Application of Bifidobacterium adolescentis B-201 in preparation of products for relieving asthenopia and protecting eyesight

Abstract

The application provides application of Bifidobacterium adolescentis B-201 in preparation of products for relieving visual fatigue and protecting vision, and belongs to the field of microbial fermentation. Bifidobacterium adolescentis B-201 provided by the application has a preservation number of CGMCC No.27234 and is preserved in the China General Microbiological Culture Collection Center on April 27, 2023. After culture fermentation and freeze-drying, the strain can be obtained in the form of bacteria powder, and after adding accessories, the bacteria powder can be used for improving eye symptoms, increasing tear secretion, reducing ocular surface disease index score and increasing near distance continuous eye use time. The strain can be used for preparing products for relieving visual fatigue and protecting vision.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation, and relates to Bifidobacterium adolescentis, specifically the application of Bifidobacterium adolescentis B-201 in the preparation of products that relieve eye fatigue and protect vision. Background Technology

[0002] The term "probiotics" was first proposed in 1953 by the German bacteriologist Werner Kollath, and translated as "beneficial to life." In 2013, the International Scientific Association for Probiotics and Prebiotics revised the definition of probiotics to: "live microorganisms that, when administered in adequate amounts, contribute to the health of the host." In addition, lactic acid bacteria, spore-forming bacteria, yeast, and algae are classified as probiotics, with lactic acid bacteria being the most commonly used. Currently, probiotic preparations are mainly divided into three categories: probiotics, prebiotics, and synbiotics. Probiotic preparations can supplement the normal microorganisms lacking in the intestines in terms of quantity or variety, adjust and maintain the balance of intestinal flora, stabilize the internal environment, enhance immunity, and promote the digestion and absorption of nutrients. Probiotics are used in probiotic probiotic preparations, fermented foods, fermented dairy products, and more.

[0003] Since Tissier first isolated Bifidobacteria from the feces of breastfed infants in 1990, the beneficial functions of Bifidobacteria for human health have received considerable attention. Subsequently, scientists have isolated Bifidobacteria from different ecological niches, such as the oral cavity, human vagina, and the feces of various mammals. Bifidobacteria belong to the Bifidobacteriaceae family of the Actinobacteria phylum. They are Gram-positive, anaerobic, non-aerobic, and have an optimal growth temperature of 37°C and an optimal growth pH of 6.0-7.0. Bifidobacteria are often curved rod-shaped, exhibiting various forms such as L, V, and Y, and frequently branch at the end. The genus *Bifidobacterium* currently includes more than 90 species.

[0004] Currently, the industrialization of Bifidobacterium is mainly divided into the following five aspects: (1) Health product market: Bifidobacterium health products exist in the form of capsules, powders, liquids, etc., for consumers to take orally. These products usually help regulate intestinal flora, help enhance immunity, help digestion, etc.; (2) Dairy product market: Bifidobacterium also has important applications in the dairy industry. Products such as yogurt, fermented milk, and cheese often add Bifidobacterium as an active strain. Its addition can provide consumers with more choices, while increasing the functionality and nutritional value of the products; (3) Dietary supplements: Dietary supplements may be Bifidobacterium products or compound products, which contain a variety of probiotic strains. These supplements can be used by consumers according to their personal needs and health goals; (4) Pharmaceutical field: In the pharmaceutical field, Bifidobacterium is used to prevent and treat intestinal-related diseases, such as diarrhea, constipation, enteritis, colitis, etc., as well as some other diseases, such as allergic rhinitis, skin diseases, etc.; (5) Animal feed: In addition to human applications, Bifidobacterium also has a wide range of applications in the field of animal feed. Adding Bifidobacteria to animal feed can improve animal gut health, promote nutrient absorption, and increase the efficiency of animal husbandry.

[0005] Eye strain has become a prevalent problem in modern life, especially with the widespread use of digital learning, remote work, and smartphones. Studies show that 90% of digital device users experience varying degrees of eye strain symptoms. In my country, the onset of eye strain is becoming more widespread and affecting younger people. The main symptoms include dry eyes, a foreign body sensation, blurred vision, eye strain, tearing, photophobia, and heavy eyelids. Mild eye strain often goes unnoticed, leading to increasingly severe symptoms. Long-term, recurring eye strain can trigger various eye diseases, potentially even cataracts. Eye strain is a syndrome involving both organic eye or systemic lesions and psychological factors. It is a group of symptoms characterized by visual disturbances, eye discomfort, and systemic symptoms after eye use, ultimately preventing normal visual tasks. Given the current prevalence of eye strain, the importance of preventing and alleviating it is increasingly prominent. In recent years, researchers have brought the "gut-eye axis" to the forefront of public attention. The relationship between the gut microbiota and the eyes is receiving increasing attention. It is well known that probiotics can effectively regulate the gut microbiota and enhance immunity.

[0006] Existing technology CN116445370A discloses a strain of Bifidobacterium breve JYBF-117 that can be formulated into a bacterial agent. Experiments have shown that the Bifidobacterium breve JYBF-117 bacterial agent can effectively alleviate dry eye syndrome caused by environmental factors or vitamin A deficiency, increase tear secretion, improve tear film stability, inhibit the expression of dry eye syndrome-related inflammatory factors IL-1β, IL-6, and TNF-α, and reduce the degree of inflammation.

[0007] Existing technology CN117643379A discloses the application of Bifidobacterium animalis subsp. lactis i797 in eye care products. This Bifidobacterium animalis subsp. lactis i797 has the effect of relieving eye fatigue. In vitro simulation experiments have demonstrated that it has good intestinal epithelial cell adhesion and can colonize in vivo for a long time to exert its effects. Zebrafish model experiments have shown that it can significantly prevent ocular cell apoptosis and can be used to prepare milk powder, yogurt, and various probiotic agents with the effect of relieving eye fatigue.

[0008] Along with the development of the probiotic industry, some potential hidden dangers and risks also exist, such as the improper use of the probiotic concept, unclear core strains, and difficulty in defining functional properties. The efficacy and safety of probiotics have always been controversial. Although adverse reactions to probiotics seem to be mild, improper use can indeed cause potential health problems. my country started relatively late in the development and utilization of probiotics, and most microbiological research has focused on the mutagenesis of fermentation strains and the improvement of metabolite production, with less attention paid to the physiological functions of the strains. Therefore, updating and developing new varieties, further improving the efficacy and safety of probiotic preparations, and standardizing the rational application of microecological preparations have become urgent issues to be addressed. Summary of the Invention

[0009] This invention addresses the problems of existing technologies by providing the application of Bifidobacterium adolescentis B-201 in the preparation of products for relieving eye fatigue and protecting vision. In this invention, Bifidobacterium adolescentis (… Bifidobacterium adolescentis B-201, with accession number CGMCC No. 27234, was deposited at the China General Microbiological Culture Collection Center on April 27, 2023. After cultivation, fermentation, and freeze-drying, the resulting bacterial powder, with the addition of excipients, can be used to improve eye symptoms, increase tear production, and extend the duration of continuous near-vision activities. This strain can be used to prepare products that relieve eye fatigue and protect vision.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides Bifidobacterium adolescentis (Bifidobacterium adolescentis) Bifidobacterium adolescentis The application of Bifidobacterium adolescentis B-201 in the preparation of products to relieve eye fatigue and protect vision. The accession number of Bifidobacterium adolescentis B-201 is CGMCC No.27234, which was deposited at the China General Microbiological Culture Collection Center on April 27, 2023.

[0011] Preferably, the product includes health supplements or pharmaceuticals.

[0012] Preferably, the medicine further includes pharmaceutical excipients.

[0013] Preferably, the pharmaceutical excipients include at least one of the following commonly used in pharmaceuticals: fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents.

[0014] Preferably, the medicine is in the form of tablets, powder, capsules or granules.

[0015] Preferably, the product further includes a microbial agent containing Bifidobacterium adolescentis B-201, or a culture, fermentation product, or freeze-dried powder containing Bifidobacterium adolescentis B-201.

[0016] Preferably, the microbial agent comprises inactivated cells of Bifidobacterium adolescentis B-201 after fermentation, fermentation broth, cell lysate, intracellular extract or metabolite.

[0017] Preferably, the microbial agent is a solid or liquid formulation.

[0018] Preferably, the bacterial agent is a freeze-dried powder containing Bifidobacterium adolescentis B-201.

[0019] Preferably, the method for preparing the freeze-dried powder includes the following steps: (1) Inoculate Bifidobacterium adolescentis B-201 into the culture medium for activation; (2) The activated strain is inoculated into the fermentation medium to obtain the seed liquid; (3) Inoculate the seed culture into the fermentation medium and carry out fermentation; (4) After fermentation, freeze-dry to obtain freeze-dried powder.

[0020] Preferably, the fermentation medium comprises, by weight, 20-30 parts sucrose, 10-15 parts lactose, 10-20 parts soybean peptone, 3-8 parts yeast powder, 10-15 parts yeast peptone, 20-25 parts Na2HPO3, 1-3 parts citric acid, 0.1-1.0 parts MgSO4·7H2O, 0.1-0.5 parts MnSO4·5H2O, 0.5-1.5 parts Tween-80, and 0.1-0.5 parts L-cysteine ​​hydrochloride.

[0021] Preferably, the fermentation medium comprises, by weight, 23.5 parts sucrose, 12.0 parts lactose, 15.0 parts soybean peptone, 5.0 parts yeast powder, 12.0 parts yeast peptone, 21.0 parts Na2HPO3, 2 parts citric acid, 0.6 parts MgSO4·7H2O, 0.3 parts MnSO4·5H2O, 1.0 part Tween-80, and 0.3 parts L-cysteine ​​hydrochloride.

[0022] Compared with the prior art, the present invention has the following beneficial effects: In this invention, Bifidobacterium adolescentis B-201 is cultured, fermented, and freeze-dried to obtain bacterial powder. After adding excipients, it can be used to improve eye symptoms, increase tear secretion, and increase the duration of continuous close-range eye use. This strain can be used to prepare products that relieve eye fatigue and protect vision.

[0023] Preservation Instructions Strain name: Bifidobacterium adolescentis B-201; Classification and nomenclature: Bifidobacterium adolescentis Bifidobacterium adolescentis ; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures; The abbreviation for the depository institution is CGMCC. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: April 27, 2023; Registered with the China National Collection Center (CGMCC) No. 27234. Detailed Implementation

[0024] The following embodiments are provided to better understand the present invention and are not intended to limit the preferred embodiments. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention. Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in the art. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0025] Example 1: Preparation of a bacterial powder Strain: Bifidobacterium adolescentis B-201, preservation number CGMCC No. 27234; Preparation method of bacterial powder: 1) Activation of bacterial strains: The strains frozen at -40℃ were inoculated into modified MRS liquid medium sterilized at 121℃ for 15 min, and cultured anaerobically at 37℃ for 24 h. The strains were then subcultured twice to obtain activated bacterial strains. 2) Optimize the preparation of the culture medium: The optimized culture medium composition is as follows: 23.5 kg sucrose, 12.0 kg lactose, 15.0 kg soybean peptone, 5.0 kg yeast extract, 12 kg yeast peptone, 21.0 kg Na2HPO3, 2.0 kg citric acid, 0.6 kg MgSO4·7H2O, 0.3 kg MnSO4·5H2O, 1.0 kg Tween-80, 0.3 kg L-cysteine ​​hydrochloride, and 1000 L distilled water.

[0026] Prepare the optimized culture medium according to the specified proportions, mix thoroughly, adjust the pH to 6.5, and sterilize at 121℃ for 15 minutes.

[0027] 3) Preparation of seed culture: The activated bacterial strain was inoculated into an optimized culture medium and anaerobically cultured at 37°C until the pH reached 4.6. This yielded the seed culture. 4) Inoculation and fermentation: Fermentation was carried out using a Bailun Bio BLBIO-10GJ fermenter. 100L of optimized culture medium was placed in the fermenter. After sterilization, the seed liquid was inoculated into the optimized culture medium at a volume fraction of 1‰. Fermentation was carried out for 18 hours under controlled fermentation conditions. Fermentation conditions were controlled as follows: in the early stage of fermentation, the temperature was kept constant at 30℃ and allowed to ferment naturally until the pH reached 5.0; then the fermentation temperature was adjusted to 37℃ and the pH was kept at 6.0 to maintain anaerobic fermentation.

[0028] The pH was controlled by adding a neutralizing agent, which was a 25% NaOH solution, during the fermentation process; the anaerobic conditions were achieved by purging nitrogen gas every two hours.

[0029] 5) Termination of fermentation: Fermentation is terminated when acid production by the cells stops, yielding a high-density fermentation broth for each strain, with a viable cell count of 2 × 10⁻⁶. 10 The concentration of cfu / ml or higher can be used to determine whether acid production has stopped, based on the fact that the pH no longer decreases and the addition of neutralizing agent ceases.

[0030] 6) Freeze-drying: a. Cell concentration: The high-density fermentation broth is concentrated into cells by centrifugation at 12000g. b. Add protectant: Add 5 times the amount of protectant solution to the bacterial concentrate; the protectant solution is composed of the following: 15kg skim milk, 12kg lactose, 1kg vitamin C, 1kg monosodium glutamate, and 1000L distilled water.

[0031] c. Drying: The bacterial suspension with added preservatives was freeze-dried (model: Sihuan LGJ-12C) to obtain freeze-dried bacterial powder, with a total viable count of 1.0 × 10⁻⁶. 11 CFU / g or higher.

[0032] Example 2: Preparation of a microbial agent Weigh 0.1g of the bacterial powder from Example 1, add 0.9g of the excipient maltodextrin, mix well, and the viable count should not be less than 1.0×10⁻⁶. 10 CFU / g.

[0033] Comparative Example 1: Preparation of a bacterial powder Bacterial strain: Bifidobacterium animalis subsp. lactis BB-12, purchased from Chr. Hansen (Beijing) Trading Co., Ltd.

[0034] Except for changing the bacterial strain to Bifidobacterium lactis subsp. BB-12, everything else is the same as in Example 1.

[0035] Comparative Example 2: Preparation of a bacterial powder The bacterial strain was Bifidobacterium breve JYBF117 from CN116445370A, purchased from the China General Microbiological Culture Collection Center, with accession number CGMCC No. 24536.

[0036] Except for changing the bacterial strain to Bifidobacterium breve JYBF117, everything else is the same as in Example 1.

[0037] Comparative Example 3: Preparation of a microbial agent Weigh 0.1g of the bacterial powder from Comparative Example 1, add 0.9g of the excipient maltodextrin, mix well, and the viable count should not be less than 1.0×10⁻⁶. 10 CFU / g.

[0038] Comparative Example 4: Preparation of a microbial agent Weigh 0.1g of the bacterial powder from Comparative Example 2, add 0.9g of the excipient maltodextrin, mix well, and ensure the viable count is not less than 1.0 × 10⁻⁶. 10 CFU / g.

[0039] Example of positive results: Probiotics help improve eye condition (1) Preparation of inoculum Inoculants of Bifidobacterium adolescentis B-201, Bifidobacterium animalis subsp. lactis BB-12, and Bifidobacterium breve JYBF117 were prepared according to Examples 2, 3, and 4, respectively.

[0040] (2) Experimental subjects 120 participants with visual fatigue were recruited from Inner Mongolia Agricultural University and randomly divided into the following three groups using a random number table method.

[0041] (3) Experimental grouping Experimental group (30 people): Administered the bacterial agent (Bifidobacterium adolescentis B-201 bacterial agent) prepared in Example 2, 1×10⁻⁶ per day. 10 CFU / g; Control group A (30 people): Administered the bacterial agent (Bifidobacterium animalis subsp. lactis BB-12) prepared in control group 3, 1×10⁻⁶ per day. 10 CFU / g; Control group B (30 people): Administered the bacterial agent (Bifidobacterium breve JYBF117) prepared in control group 4, 1×10⁻⁶ per day. 10 CFU / g; Placebo group (30 people): Received the same dose of maltodextrin, which was identical to probiotics in terms of packaging, powder form, and content.

[0042] (4) Detection method Inclusion criteria: 1) Participants were ≥18 years old; 2) Participants had symptoms such as itchy or dry eyes; 3) Participants gave informed consent.

[0043] Exclusion criteria: 1) other eye diseases; 2) mental disorders; 3) received related treatment within two weeks prior to enrollment; 4) took antibiotics within two weeks prior to enrollment.

[0044] Subjects received either probiotics or a placebo for 4 weeks. 1) Ocular symptom scoring: Before and after 4 weeks of intervention, the ocular symptoms (itchy eyes, dry eyes, eye pain, foreign body sensation, blurred vision, photophobia, tearing) of the three groups of subjects were scored on a 4-point scale according to severity: no symptoms (0 points), mild symptoms (2 points), moderate symptoms (4 points), and severe symptoms (6 points). 2) Tear secretion examination: A filter paper strip with a length of 3.5 cm and a width of 0.5 cm was placed in the fornix of the lower eyelid, taking care not to touch the cornea. After gently closing both eyes for 5 minutes, the strip was removed, and the length of infiltration was measured. 3) Ocular surface disease index scale scoring: The assessment included three aspects: ocular symptoms, visual function, and environmental factors. The final score = (total score of all questions answered × 25) / number of questions answered, with a total score of 100 points. The higher the score, the more severe the condition. Specifically, it included: All time in the past week -4 points; most time -3 points; half time -2 points; occasionally -1 point; never -0 points; N / A - not applicable.

[0045] Do you experience any of the following uncomfortable sensations? ① The eyes are sensitive to light; ②It feels like there's sand in my eyes; ③ Eye pain or soreness; ④ The objects are blurry; ⑤ Deterioration of eyesight.

[0046] Are you experiencing eye discomfort that would affect your ability to participate in the following activities? ⑥ Reading; ⑦ Driving at night; ⑧ Use a computer or mobile phone; 9. Watching TV.

[0047] Do your eyes feel uncomfortable in the following environments? ⑩ In windy / windy environments; In a relatively dry location or environment; In a place where the air conditioning is on.

[0048] 4) Efficacy criteria: Disappearance of eye symptoms and continuous close-range eye use time >45 minutes are considered significant effects; relief of eye symptoms and continuous close-range eye use time >30 minutes are considered effective; no change or worsening of eye symptoms.

[0049] Statistical data were collected using SPSS 22.0; P <0.05 is statistically significant.

[0050] (5) Experimental results 5.1 Comparison of ocular symptom scores Analysis of the subjects' ocular symptoms revealed that probiotic intervention effectively improved their ocular condition scores (including itchy eyes, eye pain, dry eyes, foreign body sensation, blurred vision, photophobia, and tearing, see Table 1). The results showed that compared to the control group and the placebo group, the experimental group (B-201 probiotic) intervention significantly alleviated the subjects' ocular symptoms. P <0.05), thus relieving eye fatigue and protecting eyesight.

[0051] Table 1 Comparison of ocular symptom scores before and after intervention in subjects

[0052] Note: Data in the same column with the same superscript letter indicates no significant difference (P>0.05), while data with different superscript letters indicates significant difference (P<0.05).

[0053] 5.2 Assessment of tear secretion Analysis of tear secretion in the subjects' eyes revealed that probiotic intervention effectively increased tear secretion. P <0.05 (see Table 2). The results indicate that compared with the control group and the placebo group, the intervention in the experimental group (B-201 bacterial agent) can significantly improve the tear secretion of the subjects, thereby relieving eye fatigue and protecting vision.

[0054] Table 2 Comparison of tear secretion before and after intervention in subjects

[0055] Note: Data in the same column with the same superscript letter indicates no significant difference (P>0.05), while data with different superscript letters indicates significant difference (P<0.05).

[0056] 5.3 Comparison of Ocular Surface Disease Index Scores Analysis of the ocular surface disease index of the subjects revealed that probiotic intervention effectively reduced the ocular surface disease index scores (including ocular symptoms, visual function, and environmental factors, see Table 3). The results showed that compared with the control group and the placebo group, the experimental group (B-201 probiotic) significantly reduced the ocular condition of the subjects. P <0.05), thus relieving eye fatigue and protecting eyesight.

[0057] Table 3 Comparison of OSDI before and after intervention in subjects

[0058] Note: Data in the same column with the same superscript letter indicates no significant difference (P>0.05), while data with different superscript letters indicates significant difference (P<0.05).

[0059] 5.4 Comparison of eye symptoms and continuous near-vision time Analysis of the subjects' ocular symptoms and continuous near-vision time revealed that probiotic intervention effectively improved the efficacy of treatment in the number of subjects (see Table 4). This indicates that compared with the control group and the placebo group, the experimental group (B-201 probiotic) intervention significantly improved the subjects' ocular symptoms and increased the continuous near-vision time. P <0.05), thus relieving eye fatigue and protecting eyesight.

[0060] Table 4 Comparison of the efficacy of ocular symptoms and continuous near-vision time before and after intervention in subjects

[0061] Note: The numbers in parentheses represent the percentage of the total.

[0062] (6) Conclusion The probiotic preparation of Bifidobacterium adolescentis B-201 can significantly improve eye symptoms (including itchy eyes, eye pain, dry eyes, foreign body sensation, blurred vision, photophobia and tearing), increase tear secretion, reduce ocular surface disease index scores (including eye symptoms, visual function, and environmental factors), and increase the duration of continuous close-range eye use, thereby effectively relieving eye fatigue and protecting vision.

[0063] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. Bifidobacterium adolescentis ( Bifidobacterium adolescentis The application of B-201 in the preparation of health food products that relieve visual fatigue is characterized by, The Bifidobacterium adolescentis B-201, with accession number CGMCC No. 27234, was deposited at the China General Microbiological Culture Collection Center on April 27, 2023.

2. The application according to claim 1, characterized in that, The health food also includes microbial agents, which contain Bifidobacterium adolescentis B-201, or cultures, ferments, or freeze-dried powders containing Bifidobacterium adolescentis B-201.

3. The application according to claim 2, characterized in that, The microbial agent includes inactivated cells or fermentation broth of Bifidobacterium adolescentis B-201 after fermentation.

4. The application according to claim 3, characterized in that, The microbial agent can be a solid or liquid formulation.

5. The application according to claim 2, characterized in that, The bacterial agent is a freeze-dried powder containing Bifidobacterium adolescentis B-201.

6. The application according to claim 5, characterized in that, The method for preparing the freeze-dried powder includes the following steps: (1) Inoculate Bifidobacterium adolescentis B-201 into the culture medium for activation; (2) The activated strain is inoculated into the fermentation medium to obtain the seed liquid; (3) Inoculate the seed culture into the fermentation medium and carry out fermentation; (4) After fermentation, freeze-dry to obtain freeze-dried powder.

7. The application according to claim 6, characterized in that, The fermentation medium comprises, by weight, 20-30 parts sucrose, 10-15 parts lactose, 10-20 parts soybean peptone, 3-8 parts yeast powder, 10-15 parts yeast peptone, 20-25 parts Na2HPO3, 1-3 parts citric acid, 0.1-1.0 parts MgSO4·7H2O, 0.1-0.5 parts MnSO4·5H2O, 0.5-1.5 parts Tween-80, and 0.1-0.5 parts L-cysteine ​​hydrochloride.

8. The application according to claim 7, characterized in that, The fermentation medium comprises, by weight, 23.5 parts sucrose, 12.0 parts lactose, 15.0 parts soybean peptone, 5.0 parts yeast extract, 12.0 parts yeast peptone, 21.0 parts Na2HPO3, 2 parts citric acid, 0.6 parts MgSO4·7H2O, 0.3 parts MnSO4·5H2O, 1.0 part Tween-80, and 0.3 parts L-cysteine ​​hydrochloride.

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