Amniotic fluid-derived lactobacillus crispatus and application thereof

Abstract

The invention discloses an amniotic fluid sourced lactobacillus crispatus and application thereof. The lactobacillus crispatus is preserved in the China General Microbiological Culture Collection Center on July 3, 2024, and the preservation number of the lactobacillus crispatus is CGMCC (China General Microbiological Culture Collection Center) No.31148. The lactobacillus crispatus has the advantages that the lactobacillus crispatus is preserved in the China General Microbiological Culture Collection Center on July 3, 2024; the new lactobacillus crispatus strain obtained through screening comes from amniotic fluid, can effectively remove free radicals, is high in oxidation resistance, hydrophobicity and self-aggregation capacity and can adhere to and be planted in a living body, and a product developed by utilizing the new strain has the effects of regulating gastrointestinal flora and resisting oxidation and is wide in application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Lactobacillus crispatus derived from amniotic fluid and application thereof. Background Art

[0002] Free radicals are highly active molecules produced during cell metabolism. When the body's antioxidant capacity cannot resist the number of free radicals, high concentrations of free radicals can damage cell membranes, proteins, and DNA. An imbalance in the ratio between free radicals and antioxidants will cause oxidative stress, leading to cell dysfunction and death. As humans delve deeper into the mechanisms of chronic diseases and aging, they have found that they are closely related to oxidative stress. Related studies have shown that in cardiovascular diseases, free radicals, especially reactive oxygen species (ROS), can oxidize low-density lipoprotein (LDL) into oxidized low-density lipoprotein (oxLDL), which may amplify LDL atherosclerosis and increase the risk of coronary heart disease and stroke. In addition, cancer, neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease), and chronic inflammatory diseases (such as rheumatoid arthritis and inflammatory bowel disease) are also closely related to oxidative damage from free radicals.

[0003] Antioxidants are a class of molecules that can inhibit or prevent oxidation. They reduce oxidative stress and prevent cell damage by neutralizing free radicals, and play a key role in reducing oxidative stress. At present, antioxidants are divided into two categories: synthetic antioxidants and natural antioxidants. Synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) may cause side effects or even toxicity if consumed in excess, causing damage to organs such as the liver and kidneys, and may disrupt the balance between natural free radicals and antioxidants in the body, affecting the normal physiological functions of the human body, and posing certain safety issues. Natural antioxidants such as rosemary extract and tea polyphenols have a relatively single source and high separation and purification costs. In addition, during the extraction process, the toxicity and residue of the solvent, as well as the impurities and unknown components contained in the extract may also pose safety issues. In contrast, probiotics, as a natural and safe source of antioxidants, directly or indirectly reduce oxidative stress by producing antioxidant enzymes and metabolites, showing unique advantages.

[0004] At present, the research on the antioxidant capacity of probiotics is mainly focused on Lactobacillus and Bifidobacterium. Related studies have shown that Bifidobacterium probiotics show the best growth conditions in anaerobic environments, and they mainly colonize in the large intestine and other low-oxygen environments. Compared with Bifidobacterium probiotics, Lactobacillus probiotics can survive in different environments from the stomach to the large intestine, including acidic gastric juice and the small intestine with high oxygen content, and they show stronger environmental adaptability. In addition, Lactobacillus casei derived from fermented dairy products and some plant-based fermented foods can exert antioxidant effects through their antioxidant enzymes, such as catalase and superoxide dismutase. Therefore, Lactobacillus probiotics show unique antioxidant potential and application prospects among probiotics with their antioxidant enzymes and a wide range of living environments. Among them, Lactobacillus crispatus, as a probiotic of the genus Lactobacillus, often colonizes in the human oral cavity, intestines and female vagina, as well as the intestines and crops of chickens. Currently, there are few studies on the antioxidant capacity of Lactobacillus crispatus, and its research mainly focuses on its anti-inflammatory application in urogenital health, such as the prevention and treatment of bacterial vaginosis, and most strains are derived from the female reproductive tract. Studies have shown that the flora in amniotic fluid may enhance the production and activity of antioxidant enzymes by regulating the gene expression of fetal cells, thereby enhancing the antioxidant capacity of fetal cells, suggesting that Lactobacillus crispatus from amniotic fluid may have the potential to develop new antioxidant probiotic products, but no Lactobacillus crispatus strains with antioxidant capacity derived from amniotic fluid have been found so far.

[0005] Therefore, screening Lactobacillus crispatus with strong antioxidant capacity is of great significance for broadening its application scope in antioxidant and developing functional probiotic foods and health products. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a strain of Lactobacillus crispatus derived from amniotic fluid and its application. The new strain of Lactobacillus crispatus screened by the present invention is derived from amniotic fluid, can effectively remove free radicals, has strong antioxidant ability, high hydrophobicity, strong self-aggregation ability, can adhere to and colonize in the body, and the products developed using the new strain have the effects of regulating gastrointestinal flora and anti-oxidation, and have broad application prospects.

[0007] In a first aspect of the present invention, the present invention provides a microorganism. According to an embodiment of the present invention, the microorganism is Lactobacillus crispatus, which was deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration on July 3, 2024, with a deposit number of CGMCC No.31148.

[0008] Collection information:

[0009] Strain name: LaBA02-3

[0010] Taxonomic nomenclature: Lactobacillus crispatus

[0011] Deposit date: July 3, 2024

[0012] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0013] Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101

[0014] Deposit number: CGMCC No.31148

[0015] The new strain of Lactobacillus crispatus screened by the present invention is derived from amniotic fluid, can effectively remove free radicals, has strong antioxidant ability, high hydrophobicity, strong self-aggregation ability, can adhere to and colonize in organisms, and the products developed using the new strain have the effects of regulating gastrointestinal flora and anti-oxidation, and have broad application prospects.

[0016] In a second aspect of the present invention, the present invention provides a microbial agent. According to an embodiment of the present invention, the microorganism is included.

[0017] Those skilled in the art will appreciate that the features and advantages described above for microorganisms are also applicable to the microbial agent and will not be described in detail here.

[0018] In a third aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the aforementioned microorganism or the aforementioned microbial agent. The pharmaceutical composition according to an embodiment of the present invention can be used to prevent or treat diseases related to gastrointestinal flora imbalance or diseases related to oxidative stress.

[0019] In a fourth aspect of the present invention, the present invention proposes the use of the aforementioned microorganism, the aforementioned microbial agent or the aforementioned pharmaceutical composition in the preparation of a drug. According to an embodiment of the present invention, the drug has at least one of the following uses: preventing and / or treating diseases related to gastrointestinal flora imbalance; preventing and / or treating diseases related to oxidative stress.

[0020] Those skilled in the art will appreciate that the features and advantages described above for microorganisms, microbial agents or pharmaceutical compositions are also applicable to this use and will not be described in detail herein.

[0021] In a fifth aspect of the present invention, the present invention provides a food, health product or feed. According to an embodiment of the present invention, the food, health product or feed helps to regulate the gastric and / or intestinal flora and / or helps to resist oxidation, including: the aforementioned microorganism or the aforementioned microbial agent.

[0022] Those skilled in the art will appreciate that the features and advantages described above for microorganisms and microbial agents are also applicable to the food, health products or feed, and will not be elaborated herein.

[0023] In a sixth aspect of the present invention, the present invention proposes the use of the aforementioned microorganism or the aforementioned microbial agent in the preparation of food, health products or feed. According to an embodiment of the present invention, the food, health products or feed has at least one of the following functions: regulating gastric and / or intestinal flora; anti-oxidation.

[0024] Those skilled in the art will appreciate that the features and advantages described above for microorganisms and microbial agents are also applicable to this use and will not be described in detail here.

[0025] In a seventh aspect of the present invention, the present invention provides an antioxidant product, which according to an embodiment of the present invention comprises: the aforementioned microorganism and the aforementioned microbial agent.

[0026] Those skilled in the art will appreciate that the features and advantages described above for microorganisms and microbial agents are also applicable to the antioxidant product and will not be elaborated herein.

[0027] In an eighth aspect of the present invention, the present invention provides a method for improving the antioxidant activity of a sample. According to an embodiment of the present invention, the method includes: contacting the sample with the aforementioned microorganism, the aforementioned microbial agent, or the aforementioned antioxidant product. According to the method for improving the antioxidant activity of a sample according to an embodiment of the present invention, the aforementioned microorganism effectively removes free radicals to improve the antioxidant capacity of the sample.

[0028] In a ninth aspect of the present invention, the present invention proposes the use of the aforementioned microorganisms, the aforementioned microbial agents or the aforementioned antioxidant products in the preparation of products. According to an embodiment of the present invention, the product is used for antioxidant.

[0029] Those skilled in the art will appreciate that the features and advantages described above for microorganisms, microbial agents or the aforementioned antioxidant products are also applicable to this application and will not be described in detail herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] 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:

[0031] Figure 1 This is a colony morphology diagram of Lactobacillus crispatus CGMCC NO.31148 in Example 1 of the present invention;

[0032] Figure 2 The genome structure diagram of Lactobacillus crispatus CGMCC NO.31148 in Example 1 of the present invention;

[0033] Figure 3 This is a diagram showing the result of constructing a phylogenetic tree of Lactobacillus crispatus CGMCC NO.31148 and strains from different sources in Example 1 of the present invention;

[0034] Figure 4 The average nucleotide identity (ANI) heat map of Lactobacillus crispatus CGMCC NO.31148 in Example 1 of the present invention;

[0035] Figure 5 This is a graph showing the results of investigating the ability of Lactobacillus crispatus CGMCC NO.31148 in Example 2 of the present invention to scavenge diphenyltrinitrophenylhydrazine (DPPH) free radicals, **: p<0.01, ***: p<0.001, ****: p<0.0001;

[0036] Figure 6 This is a graph showing the results of investigating the ability of Lactobacillus crispatus CGMCC NO.31148 to scavenge hydroxyl radicals in Example 2 of the present invention, **: p<0.01, ***: p<0.001;

[0037] Figure 7 This is a graph showing the results of investigating the ability of Lactobacillus crispatus CGMCC NO.31148 to scavenge superoxide anion radicals in Example 2 of the present invention, wherein, **: p<0.01, ***: p<0.001;

[0038] Figure 8 This is a graph showing the results of the surface hydrophobicity determination of Lactobacillus crispatus CGMCC NO.31148 in Example 3 of the present invention, p<0.05;

[0039] Fig. 9 This is a graph showing the percentage of self-aggregation ability of Lactobacillus crispatus CGMCC NO.31148 in Example 4 of the present invention at the eighth hour, p<0.05. DETAILED DESCRIPTION

[0040] 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 construed as limiting the present invention.

[0041] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0042] 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.

[0043] Terms and Definitions

[0044] As used herein, the term "Lactobacillus crispatus" refers to a lactic acid bacterium belonging to the phylum Firmicutes, class Bacilli, order Lactobacillales, family Lactobacillaceae, and genus Lactobacillus. It is a facultative anaerobic, Gram-positive bacillus with an elongated, curved, slender rod shape.

[0045] As used herein, the term "oxidative stress" refers to a state in which free radical production in the body exceeds the clearance capacity of the antioxidant system, leading to cellular damage.

[0046] Bacteria

[0047] The present invention provides a microorganism. According to an embodiment of the present invention, the microorganism is Lactobacillus crispatus, which was deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration on July 3, 2024, with a deposit number of CGMCC No.31148.

[0048] According to an embodiment of the present invention, the 16S rDNA sequencing result of the microorganism is shown as SEQ ID NO:1.

[0049] The new strain of Lactobacillus crispatus screened by the present invention is derived from amniotic fluid, can effectively remove free radicals, has strong antioxidant ability, high hydrophobicity, strong self-aggregation ability, can adhere to and colonize in organisms, and the products developed using the new strain have the effects of regulating gastrointestinal flora and anti-oxidation, and have broad application prospects.

[0050] In this document, the term "Lactobacillus crispatus LaBA02-3" is synonymous with "Lactobacillus crispatus CGMCC NO. 31148".

[0051] Microbial Agents

[0052] The present invention provides a microbial agent, which includes the aforementioned microorganisms according to an embodiment of the present invention.

[0053] Those skilled in the art will appreciate that the features and advantages described above for microorganisms are also applicable to the microbial agent and will not be described in detail here.

[0054] It should be noted that the microbial agent of the present invention may be a liquid microbial agent, including but not limited to fermentation products, etc.; it may also be a solid microbial agent, including but not limited to freeze-dried powder, etc.

[0055] It should be noted that, in the microbial agent of the present invention, the Lactobacillus crispatus CGMCC NO.31148 exists in the form of living cells and / or non-living cells.

[0056] As used herein, "living cells" refer to cells that have the ability to metabolize, reproduce or replicate.

[0057] Exemplarily, the living cells may be immobilized cells. Herein, "immobilized cells" refer to living cells that are fixed on a carrier and can carry out life activities such as growth, development, reproduction, inheritance and metabolism within a certain spatial range.

[0058] In this article, "non-living cells" refer to cells that do not have the ability to metabolize, reproduce and replicate, including but not limited to dry bacteria. Exemplarily, the microbial agent is a freeze-dried powder.

[0059] In some specific embodiments, the Lactobacillus crispatus CGMCC NO.31148 exists in the form of living cells, dried bacteria, immobilized cells or any other forms.

[0060] In some specific embodiments, the dry bacteria are obtained by freeze-drying the Lactobacillus crispatus CGMCC NO.31148.

[0061] In some specific embodiments, the microbial agent may also contain strains acceptable to medicines.

[0062] In some specific embodiments, the microbial agent further includes an excipient or carrier acceptable in medicines.

[0063] In some specific embodiments, the microbial agent may also contain strains acceptable to food and feed.

[0064] In some specific embodiments, the microbial agent further includes an auxiliary material or carrier acceptable in food, or an auxiliary material or carrier acceptable in animal feed.

[0065] 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.

[0066] In this context, "adjuvant or carrier acceptable in animal feed" refers to a substance or composition that can be consumed by animals, which may be adjusted according to the animal feed requirements of different countries.

[0067] Pharmaceutical composition

[0068] The present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the aforementioned microorganism or the aforementioned microbial agent. The pharmaceutical composition according to an embodiment of the present invention can be used to prevent or treat diseases related to gastrointestinal flora imbalance or diseases related to oxidative stress.

[0069] According to an embodiment of the present invention, the pharmaceutical composition further includes a pharmaceutically acceptable excipient or carrier. Such as but not limited to one or more solvents, dispersion media, diluents, dispersing aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, adhesives, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, colorants, etc. Excipients such as starch, lactose or dextrin. Pharmaceutically acceptable excipients are well known in the art. Examples of diluents may include but are not limited to calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar and / or combinations thereof.

[0070] According to an embodiment of the present invention, the pharmaceutical composition is presented in one of the forms including but not limited to pills, powders, capsules, tablets, granular powders, film-coated agents, orally soluble granules, sachets, dragees or liquids. The pharmaceutical composition of the present disclosure can be prepared by methods well known in the pharmaceutical field.

[0071] Use in the preparation of medicines

[0072] The present invention proposes the use of the aforementioned microorganism, the aforementioned microbial agent or the aforementioned pharmaceutical composition in the preparation of a drug. According to an embodiment of the present invention, the drug has at least one of the following uses: preventing and / or treating diseases related to gastrointestinal flora imbalance; preventing and / or treating diseases related to oxidative stress.

[0073] Those skilled in the art will appreciate that the features and advantages described above for microorganisms, microbial agents or pharmaceutical compositions are also applicable to this use and will not be described in detail herein.

[0074] Food, health products or feed

[0075] The present invention provides a food, health product or feed. According to an embodiment of the present invention, the food, health product or feed helps to regulate gastric or intestinal flora and / or helps to resist oxidation, and includes: the aforementioned microorganism or the aforementioned microbial agent.

[0076] According to a specific embodiment of the present invention, the health care product or food further comprises an auxiliary material acceptable to the health care product or food.

[0077] For example, it can be a solubilizer, a co-solvent, an isotonicity regulator, a pH regulator (hydrochloric acid, sulfuric acid, lactic acid, malic acid, acetic acid, citric acid, phosphoric acid, sodium hydroxide, sodium carbonate, sodium bicarbonate, disodium bicarbonate, sodium citrate, arginine), an antioxidant (L-cysteine ​​hydrochloride, sodium sulfite, sodium bisulfite, propyl gallate, glutathione sodium thiosulfate, thiourea, thioglycolic acid, sodium metabisulfite, potassium metabisulfite, vitamin E), an emulsifier (lecithin, soy lecithin, poloxamer 68, cholesterol, glycerol monooleate), an adsorbent, a complexing agent (disodium ethylenediaminetetraacetate, calcium disodium ethylenediaminetetraacetate), a sustained-release agent, an excipient, a binder, a diluent, a disintegrant, a retarder, a thickener, a suspending agent or a stabilizer, etc., and the specific type is not limited.

[0078] Exemplarily, the health care product or food acceptable excipients include at least one of salt (organic salt or inorganic salt), carbohydrates (lactose, starch, dextrin, microcrystalline cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose), yeast, povidone, cross-linked polyvinylpyrrolidone, silicon dioxide, vegetable oil, animal oil, Tween 80, Tween 60, poloxamer, meglumine, organic acid, amino acid, vitamin (vitamin C), glycerol, sorbitol, xylitol and mannitol.

[0079] Illustratively, the aforementioned foods include but are not limited to: biscuits, yogurt, solid beverages, milk powder, cheese, soy products, candies, etc.

[0080] Illustratively, the aforementioned health care products include but are not limited to: health care products for humans and health care products for animals.

[0081] According to a specific embodiment of the present invention, the feed further comprises an adjuvant or carrier acceptable in animal feed.

[0082] Exemplarily, the acceptable excipients or carriers in the animal feed include at least one of corn bran, wheat bran, rice bran, soybean meal, fish meal, blood meal, feather meal, calcium carbonate, calcium hydrogen phosphate, sulfate, sodium chloride, vitamins, lysine, methionine, vegetable oil, animal fat, cellulose, lignin, ethoxyquin, propionate, benzoate, colorant, sweetener, guar gum, xanthan gum, bulking agent, cellulase, and protease.

[0083] Exemplarily, the aforementioned feed includes but is not limited to: pig feed, poultry feed (such as chickens and ducks), ruminant feed (such as cattle and sheep), aquatic feed (such as fish and shrimp), pet feed (such as dogs and cats), special animal feed (such as rabbits and foxes), etc.

[0084] Those skilled in the art will appreciate that the features and advantages described above for microorganisms and microbial agents are also applicable to the food, health products or feed, and will not be elaborated herein.

[0085] Use in preparing food, health products or feed

[0086] The present invention proposes the use of the aforementioned microorganism or the aforementioned microbial agent in the preparation of food, health products or feed. According to an embodiment of the present invention, the food, health products or feed has at least one of the following functions: regulating gastric and / or intestinal flora; anti-oxidation.

[0087] Those skilled in the art will appreciate that the features and advantages described above for microorganisms and microbial agents are also applicable to this use and will not be described in detail here.

[0088] Antioxidant products

[0089] The present invention provides an antioxidant product. According to an embodiment of the present invention, the product comprises: the aforementioned microorganism and the aforementioned microbial agent.

[0090] Those skilled in the art will appreciate that the features and advantages described above for microorganisms and microbial agents are also applicable to the antioxidant product and will not be elaborated herein.

[0091] Methods for improving the antioxidant activity of samples

[0092] The present invention provides a method for improving the antioxidant activity of a sample. According to an embodiment of the present invention, the method includes: contacting the sample with the aforementioned microorganism, the aforementioned microbial agent, or the aforementioned antioxidant product. According to the method for improving the antioxidant activity of a sample according to an embodiment of the present invention, the aforementioned microorganism effectively removes free radicals to improve the antioxidant capacity of the sample.

[0093] According to an embodiment of the present invention, the sample is a biological sample or a cell culture medium;

[0094] According to an embodiment of the present invention, the biological sample contains cells, including but not limited to: blood samples, tissue samples, bone marrow samples, saliva samples, urine samples, stool samples, cerebrospinal fluid samples, alveolar lavage fluid, cell culture, biopsy samples, amniotic fluid samples, semen samples, and cervical smears.

[0095] application

[0096] The present invention proposes the use of the aforementioned microorganism, the aforementioned microbial agent or the aforementioned antioxidant product in the preparation of a product. According to an embodiment of the present invention, the product is used for antioxidant.

[0097] According to an embodiment of the present invention, the antioxidant products include but are not limited to: reagents for biological sample detection, cell culture medium;

[0098] According to an embodiment of the present invention, the biological sample contains cells, including but not limited to: blood samples, tissue samples, bone marrow samples, saliva samples, urine samples, stool samples, cerebrospinal fluid samples, alveolar lavage fluid, cell culture, biopsy samples, amniotic fluid samples, semen samples, and cervical smears.

[0099] Those skilled in the art will appreciate that the features and advantages described above for microorganisms, microbial agents or the aforementioned antioxidant products are also applicable to this application and will not be described in detail herein.

[0100] 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.

[0101] Example 1: Screening, separation, purification and identification of Lactobacillus crispatus CGMCC NO.31148

[0102] 1. Sample source

[0103] The strain used in the present invention is isolated from amniotic fluid samples collected from pregnant women undergoing cesarean section and excrement (meconium samples) of the first bowel movement within 24 to 48 hours after birth of healthy newborns in the obstetrics department of Peking University Third Hospital in Beijing.

[0104] 2. Isolation and purification of strains

[0105] Add the amniotic fluid sample and meconium sample to sterile phosphate buffered saline (PBS) buffer and mix thoroughly. -6 , 10 -7 , 10 -8 Gradient dilution was performed, and 100uL of the bacterial solution was spread on MRS agar medium containing nystatin, and each dilution was repeated three times, and anaerobic culture was carried out at 37℃ for 48h. After the anaerobic culture, a single colony plate with 50 to 150 colonies was selected from the MRS agar medium, and then a single colony was picked from the single colony plate and continued to be isolated and cultured on MRS agar medium containing nystatin for 48h to obtain a purified strain. The obtained strains were all frozen and stored at -80℃ in MRS broth medium containing 40% glycerol.

[0106] The strain isolated and purified from the amniotic fluid sample was numbered LaBA02-3.

[0107] The strains isolated and purified from meconium samples were numbered BiB2A14-3, BiBF10-4, reBF17-3, reBF17-6, BiB1F19-2, reBF23-20, reBF24-5, reBF24-6, reBF26-6, reBF26-11, and reBF20-3.

[0108] Among them, the formula of MRS agar medium containing nystatin: 10g soy peptone, 10g beef extract, 5g yeast powder, 20g glucose, 1mL Tween 80, 2g sodium dihydrogen phosphate, 5g anhydrous sodium acetate, 2g triammine citrate, 0.05g manganese sulfate, 0.1g magnesium sulfate, 1L distilled water, adjust the pH to about 6.2, 15g agar, sterilize at 121℃ for 15min.

[0109] 3. Identification of strains

[0110] (1) PCR amplification and sequencing analysis of 16S rDNA gene

[0111] Take 1 mL of the above-mentioned isolated and purified bacteria cultured for 12-48 hours to a 1.5 mL EP tube, centrifuge at 8000 r / min for 3 minutes, discard the supernatant, add 1 mL of sterile water, mix thoroughly, centrifuge (8000 r / min, 3 minutes), discard the supernatant, extract DNA from the bacterial precipitate, and then use the universal primers of the 16S rRNA gene to perform PCR amplification on the extracted DNA to obtain PCR products. The PCR amplification product was identified by 1.2% agarose gel electrophoresis and sent to Shanghai Shenggong Biotechnology Co., Ltd. for nucleic acid sequence determination, and the base sequence in the nucleic acid sequence determination result was entered into the NCBI database for BLAST sequence comparison.

[0112] The results of the colony morphology of Lactobacillus crispatus CGMCC NO.31148 are shown in Figure 1 .

[0113] The results showed that: (1) the colonies of CGMCC NO.31148 strain from amniotic fluid samples were round or nearly round on MRS agar medium, with a diameter of about 1-2 mm, neat edges, smooth surface, milky white color, relatively moist texture, slightly sticky, and opaque or slightly transparent; (2) the sequence of CGMCC NO.31148 strain from amniotic fluid samples had a homology of more than 99% with the 16SrDNA sequence of Lactobacillus crispatus. The strains BiB2A14-3, BiBF10-4, reBF17-3, reBF17-6, BiB1F19-2, reBF23-20, reBF24-5, reBF24-6, reBF26-6, reBF26-11, and reBF20-3 from meconium samples had a homology of more than 99% with the 16SrDNA sequence of Bifidobacterium animalis subsp. lactis.

[0114] The 16S rDNA sequencing result of Lactobacillus crispatus CGMCC NO.31148 is shown in SEQ ID NO:1.

[0115]

[0116] (2) Whole genome identification and analysis

[0117] The whole genomes of the above strains were sequenced and analyzed to obtain more comprehensive genetic information, including genome size, GC content, coding sequence (CDS), rRNA and tRNA genes, etc.

[0118] The genome structure of Lactobacillus crispatus CGMCC NO.31148 is shown in Figure 2 .

[0119] The results showed that the whole genome size of Lactobacillus crispatus CGMCC NO.31148 strain was 2.28Mbp; the GC content was 36.91%; the coding sequence was as follows: a total of 2380 coding sequences (CDS), 3 rRNA genes, 57 tRNA genes, and 1 tmRNA gene were annotated. In addition, 1 repeat region was also found. It is worth noting that some antioxidant genes were annotated in the genome of this strain, such as nfrA1, npr, trxB, tpx, pgl, nrdE2, fpgS, iolS and hydroxypyruvate reductase genes, which play an important role in cellular antioxidant stress response and maintaining cellular redox balance ( Figure 2 ).

[0120] (3) Phylogenetic analysis

[0121] According to the 16S rDNA sequencing results, whole genome identification and analysis results of Lactobacillus crispatus CGMCC NO.31148 strain, the Lactobacillus crispatus genome data was downloaded from the Genome database of NCBI, and the Prokka (v1.14.6) software was used to perform functional annotation of the Lactobacillus crispatus CGMCC NO.31148 genome. The OrthoFinder (v2.5.5) software was used to search for homologous genes and construct a phylogenetic tree based on the above genome functional annotation results. First, DIAMOND (v2.1.9) software was used to search for similar sequences of Lactobacillus crispatus CGMCC NO.31148, and then MAFFT (v7.525) software was used for multiple sequence alignment. Finally, FastTree (v2.1.11) software was used to calculate and infer the phylogenetic tree. The above analysis process adopted the default parameters of each software, and the relationship between Lactobacillus crispatus CGMCC NO.31148 strain and other known strains was determined based on the construction of the phylogenetic tree.

[0122] The results of phylogenetic tree construction of Lactobacillus crispatus CGMCC NO.31148 and strains from different sources are shown in Figure 3 .

[0123] The results showed that in terms of genetic relationship, Lactobacillus crispatus CGMCC NO.31148 had a close relationship with strains from feces, intestines, oral cavity and vagina, especially with strains from vagina; in the phylogenetic tree, these strains formed a close branch with Lactobacillus crispatus CGMCC NO.31148. In contrast, strains from urine and unknown sources were distantly related to CGMCC NO.31148 ( Figure 3 ).

[0124] (4) Average nucleotide identity (ANI) analysis

[0125] FastANI (v1.34) software was used to set the fragment length to 1000 (--fragLen 1000) to calculate the average nucleotide consistency between Lactobacillus crispatus CGMCC NO.31148 and the public genomes of other Lactobacillus crispatus involved in the above phylogenetic analysis, and to evaluate the genetic similarity between Lactobacillus crispatus CGMCC NO.31148 strain and other Lactobacillus crispatus involved in the above phylogenetic analysis.

[0126] The heat map results of average nucleotide identity (ANI) analysis of Lactobacillus crispatus CGMCC NO.31148 are shown in Figure 4 .

[0127] The results showed that the ANI values ​​of Lactobacillus crispatus CGMCC NO.31148 and AB70 (the strain was isolated from the vaginal fluid of Koreans and has strong antibacterial activity against vaginal pathogens) were the highest, reaching 99.7%, indicating that the two strains have a high degree of similarity in genome. Other strains similar to Lactobacillus crispatus CGMCC NO.31148 include SJ_3C_US (the strain was isolated from the human vagina), 2029, FB077_07 (the strain was isolated from the human vagina) and FB049_03 (the strain was isolated from the human vagina). The ANI values ​​of these strains all exceeded 99%, indicating that they have a high degree of consistency at the genome level, which further reflects their close relationship in evolution and function ( Figure 4 ).

[0128] The final identification determined that the isolated strain was a Lactobacillus crispatus LaBA02-3, named Lactobacillus crispatus LaBA02-3 (hereinafter referred to as "Lactobacillus crispatus CGMCC NO.31148"), which was deposited in the "General Microbiology Center of China National Culture Collection Administration" on July 3, 2024, with the deposit number CGMCCNO.31148.

[0129] Example 2: Evaluation of the antioxidant capacity of Lactobacillus crispatus CGMCC NO.31148

[0130] The inventors investigated the antioxidant capacity of Lactobacillus crispatus CGMCC NO.31148, specifically the ability of the strain to scavenge diphenyltrinitrophenylhydrazine free radical (DPPH), hydroxyl free radical, and superoxide anion free radical. The specific investigation method is as follows:

[0131] 1. Selection of test strains

[0132] The commercial strain of Bifidobacterium animalis ssp. lactisBB-12 was selected as the positive control and purchased from Chr. Hansen; other participating strains BiB2A14-3, BiBF10-4, reBF17-3, reBF17-6, BiB1F19-2, reBF23-20, reBF24-5, reBF24-6, reBF26-6, reBF26-11, and reBF20-3 were other strains screened and numbered from meconium samples in the same batch as Lactobacillus crispatus CGMCC NO.31148.

[0133] 2. Preparation of Lactobacillus crispatus CGMCC NO.31148 bacterial suspension

[0134] After thawing, Lactobacillus crispatus CGMCC NO.31148 stored in a -80℃ ultra-low temperature refrigerator was streaked on an MRS agar plate with an inoculation loop, cultured anaerobically in a constant temperature incubator at 37℃ for 48h, and a single colony was picked and placed in MRS liquid culture medium, cultured anaerobically in a constant temperature incubator at 37℃ for 48h, subcultured to the fourth generation, centrifuged (8000r / min, 10min, 4℃), discarded the supernatant, collected the bacterial precipitate, washed the bacterial precipitate twice with sterile saline and resuspended in PBS solution to 1×10 8 CFU / mL, the bacterial suspension is ready for use.

[0135] 3. Determination of DPPH free radical scavenging rate of Lactobacillus crispatus CGMCC NO.31148

[0136] Weigh 0.008g of 1,1-diphenyl-2-picrylphenylhydrazine free radical, dissolve it in 100mL of anhydrous ethanol, prepare a DPPH solution with a concentration of 0.2mmol / L, mix well, wrap it in tin foil and store it away from light. Take 0.5mL of the above bacterial suspension and 0.5mL of PBS and mix them with 0.5mL of 0.2mmol / L DPPH solution respectively, react at room temperature for 30min away from light, centrifuge at 8000r / min for 10min at 4℃, and use a microplate reader to measure the OD value at 517nm, which is recorded as A517(样品) Then, 0.5 mL of PBS solution and 0.5 mL of anhydrous ethanol were mixed, reacted at room temperature for 30 min in the dark, centrifuged (8000 r / min, 10 min, 4°C), and the OD value was measured at 517 nm using an enzyme marker, which was recorded as A. 517(对照) , the scavenging rate of the strain to DPPH free radicals was calculated according to formula 1:

[0137] DPPH free radical scavenging rate % = [1-A 517(样品) / A 517(对照) ]×100 formula 1

[0138] The results of the investigation on the ability of Lactobacillus crispatus CGMCC NO.31148 to scavenge diphenyltrinitrophenylhydrazine (DPPH) free radicals are shown in Figure 5 .

[0139] DPPH is a stable organic nitrogen free radical that can absorb electrons from antioxidants, causing the sample to change color. The DPPH free radical scavenging rate is positively correlated with the antioxidant capacity of the strain. The results showed that: (1) The DPPH free radical scavenging rate of Lactobacillus crispatus CGMCC NO.31148 was 37.73% ± 1.29%, which was significantly higher than that of most experimental strains (p < 0.05). (2) Compared with the DPPH free radical scavenging rate of the commercial positive control strain BB-12 of 26.45% ± 0.91%, the DPPH free radical scavenging rate of Lactobacillus crispatus CGMCC NO.31148 was significantly increased ( Figure 5 ). The results showed that Lactobacillus crispatus CGMCCNO.31148 strain had obvious advantages in scavenging DPPH free radicals and had strong antioxidant capacity.

[0140] 4. Determination of the ability of strains to scavenge hydroxyl free radicals

[0141] Take 0.5 mL of the above bacterial suspension, 0.5 mL of 2.5 mmol / L 1,10-phenanthroline, 0.5 mL of PBS, and 0.5 mL of 2.5 mmol / L FeSO4, mix well, then add 0.5 mL of 20 mmol / L H2O2, mix thoroughly, incubate at 37°C in dark, in a water bath for 1.5 h, and use an ELISA reader to measure the OD value at 536 nm, which is recorded as A. 536(样品) Then take 0.5mL PBS, 0.5mL 2.5mmol / L 1,10-phenanthroline, 0.5mL PBS, 0.5mL 2.5mmol / L FeSO4, mix well, add 0.5mL 20mmol / L H2O2, mix thoroughly in a 5ml EP tube, place in a light-proof water bath at 37℃ for 1.5h, and use an enzyme marker to measure the OD value at 536nm, which is recorded as A. 536(空白)Take 0.5 mL of the above bacterial suspension, 0.5 mL of 2.5 mmol / L 1,10-phenanthroline, 0.5 mL of PBS, and 0.5 mL of 2.5 mmol / L FeSO4, mix well, add 0.5 mL of distilled water, mix thoroughly in a 5 mL EP tube, and place in a light-proof water bath at 37°C for 1.5 h. Use an enzyme marker to measure the OD value at 536 nm, which is recorded as A. 536(对照) , the scavenging rate of the strain for hydroxyl free radicals was calculated according to Formula 2:

[0142] Hydroxyl free radical scavenging rate % = ((A 536(样品) -A 536(空白) ) / (A 536(对照) -A 536(空白) ))×100 Formula 2 Evaluation results of the scavenging ability of Lactobacillus crispatus CGMCC NO.31148 on hydroxyl free radicals are shown in Figure 6 .

[0143] The results showed that hydroxyl radicals are extremely destructive reactive oxygen species that can cause lipid peroxidation, DNA damage, and oxidative damage to other biological molecules. Excessive hydroxyl radicals can lead to cell dysfunction and even cause various diseases. Therefore, the determination of the hydroxyl radical scavenging ability of the strain is an important indicator for evaluating its antioxidant capacity. The hydroxyl radical scavenging rate of the commercial positive control strain BB-12 was 16.58%±1.19%. The hydroxyl radical scavenging rates of each experimental strain ranged from 0.87% to 31.30%, showing strain specificity. The hydroxyl radical scavenging rate of Lactobacillus crispatus CGMCC NO.31148 was 20.93%±1.43%, which was significantly higher than that of most experimental strains such as BiBF10-4 (p<0.05) and the commercial positive control strain BB-12 (p<0.05) ( Figure 6 ). The results showed that Lactobacillus crispatus CGMCC NO.31148 strain showed high activity in scavenging hydroxyl free radicals and had strong antioxidant capacity.

[0144] 5. Determination of the ability of strains to scavenge superoxide anion free radicals

[0145] Take 3.4mL Tris-HCl solution (50mmol / L, pH=8.2) and 0.5mL pyrogallol solution (50mmol / L), add 1mL of bacterial suspension, mix well and place in a 25℃ metal bath for 4min, then add 0.1mL HCl solution (8mol / L) to terminate the reaction, use a microplate reader to measure the OD value at 320nm, and the scavenging rate of the strain for superoxide anion radicals is calculated according to Formula 3:

[0146] Superoxide anion free radical scavenging rate (%) = (1-A1 / A0) × 100 Formula 3

[0147] Among them, A0 is the absorbance at 320 nm when an equal volume of water replaces the sample; A1 is the absorbance at 320 nm of the experimental group.

[0148] The results of the evaluation of the ability of Lactobacillus crispatus CGMCC NO.31148 to scavenge superoxide anion free radicals are shown in Figure 7 .

[0149] The results showed that superoxide anion is an important reactive oxygen species that can serve as a signal molecule for apoptosis or aging. Excessive superoxide anion can cause cell damage. Therefore, evaluating the superoxide anion scavenging ability of the strain is an important means to evaluate its antioxidant activity. By measuring the superoxide anion scavenging effect of the strain, its potential in preventing oxidative stress can be effectively judged. The superoxide anion scavenging rate of each experimental strain ranged from -9.12% to 35.00%. The superoxide anion scavenging rate of Lactobacillus crispatus CGMCC NO.31148 was 10.51% ± 5.73%, which was significantly higher than the superoxide anion scavenging rates of the two experimental strains reBF20-3 and reBF24-5 (p < 0.05), and slightly higher than the commercial positive control strain BB-12 (7.52% ± 2.58%) ( Figure 7 ). The results showed that Lactobacillus crispatus CGMCC NO.31148 had a strong potential in scavenging superoxide anions.

[0150] The above results show that Lactobacillus crispatus CGMCC NO.31148 has obvious advantages in scavenging DPPH free radicals, hydroxyl free radicals and superoxide anions, and has strong antioxidant capacity and potential to prevent oxidative stress.

[0151] Example 3: Evaluation of the surface hydrophobicity of Lactobacillus crispatus CGMCC NO.31148

[0152] Related studies have shown that the adhesion ability of bacteria is positively correlated with the hydrophobicity of their surface. The higher the hydrophobicity of the probiotic biofilm, the easier it is to form a biofilm and adhere to the surface of the human gastrointestinal tract, enhance the survival rate in the intestinal environment, and fight against pathogens through competitive exclusion mechanisms, thereby exerting a probiotic effect. Strains with a hydrophobicity of less than 30.0% are hydrophilic, strains with a hydrophobicity between 30.0% and 60.0% are moderately hydrophobic, and strains with a hydrophobicity greater than 60.0% are highly hydrophobic.

[0153] In order to evaluate the adhesion ability of the strain on the surface of the host intestine and other surfaces, and then predict its colonization potential and the possibility of exerting a probiotic effect, the inventors used the microbial adhesion hydrocarbon method (BATH) to measure the surface hydrophobicity of Lactobacillus crispatus CGMCC NO.31148, and thus evaluated its surface hydrophobicity. The specific method is as follows:

[0154] 1. Selection of test strains

[0155] The commercial strain of Bifidobacterium animalis subspecies Lactobacillus BB-12 was selected as the positive control and purchased from Chr. Hansen; the other test strains reBF23-20, reBF24-6, and reBF26-6 were other strains screened and numbered from meconium in the same batch as Lactobacillus crispatus CGMCC NO.31148.

[0156] 2. Determination of surface hydrophobicity of Lactobacillus crispatus CGMCC NO.31148

[0157] The OD value of the bacterial suspension described in Example 2 was measured at 600 nm using an ELISA reader and recorded as A0. 1.5 mL of the bacterial suspension described in Example 2 was mixed with 100 μL of trichlorotoluene, vortexed for 10 min, and allowed to stand at room temperature for 30 min. The lower aqueous phase was aspirated and the OD value was measured at 600 nm using an ELISA reader and recorded as A. The surface hydrophobicity of the strain was calculated according to Formula 4:

[0158] Surface hydrophobicity (%) = ((A0-A) / A0) × 100 Formula 4

[0159] The results of the surface hydrophobicity determination of Lactobacillus crispatus CGMCC NO.31148 in Example 3 are shown in Figure 8 .

[0160] The results showed that the commercial positive control strain BB-12, as a positive control strain, had a surface hydrophobicity of 77.94% ± 0.61%, and its high surface hydrophobicity indicated that it had good adhesion properties. The surface hydrophobicity of Lactobacillus crispatus CGMCCNO.31148 was 80.91% ± 1.27%, which was not significantly different from the commercial positive control strain BB-12.

[0161] The above results show that the surface hydrophobicity of Lactobacillus crispatus CGMCC NO.31148 is comparable to that of the positive control strain BB-12, indicating that it has extremely strong adhesion ability and has the potential to colonize in the human body and play a significant probiotic role.

[0162] Example 4: Evaluation of the self-aggregation ability of Lactobacillus crispatus CGMCC NO.31148

[0163] Related studies have shown that the adhesion ability of bacteria is positively correlated with their self-aggregation ability. Self-aggregation ability can evaluate the adhesion and colonization ability of strains in the body, which is crucial for the strains to play a probiotic role. Self-aggregation ability is generally divided into three categories: low (16%-35%), medium (35%-50%) and high (more than 50%).

[0164] In order to evaluate the colonization potential of the strain in the host, the inventors evaluated the self-aggregation ability of Lactobacillus crispatus CGMCC NO.31148. The specific method is as follows:

[0165] 1. Selection of test strains

[0166] The commercial strain of Bifidobacterium animalis subspecies Lactobacillus BB-12 was selected as the positive control and purchased from Chr. Hansen; the other test strains reBF23-20, reBF24-6, and reBF26-6 were other strains screened and numbered from meconium in the same batch as Lactobacillus crispatus CGMCC NO.31148.

[0167] 2. Determination of the self-aggregation ability of Lactobacillus crispatus CGMCC NO.31148d

[0168] The OD value of the bacterial suspension described in Example 2 was measured at 600 nm using an ELISA instrument and recorded as A0. The bacterial suspension described in Example 2 was incubated at 37°C for 2 h, 4 h, 6 h, and 8 h, and three parallel bacterial suspension samples were prepared for each time period. The OD value of the supernatant of the bacterial suspension samples described in Example 2 incubated for different times was measured at 600 nm using an ELISA instrument and recorded as A t , the self-aggregation ability of the strain was calculated according to Formula 5:

[0169] Self-aggregation ability (%) = ((A0-A t ) / A0)×100 formula 5

[0170] The results of the five strains with significant antioxidant properties and their self-aggregation abilities at 2h, 4h, 6h and 8h are shown in Table 1.

[0171] Table 1 Results of the self-aggregation ability of 5 strains with significant antioxidant properties

[0172]

[0173] Note: Different lowercase letters in the table indicate significant differences in the self-aggregation abilities of different strains under the same time conditions (p < 0.05).

[0174] The results of the self-aggregation ability percentage of Lactobacillus crispatus CGMCC NO.31148 in the eighth hour are shown in Fig. 9 .

[0175] The results showed that Lactobacillus crispatus CGMCC NO.31148 was extremely outstanding in self-aggregation ability, which was significantly higher than other strains in each time period (p<0.05), indicating that it has strong self-aggregation ability. As the static time passed, the bacteria grew and the self-aggregation ability of the strain continued to increase. After 8 hours of static standing, Lactobacillus crispatus CGMCC NO.31148 showed a self-aggregation ability of more than 50%, with high self-aggregation, and significantly higher than the commercial positive control strain BB-12 (p<0.05).

[0176] The above results show that Lactobacillus crispatus CGMCC NO.31148 has a very strong self-aggregation ability, which is conducive to the colonization, reproduction and survival of the strain in the human body, thereby exerting its probiotic properties.

[0177] 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 microorganism, characterized in that The microorganism is Lactobacillus crispatus, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on July 3, 2024, with the deposit number CGMCC No.31148.

2. A microbial agent, characterized in that: The microorganism according to claim 1.

3. A pharmaceutical composition, characterized in that include: The microorganism according to claim 1 or the microbial agent according to claim 2; Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient or carrier; Optionally, the pharmaceutical composition is in the form of one of pills, powders, capsules, tablets, granular powders, film-coated tablets, orally disintegrating granules, sachets, dragees or liquids.

4. Use of the microorganism according to claim 1, the microbial agent according to claim 2 or the pharmaceutical composition according to claim 3 in the preparation of a drug, wherein the drug has at least one of the following uses: Prevent and / or treat diseases related to gastrointestinal flora imbalance; Prevention and / or treatment of oxidative stress related diseases.

5. A food, health product or feed, characterized in that: The food, health product or feed helps to regulate the gastric and / or intestinal flora and / or helps to resist oxidation, and includes: the microorganism according to claim 1 or the microbial agent according to claim 2.

6. Use of the microorganism according to claim 1 or the microbial agent according to claim 2 in the preparation of food, health products or feed, wherein the food, health products or feed has at least one of the following functions: Modulate gastric and / or intestinal flora; Antioxidant.

7. An antioxidant product, characterized in that: include: The microorganism according to claim 1 and the microbial agent according to claim 2.

8. A method for improving the antioxidant activity of a sample, characterized in that: include: contacting the sample with the microorganism according to claim 1, the microbial agent according to claim 2, or the antioxidant preparation according to claim 7; Optionally, the sample is a biological sample or a cell culture medium; Optionally, the biological sample comprises cells, including but not limited to: blood sample, tissue sample, bone marrow sample, saliva sample, urine sample, stool sample, cerebrospinal fluid sample, bronchoalveolar lavage fluid, cell culture, biopsy sample, amniotic fluid sample, semen sample, cervical smear.

9. Use of the microorganism according to claim 1, the microbial agent according to claim 2 or the antioxidant product according to claim 7 in the preparation of a product for anti-oxidation.

10. The use according to claim 9, characterized in that: The products include but are not limited to: medicines, food, health products, feed, cosmetics, antioxidant products; Optionally, the antioxidant products include but are not limited to: biological sample detection reagents, cell culture media; Optionally, the biological sample comprises cells, including but not limited to: blood sample, tissue sample, bone marrow sample, saliva sample, urine sample, stool sample, cerebrospinal fluid sample, bronchoalveolar lavage fluid, cell culture, biopsy sample, amniotic fluid sample, semen sample, cervical smear.

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