Lactobacillus plantarum 9A-2 and application thereof in preparation of products for regulating intestinal tracts and protecting eyes

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus plantarum 9A-2 and application thereof in preparation of products for regulating intestinal tracts and protecting eyes. A probiotic product is successfully researched and developed on the basis of the lactobacillus plantarum, and verification shows that the lactobacillus plantarum can continuously provide endophytic essential isorhamnetin, chromium and vitamin H in the intestinal reproduction and growth process while regulating intestinal flora balance and promoting digestion, and also has the effect of relieving visual fatigue; particularly, visual fatigue caused by eye related diseases is relieved. The endophytic essential quantity of isorhamnetin, chromium and vitamin H can be continuously provided in the intestinal reproduction and growth process by feeding 3 billion strains and 200 milligrams of metabolites of the strain every day, visual fatigue caused by eye related diseases can be prevented and relieved, and the body health can be kept.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Lactobacillus plantarum 9A-2 and application thereof in preparing intestinal regulating and eye protecting products. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] In recent years, studies have found that intestinal flora not only affects intestinal health, but may also affect eye health through the gut-ocular axis. The gut-ocular axis is a neural network connecting the intestinal nervous system and the eye nerves. Intestinal flora imbalance and increased intestinal permeability may send inflammatory signals from the intestine to the eyes through the gut-ocular axis, promoting the occurrence of eye diseases. Intestinal flora imbalance may be related to the occurrence of ophthalmic diseases such as uveitis, age-related macular degeneration, and dry eye.

[0004] The relationship between probiotics and eye health is an emerging research field. Probiotics are a type of active microorganisms that are beneficial to the human body. They can regulate intestinal flora through a variety of mechanisms, enhance intestinal health, and play an important role in maintaining intestinal flora balance and promoting eye health. Patent CN117643379A discloses the use of animal Bifidobacterium lactis subspecies i797 in the preparation of eye protection products. It has been verified by animal and cell experiments that it has the effect of relieving visual fatigue. The animal Bifidobacterium in the above patent has a single function, and its effect on other eye diseases is unknown. Therefore, it is clinically important to prevent and relieve ophthalmic diseases such as uveitis, age-related macular degeneration, and dry eye by taking oral prebiotics. Summary of the invention

[0005] The present invention separates a strain of Lactobacillus plantarum from fresh seabuckthorn fruit pulp. Studies have shown that the strain can regulate the balance of intestinal flora and promote digestion, and can sustainably provide endogenously required isorhamnetin, chromium, and vitamin H during the intestinal reproduction and growth process, and also has the effects of preventing, alleviating, and treating eye diseases.

[0006] Based on the above research results, the present invention provides the following technical solutions: The first aspect of the present invention provides plant lactobacillus ( Lactiplantibacillus plantarum )9A-2, the strain was deposited in Guangdong Microbiological Culture Collection Center (address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou), the deposit date is July 8, 2024, and the deposit number is GDMCC NO: 64832.

[0007] The strain provided by the present invention is identified as Lactobacillus plantarum by 16srRNA method and is a probiotic strain approved for use by the National Health Commission.

[0008] The second aspect of the present invention provides derivatives of the above-mentioned Lactobacillus plantarum 9A-2, including one or more of its live bacteria, inactivated bacteria, fermentation broth, exosomes or metabolites.

[0009] The third aspect of the present invention provides a microbial agent comprising an active ingredient, wherein the active ingredient comprises the above-mentioned Lactobacillus plantarum 9A-2 or its inactivated bacteria, fermentation broth, and metabolites.

[0010] A fourth aspect of the present invention provides a method for preparing the above-mentioned microbial agent, the preparation method comprising: S1. Activation of bacterial strains: inoculating the Lactobacillus plantarum 9A-2 into an activation medium, and culturing at 35-40° C. for 16-20 h to obtain an expanded bacterial strain solution; S2. Preparation of primary bacterial seed solution: inoculate the expanded bacterial seed solution prepared in step S1 into the primary seed culture solution, and culture at 35-40° C. for 16-20 h to obtain the primary bacterial seed solution; S3, seed tank strain preparation: the primary strain liquid prepared in step S2 is inoculated into a seed tank containing a seed culture medium under sterile conditions, the inoculation amount is 0.5-5%, the stirring culture is 110-130 r / min, the pH is natural, the temperature is 30-40°C, the tank pressure is 0.03-0.06 MPa, and the culture time is 11-13 h to obtain the seed tank strain; S4, plant lactobacillus fermentation: inoculate the seed tank strain into a fermentation tank containing sterilized fermentation culture medium, with an inoculation amount of 0.5-5%, 35-39°C, 70-90 r / min, tank pressure of 0.04-0.06 MPa, pH of 5.9-6.1, and ferment for 24-26 h; cool to 19-21°C, add sterile glycerol of 1.9-2.1% of the volume of the fermentation liquid into the fermentation tank, raise the temperature to 119-121°C and maintain for 5-7 s for inactivation treatment.

[0011] After step S4 is completed, the obtained fermentation liquid is centrifuged to collect the inactivated wet bacterial sludge metabolites, and the inactivated wet bacterial sludge metabolites are freeze-dried to obtain freeze-dried powder of the inactivated bacterial metabolites.

[0012] A fifth aspect of the present invention provides the use of the above-mentioned Lactobacillus plantarum 9A-2 or a derivative or microbial agent of Lactobacillus plantarum 9A-2 in the preparation of an eye protection product.

[0013] A sixth aspect of the present invention provides use of the above-mentioned Lactobacillus plantarum 9A-2 or a derivative or microbial agent of Lactobacillus plantarum 9A-2 in the preparation of a product for regulating the intestinal tract.

[0014] The intestinal regulation includes regulating intestinal flora, promoting intestinal peristalsis and increasing the content of beneficial ingredients in the intestine; the beneficial ingredients are one or more of isorhamnetin, chromium and vitamin H.

[0015] A seventh aspect of the present invention provides a product comprising the above-mentioned Lactobacillus plantarum 9A-2 or a derivative or microbial agent of Lactobacillus plantarum 9A-2 and an acceptable adjuvant or auxiliary material.

[0016] The beneficial effects of the present invention are: The above technical scheme screened and obtained a strain of Lactobacillus plantarum 9A-2, and a probiotic product was successfully developed based on the Lactobacillus plantarum. It has been verified that the bacteria can regulate the balance of intestinal flora and promote digestion, while providing endogenously required isorhamnetin, chromium, and vitamin H in the process of intestinal reproduction and growth, and also has the effect of relieving visual fatigue, especially relieving visual fatigue caused by eye-related diseases. Daily administration of 30 billion strains of the present invention and 200 mg of metabolites can sustainably provide endogenously required amounts of isorhamnetin, chromium, and vitamin H in the process of intestinal reproduction and growth, which can prevent and relieve visual fatigue caused by eye-related diseases and maintain the health of the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0018] Figure 1 The anterior segment of the rats in each group in Example 1 is shown in Figure 1, where (a) is the control group, with no inflammatory reaction; (b) is the model group, with dilated and tortuous iris blood vessels, hypopyon, less round pupil, and weakened or absent retinal red light reflex; (c) is the experimental group 1, with mild congestion of iris blood vessels, inflammatory reaction, and slightly delayed light reflex; (d) is the experimental group 2, with mild congestion of iris blood vessels, and mild inflammatory reaction; (e) is the experimental group 3, with inflammation disappearing and iris blood vessels returning to normal; Figure 2 is the relative expression levels of IL-4, IL-15 and IFN-γ in the serum of each group of rats in Example 1; Figure 3 Comparison of intraocular pressure of mice in each group in Example 1; Figure 4 (a) is the retinal thickness of each group of mice in Example 1, Figure 4 (b) shows the number of RGCs in each group of mice in Example 1; Figure 5 The levels of SOD, GSH-Px and NOS in each group of mice in Example 1; Figure 6is the relative expression level of NLRP3, Caspase-1, and ASC proteins in the retina of each group of mice in Example 1; Figure 7 The free radical metabolism results of the lens of each group of mice in Example 1; Figure 8 The free radical metabolism results of plasma in each group of mice in Example 1; Fig. 9 The metabolomics analysis results of mouse feces in Example 1. DETAILED DESCRIPTION

[0019] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0020] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0021] In a typical embodiment of the present invention, a plant lactobacillus ( Lactiplantibacillus plantarum )9A-2, deposited in Guangdong Microbiological Culture Collection Center (address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou), the deposit date is July 8, 2024, and the deposit number is GDMCC NO: 64832.

[0022] The bacterium was isolated from fresh seabuckthorn fruit pulp and identified as Lactobacillus plantarum by 16srRNA method. The identification sequence of the bacterium is as follows:

[0023] The strain provided by the present invention is identified as Lactobacillus plantarum by 16srRNA method and is a probiotic strain approved for use by the National Health Commission.

[0024] In another specific embodiment of the present invention, a derivative of the above-mentioned Lactobacillus plantarum 9A-2 is provided, including one or more of its live bacteria, inactivated bacteria, fermentation broth, exosomes or metabolites.

[0025] In another specific embodiment of the present invention, a microbial agent is provided, comprising an active ingredient, wherein the active ingredient comprises the above-mentioned Lactobacillus plantarum 9A-2 or its inactivated bacteria, fermentation broth, and metabolites.

[0026] More specifically, the invention also includes auxiliary materials, which include one or more of lactulose, maltodextrin, anhydrous glucose, trehalose, broccoli powder and L-carnitine.

[0027] More specifically, the microbial agent is freeze-dried powder.

[0028] In another specific embodiment of the present invention, a method for preparing the above-mentioned microbial agent is provided, and the preparation method comprises: S1. Bacterial activation: the plant lactobacillus ( Lactiplantibacillus plantarum ) 9A-2 was inoculated into the activation medium and cultured at 35-40°C for 16-20 h to obtain the expanded bacterial culture liquid; The activation medium is composed of: based on the weight of water added, 5-20 parts of D-glucose, 1-10 parts of maltitol, 1-10 parts of beef extract powder, 0.1-2 parts of sodium acetate, 0.1-1 parts of Tween-80, 0.1-1 parts of potassium dihydrogen phosphate, 1000 parts of water, and the pH is adjusted to 7; More specifically, the composition of the activation medium is as follows: based on the weight of added water, 17 parts of D-glucose, 1 part of maltitol, 2 parts of beef extract powder, 0.4 parts of sodium acetate, 0.5 parts of Tween-80, 0.2 parts of potassium dihydrogen phosphate, 1000 parts of water, and the pH is adjusted to 7; S2. Preparation of primary bacterial seed solution: inoculate the expanded bacterial seed solution prepared in step S1 into the primary seed culture solution, and culture at 35-40° C. for 16-20 h to obtain the primary bacterial seed solution; The composition of the primary seed culture solution is as follows: based on the weight of added water, 1-10 parts of D-glucose, 1-10 parts of yeast peptone, 1-3 parts of beef extract powder, 1-5 parts of histidine, 0.5-1 parts of sodium acetate, 0.5-1.5 parts of Tween-80, 1000 parts of water, and the pH value is natural; More specifically, the composition of the primary seed culture solution is as follows: based on the weight of added water, 6 parts of D-glucose, 5 parts of yeast peptone, 1 part of beef extract powder, 2 parts of histidine, 0.7 parts of sodium acetate, 1 part of Tween-80, 1000 parts of water, and the pH value is natural; S3, seed tank strain preparation: the primary strain liquid prepared in step S2 is inoculated into a seed tank containing a seed culture medium under sterile conditions, the inoculation amount is 0.5-5%, the stirring culture is 110-130 r / min, the pH is natural, the temperature is 30-40°C, the tank pressure is 0.03-0.06 MPa, and the culture time is 11-13 h to obtain the seed tank strain; The composition of the seed culture medium is as follows: D-glucose 1-5%, yeast peptone 1-5%, dipotassium hydrogen phosphate 0.1-0.5%, calcium propionate 1-5%, sodium acetate 0.1-1%, L-theanine 1-5%, Tween-80 0.05-0.15%, polyether defoamer 0.05-0.15%, water 90-95%, pH value natural; More specifically, the composition of the seed culture medium is as follows: D-glucose 4%, yeast peptone 1.4%, dipotassium hydrogen phosphate 0.4%, calcium propionate 1%, sodium acetate 0.7%, L-theanine 1%, Tween-80 0.1%, polyether defoamer 0.1%, water 91.3%, pH natural; S4, plant lactobacillus fermentation: inoculate the seed tank strain into a fermentation tank containing sterilized fermentation culture medium, the inoculation amount is 0.5-5%, 35-39°C, 70-90 r / min, tank pressure is 0.04-0.06 MPa, pH is 5.9-6.1, and fermentation is carried out for 24-26 h; cool to 19-21°C, add sterile glycerol of 1.9-2.1% of the volume of the fermentation liquid into the fermentation tank, raise the temperature to 119-121°C and maintain for 5-7 s for inactivation treatment; The fermentation culture solution is composed of: 1-5% D-glucose, 0.5-1.5% yeast peptone, 1-2% erythritol, 0.5-1.5% bitter melon peptide powder, 1-2% yeast extract powder, 0.5-1.5% sodium acetate, 0.1-0.3% Tween-80, 0.1-1% leucine, 0.5-1.5% dipotassium hydrogen phosphate, 0.05-0.15% polyether defoamer, and 88-89% water; More specifically, the fermentation broth is composed of: 4% D-glucose, 1% yeast peptone, 1.5% erythritol, 1% bitter melon peptide powder, 1.5% yeast extract powder, 0.8% sodium acetate, 0.2% Tween-80, 0.6% leucine, 1% dipotassium hydrogen phosphate, 0.1% polyether defoamer, and 88.3% water.

[0029] In some examples of this embodiment, after step S4 is completed, the obtained fermentation broth is centrifuged to collect the inactivated wet bacterial sludge metabolites, and the inactivated wet bacterial sludge metabolites are freeze-dried (the final moisture content is preferably controlled to be 4%) to obtain freeze-dried powder of inactivated bacterial metabolites.

[0030] More specifically, the freeze-dried suspension of bacterial sludge metabolites was prepared by taking 1000 g of inactivated wet bacterial sludge metabolites, adding 4000 mL of sterile saline, 600 g of lactulose, 1200 g of broccoli powder, and 30 g of L-carnitine.

[0031] First, take 2500 mL of normal saline in the formula, add lactulose and stir to dissolve, then add 1000 g of inactivated wet bacterial sludge metabolites, and then add the remaining normal saline and broccoli powder and auxiliary materials in the formula and stir thoroughly to obtain a suspension. The suspension is freeze-dried and the moisture content is finally controlled to 4%, which becomes freeze-dried powder of inactivated bacterial metabolites.

[0032] Another specific embodiment of the present invention provides the use of the above-mentioned Lactobacillus plantarum 9A-2 or a derivative or microbial agent of Lactobacillus plantarum 9A-2 in the preparation of eye protection products.

[0033] The eye protection product can be food or medicine.

[0034] The term "food" used in the present invention can be in any form that can be eaten, including but not limited to beverages, dairy products, candy products, health foods, special dietary foods, special medical formula foods, etc.

[0035] The medicine may comprise a carrier, an excipient and a diluent, and its dosage form is an oral preparation in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, or an injection in the form of a sterile injection solution.

[0036] The product can be a medicine, and the medicine can also contain an appropriate amount of commonly used carriers, excipients and diluents. Moreover, according to the usual method, it can be made into a dosage form in the form of an oral agent such as a powder, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, and sterile injection solutions. The non-pharmaceutical active ingredients such as carriers, excipients and diluents that can be included in the present invention are well known in the art, and those of ordinary skill in the art can determine that they meet clinical standards.

[0037] The carrier, excipient and diluent of the present invention include, but are not limited to, lactose, glucose, sorbitol, mannitol, xylitol, maltitol, starch, gum arabic, gelatin, calcium phosphate, cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, and mineral oil, etc. No specific limitation is made here.

[0038] The object of application of the eye protection product is a human or a non-human mammal. The non-human mammal may be a rat, a mouse, a rabbit, a dog, etc.

[0039] In some examples of this implementation mode, the eye protection product is for relieving visual fatigue, which is visual fatigue caused by improper use of the eyes or eye-related diseases.

[0040] The term "visual fatigue", also known as eye fatigue, is a common symptom of eye discomfort. The symptoms of visual fatigue are varied, and the most common ones are the inability to use the eyes for a long time at close range, pain in the eyes and around the eye sockets, blurred vision, dry eyes, tears, etc. In severe cases, headaches and dizziness may occur. In addition, symptoms such as dry, astringent, itchy eyes, foreign body sensation, eye socket discomfort, and eye swelling may also occur. In severe cases, they may even be accompanied by headaches, nausea, and vomiting. The causes of visual fatigue are varied, and the main influencing factors include excessive use of the eyes, dry eyes, presbyopia, conjunctivitis, trachoma, keratitis, uveitis and other factors.

[0041] In some examples of this embodiment, the eye-related diseases include uveitis, macular degeneration, dry eyes, blepharitis, cataracts.

[0042] Specifically, experiments on EAU model rats have shown that it can significantly enhance the anti-inflammatory effect of rats with autoimmune uveitis, experiments on zebrafish model have shown that it can significantly prevent ocular cell apoptosis, experiments on DBA / 2J mouse model have shown that it can lower intraocular pressure, improve retinal thickness, and alleviate oxidative damage to retinal tissue, and experiments on cataract mouse model have shown that it can significantly delay the progression of cataract disease.

[0043] Another specific embodiment of the present invention provides the use of the above-mentioned Lactobacillus plantarum 9A-2 or a derivative or microbial agent of Lactobacillus plantarum 9A-2 in the preparation of a product for regulating the intestinal tract.

[0044] The intestinal regulation includes regulating intestinal flora, promoting intestinal peristalsis and increasing the content of beneficial ingredients in the intestine; the beneficial ingredients are one or more of isorhamnetin, chromium and vitamin H.

[0045] The intestinal tract regulating product of the present invention may be food or medicine, or may be food or medicine for eye protection; the food is health food.

[0046] Specifically, the intestinal tract regulating product is administered to humans or non-human mammals, such as rats, mice, rabbits, dogs, etc.

[0047] Another specific embodiment of the present invention provides a product, comprising the above-mentioned Lactobacillus plantarum 9A-2 or a derivative or microbial agent of Lactobacillus plantarum 9A-2 and an acceptable adjuvant or auxiliary material.

[0048] The product is one of food and medicine.

[0049] In some examples of this embodiment, the auxiliary agent or adjuvant may be a carrier, excipient, diluent, etc. commonly used in pharmacy. Moreover, according to the usual method, it can be made into an oral preparation in the form of powder, granule, capsule, tablet, pill, extract, liquid preparation, sugar or jelly, etc. The non-pharmaceutical active ingredients such as carrier, excipient, diluent, etc. that may be included are well known in the art, and a person of ordinary skill in the art can determine whether they meet the standards. In another specific embodiment of the present invention, the carrier, excipient and diluent include but are not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0050] In the examples, the "%" of the components in each culture medium (liquid) is the mass volume percentage, and the unit is g / mL. In the examples, the density of Tween-80 is 1.08 g / cm 3 .

[0051] The equipment used in production are as follows: culture incubator, clean bench, constant temperature incubator, various test tubes, triangular flasks, seed tanks, fermentation tanks, vacuum freeze dryer, three-dimensional mixer, low-temperature crusher, vacuum packaging machine.

[0052] Example 1 A method for preparing a postbiotic preparation with eye protection effect comprises the following steps: (1) Bacteria activation: Bacterial strain culture: Expansion culture of bacterial strains preserved in -80℃ refrigerator.

[0053] Preparation of culture medium: 17 g D-glucose, 1 g maltitol, 2 g beef extract powder, 0.4 g sodium acetate, 0.5 mL Tween-80, 0.2 g potassium dihydrogen phosphate, 1000 mL tap water, pH adjusted to 7.0.

[0054] Operation: Add D-glucose and other raw materials in the above formula into 1000 mL of water in the formula without any particular order, stir for 10 minutes to dissolve, then divide into test tubes, each test tube contains 5 mL, seal with kraft paper stopper, place in a medical sterilizer and sterilize at 0.1-0.12 MPa for 20 minutes, and the culture medium is ready.

[0055] Inoculation: Under sterile conditions (clean bench), open the -80℃ low-temperature freezer to store the frozen tube of bacteria, and dissolve it at room temperature. Then take one sterilized test tube culture medium, add 0.5 mL of the dissolved bacterial solution into the test tube, seal the tube mouth with a rubber stopper and kraft paper, shake it gently by hand, and place it in a constant temperature incubator at 37℃ for 16 hours. This is called expansion culture medium.

[0056] (2) Preparation of primary bacterial culture liquid: Primary seed culture formula: D-glucose 6 g, yeast peptone 5 g, beef extract powder 1 g, histidine 2 g, sodium acetate 0.7 g, Tween-80 1 mL, tap water 1000 mL, pH natural.

[0057] Operation: Add the above raw materials into 1000 mL of tap water without any distinction, start the blender, and stir for 10 minutes to dissolve them all. This is called the primary seed culture solution.

[0058] Add the stirred and dissolved first-level seed culture solution into the conical flask according to the dosage, then wrap the bottle mouth with 6 layers of gauze and one layer of kraft paper, and put it into the sterilizer for sterilization. Before sterilization, first open the exhaust valve on the sterilizer and close it when a small amount of steam is discharged. When the pressure on the sterilizer reaches 0.05 MPa, open the exhaust valve on the sterilizer to exhaust for 6-8 minutes, then close the exhaust valve and continue heating. When the steam pressure in the sterilizer reaches 0.1-0.12 MPa, start timing. The sterilization time is 20 minutes. After completion, leave the heat source and let it cool down naturally. When the pressure gauge on the sterilizer returns to zero, open the sterilizer, take out the sterilized culture medium and put it into the clean bench for natural cooling. When the temperature drops to 37°C, transfer the above cultured expansion culture solution to the conical flask containing the sterilized culture solution. The inoculation amount is 0.5%, that is, 100 mL of first-level seed culture solution and 0.5 mL of expansion culture solution. mL. After completion, seal the bottle mouth with the original gauze and kraft paper, shake it slightly by hand to mix the inoculated bacteria and culture solution evenly, and then place it in a constant temperature box and culture it at 37℃ for 24 hours. This is called the primary culture solution.

[0059] (3) Preparation of seed tank strains: Sterilization of fermentation equipment, pipelines and sterile filtration systems: First, open the valves of all inlet and outlet pipelines and sterile air pipelines, and pass 0.12-0.14 MPa steam. Allow the steam to connect with the pipeline valves and discharge a small amount of steam. Ventilate for 40 minutes, then close the valves of all inlet and outlet pipelines and set aside.

[0060] Sterilization of empty seed tanks and fermentation tanks: Close all valves, open the drain valve at the bottom of the tank and the drain valve in the tank interlayer, open the direct steam valve to introduce 0.12-0.14 MPa steam into the pipe, start timing when the temperature in the tank reaches 121°C, the sterilization time is 40 min, then close the drain valve at the bottom of the tank and the drain valve in the interlayer, and the temperature in the tank will naturally drop to 37°C for standby use.

[0061] Seed culture medium formula: D-glucose 4%, yeast peptone 1.4%, dipotassium hydrogen phosphate 0.4%, calcium propionate 1%, sodium acetate 0.7%, L-theanine 1%, Tween-80 0.1%, polyether defoamer 0.1%, tap water 91.3%, natural pH.

[0062] Specific operation: First, pour the tap water used in the formula into the sterilized seed tank, start the mixer, add the raw materials required in the formula respectively (the order of addition is not important), then stir continuously and pass steam for heating. First, use the steam in the tank interlayer to heat to 95°C, then switch to direct steam heating. When the temperature in the tank reaches 121°C, maintain it for 20 minutes to achieve the sterilization effect, then turn off the steam, and start cooling through the tap water in the tank interlayer. When the temperature in the tank reaches 37°C, it is ready for use. This is called seed culture medium. Then inoculate the above-cultured first-level bacterial culture solution into the seed tank under sterile conditions. The inoculation amount is 3% of the culture medium in the tank. 100 kg of seed tank culture medium can be inoculated with 3 kg of first-level bacterial solution. Then cover the inoculation cap on the tank and start stirring and culturing. The culture conditions are: temperature 37°C, natural pH value, stirring speed 120 r / min, tank pressure 0.05 MPa, and culture time 12 h to meet the requirements. If the tank body is lowered, sterile air can be introduced to maintain the tank pressure. If the tank pressure is too high, the air release valve on the tank top can be used to adjust it. This is called seed tank culture.

[0063] (4) Lactobacillus plantarum fermentation: Culture medium formula: D-glucose 4%, yeast peptone 1%, erythritol 1.5%, bitter melon peptide powder 1%, yeast extract powder 1.5%, sodium acetate 0.8%, Tween-80 0.2%, leucine 0.6%, dipotassium hydrogen phosphate 1%, polyether defoamer 0.1%, tap water 88.3%.

[0064] Specific operation: First, add the tap water used in the formula into the sterilized fermentation tank, start the mixer, add the raw materials used in the formula into the fermentation tank (the order of addition is not important), and then pass steam for heating. First, use the interlayer steam to heat to 95°C, and then switch to direct steam heating. When the temperature in the tank reaches 121°C, start timing, and keep it for 30 minutes to achieve the sterilization effect, and then cool it down with the tap water in the interlayer. When the temperature in the tank drops to 37°C, start to inoculate the strain. Before inoculation, first increase the tank pressure of the seed tank to 0.1 MPa, and keep the tank pressure of the fermentation tank at 0.05MPa, then open the inoculation pipeline valve, and transport the seeds cultured in the seed tank to the fermentation tank by pressure difference, and then close the inoculation channel valve.

[0065] (5) Centrifugation of fermentation broth: The fermentation liquid is pumped into a storage tank for centrifugation. Operation: Start the centrifuge, adjust the speed to 10000 r / min, centrifuge at a feed rate of 10kg / min, collect the inactivated wet bacterial sludge metabolites after centrifugation, and freeze-dry the collected inactivated wet bacterial sludge metabolites.

[0066] (6) Freeze-drying of bacterial sludge metabolites: Preparation of bacterial suspension: Take 1000 g of inactivated wet bacterial sludge metabolites, add 4000 mL of sterile saline, 600 g of lactulose, 1200 g of broccoli powder, and 30 g of L-carnitine.

[0067] Operation: First, take 2500 mL of normal saline in the formula, add lactulose and stir to dissolve, then add 1000 g of inactivated wet bacterial sludge metabolites, and then add the remaining normal saline and broccoli powder and auxiliary materials in the formula and stir thoroughly to obtain a suspension. The suspension is freeze-dried and the moisture content is finally controlled to 4%, which becomes freeze-dried powder of inactivated bacterial metabolites.

[0068] After testing, the freeze-dried powder of inactivated bacterial metabolites contained 24% extracellular polysaccharide, 6% polypeptide, 22% amino acid and 150 billion bacteria per gram.

[0069] The inactivated bacteria of the freeze-dried powder will be lost or broken during the dilution process. Although the bacteria are broken, the effect of the strain will not be weakened.

[0070] The freeze-dried powder can be widely used in food, baked food, solid beverages, liquid beverages, and compressed candies.

[0071] The freeze-dried powder prepared by the above method was subjected to the following effect verification.

[0072] 1. Anti-inflammatory effect of probiotics on rats with autoimmune uveitis Autoimmune uveitis (EAU) model rats were used as experimental subjects to observe the effect of the probiotic product prepared in Example 1 on autoimmune uveitis in rats.

[0073] Experimental scheme: Rats with autoimmune uveitis (EAU) model were divided into a model group and an experimental group, and normal rats were used as a control group. The control group and the model group were given 0.5 mL of normal saline by gavage while being fed normally, and the experimental group was gavaged with a probiotic product (i.e., the lyophilized powder prepared in Example 1) while being fed normally. The preparation method of the probiotic product solution is as follows: the probiotic product (i.e., the lyophilized powder prepared in Example 1) was dissolved in 0.5 mL of sterile water, wherein the added mass of the probiotic product (i.e., the lyophilized powder prepared in Example 1) was 15 mg (experimental group 1), 20 mg (experimental group 2), and 25 mg (experimental group 3), respectively. The above-mentioned probiotic product solution (0.5 mL) was gavaged into mice once a day.

[0074] After 30 days of feeding, the slit lamp examination results of rats in each group were as follows: Figure 1 As shown, according to Figure 1 It can be seen that probiotic products can significantly reduce the inflammatory response of rats and protect the eye health of the body.

[0075] Each rat was anesthetized by intraperitoneal injection of 10% chloral hydrate to extract peripheral serum. After anesthesia, the rats were fixed, the hair was clipped, the abdominal skin and peritoneum were cut, the tissues around the abdominal aorta were separated, and 5 mL of blood was collected from the abdominal aorta and placed in a heparin anticoagulant tube (sterile operation). The tubes were allowed to stand at room temperature for 30 min, centrifuged at 3000 r / min for 15 min, and the upper layer of serum was taken. The serum was inactivated for complement at 56°C for 30 min and stored in a -20°C refrigerator for later use. Enzyme-linked immunosorbent assay (ELISA) was used to measure the expression of IL-4, IL-15, and IFN-γ in the serum. Test results Figure 2 shown.

[0076] IL-4 is an inflammation-protective cytokine. It usually reaches its peak in the late stage of inflammation and plays a role in counteracting pro-inflammatory factors and inhibiting Th1 cell responses, thereby protecting the body. IL-15 and IFN-γ are pro-inflammatory factors. Figure 2 It can be seen that probiotics can increase the level of IL-4 in rat serum, reduce the levels of IL-15 and IFN-γ in serum, and inhibit the formation of pro-inflammatory factors in a dose-dependent manner.

[0077] 2. Effect of probiotic products on alleviating visual fatigue in zebrafish Wild-type AB zebrafish of 1 dpf were used as experimental subjects to observe the effect of the probiotic product (i.e., the freeze-dried powder prepared in Example 1) on apoptotic cells around the eye of zebrafish.

[0078] Experimental scheme: 1dpf wild-type AB strain zebrafish were divided into a control group and an experimental group. The control group was placed in a 6-well plate and given 1 mL of water. The experimental group was placed in a 6-well plate and given 1 mL of probiotic product (i.e., freeze-dried powder prepared in Example 1) solution (20 mg freeze-dried powder was added to 1 ml of sterile water) and Bifidobacterium breve probiotic solution (Bifidobacterium breve was produced by Morinaga Milk Industry of Japan, the probiotic content in the freeze-dried powder was 100 billion / g, and 30 mg was added to 1 ml of sterile water to prepare). Each group was given water-soluble mycophenolic acid morpholine ethyl to establish a zebrafish visual fatigue model. After treatment at 28°C for 24 h, it was stained with acridine orange (AO). After staining, it was placed under a fluorescence microscope to take pictures, and the software was used to analyze and collect data to evaluate whether the probiotics have the effect of relieving visual fatigue. The results are shown in Table 1.

[0079] Table 1 Experimental results on the dosage of bacterial solution and its efficacy in relieving visual fatigue in zebrafish

[0080] Note: “-” in the table means none.

[0081] According to Table 1, probiotics have a significant effect in relieving visual fatigue, while Bifidobacterium breve has no obvious effect in relieving visual fatigue.

[0082] 3. Protective effect of probiotics on retinal ganglion cells in glaucoma mice 10-month-old DBA / 2J mice with successful modeling were selected, and C57BL / 6J mice were used as the control group. The DBA / 2J mice were divided into a model group and an experimental group. The mice in the control group and the model group were gavaged with 0.5 mL of normal saline, and the experimental group was gavaged with a sterile aqueous solution of a probiotic product (i.e., the freeze-dried powder prepared in Example 1). The aqueous solution of the probiotic product (i.e., the freeze-dried powder prepared in Example 1) was prepared as follows: the probiotic product (i.e., the freeze-dried powder prepared in Example 1) was dissolved in 0.5 mL of sterile water, wherein the added mass of the probiotic product (i.e., the freeze-dried powder prepared in Example 1) was 15 mg (experimental group 1), 20 mg (experimental group 2), and 25 mg (experimental group 3), respectively, and the above probiotic product solution (0.5 mL) was gavaged into the mice. The above mice were gavaged once a day for 8 consecutive weeks.

[0083] (1) The intraocular pressure of mice was measured before oral gavage and at 2, 4, and 8 weeks after gavage. The results are shown in Figure 3 .

[0084] (2) Eight weeks after oral gavage, the mice were killed by cervical dislocation after intraperitoneal injection of 40 mg / kg sodium pentobarbital. The eyeballs were removed, and the retinal tissue was peeled off under a microscope and stored in a -20°C refrigerator. 1 mg of retinal tissue from each group of mice was used to make slices, fixed with paraformaldehyde, dehydrated, and embedded in slices. The slices were stained with hematoxylin, rinsed after 5-10 min, differentiated with ethanol, soaked for 15 minutes, and then counterstained with eosin. The slices were dehydrated again with ethanol, soaked in xylene I and II, and sealed with neutral resin. The number of mouse RGCs was observed under an inverted microscope; starting from the optic disc, bright fields on both sides were taken to analyze the thickness of the mouse retina. The results are shown in the figure. Figure 4 .

[0085] (3) 1 mg of retinal tissue from each group of mice was taken, rinsed with physiological saline, minced and ground in a mortar to prepare a homogenate, centrifuged at 3500 r / min for 10 min, and the levels of SOD, GSH-Px, and NOS were determined by colorimetry. The results are shown in Table 1. Figure 5 .

[0086] (4) Western blot was used to detect the relative expression levels of NLRP3, Caspase-1, and ASC proteins in the retina of each group of mice. The results are shown in Figure 6 .

[0087] according to Figure 3 , Figure 4 , Figure 5 , Figure 6 It can be seen that probiotics can reduce intraocular pressure in mice, improve retinal thickness in glaucoma mice, alleviate oxidative damage to retinal tissue, and play an eye protection role in a dose-dependent manner.

[0088] 4. Protective effect of probiotics on cataract mice Experimental plan: Several mice were selected for modeling. The modeling method was as follows: the abdomen of the mouse was facing up, and the lower part was disinfected with iodine. The injection needle was inserted into the left lower abdomen, and the needle was pushed forward 0.5-1.0 cm. Then the needle was fixed through the abdominal muscle at a 45-degree angle, and 10% galactose solution was slowly injected at 25 mL / kg / day. The control group was injected with normal saline. The modeling time was 20 days. 40 mice with successful modeling were selected and divided into four parts, one as the model group and the other three as the experimental group. At the same time, 10 normal mice without modeling were selected as the control group. The model group and the control group were fed normally and gavaged with 0.5 mL of normal saline. The experimental group was fed normally and gavaged with probiotic product solution. The preparation method of probiotic product solution was as follows: the probiotic product was dissolved in 0.5 mL of sterilized water, and the mass percentage of probiotic product added was 15 mg (experimental group 1), 20 mg (experimental group 2), and 25 mg (experimental group 3). The probiotic product solution (0.5 mL) was gavaged into the mice once a day. After 30 days of feeding, the mice were killed by decapitation, and the lenses were removed and blood was collected.

[0089] According to the degree of lens opacity, there are five grades: grade I, no opacity; grade II, relatively opaque; grade III, obvious opacity; grade IV, nuclear opacity; grade V, complete cataract of the entire lens. The results are shown in Table 2.

[0090] The lens was added with an appropriate amount of physiological saline and ground in a glass homogenizer for 5 min to prepare a 10% tissue homogenate. The tissue homogenate and plasma were centrifuged at 3000 r / min for 10 min, and the supernatant was taken for the determination of SOD, GSH-Px, GSH and MDA. The results are shown in Figure 7 , Figure 8 .

[0091] Table 2 Lens opacity of mice in each group

[0092] According to Table 2, Figure 7 , Figure 8 It can be seen that the degree of lens opacity of cataract mice fed with probiotic products was reduced, SOD and GSH-Px in the lens and plasma increased, and MDA accumulation decreased. The results show that probiotics can significantly delay the development of cataract disease.

[0093] 5. Metabolomics analysis Metabolomics analysis of mouse feces was performed using ultra-performance liquid chromatography-tandem mass spectrometry.

[0094] Experimental plan: The mice were divided into a control group and an experimental group. The control group was gavaged with 0.5 mL of normal saline while being fed normally, and the experimental group was gavaged with 0.5 mL of probiotic product solution (20 mg of the above-mentioned inactivated bacterial metabolite freeze-dried powder dissolved in 0.5 mL of sterile water) once a day while being fed normally. After 30 days of feeding, fecal samples of mice in the control group and the experimental group were placed in 400 μL of extraction solution (acetonitrile: methanol = 1: 1), vortexed for 30 seconds, and extracted with low-temperature ultrasound for 30 minutes (5℃, 40 KHz), left to stand at -20℃ for 30 minutes, placed in a 4℃ centrifuge, centrifuged at 13000 r / min for 15 minutes, and the supernatant was taken, dried with nitrogen, and reconstituted with 120μL of reconstitution solution (acetonitrile: water = 1: 1), extracted with low-temperature ultrasound for 5 minutes (5℃, 40 KHz), and placed in a 4℃ centrifuge, centrifuged at 13000 r / min for 5 minutes, and an appropriate amount of supernatant was taken for analysis. 10 uL of sample supernatant was separated by BEHC18 column (100mm ×2.1, 1.8 µm) and then entered mass spectrometry detection. The separation flow rate was 0.40 mL / min and the column temperature was 40°C. After completion, the LC-MS raw data was transferred to the computer for processing and analysis. The MS mass spectrum information was matched with the public database to obtain the main metabolite information such as Fig. 9 As shown, the contents of the main metabolites were determined, and the results are shown in Table 3.

[0095] Isorhamnetin content determination method: high performance liquid chromatography-mass spectrometry.

[0096] Chromium content determination method: graphite furnace atomic absorption spectrometry.

[0097] Vitamin H content determination method: liquid chromatography-tandem mass spectrometry.

[0098] Table 3 Main metabolomics analysis of mice

[0099] It can be seen from Table 3 that daily administration of 20 mg of a probiotic product containing about 3 billion inactivated bacterial cells and metabolites (i.e., the freeze-dried powder prepared in Example 1) to mice can significantly increase the contents of isorhamnetin, chromium, and vitamin H in mouse feces, thereby indicating that the probiotic product provided by the present invention can sustainably provide endogenously required amounts of isorhamnetin, chromium, and vitamin H during intestinal reproduction and growth.

[0100] 6. Experiment on regulating the function of intestinal flora in mice The mice were divided into a control group and an experimental group, with 10 mice in each group. The control group was gavaged with 0.5 mL of normal saline while being fed normally, and the experimental group was gavaged with 0.5 mL of probiotic product solution (20 mg of the above-mentioned inactivated bacterial metabolite freeze-dried powder was dissolved in 0.5 mL of sterile water) while being fed normally, once a day. After 30 days of feeding, the mouse feces were aseptically collected, weighed, diluted 10 times with sterile diluent to make a uniform suspension, and then diluted 10 times in turn, and the appropriate dilution was selected and inoculated on each selective culture medium. The number of bacteria per gram of wet feces was calculated, and the logarithm was taken for statistical processing. 24 hours after the last administration of the probiotic product, rectal feces were taken and the intestinal flora was tested. The test results are shown in Table 4.

[0101] Table 4 Suitable culture conditions and detection of intestinal flora (x - ±SD)

[0102] According to Table 4, probiotics can increase the number of beneficial bacteria (Bifidobacterium and Lactobacillus), while the number of harmful bacteria (Enterobacter, Enterococcus) did not increase but showed a slight downward trend, indicating that probiotics have the function of regulating the intestinal flora of mice.

[0103] 7. Mouse gastrointestinal motility experiment The mice were divided into a control group, a model group and an experimental group, with 10 mice in each group. The control group and the model group were given 0.5 mL of sterile water by gavage once a day while feeding normally; the experimental group was given 0.5 mL of probiotic product solution (20 mg of the lyophilized powder prepared in Example 1 dissolved in 0.5 mL of sterile water) by gavage once a day while feeding normally. After 15 days of gavage, the mice in each group were fasted for 16 hours. After that, the model group and the experimental group were given compound difenoxol by gavage, and the control group was given distilled water by gavage. After 30 minutes, the mice in each group were given Arabic gum ink by gavage. After 30 minutes, the mice were killed by cervical vertebral dissection, and the abdomen was quickly cut along the midline of the abdomen to separate the mesentery. The intestinal tube was cut with the pylorus as the starting point and the ileocecal part as the end point. The small intestine was gently pulled into a straight line, and the intestinal tube length was measured as the "total length of the small intestine", and the length from the pylorus to the front of the ink was the "ink advancement length". The ink advancement rate was calculated according to the following formula. The ink advancement rate of the small intestine is shown in Table 5.

[0104]

[0105] The units of ink propulsion length and small intestine propulsion length are both centimeters.

[0106] Table 5 Small intestinal ink propulsion rate (x - ±SD)

[0107] As shown in Table 5, the intestinal motility inhibition model of mice was established by administering compound difenocool. Compared with the model group, the small intestinal ink propulsion rate of the experimental group increased, indicating that probiotics can promote small intestinal motility and digestion.

[0108] 7. Small-scale clinical trials The subjects were people aged 6-70 years old with eye diseases and ineffective medication in 8 communities in Jinan City. As of June 2005, a total of 100 people were recruited. They participated in the experiment 3 months after stopping medication and took 0.2 grams of probiotic product (i.e., freeze-dried powder prepared in Example 1) once before going to bed every day. All of the above-mentioned people signed a written informed consent form. If the participant was unable to sign, the agent (usually a family member) signed it.

[0109] Table 6 Eye protection application effect

[0110] Table 6 Description: Visual fatigue: 20 people started to collect the results after taking it for 3 days. After taking it for 3 days, 5 people felt that their intolerance to long-term vision, temporary blurred vision, double vision, dry eyes, itching, distending pain, tearing, and decreased vision had improved. After taking it for 6 days, 10 people felt that their intolerance to long-term vision, temporary blurred vision, double vision, dry eyes, itching, distending pain, tearing, and decreased vision had improved significantly. After taking it for 10 days, 15 people felt that their intolerance to long-term vision, temporary blurred vision, double vision, dry eyes, itching, distending pain, tearing, and decreased vision had improved significantly. After taking it for 15 days, 18 people felt that their intolerance to long-term vision, temporary blurred vision, double vision, dry eyes, itching, distending pain, tearing, and decreased vision had completely improved. A total of 2 people did not improve after taking Lactobacillus plantarum.

[0111] Dry eyes: 20 people started to take the medicine for 8 days and the results were counted. After taking it for 8 days, 5 people felt that their eyestrain, foreign body sensation, dryness, soreness, redness, photophobia and other conditions improved. After taking it for 16 days, 9 people felt that their eyestrain, foreign body sensation, dryness, soreness, redness, photophobia and other conditions were significantly improved. After taking it for 24 days, 14 people had extremely obvious improvement in their eyestrain, foreign body sensation, dryness, soreness, redness, photophobia and other conditions. After taking it for 30 days, 17 people had completely improved their eyestrain, foreign body sensation, dryness, soreness, redness, photophobia and other conditions. A total of 3 people did not improve after taking Lactobacillus plantarum.

[0112] Conjunctivitis: 20 people started to collect the results after taking it for 11 days. After taking it for 11 days, 4 people felt a foreign body sensation, burning sensation, heavy eyelids, increased secretions, photophobia, tearing and decreased vision, and other symptoms improved. After taking it for 22 days, 9 people felt a foreign body sensation, burning sensation, heavy eyelids, increased secretions, photophobia, tearing and decreased vision, and other symptoms were significantly improved, and immunity also increased. After taking it for 33 days, 14 people felt a foreign body sensation, burning sensation, heavy eyelids, increased secretions, photophobia, tearing and decreased vision, and other symptoms improved extremely significantly. After taking it for 45 days, 17 people had completely improved their foreign body sensation, burning sensation, heavy eyelids, increased secretions, photophobia, tearing and decreased vision. A total of 3 people did not improve after taking Lactobacillus plantarum.

[0113] Glaucoma: 20 people started to collect the results after taking it for 23 days. After taking it for 23 days, 5 people felt that their blurred vision, headache, eye swelling, narrowing of visual field and other symptoms improved. After taking it for 45 days, 9 people felt that their blurred vision, headache, eye swelling, narrowing of visual field and other symptoms were significantly improved, and their immunity also increased. After taking it for 68 days, 14 people had extremely obvious improvement in blurred vision, headache, eye swelling, narrowing of visual field and other symptoms. After taking it for 90 days, 16 people had completely improved their blurred vision, headache, eye swelling, narrowing of visual field and other symptoms. A total of 4 people did not improve after taking Lactobacillus plantarum.

[0114] Cataract: 20 people started to collect the results after taking it for 11 days. After taking it for 11 days, 3 people felt that the symptoms of blurred vision, diplopia, myopia, glare, etc. were improved. After taking it for 22 days, 7 people felt that the symptoms of blurred vision, diplopia, myopia, glare, etc. were significantly improved. After taking it for 33 days, 12 people felt that the symptoms of blurred vision, diplopia, myopia, glare, etc. were extremely improved. After taking it for 45 days, 15 people had completely improved their blurred vision, diplopia, myopia, glare, etc. symptoms. A total of 5 people did not improve after taking Lactobacillus plantarum.

[0115] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Lactobacillus plantarum ( Lactiplantibacillus plantarum ) 9A-2, characterized in that, Lactobacillus plantarum 9A-2 is deposited in Guangdong Provincial Microbiological Culture Collection Center with a preservation date of July 8, 2024 and a preservation number of GDMCC NO: 64832.

2. A derivative of Lactobacillus plantarum 9A-2 according to claim 1, characterized in that Including one or more of its live bacteria, inactivated bacteria, fermentation broth, exosomes or metabolites.

3. A microbial agent, characterized in that: The invention comprises an active ingredient, wherein the active ingredient comprises the Lactobacillus plantarum 9A-2 according to claim 1 or the derivative according to claim 2.

4. The microbial agent according to claim 3, characterized in that The microbial agent comprises inactivated Lactobacillus plantarum and fermentation liquid thereof.

5. The method for preparing the microbial agent according to claim 3 or 4, characterized in that: The preparation method comprises: S1. Activation of bacterial strains: inoculating the Lactobacillus plantarum 9A-2 into an activation medium, and culturing at 35-40° C. for 16-20 hours to obtain an expanded bacterial strain solution; S2. Preparation of primary bacterial seed solution: inoculate the expanded bacterial seed solution prepared in step S1 into the primary seed culture solution, and culture at 35-40° C. for 16-20 h to obtain the primary bacterial seed solution; S3, seed tank strain preparation: the primary strain liquid prepared in step S2 is inoculated into a seed tank containing a seed culture medium under sterile conditions, the inoculation amount is 0.5-5%, the stirring culture is 110-130 r / min, the pH is natural, the temperature is 30-40°C, the tank pressure is 0.03-0.06 MPa, and the culture time is 11-13 h to obtain the seed tank strain; S4, plant lactobacillus fermentation: inoculate the seed tank strain into a fermentation tank containing sterilized fermentation culture medium, with an inoculation amount of 0.5-5%, 35-39°C, 70-90 r / min, tank pressure of 0.04-0.06 MPa, pH of 5.9-6.1, and ferment for 24-26 h; cool to 19-21°C, add sterile glycerol of 1.9-2.1% of the volume of the fermentation liquid into the fermentation tank, raise the temperature to 119-121°C and maintain for 5-7 s for inactivation treatment.

6. The preparation method according to claim 5, characterized in that: After step S4 is completed, the obtained fermentation liquid is centrifuged to collect the inactivated wet bacterial sludge metabolites, and the inactivated wet bacterial sludge metabolites are freeze-dried to obtain freeze-dried powder of the inactivated bacterial metabolites; In step S1, the composition of the activation medium is as follows: based on the weight of added water, 5-20 parts of D-glucose, 1-10 parts of maltitol, 1-10 parts of beef extract powder, 0.1-2 parts of sodium acetate, 0.1-1 parts of Tween-80, 0.1-1 parts of potassium dihydrogen phosphate, 1000 parts of water, and the pH is adjusted to 7; In step S2, the composition of the primary seed culture solution is as follows: based on the weight of added water, 1-10 parts of D-glucose, 1-10 parts of yeast peptone, 1-3 parts of beef extract powder, 1-5 parts of histidine, 0.5-1 parts of sodium acetate, 0.5-1.5 parts of Tween-80, 1000 parts of water, and the pH value is natural; In step S3, the composition of the seed culture medium is as follows: D-glucose 1-5%, yeast peptone 1-5%, dipotassium hydrogen phosphate 0.1-0.5%, calcium propionate 1-5%, sodium acetate 0.1-1%, L-theanine 1-5%, Tween-80 0.05-0.15%, polyether defoamer 0.05-0.15%, water 90-95%, pH value natural; In step S4, the fermentation culture medium is composed of: 1-5% D-glucose, 0.5-1.5% yeast peptone, 1-2% erythritol, 0.5-1.5% bitter melon peptide powder, 1-2% yeast extract powder, 0.5-1.5% sodium acetate, 0.1-0.3% Tween-80, 0.1-1% leucine, 0.5-1.5% dipotassium hydrogen phosphate, 0.05-0.15% polyether defoamer, and 88-89% water.

7. Use of the Lactobacillus plantarum 9A-2 according to claim 1 or the derivative according to claim 2 or the microbial agent according to any one of claims 3 to 4 in the preparation of eye protection products; The product is one of a food and a medicine; The food is any one of beverages, dairy products, candy products, health foods, special dietary foods, and special medical formula foods; The function of the eye protection product is to relieve visual fatigue, which is caused by improper use of the eyes or eye-related diseases; the eye-related diseases include uveitis, macular degeneration, dry eyes, blepharitis and cataracts.

8. Use of the Lactobacillus plantarum 9A-2 according to claim 1 or the derivative according to claim 2 or the microbial agent according to any one of claims 3 to 4 in the preparation of a product for regulating the intestinal tract; The product is one of a food and a medicine; The food is any one of beverages, dairy products, candy products, health foods, special dietary foods and special medical formula foods.

9. The use according to claim 8, characterized in that The intestinal regulation includes regulating intestinal flora, promoting intestinal peristalsis and increasing the content of beneficial ingredients in the intestine; the beneficial ingredients are one or more of isorhamnetin, chromium and vitamin H.

10. A product, characterized in that The composition comprises the Lactobacillus plantarum 9A-2 of claim 1 or the derivative of Lactobacillus plantarum 9A-2 of claim 2 or the microbial agent of any one of claims 3 to 4 and an acceptable auxiliary agent or excipient; The product is one of a food and a medicine; The food is any one of beverages, dairy products, candy products, health foods, special dietary foods or special medical formula foods.

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