A strain of Pediococcus pentosaceus and its application in products relieving eye strain

Abstract

This invention discloses a strain of *Pediococcus pentosaceus* and its application in products for relieving eye strain, relating to the field of microbial technology. This invention provides a strain of *Pediococcus pentosaceus* RH3401, whose preservation number is CGMCC No. 25166. This invention also provides the application of *Pediococcus pentosaceus* RH3401 in the preparation of products for relieving eye strain. The *Pediococcus pentosaceus* RH3401 provided by this invention has a good effect in relieving eye strain, can significantly reduce apoptotic cells in the eye, and has good application prospects in the preparation of products for relieving eye strain.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Pediococcus pentosaceus and its application in products for relieving eye fatigue. Background Technology

[0002] Eye strain is a syndrome caused by abnormal eye accommodation function, insufficient or abnormal tear secretion on the ocular surface, ciliary muscle tension, etc. Typical symptoms include dry eyes, eye irritation, blurred vision, and eye soreness. In severe cases, it may be accompanied by headaches, dizziness, and difficulty concentrating. It not only reduces the efficiency of daily life and work, but long-term neglect may also induce organic eye diseases such as dry eye syndrome, increased myopia, and glaucoma.

[0003] Currently, products for relieving eye strain mainly fall into three categories: First, topical eye care products, such as artificial tears and eye patches. These products primarily relieve local discomfort by moisturizing the ocular surface and applying physical cold compresses. However, their effects are limited to the ocular surface and cannot improve eye strain at the physiological level. Furthermore, long-term use of artificial tears may damage the ocular microenvironment, and some products containing preservatives also pose a risk of corneal irritation. Second, optical aids, such as blue light blocking glasses, can only reduce the stimulation of specific wavelengths of light on the eyes and cannot address core issues such as ciliary muscle tension and decreased eye accommodation function. Third, oral dietary supplements. Traditional products often use nutrients such as lutein, zeaxanthin, and vitamin A as core ingredients, working by supplementing the nutrients needed by the retina. However, these ingredients mainly focus on retinal protection and have limited intervention effects on key aspects of eye strain, such as the regulation of tear secretion and ciliary muscle relaxation. They also suffer from problems such as significant individual differences in absorption and slow onset of action.

[0004] Currently, some probiotics have been disclosed to have the effect of relieving eye fatigue. For example, Chinese patent CN119499289A discloses the application of Bifidobacterium adolescentis B-201 in the preparation of products for relieving eye fatigue and protecting vision. The Bifidobacterium adolescentis B-201 provided in this invention has the accession number CGMCC No. 27234. After cultivation, fermentation, and freeze-drying, the resulting bacterial powder, with the addition of excipients, can be used to improve eye symptoms, increase tear secretion, reduce ocular surface disease index scores, and increase the duration of continuous near-vision use. This strain can be used to prepare products for relieving eye fatigue and protecting vision.

[0005] Chinese patent CN117210381A discloses a type of Lactobacillus paracasei, its products for relieving eye fatigue, and its applications. The Lactobacillus paracasei is Lactobacillus paracasei FG-14, with accession number CGMCC No.24331. The lutein ester yield obtained by fermenting marigolds with FG-14 is as high as 31 mg / g of fermentation broth, which can be used to prepare products for relieving eye fatigue.

[0006] However, to date, no studies have reported a link between *Pediococcus pentosaceus* and eye health, let alone explored its functional role in relieving eye strain. *Pediococcus pentosaceus* is a Gram-positive bacterium belonging to the genus *Pediococcus* in the family Lactobacillusaceae. It is widely found in fermented foods and the human gut and is recognized as a safe microorganism. Existing research indicates that *Pediococcus pentosaceus* possesses various physiological functions. In the field of gut health, it can inhibit the colonization of pathogenic bacteria (such as *Escherichia coli* and *Salmonella*), regulate the balance of gut microbiota, and relieve diarrhea or constipation. In the field of immune regulation, it can activate macrophage and T lymphocyte activity, enhancing the body's immunity. In the field of metabolic regulation, it can lower serum cholesterol and improve insulin resistance.

[0007] Based on the above situation, developing a Pediococcus pentosaceus strain with a clear function of relieving eye fatigue and applying it to related products can not only make up for the shortcomings of existing eye fatigue intervention methods, but also expand the application field of Pediococcus pentosaceus, providing new technical ideas for the research and development of eye health products, and has important academic value and market prospects. Summary of the Invention

[0008] The purpose of this invention is to provide a strain of Pediococcus pentosaceus and its application in products for relieving eye strain.

[0009] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0010] In a first aspect, the present invention provides a *Pediococcus pentosaceus* RH3401, the preservation number of which is CGMCC No. 25166.

[0011] Specifically, the 16S rDNA sequence of Pediococcus pentosaceus RH3401 is shown in SEQ ID NO:1.

[0012] Secondly, the present invention provides a method for culturing Pediococcus pentosaceus RH3401, which involves inoculating Pediococcus pentosaceus RH3401 into a culture medium for cultivation.

[0013] According to some embodiments of the present invention, the cultivation method includes:

[0014] (1) Inoculate the bacterial cells into the basic culture medium at an inoculation rate of 5%, and incubate at 37°C for 17 h to obtain a primary bacterial suspension;

[0015] (2) Transfer the primary bacterial suspension to the basic culture medium with a volume of 60%, and incubate at 37°C for 17 hours to obtain the secondary bacterial suspension;

[0016] (3) Inoculate the secondary bacterial suspension into the optimized culture medium at an inoculation rate of 5%, with a liquid volume of 60%, and incubate at 37°C for 17 h.

[0017] According to some embodiments of the present invention, the basal culture medium comprises 5 g / L yeast peptone, 5 g / L yeast extract, 20 g / L anhydrous glucose, 5 g / L citric acid monohydrate, 5 g / L sodium acetate, 2 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.25 g / L manganese sulfate, 0.2 g / L Tween 80, and pH 6.50.

[0018] According to some embodiments of the present invention, the optimized culture medium comprises 5 g / L yeast peptone, 10 g / L yeast extract, 20 g / L anhydrous glucose, 5 g / L citric acid monohydrate, 5 g / L L-malic acid, 5 g / L sodium acetate, 2 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.25 g / L manganese sulfate, 0.2 g / L Tween 80, and pH 6.50.

[0019] Thirdly, the present invention provides a preparation comprising the above-mentioned Pediococcus pentosaceus RH3401.

[0020] Specifically, the preparations include, but are not limited to, fermentation broth of Pediococcus pentosaceus RH3401, fermentation broth precipitate of Pediococcus pentosaceus RH3401, fermentation broth supernatant of Pediococcus pentosaceus RH3401, live Pediococcus pentosaceus RH3401, and freeze-dried powder of Pediococcus pentosaceus RH3401.

[0021] Specifically, the fermentation broth refers to the liquid produced after inoculating the microbial strain into a culture medium and culturing it.

[0022] More specifically, the supernatant of the fermentation broth refers to the clear liquid at the top of the fermentation broth after centrifugation; it contains abundant metabolic products from the bacterial growth and reproduction process, as well as some bacterial cell fragments.

[0023] More specifically, the fermentation broth precipitation refers to the liquid precipitate after centrifugation, including free proteins, residual bacterial cells, broken cells, and culture medium residues.

[0024] Fourthly, the present invention provides a microbial agent comprising the above-mentioned Pediococcus pentosaceus RH3401 or the above-mentioned preparation.

[0025] Specifically, the bacterial agent is a powder or liquid preparation.

[0026] Specifically, the microbial agent may be a solid, liquid, semi-solid, or any physical form containing the necessary active ingredients to achieve any of the applications, functions, or effects described in this invention, which can be obtained by those skilled in the art through current or future technologies.

[0027] In some implementations, the bacterial agent is in solid form. Specifically, it can be a lyophilized powder.

[0028] Specifically, the method for preparing freeze-dried powder can employ methods currently used in the art, or other methods that may emerge in the future to obtain a product in solid form of microorganisms or microbial cultures. It should be clarified that the method for preparing freeze-dried powder should not be a condition limiting the scope of protection of this invention.

[0029] Fifthly, the present invention provides the use of the above-mentioned Pediococcus pentosaceus RH3401 or the above-mentioned preparation or the above-mentioned bacterial agent in the preparation of products for relieving eye fatigue.

[0030] Specifically, the products mentioned include pharmaceuticals and health supplements.

[0031] Specifically, the viable count of Pediococcus pentosaceus RH3401 in the product is 1×10⁻⁶. 5 -1×10 12 CFU / mL or 1×10 5 -1×10 12 CFU / g, specifically 1×10 5 CFU / mL (CFU / g), 1×10 6 CFU / mL (CFU / g), 1×10 7 CFU / mL (CFU / g), 1×10 8 CFU / mL (CFU / g), 1×10 9 CFU / mL (CFU / g), 1×10 10 CFU / mL (CFU / g), 1×10 11 CFU / mL (CFU / g), 1×10 12 CFU / mL (CFU / g), etc., and other point values ​​within this range can be selected.

[0032] Furthermore, the viable count of Pediococcus pentosaceus RH3401 in the product is 1.25 × 10⁻⁶. 7 -5×10 7 CFU / mL or 1.25×10 7 -5×10 7 CFU / g.

[0033] According to some embodiments of the present invention, the viable count of Pediococcus pentosaceus RH3401 in the product is 1.25 × 10⁻⁶. 7 CFU / mL, 2.5×10 7 CFU / mL or 5×10 7 CFU / mL.

[0034] Furthermore, when the product is a health supplement, it also includes conventional excipients for health foods, including but not limited to fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0035] More preferably, the health food includes emulsion products, solution products, powder products, and solid products.

[0036] Specifically, when the product is a pharmaceutical product, the pharmaceutical product may also include a pharmaceutically acceptable carrier.

[0037] Furthermore, the pharmaceutically acceptable carrier is selected from one or more excipients, stabilizers, diluents, binders, preservatives, and lubricants.

[0038] In another aspect, the present invention provides a product for relieving eye strain, the product comprising the above-mentioned Pediococcus pentosaceus RH3401 or the above-mentioned preparation or the above-mentioned bacterial agent.

[0039] Specifically, the products mentioned include pharmaceuticals and health supplements.

[0040] Specifically, the viable count of Pediococcus pentosaceus RH3401 in the product is 1×10⁻⁶. 5 -1×10 10 CFU / mL or 1×10 5 -1×10 10 CFU / g, specifically 1×10 5 CFU / mL (CFU / g), 1×10 6 CFU / mL (CFU / g), 1×10 7 CFU / mL (CFU / g), 1×10 8 CFU / mL (CFU / g), 1×10 9 CFU / mL (CFU / g), 1×10 10 CFU / mL (CFU / g), etc., and other point values ​​within this range can be selected.

[0041] Furthermore, the viable count of Pediococcus pentosaceus RH3401 in the product is 1.25 × 10⁻⁶. 7 -5×10 7 CFU / mL or 1.25×107 -5×10 7 CFU / g.

[0042] According to some embodiments of the present invention, the viable count of Pediococcus pentosaceus RH3401 in the product is 1.25 × 10⁻⁶. 7 CFU / mL, 2.5×10 7 CFU / mL or 5×10 7 CFU / mL.

[0043] Furthermore, when the product is a health supplement, it also includes conventional excipients for health foods, including but not limited to fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0044] More preferably, the health food includes emulsion products, solution products, powder products, and solid products.

[0045] Specifically, when the product is a pharmaceutical product, the pharmaceutical product may also include a pharmaceutically acceptable carrier.

[0046] Furthermore, the pharmaceutically acceptable carrier is selected from one or more excipients, stabilizers, diluents, binders, preservatives, and lubricants.

[0047] The beneficial effects of this invention are as follows:

[0048] This invention provides a strain of Pediococcus pentosaceus RH3401, which has a good effect on relieving eye fatigue and can significantly reduce apoptotic cells in the eye. It has good application prospects in the preparation of products that relieve eye fatigue.

[0049] Preservation instructions:

[0050] Accession number: CGMCC No. 25166;

[0051] Classification and nomenclature: Pediococcus pentosaceus Pediococcus pentosaceus ;

[0052] Deposit date: June 23, 2022;

[0053] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections;

[0054] Abbreviation of depositary institution: CGMCC;

[0055] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0056] Figure 1 Morphological characteristics of Pediococcus pentosaceus RH3401 strain on culture medium.

[0057] Figure 2 The morphological characteristics of Pediococcus pentosaceus RH3401 under a microscope.

[0058] Figure 3 This is a phylogenetic tree analysis diagram.

[0059] Figure 4 This is a diagram showing the results of a hemolysis experiment.

[0060] Figure 5 This is a trend chart of acid resistance test for Pediococcus pentosaceus RH3401.

[0061] Figure 6 This is a trend chart of bile salt resistance test for Pediococcus pentosaceus RH3401.

[0062] Figure 7 A trend graph of simulated gastric juice for Pediococcus pentosaceus RH3401.

[0063] Figure 8 Typical fluorescence intensity of apoptotic cells in the eyes of zebrafish treated with different concentrations of Pediococcus pentosaceus.

[0064] Figure 9 Typical fluorescence intensity diagrams of apoptotic cells in the eyes of zebrafish from the normal control group, model control group, and glutathione 615 μg / ml group. Detailed Implementation

[0065] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the operating methods and equipment used in the following embodiments are conventional operating methods, and the materials and equipment used in each embodiment are the same.

[0066] Example 1

[0067] 1. Screening and Identification of Strains

[0068] 1.1 Screening of strains

[0069] (1) Enrichment: Take an appropriate amount of milk curd sample into the enrichment medium and enrich at 37℃ for 36 h;

[0070] (2) Initial screening: After the enriched sample is mixed, it is diluted into different gradients and then spread onto the separation medium. The samples are incubated at 37℃ for 48 h.

[0071] (3) Purification: Select single colonies with typical characteristics of the target strain (spherical, raised, moist, round, Gram-positive strains) and perform streak purification culture on the isolation medium. Repeat this process 4 times until the colony characteristics in the streak plate are consistent.

[0072] (4) Microscopic examination: Pick two single colonies from each purified Petri dish, smear them, Gram stain them, and observe them under a microscope to see if their color and cell shape are consistent, so as to determine whether the colonies in the Petri dish are pure cultures. If the microscopic observation results are consistent, the obtained pure culture (Petri dish colonies) is regarded as a suspected strain, and the corresponding Petri dish is numbered for identification; if the microscopic observation results are inconsistent, continue the above operation.

[0073] The results show that ( Figures 1-2 Colonies are white, round, moist, opaque, and with neat edges. The bacteria are spherical, 0.6-1.0 μm in diameter. They are Gram-positive and occur singly or in pairs.

[0074] 1.2 Identification of strains

[0075] (1) Identification of physiological and biochemical characteristics

[0076] The physiological and biochemical identification in this embodiment was completed by the China Industrial Microbial Culture Collection Center, and the results are shown in Table 1.

[0077] Table 1 Physiological and Biochemical Characteristic Identification Table

[0078]

[0079] In this context, "+" represents positive and "-" represents negative.

[0080] (3) 16S rDNA sequence identification

[0081] Two strains of bacteria that were Gram-positive and catalase-negative were selected for testing.

[0082] 16s rDNA sequence (SEQ ID NO:1):

[0083]

[0084] Sequence alignment was performed with a professional database based on the NCBI database (Update 2021.08.23). The results are shown in Table 2 and... Figure 3 .

[0085] Table 2 Sequence alignment results

[0086]

[0087] 2. Culture method of Pediococcus pentosaceus strain RH3401

[0088] The revived bacterial cells from the cryopreservation tubes were transferred to 10 mL of basal medium at a 5% inoculation rate and incubated at 37°C for 17 h to obtain a primary bacterial suspension. The primary bacterial suspension was then transferred to 100 mL of basal medium (60% capacity) and incubated at 37°C for 17 h to obtain a secondary bacterial suspension. The secondary bacterial suspension was then inoculated at a 5% inoculation rate into 300 mL of optimized medium (60% capacity) and incubated at 37°C for 17 h.

[0089] Yield after culture: 1.50%, viable count: 4.40 × 10⁻⁶ 9 CFU / mL.

[0090] Basic culture medium: yeast peptone 5 g / L, yeast extract 5 g / L, anhydrous glucose 20 g / L, citric acid monohydrate 5 g / L, sodium acetate 5 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween 80 0.2 g / L, pH 6.50. Sterilize at 115℃ for 30 min.

[0091] Optimized culture medium: Yeast peptone 5 g / L, yeast extract 10 g / L, anhydrous glucose 20 g / L, citric acid monohydrate 5 g / L, L-malic acid 5 g / L, sodium acetate 5 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween 80 0.2 g / L, pH 6.50. Sterilize at 115℃ for 30 min.

[0092] 3. Safety Evaluation

[0093] (1) Evaluation of hemolytic activity

[0094] The China Industrial Microbial Culture Collection Center was commissioned to conduct the testing.

[0095] The results show that ( Figure 4 The hemolytic activity of strain RH3401 was negative.

[0096] (2) Drug sensitivity evaluation

[0097] The China Industrial Microbial Culture Collection Center was commissioned to conduct the testing.

[0098] Drug susceptibility testing results are shown in Table 3. The results showed that Pediococcus pentosaceus strain RH3401 was sensitive to ampicillin (AM), penicillin (PEN), imipenem (IP), and chloramphenicol (CL).

[0099] Table 3. Results of Drug Sensitivity Assessment

[0100]

[0101] (3) Bacterial pathogenicity in animals

[0102] The China Industrial Microbial Culture Collection Center was commissioned to conduct the testing.

[0103] The results of bacterial pathogenicity studies in animals showed that the Pediococcus pentosaceus strain RH3401 was non-pathogenic.

[0104] 1. Results of the intraperitoneal injection test

[0105] Table 4. Effects of Pediococcus pentosaceus RH3401 culture on body weight in male mice (intraperitoneal injection)

[0106]

[0107] Results showed that there were no significant differences in initial and final body weight between the culture group and its corresponding control group in male mice. p >0.05), meaning that the Pediococcus pentosaceus RH3401 culture had no effect on the body weight of male mice during the experiment.

[0108] Table 5. Effects of Pediococcus pentosaceus RH3401 culture on body weight in female mice (intraperitoneal injection)

[0109]

[0110] Results showed that there were no significant differences in initial and final body weight between the culture group and its corresponding control group in female mice. p >0.05), meaning that the Pediococcus pentosaceus RH3401 culture had no effect on the body weight of female mice during the experiment.

[0111] Table 6. Acute toxicity of Pediococcus pentosaceus RH3401 culture in mice (intraperitoneal injection)

[0112]

[0113] Results: When mice were injected intraperitoneally with 0.2 mL of Pediococcus pentosaceus RH3401 culture stock solution once and observed for 21 consecutive days, no toxic reactions or deaths were observed in the test mice.

[0114] 2. Results of oral gavage test

[0115] Table 7. Effects of Pediococcus pentosaceus RH3401 culture on body weight in male mice (oral administration).

[0116]

[0117] Results showed that there were no significant differences in initial and final body weight between the culture group and its corresponding control group in male mice. p >0.05), meaning that the Pediococcus pentosaceus RH3401 culture had no effect on the body weight of male mice during the experiment.

[0118] Table 8 Effect of Pediococcus pentosaceus RH3401 culture on body weight of female mice (oral administration)

[0119]

[0120] Results showed that there were no significant differences in initial and final body weight between the culture group and its corresponding control group in female mice. p >0.05), meaning that the Pediococcus pentosaceus RH3401 culture had no effect on the body weight of female mice during the experiment.

[0121] Table 9. Acute toxicity of Pediococcus pentosaceus RH3401 culture in mice (oral administration).

[0122]

[0123] Results: When the original culture and concentrated culture of Pediococcus pentosaceus RH3401 were administered orally to mice at a dose of 20.0 mL / kg·BW for 3 consecutive days and observed for 21 days, no toxic reactions or deaths were observed in the test mice.

[0124] 4. Resistance of Pediococcus pentosaceus RH3401 to adverse environments in the digestive tract

[0125] (1) Acid resistance test

[0126] 1) Strain activation

[0127] Single colonies obtained by streaking RH3401 strain were inoculated into 5 mL of MRS liquid medium and cultured at 37°C for 20 h; all 5 mL of bacterial culture was inoculated into 100 mL of MRS liquid medium and cultured at 37°C for 17 h to serve as the experimental seed culture.

[0128] 2) Acid resistance test of strains

[0129] The third-generation activated strains were inoculated into MRS liquid medium at a certain ratio (bacterial solution: medium = 1 mL: 4 mL) into MRS medium at pH 3.0 and pH 2.0. After incubation at 37°C for 2 h, the strains were inoculated into fresh MRS liquid medium at pH 6.5 at a 2% inoculation rate. OD was measured every 10 min using a multi-functional microplate reader. 600 Value, until OD 600 The assay was stopped when the value increased by 0.3 units. The OD values ​​of the strain were calculated in pH 3.0, pH 2.0, and normal culture media. 600 The time required for the value to increase by 0.3 units is called the delay time.

[0130] 3) Calculation of acid resistance test delay time

[0131] The acid tolerance delay time (h) of the tested strain = LT2 - LT1.

[0132] Acid-treated culture medium OD 600 The time required for the value to increase by 0.3 units (expressed as LT2), OD of normal pH culture medium. 600 The time required for the value to increase by 0.3 units (denoted by LT1).

[0133] result:

[0134] Depend on Figure 5 It can be seen that the growth of strain RH3401 was delayed under both pH 3.0 and pH 2.0 conditions compared to normal MRS medium. As shown in Table 10, the delay time was 1.5 h under pH 2.0 conditions and only 1.16 h under pH 3.0 conditions, indicating that strain RH3401 has a strong tolerance to acid.

[0135] Table 10 Acid Resistance Test Data for RH3401

[0136]

[0137] (2) Bile salt tolerance test

[0138] 1) Strain activation

[0139] Single colonies obtained by streaking RH3401 strain were inoculated into 5 mL of MRS liquid medium and cultured at 37°C for 20 h; all 5 mL of bacterial culture was inoculated into 100 mL of MRS liquid medium and cultured at 37°C for 17 h to serve as the experimental seed culture.

[0140] 2) Bile salt tolerance test of strains

[0141] Activated third-generation Lactobacillus was inoculated at a 2% inoculation rate into MRS liquid medium containing 0.3%, 0.5%, 1.0%, and 1.5% bile salts, and into MRS medium without bile salts, respectively. OD values ​​were measured every 10 minutes using a multi-functional microplate reader. 600 Value, until OD 600 Cultivation was stopped when the value increased by more than 0.3 units. The OD values ​​of the strain were calculated in MRS media containing 0.3%, 0.5%, 1.0%, 1.5% bile salts, and without bile salts. 600 The time required for the value to increase by 0.3 units is called the delay time.

[0142] 3) Calculation of the delay time in the bile salt tolerance test

[0143] The time (h) of bile salt tolerance of the tested strains = LT4 - LT3.

[0144] Bile salt culture medium OD 600 The time required for the value to increase by 0.3 units (expressed as LT4) in bile-free medium OD 600 The time required for the value to increase by 0.3 units (denoted by LT3).

[0145] Result: From Figure 6 It can be seen that although the growth of strain RH3401 was delayed under different bile salt concentrations compared to normal MRS medium, the delay time was very short. As shown in Table 11, the delay time of strain RH3401 increased with the increase of bile salt concentration, but the delay time was less than 1 h, and the delay time under 0.3% bile salt concentration was only 0.16 h.

[0146] Table 11 Data from the bile salt tolerance test of RH3401

[0147]

[0148] (3) Simulated gastric juice test

[0149] Methods for preparing artificial gastric juice:

[0150] Take 16.4 mL of dilute hydrochloric acid and add 800 mL of distilled water; add 10 g of pepsin (1%, w / v), shake well, dilute with distilled water to 1000 mL, adjust the pH to 2.0, filter through a 0.22 μm filter membrane for sterilization, and set aside for later use.

[0151] After activating the seed culture, shake it well. Take 10 mL of the bacterial suspension and centrifuge (5000×g, 10 min, 4℃) to obtain bacterial sludge. Wash twice with PBS buffer. Resuspend the obtained bacterial sludge in 10 mL of simulated gastric juice and digest it at 37℃ for 3 h. Take samples at 0 h and 3 h respectively, and perform a series of 10-fold dilutions with sterile physiological saline. Take 1 mL of the bacterial solution of the appropriate dilution for mixed bacterial counting. Repeat each dilution twice. Incubate at 37℃ for 48 h before counting.

[0152] Survival rate (%) of the tested strain in simulated gastric juice test = lgcfuN' / lgcfuN × 100%.

[0153] The number of viable bacteria (denoted by N) of the test strain after three generations of activation in the third-generation culture medium; the number of viable bacteria (denoted by N') measured by sampling at 0h and 3h.

[0154] Results: Strains RH3401 showed good survival ability in simulated gastric fluid. After 3 hours of treatment, the logarithmic number of viable bacteria still reached 8.96, with a survival rate of 96.1% (see Table 12). Figure 7 ).

[0155] Table 12 Data from RH3401 Simulated Gastric Fluid Test

[0156]

[0157] Example 2

[0158] 1.1 Detection Principle

[0159] When the eye is under prolonged working conditions, high light intensity, and high oxygen pressure, the metabolic rate of the extraocular and ciliary muscles increases, leading to the accumulation of large amounts of peroxides and ROS (reactive oxygen species), causing oxidative stress damage to the eye. Furthermore, ROS easily oxidizes polyunsaturated fatty acids in the retina, producing large amounts of lipid peroxidation and further generating large amounts of reactive aldehydes. These reactive aldehydes bind to intracellular proteins and DNA, causing cell inflammation and apoptosis, thus inducing eye fatigue. Mycophenolic acid morpholine ethyl ester is a semi-synthetic mycophenolic acid compound isolated from Penicillium fungi with anti-metabolic effects. After oral absorption, it is rapidly hydrolyzed into mycophenolic acid. Mycophenolic acid specifically inhibits hypoxanthine mononucleotide dehydrogenase, blocking the de novo synthesis pathway of guanine nucleotides, leading to guanine nucleotide depletion, blocking DNA synthesis, and ultimately inducing apoptosis. Mycophenolic acid morpholine ethyl ester can induce apoptosis in zebrafish eye cells, mimicking human eye diseases. The efficacy of the sample in improving eye fatigue was evaluated based on changes in the fluorescence intensity of apoptotic cells in zebrafish eyes.

[0160] 1.2 Determination of Maximum Detectable Concentration (MTC)

[0161] Wild-type AB strain zebrafish, 1 day post-fertilization (1 dpf), were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Each well was treated with a water-soluble sample (Pediococcus pentosaceus RH3401 lyophilized powder, viable count 6 × 10⁻⁶). 11 CFU / g (concentration shown in Table 13) was used in both the normal control group and the model control group, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups were treated with 0.17 μg / mL of mycophenolate mofetil to establish a zebrafish visual fatigue model. After treatment at 28℃ for 1 day, the MTC of the samples on the model zebrafish was measured.

[0162] 1.3 Evaluation of efficacy in improving visual fatigue

[0163] Wild-type AB strain zebrafish with a dpf of 1 were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples (concentrations shown in Table 14) were administered in water, with a positive control group receiving 615 μg / mL glutathione. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups received 0.17 μg / mL mycophenolate mofetil to establish a zebrafish visual fatigue model. After treatment at 28℃ for 1 day, 10 zebrafish from each experimental group were randomly selected for AO staining and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software. The fluorescence intensity of apoptotic cells in the eye was analyzed, and the statistical analysis results of this index were used to evaluate the efficacy of the samples in improving visual fatigue. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software. p A value <0.05 indicates that the difference is statistically significant.

[0164] Test results:

[0165] 1.4 MTC

[0166] Under the conditions of this experiment, the MTC for improving visual fatigue with Pediococcus pentosaceus RH3401 was 500 μg / mL. See Table 13 for details.

[0167] Table 13 Experimental results of the concentration of samples that improve visual fatigue (n=30)

[0168]

[0169] 1.5 Evaluation of efficacy in improving visual fatigue

[0170] Under the experimental conditions, Pediococcus pentosaceus RH3401 showed efficacy in improving eye fatigue (Table 14). Figures 8-9 ).

[0171] Table 14. Experimental results evaluating the efficacy of the samples in improving visual fatigue (n=10)

[0172]

[0173] Note: Compared with the model control group, *** p <0.001.

[0174] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of Pediococcus pentosaceus ( Pediococcus pentosaceus RH3401, characterized in that, The preservation number of Pediococcus pentosaceus RH3401 is CGMCC No. 25166.

2. The method for culturing Pediococcus pentosaceus RH3401 according to claim 1, characterized in that, To inoculate Pediococcus pentosaceus RH3401 into the culture medium for cultivation.

3. A preparation, characterized in that, The preparation contains Pediococcus pentosaceus RH3401 as described in claim 1.

4. The preparation according to claim 3, characterized in that, The prepared products are fermentation broth of Pediococcus pentosaceus RH3401, fermentation broth precipitate of Pediococcus pentosaceus RH3401, live Pediococcus pentosaceus RH3401, or freeze-dried powder of Pediococcus pentosaceus RH3401.

5. A microbial agent, characterized in that, The bacterial agent includes Pediococcus pentosaceus RH3401 as described in claim 1 or the preparation as described in any one of claims 3-4.

6. The use of Pediococcus pentosaceus RH3401 as described in claim 1, or the preparation as described in any one of claims 3-4, or the bacterial agent as described in claim 5, in the preparation of products for relieving eye strain, characterized in that, The product in question is a medicine or health product.

7. The application according to claim 6, characterized in that, The viable count of Pediococcus pentosaceus RH3401 in the product is 1×10⁻⁶. 5 -1×10 12 CFU / mL or 1×10 5 -1×10 12 CFU / g.

8. A product for relieving eye strain, characterized in that, The product includes Pediococcus pentosaceus RH3401 as described in claim 1, or the preparation as described in any one of claims 3-4, or the bacterial agent as described in claim 5. The product is a pharmaceutical or health product.

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