Limosilactobacillus fermentum VB216 and use thereof

Abstract

Provided is a Limosilactobacillus fermentum VB216, which has the deposit number of CGMCC No. 26578 in the China General Microbiological Culture Collection Center, and has the deposit date of 20 February 2023. The Limosilactobacillus fermentum has inhibitory activities with regard to a variety of pathogenic bacteria and excellent activity for degrading arginine. The present invention can be used in the preparation of products, such as food, health-care food, medicine and feed, for preventing and / or relieving and / or treating arginine metabolism-related diseases, such as hyperarginemia, malignant tumor treatment, allergic reactions, eczema, pruritus and abdominal spasm, and has great application prospects.

Description

A fermented Lactobacillus mucilaginosus VB216 and its application Technical Field

[0001] The present invention belongs to the field of microbial technology, and in particular relates to a novel fermentative Lactobacillus mucilaginosus VB216 with good gastrointestinal tolerance, multiple pathogenic bacteria inhibitory activity and arginine degradation activity, and applications thereof. Background Art

[0002] After the outbreak of the COVID-19 pandemic, people's understanding of probiotics has increased significantly. There are more than 400 species of probiotics in the human body, of which only about 20 can colonize the human intestines and reproductive system for a long time. Lactobacillus and Bifidobacterium are the two most important types. After entering the intestine, probiotics will develop a complex relationship with the original intestinal flora, including habitat, symbiosis, partial symbiosis, competition and phagocytosis, ultimately playing 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 intestinal flora 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.

[0003] Lactobacillus fermentans, a Gram-positive facultative anaerobic bacterium belonging to the genus Lactobacillus, is a normal flora of the oral cavity, intestines, breast milk, and vagina. Lactobacillus fermentans has been reported to not only enhance local intestinal immunity but also promote nutrient absorption, regulate cholesterol and lactose metabolism, lower blood pressure, and provide antioxidant benefits. In 2011, it was included in my country's "List of Bacteria Suitable for Food Use." Consequently, Lactobacillus fermentans is often used as a candidate strain for treating a variety of conditions, including inflammatory bowel disease, antibiotic-associated diarrhea, atopic dermatitis, and vaginitis. In the food industry, it is widely used in various fermented foods, including various types of liquor, where it not only imparts a unique flavor to the food but also shares some of the strain's beneficial properties.

[0004] Arginine is a conditionally essential amino acid that can be synthesized in the body or taken in through the diet. It is a key component of the ornithine cycle and plays an extremely important physiological, metabolic, and nutritional role in the life metabolic 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 people with underlying diseases (liver and kidney disease, asthma patients, diabetes patients, etc.), the side effects will be more serious.

[0005] Research has shown that many malignant tumors, such as malignant melanoma, liver cancer, pancreatic cancer, and breast cancer, exhibit arginine auxotrophy. This means that tumor cells are completely dependent on exogenous arginine to maintain essential biological processes. Arginine deprivation therapy is a promising new anti-tumor treatment approach that inhibits tumor cell growth by limiting the amount of arginine that tumor cells can take up from the blood. Activators such as arginine deiminase (ADI) and arginine succinate synthetase 1 (ASS1) are research hotspots for this approach. These drugs have been approved in Europe and the United States for the treatment of melanoma, glioblastoma, and hepatocellular carcinoma.

[0006] Hyperargininemia is a rare disease caused by a deficiency in arginase 1, a urea cycle metabolic disorder. It develops relatively late in childhood and worsens over time. Typical clinical manifestations include cognitive and motor impairment, progressive spastic paralysis, epilepsy, short stature, developmental delay, and early death. Currently, there is no effective treatment, primarily through dietary protein restriction to reduce arginine intake. Additionally, increasing the bypass metabolism of the waste product ammonia and promoting nitrogen excretion are also treatment options. While these measures can control blood arginine levels and slow or prevent progression, they are not a cure and their effectiveness is generally unsatisfactory. For young children, dietary restriction is not a long-term solution. Therefore, developing a treatment that can effectively reduce arginine levels without requiring protein restriction is crucial and urgent. Supplementation with probiotics with specific functional properties may become an effective alternative and a common practice, especially in the post-pandemic era.

[0007] Currently, relatively few probiotic strains possess the ability to degrade arginine, and the previously reported strains generally have weak arginine consumption capabilities. L. pontis JSB7 has been reported to consume 0.96±0.03 mmol / L of arginine, while L. fermentum JSA30 has a reported arginine consumption of 0.86±0.09 mmol / L (Patent No.: CN 106479923 A). Therefore, developing probiotics with enhanced arginine degradation capabilities is the primary technical challenge addressed by the present invention.

[0008] Summary of the Invention

[0009] The first object of the present invention is to provide a Lactobacillus fermentum strain, which is deposited with the China General Microbiological Culture Collection (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) under the deposit number CGMCC No. 26578 and the deposit date of February 20, 2023, and is named VB216. The strain has inhibitory activity against multiple pathogens and excellent arginine degradation activity.

[0010] As used herein, "Limosilactobacillus fermentum VB216" and "Lactobacillus fermentum VB216" are synonymous.

[0011] The second object of the present invention is to provide a fermentation broth comprising the aforementioned Limosilactobacillus fermentum VB216.

[0012] The third object of the present invention is to provide a fermentation supernatant comprising:

[0013] The aforementioned Limosilactobacillus fermentum VB216; and / or

[0014] The metabolites of the aforementioned Limosilactobacillus fermentum VB216.

[0015] 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.

[0016] The fourth object of the present invention is to provide a microbial agent containing the aforementioned Limosilactobacillus fermentum VB216.

[0017] According to an embodiment of the present invention, the fermented Lactobacillus mucilaginosus VB216 exists in the form of living cells, dried bacteria, immobilized cells or any other forms.

[0018] 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, inheritance and metabolism, etc.) within a certain spatial range.

[0019] According to an embodiment of the present invention, the dry bacteria are obtained by freeze-drying the fermented Lactobacillus mucilaginosus VB216.

[0020] According to an embodiment of the present invention, the microbial agent is selected from a microbial liquid agent or a microbial solid agent.

[0021] According to an embodiment of the present invention, the microbial agent further contains a strain acceptable to at least one of food, medicine, health food and feed.

[0022] As used herein, "acceptable in food" refers to substances or compositions that can be consumed by humans, which may be adjusted according to food requirements in different countries.

[0023] In this article, "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.

[0024] In this context, "acceptable in feed" refers to substances or compositions that can be consumed by animals, which may be adjusted according to feed requirements in different countries.

[0025] As used herein, "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated therewith. Preferably, "pharmaceutically acceptable" as used herein means approved by federal regulatory agencies or national governments or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0026] 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.

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

[0028] As used herein, the term "pharmaceutically acceptable excipient" includes any solvent, etc., suitable for the particular intended dosage form. Except to the extent that any conventional excipient is incompatible with the fermented Lactobacillus muciniphilus VB216 of the present disclosure, such as by producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is contemplated by the present disclosure.

[0029] A fifth object of the present invention is to provide the use of the aforementioned Limosilactobacillus fermentum 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.

[0030] According to an embodiment of the present invention, the diseases include but are not limited to hyperargininemia, malignant tumor treatment, allergic reaction, eczema, pruritus and abdominal cramps.

[0031] 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.

[0032] According to an embodiment of the present invention, the product is food, health food, feed or medicine.

[0033] According to embodiments of the present invention, the product can be applied to humans or animals.

[0034] According to an embodiment of the present invention, the product includes conventional added ingredients in the art.

[0035] According to an embodiment of the present invention, the product is a food, which can be in various dosage forms, such as powders, tablets, granules, capsules, suspensions, etc.; in the pharmaceutical combination, appropriate excipients can be added, including fillers, diluents, excipients, absorption enhancers, etc. Preferably, the food can be a fermented food (such as fermented milk, flavored fermented milk, fermented milk beverages, etc.), a milk-containing beverage, a solid beverage, cheese, milk powder, or other common food or health food.

[0036] 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 made with reference to the medicine containing probiotics in the prior art.

[0037] According to an embodiment of the present invention, the product is feed. The specific production method and formula of the feed can refer to the feed containing probiotics in the prior art. The fermented Lactobacillus mucis or its bacterial preparation of the present invention is added as a probiotic supplement.

[0038] Furthermore, the fermented Lactobacillus mucilaginosus has the following characteristics:

[0039] (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%.

[0040] (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.

[0041] (3) The hemolytic reaction, gelatin liquefaction, and indole reaction of the fermented mucus Lactobacillus of the present invention were all negative, and the metabolism did not produce ammonia and biogenic amines, indicating that the metabolic toxicity tests of the bacteria were safe.

[0042] (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 the invasion of pathogenic bacteria. Therefore, it can be used alone or in combination with other probiotics for the auxiliary treatment of various diseases, such as gynecological diseases, gastrointestinal diseases, mastitis, etc.

[0043] (5) The fermentative mucus lactobacillus of the present invention also has excellent arginine degradation ability. It can quickly degrade more than 80% of arginine within 24 hours in an MRS culture system containing 0.5% or 1% arginine. 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. These two genes are key enzyme genes involved in the ornithine cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments with reference to the following drawings, in which:

[0045] Figure 1 shows the single colony morphology of VB216 in Example 2;

[0046] FIG2 is a Gram staining microscopic image of VB216 in Example 2;

[0047] FIG3 is a hemolytic reaction of the strain VB216 in Example 3;

[0048] FIG4 is a metabolic curve of VB216 in a 1% arginine culture system in Example 5;

[0049] FIG5 is a metabolic curve of VB216 in a 0.5% arginine culture system in Example 5;

[0050] Figure 6 is a flow chart of the VB216 culture process in Example 6. DETAILED DESCRIPTION

[0051] 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.

[0052] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0053] In this document, the terms “contain”, “include” or “include” are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0054] 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.

[0055] As used herein, "prevent" and "prevent" are used interchangeably. These terms refer to an approach for obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," Lactobacillus muciniphila ferments or a product containing the same can be administered to a subject at risk for 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 yet been made.

[0056] As used herein, the terms "treat" and "alleviate" refer to methods used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or 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 individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the 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 the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0057] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0058] Example 1: Isolation and identification of fermentative Lactobacillus mucilaginosus VB216

[0059] The fermentative Lactobacillus mucilaginosus VB216 strain of the present invention was isolated from the intestines of infants. The strain was deposited with the China General Microbiological Culture Collection Center (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on ​​February 20, 2023, and is classified as Limosilactobacillus fermentum; the deposit number is CGMCC No. 26578.

[0060] 1.1 Isolation and purification of fermentative Lactobacillus mucilaginosus VB216

[0061] Fecal samples were collected from healthy infants of different ages and plated onto Lactobacillus selective culture medium using a 10-fold gradient dilution method. The plates were then incubated anaerobically at 37°C for 2-3 days. Single colonies of suspected Lactobacillus from the plates were streaked onto fresh MRS plates. After anaerobic incubation at 37°C for 2-3 days, single colonies of different morphologies were again picked from the plates for further streaking and purification. This process was repeated 2-3 times until pure colonies of a single morphology were obtained. After microscopic examination revealed no contaminating bacteria, strain identification was performed according to step 1.2.

[0062] 1.2 Identification of Lactobacillus mucilaginosus VB216

[0063] The isolated single colony was inoculated into fresh MRS medium and cultured at 37°C for 16-24 hours. An appropriate amount of the culture was then transferred to another fresh MRS medium and cultured for another 16-24 hours. This process was repeated three or more times. The resulting fermentation broth was then centrifuged, and the cells were harvested. DNA was extracted and amplified by PCR according to common bacterial gene identification methods, and the cells were sent for sequencing. Homology comparison of the obtained 16S rDNA sequence using the Blast program in GenBank revealed that the highest homology was with Lactobacillus fermentum, exceeding 99.8%. Whole-genome sequencing confirmed that the strain was a new strain of Lactobacillus fermentum, designated VB216.

[0064] The 16S rDNA sequencing result of Lactobacillus fermentans VB216 is shown in SEQ ID NO: 1:

[0065] Example 2: Characteristics of the strain of fermentation Lactobacillus mucilaginosus VB216

[0066] 2.1 Taxonomic characteristics of Lactobacillus mucifermentans VB216

[0067] Lactobacillus mucilaginosus VB216 is a facultative anaerobic lactic acid bacterium with an optimal growth temperature of 35-37°C. Its colony morphology is shown in Figure 1. The colonies are milky white, mostly perfectly round, with a moist, smooth, flat, slightly convex surface and regular edges, generally ranging from 2-5 mm in diameter. They are oxidase- and catalase-negative, Gram-positive, and exhibit rod-shaped microscopy without spores, as shown in Figure 2.

[0068] The results of the physical and chemical tests are shown in Table 1 below:

[0069] Table 1: Physical and chemical test results of Lactobacillus mucilaginosus VB216

[0070] 2.2 Gastrointestinal fluid tolerance of Lactobacillus mucilaginosus VB216

[0071] 2.2.1 Artificial gastric juice tolerance test

[0072] Methods: Lactobacillus mucilaginosus VB216 was inoculated into fresh MRS medium and cultured at 37°C for 16-20 hours. The cells were then centrifuged and harvested. The strain to be tested was then inoculated into artificial gastric fluid (pH 3.0) and cultured at 37°C for 0, 2, and 4 hours. Viable bacteria were counted on plates. The acid resistance of the strain was evaluated using the survival rate (i.e., viable bacteria count after treatment / initial viable bacteria count × 100%). The experimental results are shown in Table 2 below:

[0073] Table 2: Acid resistance results of Lactobacillus mucilaginosus VB216

[0074] The survival rate of the VB216 strain in artificial gastric juice after being treated 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 Lactobacillus mucinus VB216 of the present invention has strong gastric acid resistance.

[0075] 2.2.2 Artificial intestinal fluid tolerance test

[0076] Methods: Lactobacillus mucilaginosus VB216 was inoculated into fresh MRS medium and cultured at 37°C for 16-20 hours. The cells were then centrifuged and harvested. The strain to be tested was then inoculated into artificial intestinal fluid (pH 6.8). Viable bacteria were counted on plates after treatment at 37°C for 0, 2, and 4 hours. The survival rate (i.e., viable bacteria count after treatment / initial viable bacteria count × 100%) was used to evaluate the strain's intestinal fluid tolerance. The experimental results are shown in Table 3 below:

[0077] Table 3: Intestinal fluid tolerance results of Lactobacillus mucilaginosus VB216

[0078] The survival rate of the VB216 strain after being treated in artificial small 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 Lactobacillus mucinus VB216 of the present invention has excellent resistance to intestinal fluid.

[0079] Example 3: Safety of fermented Lactobacillus mucilaginosus VB216

[0080] 3.1 Antibiotic susceptibility testing

[0081] This example tested the resistance of fermentative Lactobacillus mucilaginosus VB216 to more than ten antibiotics, including gentamicin, clindamycin, ampicillin, tetracycline, chloramphenicol, azithromycin, amoxicillin, and cephalosporins. The resistance was determined using the broth microdilution method, following the International Organization for Standardization guidelines (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)." The standard strain Lactobacillus casei ATCC334 was used as a control, and the MIC value for each antibiotic was calculated using a data model. The experimental results are shown in Table 4 below:

[0082] Table 4: MIC values ​​of antibiotics for Lactobacillus mucilaginosus VB216

[0083] As can be seen from the above table, fermented Lactobacillus muciflorus 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.

[0084] 3.2 Metabolic toxicity testing

[0085] Hemolysis reaction: Freshly cultured Lactobacillus mucilaginosus VB216 was streaked onto a hemolysis test plate and incubated anaerobically at 37°C for approximately 2-3 days. The colonies were observed for the appearance of a transparent or translucent hemolytic ring. The results, as shown in Figure 3, indicated that no hemolytic ring appeared around the colonies, indicating a negative result, indicating that Lactobacillus mucilaginosus VB216 is non-hemolytic.

[0086] Gelatin liquefaction: To confirm protein degradation and assess bacterial cell invasion, a gelatin liquefaction test was specifically conducted. Freshly cultured Lactobacillus mucilaginosus VB216 was inoculated into a gelatin biochemical tube and incubated anaerobically at 37°C for approximately 2-3 days. The gelatin was then refrigerated at 2-8°C for 10-30 minutes and observed. Results showed that the gelatin was solid after 30 minutes of refrigeration and remained solid even after one week of observation, indicating that the gelatin liquefaction reaction with Lactobacillus mucilaginosus VB216 was negative.

[0087] Indole reaction: Freshly cultured Lactobacillus mucilaginosus VB216 was inoculated into peptone water and incubated anaerobically at 37°C for approximately 1-2 days. Then, 2-4 drops of Kovacs indole reagent were added to the culture and observed for the appearance of a red ring. The results showed that no red ring appeared in the culture, indicating that Lactobacillus mucilaginosus VB216 does not metabolize indole.

[0088] Urease: Inoculate freshly cultured Lactobacillus mucilaginosus VB216 into a urease assay biochemical tube. Incubate anaerobically at 37°C for approximately 1-2 days and observe the color reaction. A yellow color indicates a negative result, indicating that Lactobacillus mucilaginosus VB216 does not produce ammonia.

[0089] Phenylalanine deamination reaction: Inoculate freshly cultured Lactobacillus mucilaginosus VB216 into phenylalanine medium. After incubation at 37°C for approximately 1-2 days, add 2-3 drops of phenylalanine indicator and observe the color reaction within 5 minutes. A yellow color indicates a negative reaction, indicating that Lactobacillus mucilaginosus VB216 does not metabolize bioamines.

[0090] Example 4: Detection of pathogen inhibition ability of fermented Lactobacillus mucilaginosus VB216

[0091] Methods: The fermentative Lactobacillus mucilaginosus VB216 strain was inoculated into fresh MRS medium and cultured at 37°C for 16-20 hours. The bacterial residue was collected by centrifugation and prepared into a bacterial suspension with sterile water. Activated Escherichia coli CMCC44102, Staphylococcus aureus CMCC26003, Salmonella Paratyphi B CMCC50094, Shigella dysenteriae CMCC51252, Enterococcus faecalis ATCC29212, Listeria monocytogenes ATCC19114, and Candida albicans CMCC(F)98001 were inoculated onto fresh MRS plates according to national standards. The plates were allowed to dry for several minutes before wells were punched and numbered. Approximately 50 μl of fresh test bacterial solution was added to each well, while an equal amount of sterile water was added to the control group. After aerobically incubating at 37°C for 2-3 days, the wells were observed for the presence of inhibition zones and their size. The experimental results are shown in Table 5 below:

[0092] Table 5: Antibacterial ability of pathogenic bacteria

[0093] The above results show that the fermented Lactobacillus mucis 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 Escherichia coli CMCC44102, Staphylococcus aureus CMCC26003 and Salmonella paratyphi B CMCC50094 is particularly stronger.

[0094] Example 5: Detection of Lactobacillus' ability to degrade arginine in vitro

[0095] In order to compare the differences in the degradation ability of different lactobacilli on arginine, we purchased seven different species of lactobacilli from Ningbo Mingzhou Biotechnology, Shanghai Fuxiang Biotechnology, Qingdao Haibo Biotechnology, 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 from Tongcheng Beijian), etc. The experimental method is as follows: First, the freeze-dried tube bacterial liquid of the above seven lactobacilli and the fermentation mucinous Lactobacillus VB216 (bacteria 8#) of the present invention was inoculated into fresh MRS liquid culture medium, cultured at 37°C for 16-20h, and the bacterial residue was collected by centrifugation. Then, a bacterial suspension (10 8 -10 9CFU / ml) and set aside. Appropriate amounts of MRS culture medium were prepared, and appropriate amounts of arginine were added to achieve final concentrations of 1% and 0.5%, respectively. The pH was adjusted to 6.5-7.0, and the culture was sterilized at 121°C for 15 minutes. Then, each of the two culture media was aliquoted into 25ml bottles, and 30ul of the bacterial suspension of the eight lactobacilli species was inoculated into each bottle. Two bottles were placed in each group, and the culture was incubated at 37°C on a shaking incubator for two days at low speed anaerobically. Samples were taken at regular intervals, and the residual arginine content in the fermentation broth was determined by HPLC. The experimental results are shown in Figures 4 and 5.

[0096] The results show that, with the exception of fermentative Lactobacillus mucilaginosus VB216, the other seven lactobacilli strains had almost no ability to degrade arginine. However, the fermentative Lactobacillus mucilaginosus VB216 of the present invention rapidly degraded over 80% of arginine within 24 hours, maintaining a stable level thereafter, regardless of whether it was exposed to 0.5% or 1% arginine. This demonstrates that the strain has excellent arginine degradation capabilities.

[0097] In addition, also above 3 strains of fermented lactobacillus (bacteria 6#, bacteria 7# and bacteria 8#) are carried out to second generation sequencing, compare the three's differential expression at the gene transcription level, finally find that, compared with other two strains of fermented lactobacillus, fermented mucus lactobacillus VB216 of the present invention has 4 times and 5.5 times of up-regulations on the expression of genes arcA and arcC respectively, wherein arcA encodes arginine deiminase, and arcC encodes aminomethyl kinase, both of which are key enzymes participating in ornithine cycle. This also fully explains why VB216 has excellent arginine degradation ability.

[0098] Example 6: Preparation and application of fermented Lactobacillus mucilaginosus VB216 powder

[0099] According to the culture process flow chart shown in Figure 6, after the fermentation of Lactobacillus mucilaginosus VB216 glycerol tube bacteria liquid of the present invention is activated in MRS solid medium for 2-3 days, a typical single colony is selected and inoculated into a shake flask equipped with MRS liquid medium. As a first-level seed liquid, anaerobically culture is carried out at 37°C for 16-24h. Then, the culture is transplanted into a secondary seed tank (MRS medium) at a transplanting amount of 1‰-5‰. After anaerobic culture for 1 day, the culture is transferred to a fermentation tank for fermentation and centrifuged at 4-10°C, 10000rpm for 5-10min to collect the bacteria. The collected bacteria are freeze-dried to obtain the active freeze-dried bacteria powder of VB216, which can be placed at -20°C or below for preservation after strict quality and activity testing. The freeze-dried bacteria powder of VB216 obtained in this 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 Lactobacillus mucilaginosus can be present in the food, health food, feed, or pharmaceutical product as live or inactivated bacteria. The bacterial preparation or composition described herein can be used in humans or animals. The composition includes conventional additives in the field. For example, in food, the composition can be in various dosage forms, such as powders, tablets, granules, capsules, and suspensions. In pharmaceutical compositions, appropriate excipients, including fillers, diluents, excipients, and absorption enhancers, can be added. The composition has the ability to inhibit the activity of various pathogenic bacteria and degrade arginine.

[0100] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean 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 can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0101] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

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

2. A fermentation broth, characterized in that: The fermentation broth comprises the fermented Lactobacillus 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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