Abstract

The present invention relates to the field of microbial technology, and in particular to fermentation of traditional Chinese medicine using a novel Lactobacillus casei strain and its application. The present invention provides Lactobacillus casei LC-K0313, with a deposit number of CGMCC No. 28935. The Lactobacillus casei LC-K0313 has high biological activity, is resistant to gastric acid and bile salts, has the functions of inhibiting harmful bacteria, promoting beneficial bacteria, regulating the balance of intestinal flora, resisting oxidation, preventing and treating diseases, and maintaining health, and can also improve the fermentation capacity of plants and the function of fermented products.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to traditional Chinese medicine fermentation using a novel Bacillus casei strain and application thereof. Background Art

[0002] Lactobacillus casei is a non-spore-forming Gram-positive probiotic. In August 2022, the National Health Commission issued an announcement to change the genus from Lactobacillus to Lactobacillus casei, which is mainly used in probiotic products and traditional Chinese medicine fermentation. The higher the biological activity of Lactobacillus casei, the higher the output of industrial production and the stronger the efficacy, further reducing production costs and improving production efficiency. Therefore, screening highly active Lactobacillus casei has become the goal of the industry.

[0003] Lactobacillus casei needs to have a strong ability to resist gastric acid and bile salts to ensure that it can effectively pass through the stomach and small intestine to reach the large intestine to exert its effect. The increase of harmful bacteria such as Escherichia coli will lead to dysbiosis and cause various diseases. If Lactobacillus casei has a good inhibitory effect on harmful bacteria, it will accelerate the restoration of bacterial balance. Superoxide anions are harmful compounds produced in the body's oxidation reactions, which can promote aging, disease and cause health problems. Antioxidants such as the removal of superoxide anions are beneficial to maintaining the health of the body.

[0004] Chinese medicines that can be used as food and medicines can be used in food due to their safety, but their efficacy is relatively not ideal. How to improve the efficacy of Chinese medicines that can be used as food and medicines has become a difficulty in the industry. The problem of low efficacy of Chinese medicines that can be used as food and medicines can be solved through Chinese medicine fermentation technology, but finding excellent strains for fermenting Chinese medicines is the key to Chinese medicine fermentation.

[0005] Existing Lactobacillus casei has low biological activity, weak stomach acid and bile salt resistance, weak function of inhibiting harmful bacteria and regulating flora balance, weak ability of removing superoxide anions and anti-oxidation, and low fermentation ability of plants such as Chinese medicine and the function of fermented products. There is no report on Lactobacillus casei strains that have significant performance in fermenting probiotics and herbal plants such as Chinese medicine. Summary of the invention

[0006] In view of this, the present invention provides a Chinese medicine fermentation using a novel Lactobacillus casei strain and its application. The present invention provides Lactobacillus casei LC-K0313, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration Committee, with a deposit number of CGMCC No. 28935. The Lactobacillus casei LC-K0313 is superior to the Lactobacillus casei strains that have been listed on the market and other Lactobacillus casei strains screened by the inventor in terms of biological activity, ability to resist gastric acid and bile salts, inhibition of harmful bacteria, promotion of beneficial bacteria and regulation of flora balance, antioxidant, plant fermentation ability and fermentation product function, disease prevention and treatment, and health care.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The invention provides Lactobacillus casei LC-K0313, whose deposit number is CGMCC No.28935.

[0009] In some specific embodiments of the present invention, the Lactobacillus casei LC-K0313 includes one or more of its live bacteria, inactivated bacteria, fermentation broth, exosomes or metabolites.

[0010] In some specific embodiments of the present invention, the Lactobacillus casei LC-K0313 has high biological activity, acid resistance and / or bile resistance.

[0011] In some specific embodiments of the present invention, the acid-resistant pH value is comprised between 2.0 and 3.0.

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

[0013] The present invention also provides the use of the Lactobacillus casei LC-K0313 in the preparation of any of the following items:

[0014] (I), microecological products, probiotic products, synbiotics and / or postbiotic products; 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 medicines, health foods, foods for special medical purposes, functional foods, foods, feeds, feed additives or pet foods; and / or

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

[0018] The sugars include but are not limited to soft candies and / or compressed candies; 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 raw material fermentation;

[0020] The Chinese medicine fermentation includes but is not limited to the Chinese medicine fermentation with medicinal and edible properties;

[0021] The Chinese medicine includes but is not limited to one or more of American ginseng, wolfberry or yam.

[0022] The present invention also provides a product, comprising the Lactobacillus casei LC-K0313;

[0023] The product comprises any of the following:

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

[0025] (II) Products for plant fermentation;

[0026] 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 medicines, health foods, foods for special medical purposes, functional foods, foods, feeds, feed additives or pet foods; and / or

[0027] The dosage form of the microecological product, probiotic product, synbiotic product, postbiotic product and / or the product for plant fermentation includes but is not limited to one or more of powder, granule, capsule, tablet, pill, extract, liquid preparation, sugar or jelly; and / or

[0028] The sugars include but are not limited to soft candies and / or compressed candies; 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 raw material fermentation;

[0030] The Chinese medicine fermentation includes but is not limited to the Chinese medicine fermentation with medicinal and edible properties;

[0031] The Chinese medicine includes but is not limited to one or more of American ginseng, wolfberry or yam.

[0032] The invention also provides a product, which is prepared by fermenting the Lactobacillus casei LC-K0313.

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

[0034] The products 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

[0035] The dosage form of the product includes but is not limited to one or more of powder, granules, capsules, tablets, pills, extracts, liquid preparations, sugar or jelly; and / or

[0036] The sugars include but are not limited to soft candies and / or compressed candies; 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 new food raw material fermentation;

[0038] The Chinese medicine fermentation includes but is not limited to the Chinese medicine fermentation with medicinal and edible properties;

[0039] The Chinese medicine includes but is not limited to one or more of American ginseng, wolfberry or yam.

[0040] The present invention also provides the Lactobacillus casei LC-K0313; and / or

[0041] The product; and / or

[0042] Application of the product in any of the following:

[0043] (I) preparing products that inhibit the growth of harmful bacteria; and / or

[0044] (II), preparing products that promote the growth of beneficial bacteria; and / or

[0045] (III) preparing products for regulating the balance of bacterial flora; and / or

[0046] (IV), preparing antioxidant products; and / or

[0047] (V) improving the performance of plant fermentation products; and / or

[0048] (VI), preparing drugs or preparations for preventing and / or treating diseases; and / or

[0049] (VII) Preparation of health care products.

[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 bifidobacteria and / or lactobacilli; and / or

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

[0053] The antioxidant includes but is not limited to increasing superoxide dismutase and scavenging free radicals; preferably, the scavenging free radicals includes but is not limited to scavenging DPPH free radicals; and / or

[0054] The improved performance of plant fermentation products includes but is not limited to improved safety and / or enhanced immunity; and / or

[0055] The diseases to be prevented and / or treated include but are 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 health care includes but is not limited to one or more of nutritional health care, maintaining physical health, sobering up and protecting the liver, nourishing the stomach, nourishing the lungs, nourishing the skin and beauty, nourishing the ovaries, promoting the development and maturation of the intestinal tract of children, digestion and absorption, nutritional health care or growth and development;

[0057] The digestive system diseases include, but are not limited to, spleen deficiency, diarrhea, irritable bowel syndrome, ulcerative colitis, Crohn's disease, antibiotic-associated diarrhea, pseudomembranous enterocolitis, secondary diarrhea of ​​pneumonia in children, intestinal cancer, adjuvant radiotherapy and chemotherapy for tumors, abdominal distension and indigestion, constipation, fecal odor, hepatobiliary diseases, viral diarrhea, feeding intolerance, necrotizing enterocolitis, jaundice, oral ulcers, thrush, bad breath, or one or more of nutrient absorption disorders;

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

[0059] Preferably, the hepatobiliary disease includes but is not limited to one or more of fatty liver, hepatitis, cirrhosis or liver cancer;

[0060] Preferably, the hepatobiliary disease includes but is not limited to alcoholic liver disease and / or non-alcoholic liver disease;

[0061] Preferably, the hepatobiliary disease includes but is not limited to one or more of alcoholic fatty liver, alcoholic hepatitis, non-alcoholic fatty liver or non-alcoholic steatohepatitis; and / or

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

[0063] Preferably, the musculoskeletal disease includes but is 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 and relieving cough, lung nodules, pneumonia, novel 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, uterine cold, 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 diseases, hyperlipidemia, fat loss or hypertension; and / or

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

[0070] The nervous system diseases include but are not limited to one or more of insomnia, mood relief, sleep disturbance, headache, snoring control, alertness, depression or anxiety; and / or

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

[0072] Maintaining physical health includes but is not limited to one or more of removing dampness, replenishing qi, replenishing yang, replenishing blood, replenishing yin, anti-oxidation, detoxification, anti-aging, weight loss, eye protection, relieving visual fatigue, nourishing, weight management, brain health, maintaining brain health, oral health, removing oil after meals, replenishing the brain, nourishing hair, hair care, and darkening hair; and / or

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

[0074] The present invention also provides a drug or drug combination, comprising any of the following items, any other active ingredients and acceptable excipients:

[0075] (I), Lactobacillus casei LC-K0313; and / or

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

[0077] (III) the product.

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

[0079] (I), Lactobacillus casei LC-K0313; and / or

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

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

[0082] (IV), the drug or drug combination.

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

[0084] In view of the problems that the existing probiotic strains have unsatisfactory functions such as biological activity, acid resistance, bile salt resistance, antioxidant, plant fermentation and flora regulation, the present invention provides a strain of Lactobacillus casei LC-K0313, which has comprehensive performance superior to that of Lactobacillus casei on the market, and is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, with a deposit number of CGMCC No. 28935. The Lactobacillus casei LC-K0313 is superior to the Lactobacillus casei strains on the market and other Lactobacillus casei strains screened by the inventor in terms of biological activity, gastric acid and bile salt resistance, inhibition of harmful bacteria, promotion of beneficial bacteria and regulation of intestinal flora balance, antioxidant, plant fermentation ability and fermentation product function, disease prevention and treatment, health care, etc.

[0085] Biological Deposit Description

[0086] Biological material: LC-K0313, classification name: Lacticaseibacillus casei, deposited on November 10, 2023 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No. 28935.

[0087] The LC-K0313 described in the present invention is the strain with the above-mentioned deposit number of CGMCC No.28935. DETAILED DESCRIPTION

[0088] The present invention discloses fermentation of traditional Chinese medicine using a novel strain of Bacillus casei and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0089] Based on the above-mentioned deficiencies and needs of the prior art in the art, the object of the present invention is to provide a novel Lactobacillus casei LC-K0313, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration Committee, and the deposit number is CGMCC No. 28935. The Lactobacillus casei strain is superior to the Lactobacillus casei strains that have been listed on the market and other Lactobacillus casei strains screened by the inventor in terms of biological activity, ability to resist gastric acid and bile salts, inhibition of harmful bacteria, promotion of beneficial bacteria growth and regulation of flora balance, anti-oxidation, plant fermentation ability and improvement of plant fermentation product function, disease prevention and health care, etc.

[0090] The regulation of flora balance includes human flora such as intestinal flora, vaginal flora, skin flora, oral flora, stomach flora, urinary system flora, etc.

[0091] The harmful bacteria inhibited include Staphylococcus aureus, Escherichia coli and the like.

[0092] The promoting the growth of beneficial bacteria includes promoting bifidobacteria, lactobacilli and the like.

[0093] The antioxidant comprises increasing superoxide dismutase (SOD), removing superoxide anions, and further removing superoxide anions (O 2 - );

[0094] The plant fermentation capacity includes fermentation of traditional Chinese medicine, fermentation of fruits and vegetables, fermentation of new food raw materials, etc. Traditional Chinese medicine fermentation further includes fermentation of traditional Chinese medicine that is both medicine and food.

[0095] Improving the functions of plant fermentation products refers to improving the functions of the above-mentioned plants by fermenting the above-mentioned plants, further including improving safety and enhancing immunity functions, etc.

[0096] Application of the above products in disease prevention and treatment and health care.

[0097] The disease prevention and treatment and health care include the treatment of digestive system diseases, musculoskeletal system diseases, respiratory system diseases, urinary system diseases, reproductive system diseases, circulatory system diseases, endocrine system diseases, nervous system diseases, immune system diseases, nutritional health care, maintaining physical health, sobering up and protecting the liver, nourishing the stomach, nourishing the lungs, nourishing the skin and beauty, maintaining ovaries, promoting the development and maturation of the intestines in children, digestion and absorption, nutritional health care or growth and development, etc.

[0098] The digestive system diseases include spleen deficiency, diarrhea, irritable bowel syndrome, ulcerative colitis, Crohn's disease, antibiotic-associated diarrhea, pseudomembranous enterocolitis, secondary diarrhea of ​​childhood pneumonia, intestinal cancer, adjuvant radiotherapy and chemotherapy for tumors, abdominal distension and indigestion, constipation, smelly stools, hepatobiliary diseases, viral diarrhea, feeding intolerance, necrotizing enterocolitis, jaundice, oral ulcers, thrush, bad breath, and nutritional absorption disorders.

[0099] The hepatobiliary diseases include liver diseases, further including fatty liver, hepatitis, cirrhosis or liver cancer, further including alcoholic liver disease or non-alcoholic liver disease, further including alcoholic fatty liver, alcoholic hepatitis, non-alcoholic fatty liver or non-alcoholic fatty hepatitis, etc.

[0100] The musculoskeletal diseases include arthritis, osteoporosis, rickets, and further include degenerative arthritis, rheumatoid arthritis, ankylosing spondylitis, etc.

[0101] The respiratory diseases include lung moistening and cough relieving, lung nodules, pneumonia, new coronavirus infection, recurrent respiratory tract infection, antiviral infection, etc.

[0102] The urinary system diseases include kidney deficiency, further including kidney yin deficiency and kidney yang deficiency, etc.

[0103] The reproductive system diseases include vaginitis, uterine fibroids, dysmenorrhea, breast nodules, uterine cold, breast cancer, menopausal syndrome, etc.

[0104] The circulatory system diseases include cardiovascular and cerebrovascular system diseases, hyperlipidemia, hypertension, lipid-lowering and the like.

[0105] The endocrine system diseases include hyperglycemia, dysglycemia, diabetes, hyperuricemia, hyperuricemia, gout, thyroid nodules, etc.

[0106] The nervous system diseases include insomnia, mood relief, sleep abnormalities, headaches, depression, snoring control, alertness, anxiety, etc.

[0107] The immune system diseases include allergic diseases, allergies, skin diseases, immune enhancement, allergic rhinitis, etc.

[0108] The said maintaining physical health includes removing dampness, replenishing qi, replenishing yang, replenishing blood, replenishing yin, anti-oxidation, detoxification, anti-aging, weight loss, eye protection, relieving eyestrain, nourishing, weight management, brain health, maintaining brain health, oral health, removing oil after meals, nourishing the brain, nourishing hair, hair care, black hair, etc.

[0109] The beauty and skin care products include anti-sugar, moisturizing, anti-aging, whitening and the like.

[0110] The present invention also provides a microecological product, a probiotic product or a postbiotic product, comprising an active ingredient; the active ingredient comprises Lactobacillus casei LC-K0313, and the deposit number is CGMCC No. 28935. The microecological product, probiotic product or postbiotic product also comprises: an auxiliary material; the auxiliary material is selected from a pharmaceutical auxiliary material or an edible auxiliary material; the product comprises medicines, health foods, special medical purpose foods, foods, feeds, feed additives, pet foods, etc.; the dosage form of the product comprises powders, granules, capsules, tablets, pills, extracts, oral liquids, sugars, and jellies, and the sugars further comprise soft candies and compressed candies.

[0111] The present invention also provides a plant fermentation product, wherein the fermentation strain used contains at least Lactobacillus casei LC-K0313, and the deposit number is CGMCC No. 28935. The plant fermentation product also includes: auxiliary materials; the auxiliary materials are selected from pharmaceutical auxiliary materials or edible auxiliary materials; the product includes medicines, health foods, special medical purpose foods, foods, feeds, feed additives, pet foods, etc.; the dosage form of the product includes powders, granules, capsules, tablets, pills, extracts, oral liquids, sugars, and jelly, and the sugars further include soft candies and compressed candies.

[0112] The Lactobacillus casei LC-K0313 of the present invention includes living bacteria, inactivated bacteria or metabolites and the like.

[0113] Unless otherwise specified, the raw materials and reagents used in the Chinese medicine fermentation using the novel Lactobacillus casei strain and its application provided by the present invention can all be purchased from the market.

[0114] The present invention will be further described below in conjunction with embodiments:

[0115] Preparation Example Preparation of bacterial powder and powder

[0116] Preparation example description: Here, Lactobacillus casei LC-K0313 powder is used as an example to specifically describe. The preparation methods of other Lactobacillus casei LC-K0313 preparations are easy to grasp by those skilled in the art through this embodiment, and the preparation methods of other dosage forms are easy to grasp by those skilled in the art through this implementation, and are no longer described one by one here. The preparation method is not limited to the embodiments of the present invention, and the known methods that can achieve the preparation purpose are all acceptable. The preparation instructions of the embodiments are only explanations of the present invention, and are not limitations on the scope of protection of the present invention.

[0117] 1 Preparation of bacterial powder

[0118] The seed liquid of Lactobacillus casei LC-K0313 was cultured for 16 hours, and the OD 5.0 ​​was measured at 600 nm. The liquid was inoculated into MRS liquid culture medium at a volume ratio of 1%, and cultured at 37° C. for 24 hours. The obtained culture liquid was centrifuged to separate the bacteria, and the bacteria were freeze-dried in a vacuum to prepare dry bacterial powder.

[0119] 2. Preparation into powder and other dosage forms

[0120] The Lactobacillus casei LC-K0313 is made into bacterial powder, and then relevant auxiliary materials are added as needed to make various dosage forms. Preferably, the powder is made according to the number of live bacteria of the Lactobacillus casei LC-K0313 bacterial powder, so that the number of live bacteria is not less than 1×10 7 CFU / g and then bagged.

[0121] Example 1 Biological Activity Experiment

[0122] Experimental method: Lactobacillus casei LC-K0313, Lactobacillus casei-self-screening, Lactobacillus casei-commercial 1, Lactobacillus casei-commercial 2, Lactobacillus casei-commercial 3 were activated in MRS liquid medium for 8 hours, diluted with sterile water to an OD value of 1.0, inoculated into 100mL of MRS liquid medium at a 1% inoculum, and cultured at 37°C for 24 hours (3 parallels), and sampled and tested the OD value at the 24th hour. The results are shown in Table 1.

[0123] Table 1 OD values ​​of bacterial biological activity

[0124]

[0125] Conclusion: The difference between the Lactobacillus casei LC-K0313 group and other groups (P<0.01) was extremely significant. The OD value of Lactobacillus casei LC-K0313 after 24h cultivation was higher than that of other strains, indicating that the biological activity of Lactobacillus casei LC-K0313 was higher than that of other strains.

[0126] Example 2 Acid resistance test

[0127] Experimental method: The pH value of MRS liquid medium was adjusted to 2.0 and 3.0 respectively with 1mol / L HCL; sterilized at 121℃ for 15min, and activated Lactobacillus casei LC-K0313, Lactobacillus casei-self-screening, Lactobacillus casei-commercial 1, Lactobacillus casei-commercial 2, and Lactobacillus casei-commercial 3 in MRS liquid medium for 16h respectively, and diluted with sterile water to OD value of 5.0, and the inoculation amount was 2%, so that the initial viable count in the medium was the same, and cultured at 37℃ constant temperature, and the OD value was measured at 0h and 4h, wherein the survival rate of the strain (%)=Nt / N0×100%, wherein N0 represents the OD value of the strain at 0h, and Nt represents the OD value of the viable count of the strain at 4h. The results are shown in Tables 2 and 3.

[0128] Table 2 Data of bacterial strains under pH 2.0 conditions

[0129]

[0130] Table 3 Data of bacterial strains under pH 3.0 conditions

[0131]

[0132] Conclusion: Statistical analysis showed that the Lactobacillus casei LC-K0313 group was significantly different from the other groups (P<0.01).

[0133] 1. Under pH 2.0 conditions, the survival rates of the five strains of Lactobacillus casei decreased to varying degrees with the extension of time, among which Lactobacillus casei LC-K0313 had the highest survival rate, indicating that it had the strongest acid resistance under pH 2.0 conditions. 2. Under pH 3.0 conditions, the survival rate of Lactobacillus casei LC-K0313 was the highest, indicating that it had the strongest acid resistance under pH 3.0 conditions.

[0134] Example 3 Bile salt tolerance test

[0135] Experimental method: After weighing the pig bile salt, stir and dissolve it with pure water, add it to MRS broth to prepare a bile salt liquid culture medium with a concentration of 0.3%. Take Lactobacillus casei LC-K0313, Lactobacillus casei-self-screening, Lactobacillus casei-commercially available 1, Lactobacillus casei-commercially available 2, and Lactobacillus casei-commercially available 3 strains and activate them in MRS liquid culture medium for 16 hours to obtain seed liquid. The OD value of the seed liquid was diluted to 5.0, and inoculated into a liquid culture medium containing 0.3% bile salt at an inoculum volume of 2%, and cultured at 37°C for 4 hours. Samples were taken at 0h and 4h to measure OD, and the survival rate was calculated. Among them, the survival rate of the strain (%) = Nt / N0×100%, where N0 represents the OD value of the strain at 0h, and Nt represents the OD value of the number of viable bacteria of the strain at 4h. The results are shown in Table 4.

[0136] Table 4 Survival rate of bacteria under 0.3% bile salt conditions

[0137]

[0138] Conclusion: The statistical analysis showed that the difference between the Lactobacillus casei LC-K0313 group and other groups was extremely significant (P<0.01). Under the condition of 0.3% bile salt, the survival rate of the five strains of Lactobacillus casei decreased, among which Lactobacillus casei LC-K0313 had the highest survival rate, indicating that this bacterium has the strongest ability to tolerate 0.3% bile salt and can quickly adapt to high bile salt concentrations.

[0139] Example 4 Superoxide dismutase (SOD) activity detection experiment

[0140] Experimental methods:

[0141] (I) Cell culture RAW264.7 cells were added to a sterile culture dish, with a cell number of 5*10 5 pcs / dish, 37℃, 5% CO 2 Culture in a cell culture incubator for 24 h.

[0142] Cell modeling: discard the old culture medium and add 0.8mmol / L H 2 O 2 The solution was added to the culture dish to damage the cells. The control group was added with cell culture medium (DMEM high glucose medium + 10% fetal bovine serum; DMEM high glucose medium manufacturer: Bioss, product number C7076; fetal bovine serum manufacturer: Nanjing Senbeijia Biotechnology Co., Ltd., product number BC-SE-FBS07), 3 ml / culture dish, at 37°C, 5% CO 2 Culture in a cell culture incubator for 24 h.

[0143] (III) Treatment with drug addition: old culture medium was discarded, and the control group and H 2 O 2 The cell culture medium was added to the experimental group, and the fermentation supernatant diluted 25 times was added to the experimental group, 3 ml / culture dish, and incubated at 37°C, 5% CO 2 Culture in a cell culture incubator for 24 h.

[0144] in:

[0145] The fermentation liquid supernatant is obtained by fermenting Lactobacillus casei LC-K0313, Lactobacillus casei-self-screening, Lactobacillus casei-commercially available group 1, Lactobacillus casei-commercially available group 2 and Lactobacillus casei-commercially available group 3. The preparation method is specifically as follows: taking strain preservation tubes of Lactobacillus casei LC-K0313, Lactobacillus casei-self-screening, Lactobacillus casei-commercially available group 1, Lactobacillus casei-commercially available group 2 and Lactobacillus casei-commercially available group 3 to melt at room temperature, inoculating them into MRS seed culture medium at a 1% inoculation amount for activation for 16 hours, diluting them with sterile water to an OD value of 5, inoculating them into 100 mL of MRS liquid culture medium at a 1% inoculation amount, statically culturing them in an incubator at 37°C for 24 hours, sterilizing them in an autoclave at 115°C for 30 minutes, and centrifuging them for 10 minutes with a desktop centrifuge at 8000rpm after cooling to obtain a fermentation supernatant.

[0146] (IV) Sample processing: Aspirate and discard the old culture medium, add 1 ml of trypsin (manufacturer: EMD MilliporeCorp, catalog number: 3788142) to each culture dish, and after 2 minutes of enzymatic hydrolysis, use a cell scraper to collect the cells into a centrifuge tube, add the extract solution and ultrasonically disrupt them (power 200 W, ultrasonic for 20 minutes), centrifuge at 8000 rpm and 4°C for 10 minutes to obtain the sample, and place it on ice for testing.

[0147] (V) Sample determination According to the instructions of the superoxide dismutase (SOD) activity detection kit (Beijing Solebow Technology Co., Ltd., catalog number: BC0170), 70 μL of 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 were added to a 96-well plate in sequence, mixed thoroughly, and placed in a water bath at 37°C for 30 min. The absorbance value A was measured at a wavelength of 560 nm, which was recorded as A determination; the control group did not add reagent 2, but made up with distilled water. The rest of the operations were the same as those of the experimental group, and the absorbance value was recorded as A control; blank group 1 used distilled water instead of the fermentation broth supernatant, and the rest of the operations were the same as those of the experimental group, and the absorbance value was measured, which was recorded as A1 blank; blank group 2 used distilled water instead of the fermentation broth supernatant, did not add reagent 2, but made up with distilled water, and the rest of the operations were the same as those of the experimental group, and the absorbance value was measured, which was recorded as A2 blank.

[0148] ΔA assay = A assay - A control, ΔA blank = A1 blank - A2 blank.

[0149] Inhibition percentage = (ΔA blank - ΔA measurement) / ΔA blank × 100%.

[0150] 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.

[0151] Improvement rate = (SOD-H2 O 2 Group SOD) / H 2 O 2 The results are shown in Table 5.

[0152] Table 5 SOD activity detection

[0153]

[0154] Conclusion: The statistical analysis showed that the Lactobacillus casei LC-K0313 group was significantly different from the other groups (P<0.05). The experimental data showed that the superoxide dismutase (SOD) activity of the fermentation broth of Lactobacillus casei LC-K0313 was the highest, indicating that this strain had the strongest antioxidant capacity.

[0155] Example 5 Antibacterial ability experiment

[0156] Experimental method: Take 1% inoculum of Lactobacillus casei LC-K0313, Lactobacillus casei-self-selected, Lactobacillus casei-commercial 1, Lactobacillus casei-commercial 2, and Lactobacillus casei-commercial 3 strains and inoculate them into MRS broth at 37°C for activation for 16 hours. Pour 20 mL of MRS agar medium on 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 10 5-6 cfu / ml, take 100μL of the diluted bacterial solution and drop it on the solid plate, spread it with a coating rod, and put it upright for later use. Place the sterilized Oxford cup gently on the surface of the plate culture medium (3 / plate), let it stand for 5 minutes, then absorb 100μL of the activated bacterial solution and add it to the Oxford cup. After the bacterial solution is fully diffused for 2 hours, transfer the plate to a 37℃ incubator for 24 to 72 hours, and observe the phenomenon regularly.

[0157] The diameter of the inhibition zone was measured with a ruler and photographed for record. The results are shown in Table 6.

[0158] Table 6 Antibacterial zone diameter data of bacterial species against Staphylococcus aureus and Escherichia coli

[0159]

[0160] Conclusion: The statistical analysis showed that the difference between the Lactobacillus casei LC-K0313 group and other groups was extremely significant (P<0.01). 1. Comparison of the diameters of the inhibition zones of the five strains of Lactobacillus casei against Staphylococcus aureus showed that Lactobacillus casei LC-K0313 had the strongest ability to inhibit Staphylococcus aureus.

[0161] 2. By comparing the diameters of the inhibition zones of five strains of Lactobacillus casei against Escherichia coli, it was shown that Lactobacillus casei LC-K0313 had the strongest ability to inhibit Escherichia coli.

[0162] Example 6 Fermentation of Chinese medicine experiment

[0163] Experimental method: according to the solid-liquid ratio of ginseng to water of 8:100, the solid-liquid ratio of wolfberry to water of 8:100, and the solid-liquid ratio of yam to water of 15:100, water was added and boiled for 1-2 hours, the supernatant was collected by filtration, 4% crystalline fructose (W / V) was added, and the mixture was sterilized at 115℃ for 30 minutes; Lactobacillus casei LC-K0313, Lactobacillus casei-self-screening, Lactobacillus casei-commercial 1, Lactobacillus casei-commercial 2, and Lactobacillus casei-commercial 3 were activated in MRS broth at 37℃ for 16 hours. Inoculate at a volume ratio of 2%, culture at 37℃ for 48 hours, and take samples at 24h and 48h to detect OD and pH values. The results are shown in Tables 7-9.

[0164] Table 7 Experimental data of fermented American ginseng

[0165]

[0166] Conclusion: The statistical analysis showed that the difference between the Lactobacillus casei LC-K0313 group and other groups (P<0.01) was extremely significant. The experimental results showed that the OD value was the highest and the pH value was the lowest when the Lactobacillus casei LC-K0313 was used to ferment American ginseng for 24h to 48h, indicating that the bacteria grew best in the American ginseng fermentation liquid and produced the most acid.

[0167] Table 8 Fermented wolfberry experimental data

[0168]

[0169]

[0170] Conclusion: The statistical analysis showed that the difference between the Lactobacillus casei LC-K0313 group and other groups was extremely significant (P<0.01). The experimental results showed that the OD value was the highest and the pH value was the lowest when the Lactobacillus casei LC-K0313 was used to ferment wolfberry for 24h to 48h, indicating that the bacteria grew best in the wolfberry fermentation liquid and produced the most acid.

[0171] Table 9 Experimental data of fermented yam

[0172]

[0173] Conclusion: The statistical analysis showed that the difference between the Lactobacillus casei LC-K0313 group and other groups was extremely significant (P<0.01). The experimental results showed that the OD value was the highest and the pH value was the lowest when the yam was fermented with Lactobacillus casei LC-K0313 for 24h to 48h, indicating that the bacteria grew best in the yam fermentation liquid and produced the most acid.

[0174] Example 7 Immunity Enhancement Experiment

[0175] Experimental methods:

[0176] Cell culture RAW264.7 cells were added to a 96-well plate, with 1×10 cells per well. 5 , 37°C, 5% CO 2 Conditioned culture for 16 hours,

[0177] Cell modeling was performed with 0.8 mmol / L H 2 O 2 The solution was added to a 96-well plate to damage the cells. The control group was added with cell culture medium (DMEM high glucose medium + 10% fetal bovine serum; DMEM high glucose medium manufacturer: Bioss, product number C7076; fetal bovine serum manufacturer: Nanjing Senbeijia Biotechnology Co., Ltd., product number BC-SE-FBS07), 37°C, 5% CO 2 Conditioned culture for 24h.

[0178] Drug treatment: The supernatant of American ginseng fermentation broth was diluted 40 times and added to a 96-well plate, with 200 μl added to each well. 2 O 2 The cells were cultured in the control group and maintained at 37°C with 5% CO 2 Conditioned culture for 24h.

[0179] Neutral red dye staining, lysis, and detection After aspirating the culture medium, add 10% neutral red dye to each well and continue to culture for 4 hours. Aspirate the neutral red dye, add 150μL / well of neutral red lysis solution, mix for 10 minutes, and detect at a wavelength of 540nm using an enzyme reader. Phagocytic index = OD value of experimental group / OD value of control group, improvement rate (%) = (phagocytic index of experimental group - H 2 O 2 Phagocytic index (H) 2 O 2 The phagocytic index of each group × 100%. The results are shown in Table 10:

[0180] in:

[0181] The supernatant of American ginseng fermentation liquid is obtained by fermenting American ginseng extract with Lactobacillus casei LC-K0313, Lactobacillus casei-self-screening, Lactobacillus casei-commercially available group 1, Lactobacillus casei-commercially available group 2, and Lactobacillus casei-commercially available group 3. The preparation method is as follows: extracting according to a solid-liquid ratio of American ginseng to water of 8:100, adding 4% crystalline fructose according to the mass ratio to the extract and sterilizing at 115°C for 30 minutes to prepare an American ginseng culture medium; taking Lactobacillus casei LC-K0313, Lactobacillus casei-self-screening, Lactobacillus casei-commercially available group 1, Lactobacillus casei-commercially available group 2, and Lactobacillus casei-commercially available group 3. 13. Lactobacillus casei - self-selected, Lactobacillus casei - commercially available 1. Lactobacillus casei - commercially available 2. Lactobacillus casei - commercially available 3. The strain storage tube was thawed at room temperature, inoculated into MRS seed culture medium at a 1% inoculum amount for activation for 16 hours, and the seed liquid was inoculated into American ginseng culture medium at a 2% inoculum amount, and cultured in a 37°C incubator for 24 hours, sterilized at 115°C for 30 minutes, and after cooling, centrifuged at 8000rpm in a desktop centrifuge for 10 minutes to obtain American ginseng fermentation supernatant.

[0182] Table 10 Experimental data on the effect of American ginseng fermented liquid on enhancing immunity

[0183]

[0184] Conclusion: The statistical analysis showed that the difference between the Lactobacillus casei LC-K0313 group and other groups was significant (P<0.05). The cell phagocytosis index experiment proved that after 24 hours of fermentation of the Chinese medicine American ginseng by Lactobacillus casei LC-K0313, the phagocytosis index of the fermentation liquid was higher than that of the fermentation liquid of other strains, indicating that the immunity of the fermentation liquid after fermentation by this strain was more improved than that of other strains.

[0185] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Lactobacillus casei ( Lacticaseibacillus casei ) LC-K0313, characterized in that, Its deposit number is CGMCC No.28935.

2. Lactobacillus casei as claimed in claim 1 ( Lacticaseibacillus casei ) Fermentation broth of LC-K0313.

3. Lactobacillus casei as claimed in claim 1 ( Lacticaseibacillus casei ) Use of LC-K0313 or the fermentation broth as claimed in claim 2 in the preparation of any of the following: (I) Microecological products; and / or (II) Products used for plant fermentation; The microecological product is a probiotic product, a synbiotic product or a postbiotic product; The product for plant fermentation is food, feed or feed additive; The food is a health food, food for special medical purposes, functional food or pet food; The dosage form of the microecological product or the product for plant fermentation is powder, granule, capsule, tablet, pill, extract or liquid preparation; The plant fermentation is Chinese medicine fermentation, fruit and vegetable fermentation or new food raw material fermentation; The Chinese medicine fermentation is a Chinese medicine fermentation with both medicinal and edible properties; The Chinese medicine is one or more of American ginseng, wolfberry or yam.

4. A product, characterized in that comprising the Lactobacillus casei as claimed in claim 1 ( Lacticaseibacillus casei ) LC-K0313 or the fermentation broth as claimed in claim 2; The product comprises any of the following: (I) Microecological products; and / or (II) Products used for plant fermentation; The microecological products are probiotic products, synbiotic products, and postbiotic products; The product for plant fermentation is food, feed or feed additive; The food is a health food, food for special medical purposes, functional food or pet food; The dosage form of the microecological product or the product for plant fermentation is powder, granule, capsule, tablet, pill, extract or liquid preparation; The plant fermentation is Chinese medicine fermentation, fruit and vegetable fermentation or new food raw material fermentation; The Chinese medicine fermentation is a Chinese medicine fermentation with both medicinal and edible properties; The Chinese medicine is one or more of American ginseng, wolfberry or yam.

5. Lactobacillus casei as claimed in claim 1 ( Lacticaseibacillus casei ) Use of LC-K0313, the fermentation broth according to claim 2 or the product according to claim 4 in any of the following: (I) preparing products that inhibit the growth of harmful bacteria; and / or (II) Preparation of products for regulating bacterial flora balance; and / or (III), preparing antioxidant products; and / or (IV) Improving the performance of plant fermentation products; The harmful bacteria are Staphylococcus aureus and / or Escherichia coli; The flora balance is the intestinal flora balance; The anti-oxidation is to increase the activity of superoxide dismutase; The product performance is to enhance immunity.

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