Lactobacillus mucilaginosus VB216 and application thereof

Abstract

The invention discloses a lactobacillus fermentum VB216, the preservation number of the lactobacillus fermentum VB216 is CGMCC (China General Microbiological Culture Collection Center) No.26578, and the preservation date of the lactobacillus fermentum VB216 is February 20, 2023. The lactobacillus fermentum has various pathogenic bacterium inhibition activities and excellent arginine degradation activity. The arginine metabolism related diseases can be prevented and / or relieved and / or treated, such as food, health food, medicine and feed, such as products for preventing and / or relieving and / or treating hyperargininemia, malignant tumor treatment, anaphylaxis, eczema, pruritus and abdominal spasm, and the arginine metabolism related diseases have huge application prospects.

Description

[0001] Case information

[0002] This application is a divisional application of the patent application submitted to the State Intellectual Property Office of China on July 26, 2023, with patent application number CN202310921836.4 and patent name "A fermented mucus Lactobacillus VB216 and its application". Technical Field

[0003] The invention belongs to the technical field of microorganisms, and in particular relates to a novel fermented mucus lactobacillus VB216 with good gastrointestinal tolerance, multiple pathogenic bacteria inhibition activity and arginine degradation activity, and application thereof. Background Art

[0004] There are more than 400 kinds of probiotics in the human body, of which only about 20 can colonize in the human intestine and reproductive system for a long time. Lactobacillus and Bifidobacterium are the two main types. After entering the intestine, probiotics will have a complex relationship with the original flora in the intestine, such as habitat, symbiosis, partiality, competition and phagocytosis, and ultimately play a role in promoting the health of the body. If due to various factors, the beneficial bacteria in the intestine decrease and the pathogenic bacteria multiply, the dynamic balance of the original flora in the intestine will be broken, causing various physical discomforts or diseases. For these people, oral probiotics are the most direct and effective way to improve the imbalance of intestinal flora.

[0005] Fermented Lactobacillus mucus belongs to the genus Lactobacillus, is a Gram-positive facultative anaerobic bacterium, and is a normal flora of the host's oral cavity, intestines, milk and vagina. It is reported that fermented Lactobacillus mucus can not only improve the local immune function of the intestine, but also has important physiological functions such as promoting the body's absorption of nutrients, regulating cholesterol and lactose metabolism, lowering blood pressure, and anti-oxidation. It was included in my country's "List of Bacteria that Can Be Used in Food" in 2011. Therefore, fermented Lactobacillus mucus is also often used as a candidate strain for the treatment of inflammatory bowel disease, antibiotic-associated diarrhea, allergic dermatitis, female vaginitis and other diseases. In the field of food, it is mostly used in various fermented foods, including a variety of liquor fermentations, which not only gives the food a unique flavor, but also has some of the probiotic functions of the strain.

[0006] Arginine is a conditionally essential amino acid that can be synthesized in the body or taken in through diet. It is an important component of the ornithine cycle and has extremely important physiological, metabolic and nutritional functions in the life metabolism process, such as promoting muscle protein synthesis, wound recovery, and enhancing the body's immunity. However, excessive intake can also cause a variety of adverse reactions, such as shortness of breath, itching, eczema, abdominal cramps, allergic reactions, etc., especially for some people with underlying diseases (liver and kidney diseases, asthma patients, diabetics, etc.), the side effects will be more serious.

[0007] Studies have shown that many malignant tumors have arginine nutritional deficiency, such as malignant melanoma, liver cancer, pancreatic cancer, breast cancer, etc., which means that the survival of tumor cells needs to completely rely on exogenous arginine to maintain essential biological processes. Arginine deprivation therapy is a new anti-tumor treatment with great development prospects. It mainly inhibits the growth of tumor cells by limiting the uptake of sufficient arginine by tumor cells from the blood. Among them, arginine deiminase (ADI) and arginine succinate synthetase 1 (ASS1) are the research hotspots of this deprivation therapy. This type of drug has been approved in Europe and the United States for use in the treatment of melanoma, brain glioma and hepatocellular carcinoma.

[0008] In addition, as a rare disease, hyperargininemia is a urea cycle metabolic disorder caused by arginase 1 deficiency. The onset age is relatively late (early childhood) and worsens over time. Typical clinical manifestations are cognitive and motor degeneration, progressive spastic paralysis, epilepsy, short stature, developmental delay, and early death. There is currently no effective treatment. The main treatment method is to limit protein intake in the diet to reduce arginine intake. In addition, increasing the bypass metabolism of waste ammonia and promoting nitrogen excretion are also one of the measures to treat the disease. Although the above measures can control the blood arginine level in the body and slow down and prevent the deterioration of the disease, they cannot cure the disease and the treatment effect is unsatisfactory. For young children, controlling the disease by restricting diet is not a long-term solution. Therefore, it is particularly important and urgent to develop a treatment method that can effectively reduce the arginine level in the patient's body without deliberately controlling protein intake. Supplementing with probiotics with corresponding special functions may become an effective alternative and normal.

[0009] At present, there are relatively few strains with arginine degradation ability among probiotics, and the arginine consumption ability of the disclosed strains is generally weak. It is reported that the arginine consumption of L. pontis JSB7 strain can reach 0.96±0.03mmol / L, while the arginine consumption of L. fermentum JSA30 strain can reach 0.86±0.09mmol / L (patent number: CN 106479923 A). Therefore, the development of probiotics with stronger arginine degradation ability is the main technical problem solved by the present invention. Summary of the invention

[0010] The first object of the present invention is to provide a fermented mucus lactobacillus (Limosilactobacillusfermentum), which is deposited in the General Microbiological Center of China Microorganism Culture Collection Committee (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) with a deposit number of CGMCC No. 26578 and a deposit date of February 20, 2023, and is named VB216. It has multiple pathogenic bacteria inhibitory activity and excellent arginine degradation activity.

[0011] In this document, "Limosilactobacillus fermentum VB216" and "Limosilactobacillus fermentum VB216" are synonymous.

[0012] The second object of the present invention is to provide a fermentation broth, which comprises the aforementioned Limosilactobacillus fermentum VB216.

[0013] The third object of the present invention is to provide a fermentation supernatant comprising:

[0014] The aforementioned Limosilactobacillus fermentum VB216; and / or

[0015] The metabolites of the aforementioned Limosilactobacillus fermentum VB216.

[0016] It should be noted that “fermentation supernatant” refers to the supernatant after centrifugation of the fermentation broth, which may contain fermented Lactobacillus mucilaginosus VB216, or metabolites of fermented Lactobacillus mucilaginosus VB216, or both fermented Lactobacillus mucilaginosus VB216 and its metabolites.

[0017] The fourth object of the present invention is to provide a microbial agent, wherein the microbial agent contains the aforementioned Limosilactobacillus fermentum VB216.

[0018] According to an embodiment of the present invention, the fermented Lactobacillus mucilaginosus VB216 exists in the form of living cells, dry bacteria, immobilized cells or any other forms.

[0019] In this article, "living cells" refer to fermented Lactobacillus mucilaginosus VB216 that have the ability to metabolize, reproduce or replicate; "immobilized cells" refer to fermented Lactobacillus mucilaginosus VB216 fixed on a carrier, which can carry out life activities (growth, development, reproduction, heredity and metabolism, etc.) within a certain spatial range.

[0020] According to an embodiment of the present invention, the dry bacteria are obtained by freeze-drying the fermented Lactobacillus mucilaginosus VB216.

[0021] According to an embodiment of the present invention, the microbial agent is selected from a microbial liquid agent or a microbial solid agent.

[0022] According to an embodiment of the present invention, the microbial agent also contains at least one strain acceptable to food, medicine, health food and feed.

[0023] As used herein, "acceptable in food" refers to substances or compositions that can be consumed by humans, which may be adjusted according to the food requirements of different countries.

[0024] As used herein, "acceptable in health foods" refers to substances or compositions that can be consumed by humans, which may be adjusted according to the health food requirements of different countries.

[0025] In this context, "acceptable in feed" refers to substances or compositions that can be consumed by animals, which may be adjusted according to the feed requirements of different countries.

[0026] In this article, "acceptable in medicine" (or pharmaceutically acceptable) means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammals treated therewith. Preferably, "pharmaceutically acceptable" in the present invention refers to those approved by federal regulatory agencies or national governments or listed in the United States Pharmacopoeia or other generally recognized pharmacopeias for use in animals, especially humans.

[0027] According to an embodiment of the present invention, the microbial agent further contains a carrier or auxiliary material acceptable in food, medicine, health food or feed.

[0028] As used herein, the term "pharmaceutically acceptable carrier" includes any solvent, drug stabilizer, or combination thereof, which are known to those skilled in the art. Except where any conventional carrier is incompatible with the active ingredient, its use in treatment or pharmaceutical compositions is covered.

[0029] In this article, the term "pharmaceutically acceptable excipient" may include any solvent, etc., suitable for a specific target dosage form. Except for the scope of any conventional excipient being incompatible with the fermented Lactobacillus mucilaginosus VB216 of the present disclosure, such as any adverse biological effect produced or interacting with any other component of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of the present disclosure.

[0030] The fifth object of the present invention is to provide the use of the aforementioned fermented mucus lactobacillus (Limosilactobacillusfermentum) VB216, the aforementioned fermentation broth, the aforementioned fermentation supernatant or the aforementioned microbial agent in the preparation of products for preventing and / or alleviating and / or treating diseases related to arginine metabolism.

[0031] According to an embodiment of the present invention, the diseases include but are not limited to hyperargininemia, malignant tumor treatment, allergic reaction, eczema, itching and abdominal cramps.

[0032] According to an embodiment of the present invention, the malignant tumor includes but is not limited to malignant melanoma, hepatocellular carcinoma, prostate cancer, pancreatic cancer, breast cancer, glioma, small cell lung cancer, malignant pleural mesothelioma, and colorectal cancer.

[0033] According to an embodiment of the present invention, the product is food, health food, feed or medicine.

[0034] According to an embodiment of the present invention, the product can be applied to humans or animals.

[0035] According to an embodiment of the present invention, the product includes conventional added ingredients in the art.

[0036] According to an embodiment of the present invention, the product is a food, which can be in different dosage forms, such as powder, tablet, granule, capsule, suspension, etc.; in the drug combination group, some auxiliary materials can be appropriately added, including fillers, diluents, excipients, absorption promoters, etc. Preferably, the food can be a fermented food (such as fermented milk, flavored fermented milk, fermented milk beverage, etc.), milk-containing beverage, solid beverage, cheese, milk powder and other ordinary food or health food.

[0037] According to an embodiment of the present invention, the product is a medicine, and the specific production method and formula of the medicine can be carried out with reference to the medicine containing probiotics in the prior art.

[0038] According to an embodiment of the present invention, the product is feed, and the specific production method and formula of the feed can be carried out with reference to the feed containing probiotics in the prior art. The fermented mucus Lactobacillus or its bacterial preparation of the present invention is added as a probiotic supplement.

[0039] Further, the fermented mucus lactobacillus has the following characteristics:

[0040] (1) The fermented mucus lactobacillus of the present invention has a strong tolerance to the gastrointestinal tract. After being treated in artificial gastric juice at pH 3.0 for 2 hours, the survival rate can reach 84.31%, and after being treated for 4 hours, the survival rate is still 76.47%; after being treated in artificial small intestinal juice at pH 6.8 for 2 hours, the survival rate is 91.4%, and after being treated for 4 hours, the survival rate is still as high as 90.48%.

[0041] (2) The fermented mucus lactobacillus of the present invention is sensitive to multiple antibiotics such as clindamycin, ampicillin, tetracycline, chloramphenicol, azithromycin, amoxicillin, cephalosporins, and rifampicin, and does not contain exogenous antibiotic resistance genes, so it is safe to eat.

[0042] (3) The hemolytic reaction, gelatin liquefaction and indole reaction of the fermented mucus lactobacillus of the present invention are all negative, and the metabolism does not produce ammonia and biogenic amines, indicating that the metabolic toxicity tests of the bacteria are safe.

[0043] (4) The fermented mucus lactobacillus of the present invention has inhibitory activity against a variety of pathogenic bacteria, and can inhibit the growth of Escherichia coli, Staphylococcus aureus, Salmonella paratyphi B, Shigella dysenteriae, Enterococcus faecalis, Listeria monocytogenes, etc., and can protect the gastrointestinal tract, female vagina, breast tissue or other organs from invasion by pathogenic bacteria. Therefore, it can be used alone or in combination with other probiotics for the auxiliary treatment of a variety of diseases, such as gynecological diseases, gastrointestinal diseases, mastitis, etc.

[0044] (5) The fermented mucus lactobacillus of the present invention also has excellent arginine degradation ability. In the MRS culture system containing 0.5% or 1% arginine, it can quickly degrade more than 80% of arginine within 24 hours. At the same time, the second-generation sequencing results also confirmed that the strain had a 4-fold and 5.5-fold increase in the transcriptional expression of genes arcA and arcC, respectively, and these two genes are key enzyme genes involved in the ornithine cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0046] Figure 1 The single colony morphology of VB216 in Example 2;

[0047] Figure 2 This is the Gram staining microscopic examination image of VB216 in Example 2;

[0048] Figure 3 The hemolytic reaction of the strain VB216 in Example 3;

[0049] Figure 4 This is the metabolic curve of VB216 in the 1% arginine culture system in Example 5;

[0050] Figure 5 This is the metabolic curve of VB216 in the 0.5% arginine culture system in Example 5;

[0051] Figure 6 This is a flow chart of the VB216 culture process in Example 6. DETAILED DESCRIPTION

[0052] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0053] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0054] In this document, the terms “contain”, “include” or “comprise” are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0055] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0056] As used herein, "prevention" and "prevention" are used interchangeably. These terms refer to an approach to obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," fermented Lactobacillus muciniphila or a product containing it may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have been made.

[0057] As used herein, the terms "treatment" and "alleviation" both refer to the use of drugs to obtain the desired pharmacological and / or physiological effects. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit an individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0058] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0059] Example 1: Isolation and identification of fermented mucus Lactobacillus VB216

[0060] The fermented mucus lactobacillus VB216 of the present invention is isolated from the intestines of infants. The strain has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​February 20, 2023, and is classified and named: fermented mucus lactobacillus (Limosilactobacillus fermentum); the deposit number is CGMCC No. 26578.

[0061] 1.1 Isolation and purification of fermentative Lactobacillus mucilaginosus VB216

[0062] Samples were collected from feces of healthy infants of different months old, and were spread on lactobacillus selective culture medium plates by 10-fold gradient dilution method, and placed at 37℃ for anaerobic culture for 2-3 days. Single colonies of suspected lactobacillus in different forms on the plate were picked up for streaking isolation on fresh MRS plates. After anaerobic culture at 37℃ for 2-3 days, single colonies of different forms on the plate were picked up again for further streaking purification, and this was repeated 2-3 times until pure colonies of single form were obtained. After no other bacteria were found under microscopic examination, strain identification was performed according to 1.2.

[0063] 1.2 Identification of Lactobacillus mucilaginosus VB216

[0064] The isolated single colony was inoculated into a fresh MRS medium, cultured at 37°C for 16-24h, and then an appropriate amount of bacterial liquid was transferred to another fresh MRS medium, and cultured for 16-24h. This was continued for more than 3 times, and then the fermentation liquid obtained by culture was centrifuged, the bacteria were collected, and DNA was extracted and PCR sequence amplified according to the general bacterial gene identification method and sent for sequencing. The 16S rDNA sequence measured was compared for homology in GenBank using the Blast program. The results showed that the species with the highest homology was fermented Lactobacillus fermentum, which was as high as 99.8%. After whole genome sequencing, it was confirmed that the strain was a new fermented mucus Lactobacillus fermentum (Limosilactobacillus fermentum), and it was named VB216.

[0065] The 16S rDNA sequencing result of fermentative Lactobacillus mucilaginosus VB216 is shown in SEQ ID NO: 1:

[0066]

[0067] Example 2: Strain characteristics of fermentation Lactobacillus mucilaginosus VB216

[0068] 2.1 Taxonomic characteristics of Lactobacillus mucilaginosus VB216

[0069] Fermented mucus Lactobacillus VB216 is a facultative anaerobic lactic acid bacterium with an optimal growth temperature of 35-37°C. Figure 1 As shown, the colonies are milky white, mostly round, with a moist and smooth surface, flat, slightly convex, regular edges, and a diameter generally ranging from 2-5 mm. Oxidase and catalase are both negative, Gram staining is positive, and under microscopic examination, they are rod-shaped, without spores, and can be seen Figure 2 shown.

[0070] The results of the physical and chemical tests are shown in Table 1 below:

[0071] Table 1: Physical and chemical test results of Lactobacillus mucilaginosus VB216

[0072]

[0073] 2.2 Gastrointestinal fluid tolerance of fermented Lactobacillus mucilaginosus VB216

[0074] 2.2.1 Artificial gastric juice tolerance test

[0075] Method: Inoculate the fermented mucus Lactobacillus VB216 strain into fresh MRS medium, culture at 37°C for 16-20h, centrifuge and collect the bacteria. Then inoculate the strain to be tested into artificial gastric juice (pH3.0) for culture, and count the viable bacteria on the plate after treatment at 37°C for 0h, 2h and 4h, and finally evaluate the acid resistance of the strain by the survival rate (i.e. the viable bacteria after treatment / the initial viable bacteria × 100%). The experimental results are shown in Table 2 below:

[0076] Table 2: Acid resistance results of Lactobacillus mucilaginosus VB216

[0077] Processing time 0h 2h 4h Log CFU / ml 9.41 9.33 9.29 Survival rate 100％ 84.31％ 76.47％

[0078] The survival rate of the VB216 strain after being treated in artificial gastric juice for 2 hours can reach 84.31%, and the survival rate after being treated for 4 hours is still 76.47%, indicating that the fermented mucus lactobacillus VB216 of the present invention has a strong gastric acid resistance.

[0079] 2.2.2 Artificial intestinal fluid tolerance test

[0080] Method: The fermented Lactobacillus mucosa VB216 strain was inoculated into fresh MRS medium, cultured at 37°C for 16-20h, centrifuged, and the bacteria were collected. The strain to be tested was then inoculated into artificial intestinal fluid (pH 6.8) for culture, and the plate live bacteria were counted after being treated at 37°C for 0h, 2h, and 4h, and the intestinal fluid tolerance of the strain was finally evaluated by the survival rate (i.e., the number of live bacteria after treatment / the initial number of live bacteria × 100%). The experimental results are shown in Table 3 below:

[0081] Table 3: Results of intestinal fluid tolerance of Lactobacillus mucosus VB216

[0082] Processing time 0h 2h 4h Log CFU / ml 9.02 8.98 8.97 Survival rate 100％ 91.4％ 90.48％

[0083] The survival rate of the VB216 strain after being treated in the artificial intestinal fluid for 2 hours was 91.4%, and the survival rate after being treated for 4 hours was still as high as 90.48%, indicating that the fermented mucus Lactobacillus VB216 of the present invention has excellent intestinal fluid resistance.

[0084] Example 3: Safety of fermented Lactobacillus mucilaginosus VB216

[0085] 3.1 Antibiotic sensitivity testing

[0086] This example tests the resistance of fermented mucus lactobacillus VB216 to more than ten antibiotics, including gentamicin, clindamycin, ampicillin, tetracycline, chloramphenicol, azithromycin, amoxicillin, cephalosporin antibiotics, etc. The determination was carried out by microbroth dilution method, in accordance with the guidelines of the International Organization for Standardization, i.e., the detection method in ISO 10932:2010 (E) "Milk and milk products: Determination of the minimal inhibitory concentration (MIC) of antibiotics applicable to bifidobacteria and non-enterococcal lactic acid bacteria (LAB)", with the standard strain Lactobacillus casei ATCC334 as the control, and the MIC value of each antibiotic was calculated by using the data model. The experimental results are shown in Table 4 below:

[0087] Table 4: MIC values ​​of antibiotics for Lactobacillus mucilaginosus VB216

[0088]

[0089] As can be seen from the above table, fermented mucus Lactobacillus VB216 is sensitive to multiple antibiotics tested, including clindamycin, ampicillin, tetracycline, chloramphenicol, azithromycin, amoxicillin, cephalosporins, rifampicin, etc., and does not contain exogenous antibiotic resistance genes, so it is safe to eat.

[0090] 3.2 Metabolic toxicity testing

[0091] Hemolysis reaction: Spread the freshly cultured fermented mucus Lactobacillus VB216 on the hemolysis test plate, culture it anaerobically at 37℃ for about 2-3 days, and observe whether a transparent or translucent hemolysis ring appears around the colony. Figure 3 It was shown that there was no hemolytic ring around the colonies, which was negative, indicating that the fermented mucus Lactobacillus VB216 was not hemolytic.

[0092] Gelatin liquefaction: In order to confirm the ability to decompose proteins and evaluate the ability of bacteria to invade cells, the gelatin liquefaction test was specifically investigated. Freshly cultured fermented mucus Lactobacillus VB216 was inoculated into a gelatin biochemical tube and cultured anaerobically at 37°C for about 2-3 days, then taken out and placed in a refrigerator at 2-8°C for 10-30 minutes to observe the state of the gelatin. The results showed that the gelatin was solid after refrigeration for 30 minutes, and it remained solid after continuous observation for one week, indicating that the gelatin liquefaction reaction of fermented mucus Lactobacillus VB216 was negative.

[0093] Indole reaction: freshly cultured Lactobacillus mucilaginosus VB216 was inoculated into peptone water culture medium, and then 2-4 drops of Kovacs indole reagent were added after anaerobic culture at 37°C for about 1-2 days to observe whether a red ring appeared. The results showed that no red ring appeared in the bacterial solution, indicating that Lactobacillus mucilaginosus VB216 metabolism did not produce indole.

[0094] Urease: Inoculate freshly cultured fermented mucus Lactobacillus VB216 into the urease test biochemical tube, and observe the color reaction after anaerobic culture at 37°C for about 1-2 days. The results show that the culture medium is yellow, which is negative, indicating that fermented mucus Lactobacillus VB216 does not produce ammonia in metabolism.

[0095] Phenylalanine deamination reaction: Inoculate freshly cultured fermented mucus Lactobacillus VB216 into phenylalanine medium, culture at 37°C anaerobically for about 1-2 days, then add 2-3 drops of phenylalanine indicator, and observe the color reaction within 5 minutes. The results show that the bacterial solution is yellow, which is negative, indicating that fermented mucus Lactobacillus VB216 does not produce bioamines in metabolism.

[0096] Example 4: Detection of pathogen inhibition ability of fermented Lactobacillus mucilaginosus VB216

[0097] Method: The fermented mucus Lactobacillus VB216 strain was inoculated into fresh MRS medium, cultured at 37°C for 16-20h, the bacterial residue was collected by centrifugation, and a bacterial suspension was made with sterile water. The activated Escherichia coli CMCC44102, Staphylococcus aureus CMCC26003, Salmonella paratyphi B CMCC50094, Shigella dysenteriae CMCC51252, Enterococcus faecalis ATCC29212, Listeria monocytogenes ATCC19114, Candida albicans CMCC(F)98001, etc. were inoculated on a fresh MRS plate according to the national standard method, left to dry for a few minutes, and then holes were punched in the culture medium and numbered. About 50ul of fresh test bacterial solution was added to the well, and an equal amount of sterile water was added to the control group. After aerobic culture at 37°C for 2-3 days, the inhibition zone was observed around the well and the size of the inhibition zone was measured. The experimental results are shown in Table 5 below:

[0098] Table 5: Table of antibacterial ability of pathogenic bacteria

[0099]

[0100] The above results show that the fermented mucus lactobacillus VB216 of the present invention exhibits a certain inhibitory ability against the other six pathogens except Candida albicans, among which the inhibitory effect on the three pathogens of Escherichia coli CMCC44102, Staphylococcus aureus CMCC26003 and Salmonella paratyphi B CMCC50094 is particularly stronger.

[0101] Example 5: Detection of Lactobacillus in vitro arginine degradation ability

[0102] In order to compare the difference in the degradation ability of different lactobacilli to arginine, we purchased seven different species of lactobacilli from Ningbo Mingzhou Biology, Shanghai Fuxiang Biology, Qingdao Haibo Biology, Hangzhou Tiandongli Chemical Co., Ltd., etc., such as Lactobacillus rhamnosus ATCC7469 (bacteria 1#), Lactobacillus plantarum ATCC14917 (bacteria 2#), Lactobacillus casei ATCC334 (bacteria 3#), Lactobacillus reuteri DSM17938 (bacteria 4#), Lactobacillus acidophilus DSM3353 (bacteria 5#), Lactobacillus fermentum CECT5716 (bacteria 6#), Lactobacillus fermentum (bacteria 7#, commercially available Tongcheng By-Health), etc. The experimental method is as follows: First, the above 7 kinds of lactobacilli and the fermented mucus lactobacillus VB216 (bacteria 8#) freeze-dried tube bacterial liquid of the present invention were inoculated into fresh MRS liquid culture medium, cultured at 37°C for 16-20h, centrifuged to collect bacterial residues, and then made into bacterial suspension (108-109CFU / ml) with appropriate amount of sterile water for standby use. Prepare an appropriate amount of MRS culture medium, add an appropriate amount of arginine to make the final concentration of arginine 1% and 0.5% respectively, adjust the pH to 6.5-7.0, and sterilize at 121°C for 15 minutes. Then, each of the two culture media is divided into 25ml / bottle, and 30ul of the bacterial suspension of the eight lactobacilli is inoculated respectively. Each group is connected to 2 bottles, placed at 37°C, shaken in low-speed anaerobic culture for 2 days, and samples are taken regularly. The residual arginine content in the fermentation broth is determined by HPLC. The experimental results are shown in Figure 4 and Figure 5 .

[0103] The results show that, except for the fermented mucus Lactobacillus VB216, the other seven lactobacilli have almost no ability to degrade arginine. However, the fermented mucus Lactobacillus VB216 of the present invention can rapidly degrade more than 80% of arginine within 24 hours, whether in a 0.5% arginine or 1% arginine environment, and maintain a stable level thereafter, indicating that the strain has excellent degradation ability for arginine.

[0104] In addition, the above 3 strains of fermented lactobacillus (bacteria 6#, bacteria 7# and bacteria 8#) were also subjected to second generation sequencing, and the expression differences of the three at the gene transcription level were compared. It was finally found that compared with the other two strains of fermented lactobacillus, fermented mucus lactobacillus VB216 of the present invention had 4 times and 5.5 times of upregulation in the expression of genes arcA and arcC, respectively, wherein arcA encodes arginine deiminase, and arcC encodes aminomethyl kinase, both of which are key enzymes involved in ornithine cycle. This also fully explains the reason why VB216 has excellent arginine degradation ability.

[0105] Example 6: Preparation and application of fermented Lactobacillus mucilaginosus VB216 powder

[0106] according to Figure 6The culture process flow chart shown in the figure is that the fermented mucus lactobacillus VB216 glycerol tube bacterial liquid of the present invention is activated in MRS solid medium for 2-3 days, and then a typical single colony is selected to be inoculated in a shaking bottle equipped with MRS liquid medium, and as a first-level seed liquid, anaerobically cultured for 16-24h at 37°C. Then, it is transplanted to the secondary seed tank (MRS medium), and the transplanting amount is 1‰-5‰. After anaerobic culture for 1 day, it is transferred to a fermentation tank for fermentation, and centrifuged for 5-10min at 4-10°C and 10000rpm to collect the thalline. The collected thalline obtains the active freeze-dried bacterial powder of VB216 through freeze drying, and can be placed at -20°C or below for preservation after strict quality and activity detection. The freeze-dried bacterial powder of VB216 prepared in the present embodiment can be used for food, health food, feed or medicine. The food can be ordinary food or health food such as fermented food (for example, fermented milk, flavored fermented milk, fermented milk beverage, etc.), milk-containing beverage, solid beverage, cheese, milk powder, etc. The fermented mucus lactobacillus can be present in the food, health food, feed or medicine in the form of live bacteria or inactivated bacteria. The bacterial preparation or composition of the present invention can be applied to humans or animals. The composition includes conventional additives in the field. For example, in food, the composition can be in different dosage forms, such as powders, tablets, granules, capsules, suspensions, etc.; in the drug combination group, some auxiliary materials can be appropriately added, including fillers, diluents, excipients, absorption enhancers, etc. The composition has the ability to inhibit the activity of multiple pathogenic bacteria and degrade arginine.

[0107] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0108] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A fermented mucus lactobacillus (Limosilactobacillus fermentum) VB216, characterized in that: The deposit number of the fermented mucus lactobacillus (Limosilactobacillus fermentum) VB216 at the General Microbiology Center of the China Culture Collection Administration is CGMCC No.26578, and the deposit date is February 20, 2023.

2. A fermentation broth, characterized in that: The fermentation broth comprises the Limosilactobacillus fermentum VB216 according to claim 1.

3. A fermentation supernatant, characterized in that: The fermentation supernatant comprises: The fermented mucus lactobacillus (Limosilactobacillus fermentum) VB216 according to claim 1; And / or the metabolites of Limosilactobacillus fermentum VB216 according to claim 1.

4. A microbial agent, characterized in that: The microbial agent contains the Limosilactobacillus fermentum VB216 described in claim 1.

5. The microbial agent according to claim 4, characterized in that: The fermented mucus lactobacillus (Limosilactobacillus fermentum) VB216 exists in the form of living cells, dried bacteria, immobilized cells or any other form; The dry bacteria are obtained by freeze-drying the fermented Lactobacillus fermentum VB216; The microbial agent is selected from a microbial liquid agent or a microbial solid agent.

6. The microbial agent according to claim 4 or 5, characterized in that: The microbial agent also contains at least one strain acceptable to food, medicine, health food and feed.

7. The microbial agent according to claim 6, characterized in that: The microbial agent also contains carriers or auxiliary materials that are acceptable in food, medicine, health food or feed.

8. Use of the fermented mucus lactobacillus (Limosilactobacillus fermentum) VB216 according to claim 1, the fermentation broth according to claim 2, the fermentation supernatant according to claim 3 or the microbial agent according to any one of claims 4 to 7 in the preparation of a product for preventing and / or alleviating and / or treating diseases related to arginine metabolism.

9. The use according to claim 8, characterized in that: The disease is selected from at least one of hyperargininemia, malignant tumors, allergic reactions, eczema, itching and abdominal cramps; the malignant tumors include malignant melanoma, hepatocellular carcinoma, prostate cancer, pancreatic cancer, breast cancer, brain glioma, small cell lung cancer, malignant pleural mesothelioma, and colorectal cancer.

10. The use according to claim 8, characterized in that: The product is food, health food, feed or medicine.

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