Bifidobacterium longum zkb0311, polygonatum ferrmenting liquid and application thereof in preparation of product for improving eyesight

Abstract

The present application relates to the technical field of probiotic fermentation, and particularly relates to a Bifidobacterium longum ZKBb0311, a rhizoma polygonati fermentation liquor and application of the rhizoma polygonati fermentation liquor in preparation of a product for improving eyesight. Bifidobacterium longum The Bifidobacterium longum ZKBb0311 has been preserved in the China General Microbiological Culture Collection Center on May 13, 2024, and the address of the preservation center is No. 1, Xibaheyi, Chaoyang District, Beijing, and the preservation number is CGMCC No. 30549. The Bifidobacterium longum ZKBb0311 provided in the present application has good fermentation effect, and clinical tests prove that the rhizoma polygonati fermentation liquor obtained by fermenting rhizoma polygonati with the Bifidobacterium longum ZKBb0311 can significantly relieve visual fatigue, improve eyesight and improve the efficacy of rhizoma polygonati.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of probiotic fermentation, in particular to a strain of Bifidobacterium longum ZKBb0311, a rhizoma polygonati fermentation liquor and application of the strain and the fermentation liquor in preparation of a product for improving eyesight. BACKGROUND

[0002] With the advent of the information age, the frequency of using computers at work increases, and long-term mobile phone use after work. When the eyes are long-term focused on computer and mobile phone screens, the number of eye blinks is reduced, causing a corresponding reduction in tear secretion, and the strong stimulation of flickering screens on the eyes. Overuse of the eyes and lack of eye care can lead to decreased eyesight and even induce various eye diseases, such as myopia, amblyopia, and visual fatigue, causing great harm to people's physical and mental health. Recent studies have shown that traditional Chinese medicine has achieved good clinical efficacy in improving eyesight with fewer side effects.

[0003] According to traditional Chinese medicine, the occurrence of myopia is related to liver and kidney deficiency, blood deficiency, and the like. Traditional Chinese medicine treatment of myopia mainly regulates the internal environment of the body, enhances blood circulation and nutrient supply in the eyes, and thus improves eyesight. Rhizoma polygonati, sweet in taste and flat in nature, enters the spleen and lung channels, and has the effects of tonifying the spleen and lung, nourishing yin and generating fluid, and strengthening bones and muscles, and can be used to treat spleen-stomach deficiency, fatigue, weakness after illness, weakness of bones and muscles, rheumatic pain, and the like, and is known as the "King of Blood and Qi" in the traditional Chinese medicine field.

[0004] Fermentation is a processing method of traditional Chinese medicine, but traditional fermented traditional Chinese medicine is fermented under natural conditions using natural strains, and the fermentation conditions are uncontrollable. Modern fermentation uses known microorganisms to control fermentation conditions, changes the structure and content of active ingredients in traditional Chinese medicine through complex metabolic activities of microorganisms, and thus improves the efficacy and medicinal value of traditional Chinese medicine, and has a very wide application field.

[0005] CN 109349485 A provides a traditional Chinese medicine fermented beverage for eye protection and liver protection. The preparation method of the traditional Chinese medicine fermented beverage is as follows: black dates, black sesame seeds, Chinese yam, chrysanthemum, jujube, Chinese wolfberry, sea buckthorn, turmeric, cassia seed, and white peony root are crushed and then added with purified water, cellulase, and pectinase. The enzyme solution and residue are obtained by constant temperature enzymolysis. Then, the residue is extracted with purified water by ultrasonic extraction. The extract is filtered and combined with the enzyme solution. The medicinal material concentrate is obtained by vacuum concentration. The arbutus, blueberry, and cranberry are washed and then added with purified water for pulping. The filtrate is combined with honey and added to the medicinal material concentrate. The mixture is stirred uniformly to form a mixed solution. The mixed solution is sterilized by steam and then inoculated with probiotic seed solution. The fermented beverage is obtained by standing and fermentation. Finally, the fermented beverage is filled and sterilized by steam to obtain the traditional Chinese medicine fermented beverage for eye protection and liver protection. The traditional Chinese medicine fermented beverage has the following disadvantages: ① The effect of eye protection and liver protection depends on the specific formula combination. The raw material types are many, and enzyme preparations such as cellulase and pectinase need to be added. The supply chain cost is high, and the operation process is complex. ② The composition is complex. The mixture of multiple raw materials may cause interaction between components, affecting absorption or causing slight irritation. ③ The formula contains components such as cassia seed and white peony root with strong medicinal properties. Some people should be cautious when drinking. SUMMARY

[0006] In view of the technical problems of the prior art that the traditional Chinese medicine fermented beverage is prepared by enzymolysis, extraction, pulping, and fermentation of dozens of raw materials, the present application provides a Bifidobacterium longum ZKBb0311, a rhizoma polygonati fermentation liquor, and an application thereof in the preparation of a product for improving eyesight.

[0007] In the first aspect, the present application provides a Bifidobacterium longum (Bifidobacterium longum) ZKBb0311, which has been preserved in the China General Microbiological Culture Collection Center on May 13, 2024, at No. 1, Beichen West Road, Beijing City, China, with a preservation number of CGMCC No. 30549. Bifidobacterium longum

[0008] In the second aspect, the present application provides a rhizoma polygonati fermentation liquor capable of improving eyesight, which is prepared by the following method:

[0009] (1) Soaking rhizoma polygonati in water and then decocting to obtain a rhizoma polygonati extract;

[0010] (2) Inoculating Bifidobacterium longum ZKBb0311 bacterial suspension into the sterilized rhizoma polygonati extract, shaking uniformly, and then culturing at 37℃ in anaerobic conditions to obtain the rhizoma polygonati fermentation liquor.

[0011] Further, in step (1), the sliced rhizoma polygonati is soaked in 10 times the mass of water for 60 min.

[0012] Further, in step (1), the decoction condition is to maintain boiling for 120 min after boiling. ​

[0013] Further, in step (1), the filtrate is separated after decocting, and the decocting is concentrated to obtain the rhizoma polygonati extract with a crude drug content of 0.75 g / mL, wherein the crude drug content refers to the ratio of the amount of the rhizoma polygonati raw material used to the volume of the rhizoma polygonati extract obtained after concentration.

[0014] Further, in step (2), 1wt%-3wt% of the bacterial suspension of Bifidobacterium longum ZKBb0311 is inoculated into the rhizoma polygonati extract, and the bacterial suspension is prepared by the following method:

[0015] The Bifidobacterium longum ZKBb0311 is inoculated into the MRS liquid medium added with 0.05 g / L of L-cysteine hydrochloride, and is cultured anaerobically at 37 DEG C for 20 h, and is activated and passed to 2-3 generations to prepare the bacterial suspension.

[0016] In a third aspect, the application further provides a use of the rhizoma polygonati fermentation liquor in the preparation of a product for improving vision, and the product can be a health food or a medicine for relieving visual fatigue.

[0017] Further, the dosage form of the product for improving vision is an oral liquid.

[0018] The application has the following advantages:

[0019] The Bifidobacterium longum ZKBb0311 provided by the application has higher rhizoma polygonati fermentation activity than other experimental strains, and clinical tests prove that the rhizoma polygonati fermentation liquor obtained by fermentation of the Bifidobacterium longum ZKBb0311 can significantly relieve visual fatigue, improve vision, and improve the efficacy of rhizoma polygonati. Meanwhile, the application adopts a simplified production mode of single strain (Bifidobacterium longum ZKBb0311) fermentation of single medicinal component (rhizoma polygonati), and has significant advantages in process, cost, safety, and the like. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, for those skilled in the art, other drawings can also be obtained from these drawings without any creative effort.

[0021] Figure 1 is a colony morphology picture of Bifidobacterium longum ZKBb0311.

[0022] Figure 2 is a gram staining picture of Bifidobacterium longum ZKBb0311. DETAILED DESCRIPTION

[0023] In order to make the person skilled in the art better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by the person skilled in the art without creative labor should belong to the protection scope of the present application.

[0024] The reagents, materials and instruments involved in the following embodiments are all conventional commercially available products unless otherwise specified; the methods used are all conventional methods in the art unless otherwise specified.

[0025] Among them, mupirocin lithium salt and TOS medium are purchased from Qingdao Haibo Biotechnology Co., Ltd., L-cysteine hydrochloride is purchased from Sigma-Aldrich Company, USA, MRS liquid medium and MRS solid medium are purchased from Beijing Aoboxing Biotechnology Co., Ltd., DNA extraction kit is purchased from Beijing Tiangen Biotech Co., Ltd., and nine steamed and dried Huangjing is purchased from Henan Yanhuang Pharmacy.

[0026] Example 1 Isolation and identification of Bifidobacterium longum ZKBb0311

[0027] I. Sample source: collected from Changshou Village, Bama Yao Autonomous County, Hechi City, Guangxi Zhuang Autonomous Region in March 2015.

[0028] II. Strain isolation: the collected sample was diluted with physiological saline to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 times, 100 μL of the diluted solution was taken and uniformly coated on TOS medium containing 0.05 g / L mupirocin lithium salt, and then cultured in an anaerobic biochemical incubator at 37°C for 48 h, and the growth of the colonies was observed.

[0029] After the colonies were formed, single colonies were picked and three-zone streaked for three times, and repeated multiple times until the colonies were purified. The purified strain was inoculated into MRS liquid medium containing 0.05 g / L L-cysteine hydrochloride, and the culture solution and 50% sterile glycerol were mixed at a ratio of 1:1 (v / v) and stored in a-80°C refrigerator.

[0030] III. Strain identification:

[0031] (I) Colony morphology identification

[0032] ① The isolated strain was inoculated on TOS medium containing 0.05 g / L mupirocin lithium salt, and anaerobic culture was carried out for 48 h. The colony morphology and color were observed and recorded. As shown in Table 1, the colony was milky white or off-white, opaque, shiny, smooth surface, convex, soft texture, and regular edge. Figure 1

[0033] ② The colony on the medium in ① was picked for smear, fixation, initial staining with crystal violet, mordanting, decolorizing, washing with water, re-staining with safranine, drying, and then the dried colony was observed under an oil microscope at a magnification of 1000 times. The microscopic observation results of the strain are shown in Table 2. Figure 2

[0034] (II) 16S rDNA sequence determination

[0035] ① Extraction of strain genomic DNA: The culture solution of the isolated strain was centrifuged at 5000 r / min for 10 min, and the supernatant was discarded after centrifugation. The collected pure culture bacteria were used to extract the strain genomic DNA using a DNA extraction kit, and the operation was performed according to the instruction manual.

[0036] ② PCR amplification: Bacterial universal primers 27F / 1492R were used.

[0037] The PCR amplification conditions were as follows: pre-denaturation at 94℃ for 4 min; denaturation at 94℃ for 30 s; annealing at 55℃ for 35 s; extension at 72℃ for 90 s; 32 cycles, and finally extension at 72℃ for 5 min.

[0038] ③ Gene sequencing: The PCR product was subjected to 1.0% agarose gel electrophoresis, and the amplified product was subjected to gene sequencing. The sequencing work was completed by Beijing Qikang Biotechnology Co., Ltd.

[0039] The 16S rDNA sequence of the isolated strain is as follows:

[0040] ​​

[0041] After BLAST alignment, the strain was judged to be Bifidobacterium longum, and the strain was named Bifidobacterium longum ZKBb0311.

[0042] IV. Strain preservation:

[0043] Bifidobacterium longum ZKBb0311 was preserved in the China General Microbiological Culture Collection Center on May 13, 2024, the address of the preservation center is No. 3, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC No. 30549, and the classification name is Bifidobacterium longum Bifidobacterium longum .

[0044] Example 2 Preparation of Huangjing fermentation broth of Bifidobacterium longum ZKBb0311

[0045] The Huangjing fermentation broth of Bifidobacterium longum ZKBb0311 was prepared according to the following method:

[0046] (1) After slicing the nine-steamed and nine-sun Huangjing, 10 times the mass of water was added for soaking for 60 min. The soaked Huangjing and the soaking water were put into a traditional Chinese medicine cooking pot together. First, boil with high heat, then slow-cook with low heat for 120 min. The soaking water was directly used for decoction without changing water to avoid wasting effective components. During the decoction process, moderate stirring was allowed to prevent the pot from sticking, but frequent uncovering was strictly prohibited to prevent the loss of volatile components. After the decoction was completed, the filtrate was separated by filtration, and the decoction was concentrated to a crude drug content (Huangjing usage amount / volume of concentrated Huangjing extract) of 0.75 g / mL, and Huangjing extract was obtained.

[0047] (2) The Huangjing extract obtained in step (1) was sterilized at 121°C for 15 min and reserved. Bifidobacterium longum ZKBb0311 was inoculated into MRS liquid medium added with 0.05 g / L L-cysteine hydrochloride, and cultured anaerobically at 37°C for 24 h. The activated strain was passed to the 2nd generation, and a bacterial suspension (viable bacterial count of 1×10 9 CFU / mL) was prepared. The bacterial suspension was inoculated into the sterilized Huangjing extract, and the inoculation amount was 2wt%. After shaking, it was placed in a 37°C anaerobic incubator for 20 h to prepare Huangjing fermentation broth, which was named Huangjing fermentation broth of Bifidobacterium longum ZKBb0311.

[0048] Comparative example

[0049] Other probiotics were used instead of Bifidobacterium longum ZKBb0311 to ferment Huangjing to prepare Huangjing fermentation broth. Specifically as follows:

[0050] Bifidobacterium longum BB536 was inoculated into MRS liquid medium added with 0.05 g / L L-cysteine hydrochloride, and cultured anaerobically at 37°C for 20 h. The activation was passed to the 2nd generation, and a bacterial suspension (viable bacterial count: 1 x 10 9 CFU / mL) was prepared. Bifidobacterium longum BB536 is a commercial probiotic strain that is widely studied at present, and was purchased from Xima International Trade (Shanghai) Co., Ltd. The bacterial suspension of Bifidobacterium longum BB536 was inoculated into the sterilized extract of polygonatum sibiricum at an inoculation amount of 2wt%, and the extract of polygonatum sibiricum was prepared and sterilized according to the above step (1). After shaking, the mixture was placed in an anaerobic incubator at 37°C for 20 h to prepare a polygonatum sibiricum fermentation broth, which was named as Bifidobacterium longum BB536 polygonatum sibiricum fermentation broth.

[0051] Lactobacillus plantarum RZKLp100 was inoculated into MRS liquid medium, and cultured anaerobically at 37°C for 20 h. The activation was passed to the 2nd generation, and a bacterial suspension (viable bacterial count: 1 x 10 9 CFU / mL) was prepared. Lactobacillus plantarum RZKLp100 is a probiotic strain collected and preserved by the applicant, and has been disclosed in Chinese invention patent CN 118374549 B. It was found in previous studies that the Lactobacillus plantarum RZKLp100 has a good effect on fermented licorice. The bacterial suspension of Lactobacillus plantarum RZKLp100 was inoculated into the sterilized extract of polygonatum sibiricum at an inoculation amount of 2wt%, and the extract of polygonatum sibiricum was prepared and sterilized according to the above step (1). After shaking, the mixture was placed in an anaerobic incubator at 37°C for 20 h to prepare a polygonatum sibiricum fermentation broth, which was named as Lactobacillus plantarum RZKLp100 polygonatum sibiricum fermentation broth.

[0052] Lactobacillus acidophilus RZKLa0701 was inoculated into MRS liquid medium, and cultured anaerobically at 37°C for 20 h. The activation was passed to the 2nd generation, and a bacterial suspension (viable bacterial count: 1 x 10 9 CFU / mL) was prepared. Lactobacillus acidophilus RZKLa0701 is a probiotic strain collected and preserved by the applicant, and has been disclosed in Chinese invention patent CN 117305188 B. It was found in previous studies that the Lactobacillus acidophilus RZKLa0701 has a function of promoting bone growth in children. The bacterial suspension of Lactobacillus acidophilus RZKLa0701 was inoculated into the sterilized extract of polygonatum sibiricum at an inoculation amount of 2wt%, and the extract of polygonatum sibiricum was prepared and sterilized according to the above step (1). After shaking, the mixture was placed in an anaerobic incubator at 37°C for 20 h to prepare a polygonatum sibiricum fermentation broth, which was named as Lactobacillus acidophilus RZKLa0701 polygonatum sibiricum fermentation broth.

[0053] The pH value and viable cell count of the Huangqin fermentation broth of Bifidobacterium longum ZKBb0311 prepared in Example 2, the Huangqin fermentation broth of Bifidobacterium longum BB536 prepared in the comparative example, the Huangqin fermentation broth of Lactobacillus plantarum RZKLp100 and the Huangqin fermentation broth of Lactobacillus acidophilus RZKLa0701 were determined respectively, and the results are shown in Table 1 below.

[0054] Table 1 pH value and viable cell count of Huangqin fermentation broth

[0055]

[0056] It can be seen from the comparison that the viable cell count of the Huangqin fermentation broth of Bifidobacterium longum ZKBb0311 is higher, and the effect is better than that of Bifidobacterium longum BB536 and other probiotics.

[0057] If it is necessary to further improve the viable cell count of the Huangqin fermentation broth of Bifidobacterium longum ZKBb0311, the amount of Bifidobacterium longum ZKBb0311 added to the Huangqin extract can be increased, for example, the Bifidobacterium longum ZKBb0311 suspension is added at an inoculation amount of 3wt% or 4wt%.

[0058] Example 3 Clinical trial

[0059] I. Case selection criteria:

[0060] (I) Inclusion criteria:

[0061] Age 18-65 years old, long-term eye use, and voluntary subjects with visual fatigue.

[0062] (II) Exclusion criteria:

[0063] ① Patients with infectious, traumatic eye diseases, and patients who have undergone eye surgery for less than 3 months;

[0064] ② Patients with corneal, lens, vitreous, and fundus lesions, and patients with cardiovascular, cerebrovascular, liver, kidney, and hematopoietic system diseases;

[0065] ③ Patients who have long-term taken drugs, health products or other treatment methods for treating visual fatigue without success;

[0066] ④ Pregnant or lactating women;

[0067] ⑤ Subjects who do not meet the inclusion criteria, do not eat the test substance as required, or have incomplete data that affect efficacy or safety judgment.

[0068] (III) Case exclusion and dropout criteria

[0069] ① Subjects who voluntarily withdraw during the trial;

[0070] ② Poor compliance of subjects, unable to complete the entire treatment;

[0071] ③ Severe adverse reactions during treatment;

[0072] ④ Patients with disease progression, death during the study period.

[0073] II. Test plan:

[0074] (I) Test grouping: Randomized, double-blind study. All 360 subjects were randomly assigned to treatment group 1, treatment group 2, treatment group 3, treatment group 4, treatment group 5 and blank control group, 60 cases in each group.

[0075] (II) Treatment method:

[0076] ① Each person in treatment group 1 takes 50 mL of long Bifidobacterium ZKBb0311 Huangjing fermentation broth in the morning, noon and evening every day, and the treatment course lasts for 60 days. The preparation method of long Bifidobacterium ZKBb0311 Huangjing fermentation broth is the same as that of Example 2.

[0077] ② Each person in treatment group 2 takes 50 mL of long Bifidobacterium BB536 Huangjing fermentation broth in the morning, noon and evening every day, and the treatment course lasts for 60 days. The preparation method of long Bifidobacterium BB536 Huangjing fermentation broth is the same as that of the comparative example.

[0078] ③ Each person in treatment group 3 takes 50 mL of plant lactobacillus RZKLp100 Huangjing fermentation broth in the morning, noon and evening every day, and the treatment course lasts for 60 days.

[0079] The preparation method of plant lactobacillus RZKLp100 Huangjing fermentation broth is the same as that of the comparative example.

[0080] ④ Each person in treatment group 4 takes 50 mL of acidophilic lactobacillus RZKLa0701 Huangjing fermentation broth in the morning, noon and evening every day, and the treatment course lasts for 60 days.

[0081] The preparation method of acidophilic lactobacillus RZKLa0701 Huangjing fermentation broth is the same as that of the comparative example.

[0082] ⑤ Each person in treatment group 5 takes 50 mL of unfermented Huangjing oral liquid in the morning, noon and evening every day, and the treatment course lasts for 60 days.

[0083] The preparation method of Huangjing oral liquid is:

[0084] After cutting the nine steamed and nine dried Rhizoma Polygonati, 10 times of water by weight was added to soak for 60 min. The soaked Rhizoma Polygonati and the soaking water were put into a traditional Chinese medicine cooking pot. Boiling was performed at high heat, and then slow cooking was performed at low heat for 120 min. The soaking water was directly used for decocting without changing water to avoid waste of effective components. During the decocting process, proper stirring was performed to prevent sticking to the pot, but frequent uncovering was avoided to prevent loss of volatile components. After the decocting process, the filtrate was separated by filtration, and was concentrated to a crude drug content (Rhizoma Polygonati usage amount / extracted Rhizoma Polygonati liquid volume) of 0.75 g / mL. The Rhizoma Polygonati extract was sterilized at 121℃ for 15 min to obtain the Rhizoma Polygonati oral liquid.

[0085] ⑥The blank control group took 50 mL of drinking water each time per day, and the treatment course lasted for 60 days.

[0086] III. Observation index and results:

[0087] (1) Questionnaire survey: the types of visual fatigue symptoms and the improvement of symptoms of the subjects were inquired before and after the test. The visual fatigue symptoms included eight symptoms of eye pain, eye swelling, photophobia, blurred vision, dry eyes, foreign body sensation, tearing, and general malaise. When at least three symptoms were improved and other symptoms were not worsened, it was determined to be effective.

[0088] The judgment standard of symptom improvement was based on the change of symptom score of the subjects before and after the test. The decrease of the score of a single type of symptom represented the improvement of the symptom. The symptom score standard was shown in Table 2.

[0089] Table 2 Improvement score table of subjects

[0090]

[0091] Note: "occasionally" and "occasionally" refer to 1-2 times / 2 days; "sometimes" refers to 1-3 times / day; "often" refers to >3 times / day.

[0092] The results are shown in Table 3.

[0093] Table 3 Improvement effect of visual fatigue before and after treatment (unit: cases)

[0094]

[0095] Compared with before treatment, the effective cases of visual fatigue improvement in the treatment group 1 after 60 days of treatment were 54 cases, the ineffective cases were 6 cases, and the effective rate was 90.0%; the effective cases of visual fatigue improvement in the treatment group 2 after 60 days of treatment were 39 cases, the ineffective cases were 21 cases, and the effective rate was 65.0%; the effective cases of visual fatigue improvement in the treatment group 3 after 60 days of treatment were 36 cases, the ineffective cases were 24 cases, and the effective rate was 60.0%; the effective cases of visual fatigue improvement in the treatment group 4 after 60 days of treatment were 35 cases, the ineffective cases were 25 cases, and the effective rate was 58.33%; the effective cases of visual fatigue improvement in the treatment group 5 were 32 cases, the ineffective cases were 28 cases, and the effective rate was 53.3%; the effective cases of visual fatigue improvement in the blank control group were 2 cases, the ineffective cases were 58 cases, and the effective rate was 3.33%. By comparing the effective rate data of the treatment group 1 to the treatment group 5, it can be found that the effective rate of Bifidobacterium longum ZKBb0311 Huangjing fermentation broth in improving visual fatigue is significantly higher than that of Huangjing oral liquid without fermentation ( P <0.05) and Huangjing fermentation broth of other strains P <0.05), indicating that Bifidobacterium longum ZKBb0311 fermented Huangjing can effectively enhance the improvement effect of Huangjing on visual fatigue.

[0096] (2) Visual acuity test: Before and after the test, the subjects were tested for visual acuity using a standard logarithmic visual acuity chart. If the subject's visual acuity after the test increased by two lines or more, i.e. the five-point score increased by 0.2 points or more, it was considered effective.

[0097] The results are shown in Table 4 below.

[0098] Table 4 Statistics of visual acuity improvement of subjects (unit: cases)

[0099]

[0100] Compared with before treatment, the effective cases of visual improvement after 60 days of treatment in the treatment group one were 56 cases, the ineffective cases were 4 cases, and the effective rate was 93.33%; the effective cases of visual improvement after 60 days of treatment in the treatment group two were 42 cases, the ineffective cases were 18 cases, and the effective rate was 70.0%; the effective cases of visual improvement after 60 days of treatment in the treatment group three were 39 cases, the ineffective cases were 21 cases, and the effective rate was 65.0%; the effective cases of visual improvement after 60 days of treatment in the treatment group four were 34 cases, the ineffective cases were 26 cases, and the effective rate was 56.670%; the effective cases of visual fatigue improvement in the treatment group five were 34 cases, the ineffective cases were 26 cases, and the effective rate was 56.57%; the effective cases of visual improvement in the blank control group were 1 case, the ineffective cases were 59 cases, and the effective rate was 1.69%. By comparing the effective rate data of the treatment group one to the treatment group five, it can be found that the effective rate of the long bifidobacterium ZKBb0311 Huangjing fermentation liquid in improving visual fatigue is significantly higher than that of the Huangjing oral liquid without fermentation P <0.05) and Huangjing fermentation liquids of other strains P <0.05), indicating that Huangjing fermented by long bifidobacterium ZKBb0311 can effectively enhance the improvement effect of Huangjing on visual fatigue.

[0101] (Three) Safety observation:

[0102] No adverse reactions occurred in the six groups of subjects during the treatment.

[0103] The core genome of long bifidobacterium has more carbohydrate utilization genes and more proteolytic genes than the average level of bifidobacterium, so long bifidobacterium has strong carbon and nitrogen utilization ability. The present application uses this characteristic and selects long bifidobacterium as a fermentation strain to ferment Huangjing. Compared with different species of probiotic strains such as Lactobacillus plantarum and Lactobacillus acidophilus, the fermentation ability of long bifidobacterium is stronger, and the effect of the obtained fermentation liquid on relieving visual fatigue and enhancing visual acuity is also better. Compared with the same mature commercial strain long bifidobacterium BB536, the Huangjing fermentation liquid obtained by using long bifidobacterium ZKBb0311 has a significantly improved effect on improving visual acuity, indicating that long bifidobacterium ZKBb0311 has a stronger Huangjing fermentation effect. In summary, the long bifidobacterium ZKBb0311 screened in the present application has a better Huangjing fermentation effect than existing strains.

[0104] Example 4 Typical case

[0105] Case 1: Li, a male college student, 20 years old, long-term computer game playing, leading to decreased visual acuity, blurred vision, and easy eye fatigue. After taking long bifidobacterium ZKBb0311 Huangjing fermentation liquid (preparation method same as example 2) for one month, the visual acuity improved, and the visual fatigue decreased. After continuing to take for two months, the visual acuity recovered.

[0106] Case 2: Liu, male, programmer, 31 years old, myopia 600 degrees, work to long-term stare at the computer screen, and use computer for a long time easy to tear, dry eyes, sore, every day work one or two hours need to rest the eyes, long-term impact on work. Take long bifidobacterium ZKBb0311 rhizoma polygonati fermentation broth (preparation method same as example 2) two months later, vision has improved, work a morning will not appear eyes tear and dry eyes.

[0107] Case 3: Guan, female, accountant, 50 years old, due to the nature of work long-term use computer, lead to eyesight decline, blurred vision, long-term work eyes swelling pain, serious influence work and life. Take long bifidobacterium ZKBb0311 rhizoma polygonati fermentation broth (preparation method same as example 2) two months later, vision has recovered, long work blurred vision weakened, eyes swelling pain weakened.

[0108] Although the present application has been described in detail with reference to the preferred embodiments, it should be understood that the application is not limited to those preferred embodiments. Without departing from the spirit and essential characteristics of the application, one of ordinary skill can make other variations and modifications of the application under the framework of variations in form and details for the purpose of practicing the application disclosed in the technical range of the present application. Any skilled person in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A fermentation liquid of Polygonatum sibiricum, characterized in that, Prepared using the following method: (1) Soak Polygonatum in water, decoct and filter to separate the filtrate, and decoct and concentrate to obtain Polygonatum extract with a crude drug content of 0.75 g / mL; (2) Inoculate 1wt%~3wt% of Bifidobacterium longum into the extract of Polygonatum sibiricum. Bifidobacterium longum The bacterial suspension of ZKBb0311, after being shaken well, is cultured anaerobically at 37°C to obtain the product. The bacterial suspension was prepared by the following method: Bifidobacterium longum ZKBb0311 was inoculated into MRS liquid medium supplemented with 0.05 g / L L-cysteine ​​hydrochloride, anaerobic cultured at 37℃ for 20 h, activated and passaged to 2-3 generations to prepare bacterial suspension; Bifidobacterium longum ZKBb0311 was deposited on May 13, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30549.

2. The Polygonatum sibiricum fermentation broth as described in claim 1, characterized in that, In step (1), after the Polygonatum sibiricum slices are steamed and dried nine times, they are soaked in 10 times their weight of water for 60 minutes.

3. The Polygonatum sibiricum fermentation broth as described in claim 1, characterized in that, In step (1), the simmering conditions are to boil and then keep boiling for 120 minutes.

4. The use of the Polygonatum odoratum fermentation liquid as described in any one of claims 1 to 3 in the preparation of products that improve eyesight.

5. The application as described in claim 4, characterized in that, The product for improving eyesight is in the form of an oral liquid.

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