Bifidobacterium longum ZKBb0311, rhizoma polygonati fermentation liquor and application of bifidobacterium longum ZKBb0311 in preparation of products for improving eyesight

Abstract

The invention relates to the technical field of probiotic fermentation, in particular to bifidobacterium longum ZKBb0311, rhizoma polygonati fermentation liquor and application of the bifidobacterium longum ZKBb0311 to preparation of products for improving eyesight. The Bifidobacterium longum ZKBb0311 is preserved in the China General Microbiological Culture Collection Center on May 13, 2024, the preservation address is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.30549. The Bifidobacterium longum ZKBb0311 disclosed by the invention has the advantages that the Bifidobacterium longum ZKBb0311 can be applied to the field of agricultural production, and the production cost is low; the bifidobacterium longum ZKBb0311 provided by the invention has a good fermentation effect, and clinical tests prove that the polygonatum sibiricum fermentation liquor obtained by fermenting polygonatum sibiricum by using the bifidobacterium longum ZKBb0311 can obviously relieve asthenopia, improve eyesight and improve the curative effect of polygonatum sibiricum.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotic fermentation, and in particular to a strain of Bifidobacterium longum ZKBb0311, a Polygonatum sibiricum fermentation liquid and applications thereof in preparing a product for improving vision. Background Art

[0002] With the advent of the information age, we're increasingly using computers at work, and spending extended periods looking at our phones after get off work. This leads to decreased blinking and tear production when our eyes are focused on screens like computers and phones. At the same time, flickering screens can be a strong eye irritant. Excessive eye use and a lack of proper eye care can lead to decreased vision and even various eye diseases, such as myopia, amblyopia, and visual fatigue, severely harming our physical and mental health. Recent studies have demonstrated the effectiveness of Traditional Chinese Medicine in improving vision with minimal side effects.

[0003] Traditional Chinese Medicine (TCM) believes that myopia is related to liver and kidney deficiency, as well as qi and blood deficiency. Traditional Chinese medicine treatments for myopia primarily regulate the body's internal environment, enhancing blood circulation and nutrient supply to the eyes, thereby improving vision. Polygonatum sibiricum, sweet in flavor and neutral in nature, enters the spleen and lung meridians, tonifying the spleen and moistening the lungs, nourishing yin and promoting fluid production, and strengthening muscles and bones. It can be used to treat spleen and stomach qi deficiency, fatigue, post-illness weakness, weak muscles and bones, and rheumatic pain. In TCM, it enjoys the reputation of "King of Qi and Blood Tonics."

[0004] Fermentation is a method of preparing traditional Chinese medicine. However, traditional fermented traditional Chinese medicine is fermented under natural conditions using natural bacteria, and the fermentation conditions are uncontrollable. Modern fermentation uses known microorganisms to control fermentation conditions. Through the complex metabolic activities of microorganisms, the structure and content of active ingredients in traditional Chinese medicine are changed, thereby improving the efficacy and medicinal value of traditional Chinese medicine. Its application field is very wide.

[0005] CN 109349485 A provides a traditional Chinese medicine fermented beverage for benefiting the eyes and protecting the liver. The preparation method of the traditional Chinese medicine fermented beverage comprises the following steps: crushing black dates, black sesame seeds, Chinese yam, chrysanthemum, Hovenia dulcis fruit, wolfberry, sea buckthorn, polygonatum, cassia seed, and angelica dahurica, adding purified water, cellulase, and pectinase, and performing constant temperature enzymolysis to obtain an enzymatic hydrolyzate and a filter residue; then ultrasonically extracting the filter residue with purified water, filtering the extract, combining it with the enzymatic hydrolyzate, and vacuum concentrating to obtain a medicinal material concentrate; washing mulberries, blueberries, and cranberries, pulping them with purified water, filtering them, and adding them together with honey to the medicinal material concentrate, stirring them evenly to form a mixed solution; steam-sterilizing the mixed solution, adding a probiotic seed solution, and standing for fermentation to obtain a fermentation solution; and finally filling and steam-sterilizing to obtain the traditional Chinese medicine fermented beverage for benefiting the eyes and protecting the liver. The shortcomings of this fermented Chinese herbal beverage are: ① Its eye-benefiting and liver-protecting effects rely on a specific formula combination, involving a wide variety of raw materials and the addition of enzymes such as cellulase and pectinase, resulting in high supply chain costs and complex operating procedures. ② The complex ingredients, combined with multiple ingredients, may cause interactions between them, affecting absorption or causing mild irritation. ③ The formula contains strong ingredients such as Cassia seed and Angelica dahurica, so some people should consume it with caution. Summary of the Invention

[0006] In response to the technical problems in the existing technology of preparing Chinese medicine fermented drinks by using more than a dozen raw materials through enzymatic hydrolysis, extraction, pulping and fermentation, such as the large variety of raw materials, complex operation procedures and complex ingredients, the present invention provides a strain of Bifidobacterium longum ZKBb0311, Polygonatum sibiricum fermentation liquid and their application in the preparation of products for improving vision.

[0007] In a first aspect, the present invention provides a strain of Bifidobacterium longum ( Bifidobacterium longum ) ZKBb0311 was deposited in the General Microbiology Center of China Culture Collection Administration on May 13, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 30549.

[0008] In a second aspect, the present invention provides a Polygonatum sibiricum fermented liquid capable of improving eyesight, which is prepared by the following method: (1) Soak the polygonatum in water and then boil it to obtain the polygonatum extract; (2) Inoculate the bacterial suspension of Bifidobacterium longum ZKBb0311 into the sterilized Polygonatum sibiricum extract, shake well, and then culture anaerobically at 37°C to obtain the product.

[0009] Furthermore, in step (1), the nine-steamed and nine-dried Polygonatum odoratum is sliced ​​and then added with 10 times the weight of water and soaked for 60 minutes.

[0010] Furthermore, in step (1), the decoction condition is to boil the mixture and then keep boiling for 120 minutes.

[0011] Furthermore, in step (1), after decoction, the filtrate is filtered and separated, and the decoction is concentrated to obtain a polygonatum extract having a crude drug content of 0.75 g / mL, wherein the crude drug content refers to the ratio of the amount of the polygonatum raw material used to the volume of the polygonatum extract obtained after concentration.

[0012] Furthermore, in step (2), 1 wt% to 3 wt% of a bacterial suspension of Bifidobacterium longum ZKBb0311 is inoculated into the polygonatum extract, and the bacterial suspension is prepared by the following method: Bifidobacterium longum ZKBb0311 was inoculated into MRS liquid medium supplemented with 0.05 g / L L-cysteine ​​hydrochloride, cultured anaerobically at 37°C for 20 h, activated and propagated to the 2nd or 3rd generation, and a bacterial suspension was prepared.

[0013] In a third aspect, the present invention further provides a use of the above-mentioned Polygonatum sibiricum fermentation liquid in the preparation of a product for improving vision, wherein the product may be a health food or medicine for relieving visual fatigue.

[0014] Furthermore, the product for improving vision is in the form of an oral solution.

[0015] The beneficial effects of the present invention are: The Bifidobacterium longum ZKBb0311 provided by the present invention has higher Polygonatum sibiricum fermentation activity than other experimental strains. Clinical trials have demonstrated that the Polygonatum sibiricum fermentation broth obtained using this Bifidobacterium longum ZKBb0311 significantly alleviates visual fatigue, improves vision, and enhances the efficacy of Polygonatum sibiricum. Furthermore, the present invention employs a simplified production model using a single strain (Bifidobacterium longum ZKBb0311) to ferment a single medicinal ingredient (Polygonatum sibiricum), offering significant advantages in terms of process, cost, and safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0017] Figure 1 This is a picture of the colony morphology of Bifidobacterium longum ZKBb0311.

[0018] Figure 2 This is a Gram staining image of Bifidobacterium longum ZKBb0311. DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0020] Unless otherwise specified, the reagents, materials, and instruments involved in the following examples are all conventional commercial products; unless otherwise specified, the methods used are all conventional methods in the art.

[0021] Among them, mupirocin lithium salt and TOS culture medium were purchased from Qingdao Haibo Biotechnology Co., Ltd., L-cysteine ​​hydrochloride was purchased from Sigma-Aldrich, USA, MRS liquid culture medium and MRS solid culture medium were purchased from Beijing Aoboxing Biotechnology Co., Ltd., DNA extraction kit was purchased from Beijing Tiangen Biochemical Technology Co., Ltd., and nine-steamed and nine-dried polygonatum was purchased from Henan Yanhuang Pharmacy.

[0022] Example 1 Isolation and identification of Bifidobacterium longum ZKBb0311 1. Sample source: Samples were collected from Changshou Village, Bama Yao Autonomous County, Hechi City, Guangxi Zhuang Autonomous Region in March 2015.

[0023] 2. Strain Isolation: The collected samples were diluted with physiological saline to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 100 μL of the dilution solution was taken and evenly spread on TOS medium containing 0.05 g / L mupirocin lithium salt, and cultured anaerobically in a biochemical incubator at 37°C for 48 h to observe the growth of the colonies.

[0024] After colonies have formed, single colonies are picked and streaked three times. Repeat the streaking process several times until the colonies are purified. The purified strain is inoculated into MRS liquid medium supplemented with 0.05 g / L L-cysteine ​​hydrochloride. The culture medium is mixed with 50% sterile glycerol at a ratio of 1:1 (v / v) and stored at -80°C.

[0025] 3. Strain identification: (1) Colony morphology identification ①Inoculate the isolated strain onto TOS medium containing 0.05 g / L mupirocin lithium salt, culture anaerobically for 48 h, and observe and record the morphology and color of the colonies. Figure 1As shown, the colonies are milky white or off-white, opaque, shiny, smooth, raised, soft in texture, and have neat edges.

[0026] ②Pick the colonies on the medium in ① and smear, fix, stain with crystal violet, mordant, decolorize, wash with water, counterstain with safranin, dry, and then examine the dried colonies under an oil microscope with a magnification of 1000 times. Figure 2 Microscopic observation revealed that the bacteria were irregular rod-shaped, with shapes such as straight, curved, match-head, and dumbbell, arranged singly or in pairs, and were Gram-positive.

[0027] (II) 16S rDNA sequence determination ① Extract genomic DNA of the strain: Take the culture medium of the isolated strain and centrifuge it at 5000 r / min for 10 min. After centrifugation, discard the supernatant and collect the pure culture bacteria to be tested. Use a DNA extraction kit to extract the genomic DNA of the strain and follow the instructions.

[0028] ②PCR amplification: using bacterial universal primers 27F / 1492R.

[0029] PCR amplification conditions were as follows: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s; annealing at 55°C for 35 s; extension at 72°C for 90 s; after 32 cycles, the final extension was at 72°C for 5 min.

[0030] ③Gene sequencing: PCR products were subjected to 1.0% agarose gel electrophoresis, and gene sequencing was performed on the amplified products. The sequencing work was completed by Beijing Qingke Biotechnology Co., Ltd.

[0031] The 16S rDNA sequence of the isolated strain is as follows:

[0032] The strain was identified as Bifidobacterium longum by BLAST comparison and was named Bifidobacterium longum ZKBb0311.

[0033] IV. Strain Preservation: Bifidobacterium longum ZKBb0311 was deposited in the General Microbiology Center of China Culture Collection Administration on May 13, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 30549 and the classification name is Bifidobacterium longum Bifidobacterium longum .

[0034] Example 2 Preparation of Polygonatum sibiricum Fermentation Broth from Bifidobacterium longum ZKBb0311 The fermentation liquid of Bifidobacterium longum ZKBb0311 and Polygonatum sibiricum was prepared as follows: (1) Slice the nine-steamed and nine-dried Polygonatum odoratum and soak it in 10 times its weight of water for 60 minutes. Place the soaked Polygonatum odoratum and the soaking water into a traditional Chinese medicine cooking pot. First, boil it over high heat, then simmer it over low heat for 120 minutes. The soaking water is directly used for decoction without changing the water to avoid wasting the active ingredients. During the decoction process, stir the pot moderately to prevent sticking, but avoid opening the lid frequently to prevent the loss of volatile components. After decoction, filter and separate the filtrate, and steam and concentrate it until the crude drug content (amount of Polygonatum odoratum used / volume of concentrated Polygonatum odoratum extract) is 0.75 g / mL to obtain the Polygonatum odoratum extract.

[0035] (2) The polygonatum extract obtained in step (1) was sterilized at 121°C for 15 min and then used. Bifidobacterium longum ZKBb0311 was inoculated into MRS liquid culture medium supplemented with 0.05 g / L L-cysteine ​​hydrochloride, cultured anaerobically at 37°C for 24 h, activated and propagated to the second generation, and a bacterial suspension (viable bacteria count of 1×10 9 The bacterial suspension was inoculated into the sterilized polygonatum extract at an inoculum size of 2 wt %. After shaking, the mixture was placed in an anaerobic incubator at 37 °C for 20 h to obtain the polygonatum fermentation broth, which was named Bifidobacterium longum ZKBb0311 polygonatum fermentation broth.

[0036] Comparative Example Other probiotics were used instead of Bifidobacterium longum ZKBb0311 to ferment Polygonatum sibiricum to prepare Polygonatum sibiricum fermentation liquid. The details are as follows: Bifidobacterium longum BB536 was inoculated into MRS liquid medium supplemented with 0.05 g / L L-cysteine ​​hydrochloride and cultured anaerobically at 37°C for 20 h. After activation and passage to the second generation, a bacterial suspension was prepared (the number of viable cells was 1×10 9CFU / mL). Bifidobacterium longum BB536 is a commercial probiotic strain that has been widely studied. It was purchased from Xima International Trading (Shanghai) Co., Ltd. The bacterial suspension of Bifidobacterium longum BB536 was inoculated into the sterilized Polygonatum sibiricum extract at an inoculum rate of 2 wt%. The preparation method and sterilization process of the Polygonatum sibiricum extract were the same as those in step (1) above. After shaking, the extract was placed in an anaerobic incubator at 37°C and cultured for 20 h to obtain the Polygonatum sibiricum fermentation liquid, which was named Bifidobacterium longum BB536 Polygonatum sibiricum fermentation liquid.

[0037] Lactobacillus plantarum RZKLp100 was inoculated into MRS liquid medium and cultured anaerobically at 37°C for 20 h. After activation and passage to the second generation, a bacterial suspension was prepared (the number of viable cells was 1×10 9 CFU / mL). Lactobacillus plantarum RZKLp100 is a probiotic collected and preserved by the applicant and has been disclosed in the Chinese invention patent CN 118374549 B. Previous studies have found that Lactobacillus plantarum RZKLp100 has a good effect on the fermentation of licorice. The Lactobacillus plantarum RZKLp100 bacterial suspension was inoculated into the sterilized Polygonatum sibiricum extract at an inoculum rate of 2wt%. The preparation method and sterilization process of the Polygonatum sibiricum extract were the same as those in the above step (1). After shaking, the extract was placed in an anaerobic incubator at 37°C and cultured for 20 h to obtain the Polygonatum sibiricum fermentation liquid, which was named Lactobacillus plantarum RZKLp100 Polygonatum sibiricum fermentation liquid.

[0038] Lactobacillus acidophilus RZKLa0701 was inoculated into MRS liquid medium and cultured anaerobically at 37°C for 20 h. After activation and passage to the second generation, a bacterial suspension was prepared (the number of viable bacteria was 1×10 9 CFU / mL). Lactobacillus acidophilus RZKLa0701 is a probiotic collected and preserved by the applicant and has been disclosed in the Chinese invention patent CN 117305188 B. Previous studies have found that the bacterial powder of Lactobacillus acidophilus RZKLa0701 has the function of promoting children's bone growth. The Lactobacillus acidophilus RZKLa0701 bacterial suspension was inoculated into the sterilized Polygonatum sibiricum extract at an inoculum rate of 2wt%. The preparation method and sterilization process of the Polygonatum sibiricum extract were the same as those in the above step (1). After shaking, the extract was placed in an anaerobic incubator at 37℃ and cultured for 20 hours to obtain Polygonatum sibiricum fermentation liquid, which was named Lactobacillus acidophilus RZKLa0701 Polygonatum sibiricum fermentation liquid.

[0039] The pH values ​​and viable counts of the Bifidobacterium longum ZKBb0311 Polygonatum sibiricum fermentation broth prepared in Example 2, the Bifidobacterium longum BB536 Polygonatum sibiricum fermentation broth prepared in the comparative example, the Lactobacillus plantarum RZKLp100 Polygonatum sibiricum fermentation broth, and the Lactobacillus acidophilus RZKLa0701 Polygonatum sibiricum fermentation broth were measured respectively. The results are shown in Table 1 below.

[0040] Table 1 pH value and viable bacterial count of Polygonatum sibiricum fermentation broth

[0041] By comparison, it can be seen that the number of live bacteria in the fermented broth of Bifidobacterium longum ZKBb0311 and Polygonatum sibiricum is higher, and its effect is better than that of Bifidobacterium longum BB536 and other probiotics.

[0042] If it is necessary to further increase the number of live bacteria in the Polygonatum sibiricum fermentation broth, this can be achieved by increasing the amount of Bifidobacterium longum ZKBb0311 added to the Polygonatum sibiricum extract, for example, adding Bifidobacterium longum ZKBb0311 bacterial suspension at an inoculum rate of 3wt% or 4wt%.

[0043] Example 3 Clinical Trial 1. Case selection criteria: (I) Case inclusion criteria: Volunteer subjects aged 18-65 years who use their eyes for a long time and are prone to visual fatigue.

[0044] (II) Case exclusion criteria: ① Patients with infectious or traumatic eye diseases and those who have undergone eye surgery within 3 months; ② Patients with internal and external eye diseases such as corneal, lens, vitreous body, fundus lesions, and those with cardiovascular, cerebrovascular, liver, kidney, and hematopoietic system diseases; ③ Those who have been taking long-term medications, health products or other treatments related to vision treatment and have not been able to stop; ④ Pregnant or breastfeeding women; ⑤ Those who do not meet the inclusion criteria, do not consume the test substance as required, or have incomplete information that affects the efficacy or safety judgment.

[0045] (3) Case exclusion and dropout criteria ① The subject voluntarily requests to withdraw during the trial; ② The subjects have poor compliance and cannot complete all treatments; ③ Those who experience severe adverse reactions during treatment; ④ Patients whose condition worsened or died during the study period.

[0046] 2. Test plan: (I) Trial Grouping: A randomized, double-blind study was conducted. All 360 subjects were randomly assigned to treatment group 1, treatment group 2, treatment group 3, treatment group 4, treatment group 5, and a blank control group, with 60 subjects in each group.

[0047] (2) Treatment methods: ① Treatment Group 1: Each person took 50 mL of Bifidobacterium longum ZKBb0311 Polygonatum sibiricum fermented liquid in the morning, afternoon, and evening for 60 days. The preparation method of Bifidobacterium longum ZKBb0311 Polygonatum sibiricum fermented liquid was the same as that in Example 2.

[0048] ② Each participant in the second treatment group took 50 mL of Bifidobacterium longum BB536 and Polygonatum sibiricum fermented liquid each morning, noon, and evening for 60 days. The preparation method of Bifidobacterium longum BB536 and Polygonatum sibiricum fermented liquid was the same as that of the comparative example.

[0049] ③ Each person in the three treatment groups took 50 mL of Lactobacillus plantarum RZKLp100 Polygonatum sibiricum fermentation liquid in the morning, noon and evening every day, and the treatment course lasted for 60 days.

[0050] The preparation method of the Lactobacillus plantarum RZKLp100 Polygonatum sibiricum fermentation broth is the same as that of the comparative example.

[0051] ④ Each person in the four treatment groups took 50 mL of Lactobacillus acidophilus RZKLa0701 Polygonatum sibiricum fermented liquid in the morning, noon and evening every day for 60 days.

[0052] The preparation method of Lactobacillus acidophilus RZKLa0701 Polygonatum sibiricum fermentation liquid is the same as that of the comparative example.

[0053] ⑤ Each person in the five treatment groups took 50 mL of unfermented Polygonatum oral liquid in the morning, noon and evening every day, and the treatment course lasted for 60 days.

[0054] The preparation method of Polygonatum oral liquid is as follows: After nine-steaming and nine-drying Polygonatum sibiricum slices were sliced ​​and soaked in 10 times its weight of water for 60 minutes. The soaked Polygonatum sibiricum and the soaking water were placed in a traditional Chinese medicine decoction pot and first boiled over high heat, then simmered over low heat for 120 minutes. The soaking water was used directly for decoction without changing the water to avoid wasting the active ingredients. Moderate stirring was allowed during the decoction to prevent sticking, but frequent opening of the lid was avoided to prevent the loss of volatile components. After decoction, the filtrate was filtered and concentrated by steaming to a crude drug content (Polygonatum sibiricum dosage / Polygonatum sibiricum extract volume) of 0.75 g / mL to obtain the Polygonatum sibiricum extract. The Polygonatum sibiricum extract was sterilized at 121°C for 15 minutes to obtain Polygonatum sibiricum oral solution.

[0055] ⑥ Each person in the blank control group took 50 mL of drinking water in the morning, noon and evening every day, and the treatment lasted for 60 days.

[0056] 3. Observation indicators and results: (1) Questionnaire Survey: Before and after the trial, participants were asked about the types of visual fatigue symptoms and their improvement. Visual fatigue symptoms include eight symptoms: eye pain, eye swelling, photophobia, blurred vision, dry eyes, foreign body sensation, tearing, and general discomfort. The treatment was considered effective if at least three of the eight symptoms improved and the other symptoms did not worsen.

[0057] Symptom improvement was determined based on the change in the subject's symptom score before and after the start of the trial. A decrease in a single symptom score represented improvement. The symptom scoring criteria are shown in Table 2 below.

[0058] Table 2 Subjects' improvement score sheet

[0059] Note: “Occasionally” or “occasionally” refers to 1-2 times / 2 days; “occasionally” refers to 1-3 times / day; “often” refers to >3 times / day.

[0060] The results are shown in Table 3 below.

[0061] Table 3 Improvement effect of visual fatigue in each group before and after treatment (unit: case)

[0062] Compared with before treatment, after 60 days of treatment, the number of effective cases of visual fatigue improvement in treatment group one was 54, and the number of ineffective cases was 6, with an effective rate of 90.0%; after 60 days of treatment, the number of effective cases of visual fatigue improvement in treatment group two was 39, and the number of ineffective cases was 21, with an effective rate of 65.0%; after 60 days of treatment, the number of effective cases of visual fatigue improvement in treatment group three was 36, and the number of ineffective cases was 24, with an effective rate of 60.0%; after 60 days of treatment, the number of effective cases of visual fatigue improvement in treatment group four was 35, and the number of ineffective cases was 25, with an effective rate of 58.33%; after 60 days of treatment, the number of effective cases of visual fatigue improvement in treatment group five was 32, and the number of ineffective cases was 28, with an effective rate of 53.3%; after 60 days of treatment, the number of effective cases of visual fatigue improvement in the blank control group was 2, and the number of ineffective cases was 58, with an effective rate of 3.33%. By comparing the efficacy data of treatment group one to treatment group five, it can be found that the efficacy of Bifidobacterium longum ZKBb0311 Polygonatum sibiricum fermented liquid in improving visual fatigue is significantly higher than that of unfermented Polygonatum sibiricum oral liquid ( P <0.05 and other strains of Polygonatum sibiricum fermentation broth ( P <0.05), indicating that Bifidobacterium longum ZKBb0311 fermented polygonatum can effectively enhance the improvement effect of polygonatum on visual fatigue.

[0063] (II) Vision Examination: Before and after the test, the subjects' vision will be examined using a standard logarithmic vision chart. The test will be considered effective if the subject's vision after the test improves by at least two lines compared to before the test, i.e., if the five-point record increases by at least 0.2 points.

[0064] The results are shown in Table 4 below.

[0065] Table 4 Statistics of subjects’ visual acuity improvement (unit: case)

[0066] Compared with before treatment, after 60 days of treatment, the number of cases with effective visual improvement in treatment group one was 56, and the number of cases with ineffectiveness was 4, with an effective rate of 93.33%; after 60 days of treatment, the number of cases with effective visual improvement in treatment group two was 42, and the number of cases with ineffectiveness was 18, with an effective rate of 70.0%; after 60 days of treatment, the number of cases with effective visual improvement in treatment group three was 39, and the number of cases with ineffectiveness was 21, with an effective rate of 65.0%; after 60 days of treatment, the number of cases with effective visual improvement in treatment group four was 34, and the number of cases with ineffectiveness was 26, with an effective rate of 56.670%; after 60 days of treatment, the number of cases with effective visual improvement in treatment group five was 34, and the number of cases with ineffectiveness was 26, with an effective rate of 56.57%; in the blank control group, the number of cases with effective visual improvement was 1, and the number of cases with ineffectiveness was 59, with an effective rate of 1.69%. By comparing the efficacy data of treatment group one to treatment group five, it can be found that the efficacy of Bifidobacterium longum ZKBb0311 Polygonatum sibiricum fermented liquid in improving visual fatigue is significantly higher than that of unfermented Polygonatum sibiricum oral liquid ( P <0.05 and other strains of Polygonatum sibiricum fermentation broth ( P <0.05), indicating that fermentation of Polygonatum sibiricum with Bifidobacterium longum ZKBb0311 can effectively enhance the improvement effect of Polygonatum sibiricum on visual fatigue.

[0067] (3) Safety observation: No adverse reactions occurred in the six groups of subjects during the treatment.

[0068] The core genome of Bifidobacterium longum has carbohydrate utilization genes and more proteolytic genes that are higher than the average level of Bifidobacterium, so Bifidobacterium longum has a strong carbon and nitrogen utilization ability. The present invention takes advantage of this characteristic and selects Bifidobacterium longum as a fermentation strain to ferment Polygonatum. Compared with different probiotic strains such as Lactobacillus plantarum and Lactobacillus acidophilus, the ability to ferment Polygonatum is stronger, and the resulting fermentation liquid is also better in alleviating visual fatigue and enhancing vision. Compared with the mature commercial strain Bifidobacterium longum BB536 of the same species, the Polygonatum fermentation liquid obtained using Bifidobacterium longum ZKBb0311 has a significantly improved effect on improving vision, indicating that Bifidobacterium longum ZKBb0311 has a stronger Polygonatum fermentation effect. In summary, the Bifidobacterium longum ZKBb0311 obtained by screening the present invention has achieved a Polygonatum fermentation effect that is better than that of existing strains.

[0069] Example 4 Typical Case Case 1: Mr. Li, a 20-year-old male university student, had long-term computer game play, which resulted in decreased vision, blurred vision, and eye fatigue. After taking Bifidobacterium longum ZKBb0311 and Polygonatum sibiricum fermentation broth (prepared as in Example 2) for one month, his vision improved and his visual fatigue lessened. After two months of continued use, his vision had recovered.

[0070] Case 2: Mr. Liu, a 31-year-old male programmer with 600-degree myopia, had to stare at a computer screen for extended periods at work. This caused him to tear, dry eyes, and soreness. He needed to rest his eyes after working for an hour or two each day, which affected his work over a long period of time. After two months of taking Bifidobacterium longum ZKBb0311 and Polygonatum sibiricum fermentation broth (prepared using the same method as in Example 2), his vision improved, and he could work all morning without tearing or dryness.

[0071] Case 3: Guan, a 50-year-old female accountant, suffered from vision loss, blurred vision, and eye pain from prolonged work due to her work-related computer use, which seriously affected her work and personal life. After two months of taking Bifidobacterium longum ZKBb0311 Polygonatum sibiricum fermentation broth (prepared using the same method as in Example 2), her vision improved, and the blurred vision and eye pain after long hours of work were alleviated.

[0072] Although the present invention has been described in detail with reference to the accompanying drawings and in conjunction with preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, persons of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any changes or substitutions that can be easily conceived by persons skilled in the art within the technical scope disclosed in the present invention shall be within the scope of protection of the present invention.

Claims

1. A strain of Bifidobacterium longum ZKBb0311, characterized in that: Bifidobacterium longum ( Bifidobacterium longum ) ZKBb0311 was deposited in the General Microbiology Center of China Culture Collection Administration on May 13, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 30549.

2. A Polygonatum sibiricum fermentation broth, characterized in that: Prepared by the following method: (1) Soak the polygonatum in water and then boil it to obtain the polygonatum extract; (2) Inoculate the bacterial suspension into the sterilized Polygonatum sibiricum extract, shake well, and then incubate anaerobically at 37°C to obtain the product; Wherein, the bacterial suspension is the bacterial suspension of Bifidobacterium longum ZKBb0311 as claimed in claim 1.

3. The Polygonatum sibiricum fermentation broth according to claim 2, wherein In step (1), the nine-steamed and nine-dried Polygonatum odoratum is sliced ​​and then added with 10 times the weight of water and soaked for 60 minutes.

4. The Polygonatum sibiricum fermentation broth according to claim 2, wherein In step (1), the decoction condition is to boil the food and then keep boiling for 120 minutes.

5. The Polygonatum sibiricum fermentation broth according to claim 2, wherein In step (1), after decoction, the filtrate is filtered and separated, and the decoction is concentrated to obtain a polygonatum extract with a crude drug content of 0.75 g / mL.

6. The Polygonatum sibiricum fermentation liquid according to claim 2, wherein In step (2), 1 wt% to 3 wt% of a bacterial suspension of Bifidobacterium longum ZKBb0311 is inoculated into the polygonatum extract, and the bacterial suspension is prepared by the following method: Bifidobacterium longum ZKBb0311 was inoculated into MRS liquid medium supplemented with 0.05 g / L L-cysteine ​​hydrochloride, cultured anaerobically at 37°C for 20 h, activated and propagated to the 2nd or 3rd generation, and a bacterial suspension was prepared.

7. Use of the polygonatum fermentation liquid according to any one of claims 2 to 6 in preparing a product for improving vision.

8. The use according to claim 7, characterized in that The product for improving vision is in the form of an oral solution.

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