CN117643379A - Application of bifidobacterium animalis subspecies i797 in preparing eye-protection products - Google Patents
Application of bifidobacterium animalis subspecies i797 in preparing eye-protection products Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an application of bifidobacterium animalis subspecies i797 in preparing eye-protection products, and belongs to the field of microbial engineering. The strain can be used for preparing eye-protecting products. The bifidobacterium animalis subspecies i797 provided by the invention has the effect of relieving visual fatigue, and in-vitro simulation experiments prove that the bifidobacterium animalis subspecies i797 has good intestinal epithelial cell adhesion effect and can play a role in long-term field planting; the zebra fish model experiment proves that the zebra fish can obviously prevent the apoptosis of eye cells and can be used for preparing milk powder, yoghourt and various probiotics with the effect of relieving visual fatigue.
Description
Technical Field
The invention belongs to the field of microbial engineering, and relates to a novel application of bifidobacterium animalis subspecies lactis, in particular to an application of bifidobacterium animalis subspecies i797 in preparing eye-protection products.
Background
Visual fatigue, or eye fatigue, is a common disease of eyes, which causes dry eyes, unsmooth eyes, acid swelling of eyes, blurred vision and even vision decline to directly influence the work and life of people. The eye fatigue is mainly caused by that when people pay attention to watch screens of electronic products such as televisions, computers or mobile phones, eye blinking times are reduced, tear secretion is correspondingly reduced, and meanwhile, eyes are strongly stimulated by blinking screens. Eye fatigue can also cause and exacerbate various eye diseases.
The modern people's work life leisure mode brings the excessive eye problem, and asthenopia often puzzles students, office workers. The best way to relieve eye strain is to rest the eyes. But intense learning and work often does not allow the eyes to get enough rest. A series of products for protecting eyes and relieving visual fatigue are generated.
However, most products for relieving visual fatigue are eye drops and eye patches, and only short-time relief is brought to visual fatigue, and probiotics can be planted in intestinal tracts, so that the products have a continuous effect. However, probiotics have strain specificity, and not all probiotic strains have the effects of relieving asthenopia and preventing apoptosis of eye cells.
The animal bifidobacterium lactis is a kind of special anaerobic gram-positive bacteria which does not generate spores, and belongs to actinomycota, bifidobacterium and lactis on the basis of system taxonomies. The representative strain BB-12 is the probiotic strain with the most wide application and most published related papers at present, and has the effects of improving immunity, regulating intestinal and oral health and relieving upper respiratory tract infection in vitro and crowd experiments. However, no research on application of bifidobacterium animalis subspecies to relieving visual fatigue exists at present.
Disclosure of Invention
The invention aims to provide application of bifidobacterium animalis subspecies i797 in preparing eye protection products, which can relieve visual fatigue and widen the application range of bifidobacterium animalis subspecies.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
use of bifidobacterium animalis subspecies i797 in the manufacture of an eye care product.
As one limitation, the eye-care product is a food or a medicine having an effect of relieving asthenopia.
As another limitation, the eye care product includes milk powder, yogurt and probiotics having asthenopia relieving effects.
As a third limitation, the viable count of the bifidobacterium animalis subspecies i797 contained in the eye-protection product is 2 multiplied by 10 6 -5×10 10 CFU/g or CFU/mL.
In the invention, the following components are added:
the bifidobacterium animalis subspecies i797 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 18403 and the Latin name of Bifidobacterium animalis subsp.
By adopting the technical scheme, compared with the prior art, the invention has the following technical progress:
the application of the bifidobacterium animalis subspecies i797 in eye protection products provided by the invention has the effect of relieving visual fatigue, and in-vitro simulation experiments prove that the bifidobacterium animalis subspecies i797 has good intestinal epithelial cell adhesion effect and can play a role in long-term field planting; the zebra fish model experiment proves that the zebra fish can obviously prevent the apoptosis of eye cells and can be used for preparing milk powder, yoghourt and various probiotics with the effect of relieving visual fatigue.
Detailed Description
The invention is further illustrated by the following examples. It should be understood that the described embodiments are only for explaining the present invention and do not limit the present invention.
The experimental methods used in the examples described below are conventional in the art unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
In the following examples:
the bifidobacterium animalis subspecies i797 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 18403 and the Latin name of Bifidobacterium animalis subsp.lactis in 8-20 days of 2019;
the bifidobacterium longum subspecies i772 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 19856 and the Latin name of Bifidobacterium longum subsp.longum in the year 2020, 5 and 20;
bifidobacterium breve i1088, which has been deposited in China general microbiological culture Collection center (CGMCC) with accession number of CGMCC No.27281 and Latin name of Bifidobacterium breve in 2023, 5 and 8;
bifidobacterium bifidum i771, which has been preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 19855 and Latin name of Bifidobacterium bifidum in the year 5 and 20 of 2020;
commercial strain 1, provided by the junlebao microbiological deposit center (JMCC), isolated from commercial yogurt, identified as bifidobacterium animalis;
commercial strain 2, provided by the junlebao collection of microorganisms (JMCC), isolated from commercial ready-to-eat probiotic powder, was identified as lactobacillus rhamnosus.
In the following examples:
modified solid MRS medium: 10.0g/L of peptone, 10.0g/L of beef powder, 5.0g/L of yeast powder, 2.0g/L of diammonium hydrogen citrate, 20.0g/L of glucose, 1mL/L of tween 80, 5.0g/L of sodium acetate, 2.0g/L of dipotassium hydrogen phosphate, 0.5g/L of magnesium sulfate, 0.25g/L, L-cysteine 0.5g/L of manganese sulfate, 15.0g/L of agar and 1L of distilled water, and the pH value is 6.2-6.5;
improved liquid MRS medium: 10.0g/L of peptone, 10.0g/L of beef powder, 5.0g/L of yeast powder, 2.0g/L of diammonium hydrogen citrate, 20.0g/L of glucose, 80 1mL/L of tween, 5.0g/L of sodium acetate, 2.0g/L of dipotassium hydrogen phosphate, 0.5g/L of magnesium sulfate, 0.25g/L, L-cysteine 0.5g/L of manganese sulfate and distilled water 1L, pH have a value of 6.2 to 6.5;
artificial gastric juice: naCl 0.2g/100mL and pepsin 0.35g/100mL, adjusting the pH value to 3.0 by using HCl with the concentration of 1mol/L, and filtering and sterilizing for later use;
artificial intestinal juice: mixing pancreatic liquid (sodium bicarbonate 1.1g/100mL, naCl 0.2g/100mL and trypsin 0.1g/100mL, adjusting pH to 8.0, filtering and sterilizing for later use) and bile liquid (bile salt 0.9g/100mL, adjusting pH to 8.0, filtering and sterilizing for later use) at a volume ratio of 2:1;
high density medium: 10g/L of soybean peptone, 10g/L of beef extract, 5g/L of yeast extract powder, 2g/L of diammonium hydrogen citrate, 5g/L of anhydrous sodium acetate, 2g/L of dipotassium hydrogen phosphate, 1g/L of tween 80, 0.2g/L of magnesium sulfate, 0.054g/L of manganese sulfate, 5g/L of calcium carbonate, 30g/L of anhydrous glucose and 5g/L of L-cysteine hydrochloride, and the pH value is 7.2;
lyoprotectant: 12% of skim milk powder, 1% of trehalose, 3% of glycerol, 1% of sodium glutamate and 83% of water in percentage by mass, and the pH value is 5.6;
mycophenolate mofetil (lot number: H14J 6335), available from Shanghai Ala Biochemical technologies Co., ltd;
acridine orange (lot number: C12894919), available from Shanghai Micin Biochemical technologies Co., ltd.
Example use of bifidobacterium animalis subspecies lactis i797 in eye care products
The embodiment discloses application of bifidobacterium animalis subspecies i797 in preparing probiotics and relieving asthenopia, and the specific verification experiment is as follows:
1. gastrointestinal fluid tolerance
Inoculating bifidobacterium animalis subspecies i797 strain, commercial strain 1 and commercial strain 2 to an improved liquid MRS culture medium, respectively activating for 3 generations, respectively inoculating 1mL to 9mL of artificial gastric juice with pH value of 3.0 subjected to filtration sterilization treatment, respectively inoculating to the improved solid MRS culture medium after shaking uniformly and culturing at 37 ℃ for 0h and 2h, respectively sampling, inoculating to the improved solid MRS culture medium, measuring the viable count by a plate pouring method (1 mupirocin lithium salt is added to each 100mL of the improved liquid MRS culture medium before plate pouring), respectively inoculating to 1mL of culture solution after culturing for 2h in 9mL of artificial gastric juice with pH value of 8.0 subjected to filtration sterilization treatment, respectively inoculating to the improved solid MRS culture medium after culturing for 0h and 4h, measuring the viable count by a plate pouring method, calculating the survival rate of the strain, and calculating the following formula:
gastric juice survival (%) = (cfu N1/cfu N0) ×100%
Wherein N1 is the number of viable bacteria cultured for 2h by gastric juice, and N0 is the number of viable bacteria cultured for 0h by gastric juice
Intestinal juice survival rate (%) = (cfu N2/cfu N1) ×100%
Wherein N2 is the number of viable bacteria cultured for 4h through intestinal juice (namely the number of viable bacteria cultured for 2h through gastric juice and then 4h through intestinal juice), and N1 is the number of viable bacteria cultured for 0h through intestinal juice (namely the number of viable bacteria cultured for 2h through gastric juice)
Digests survival (%) = (cfu N2/cfu N0) ×100%
Wherein the digestive juice is gastric juice and intestinal juice, N2 is viable count of gastric juice cultured for 2 hours and then intestinal juice cultured for 4 hours, and N0 is viable count of gastric juice cultured for 0 hour
The strain survival results are shown in table 1:
TABLE 1 results of test for gastrointestinal fluid tolerance by strains
| Strain | Gastric juice survival rate | Survival rate of intestinal juice | Digestive juice survival rate |
| Bifidobacterium animalis subspecies i797 | 6.4％ | 116％ | 7.4％ |
| Commercial Strain 1 | 6.8％ | 66.7％ | 4.5％ |
| Commercial Strain 2 | 96％ | 0.13％ | 0.13％ |
The method has good gastric acid and intestinal juice tolerance capability, can ensure that probiotics can reach intestinal tracts to play a role, can resist acid stress and alkali stress, is a key index of whether the probiotics can reach human intestinal tracts in a live bacterial mode and play a role, and can ensure that the survival rate of bifidobacterium animalis subspecies i797 in gastric juice is 6.4 percent, the viable count in the intestinal juice is not reduced but slightly increased, the survival rate reaches 7.4 percent after 2h gastric juice and 4h intestinal juice treatment, and compared with two commercial strains, the method has better digestive juice survival rate, and can ensure that the sufficient viable count reaches the intestinal tracts to play the probiotic role.
2. Intestinal epithelial cell adhesion ability
Inoculating Bifidobacterium animalis lactosub-species i797 strain, commercial strain 1 and commercial strain 2 into modified liquid MRS culture medium, activating for 3 generations, respectively, taking 1mL, centrifuging at 4000rmp for 5min, and collecting bacterial suspension with concentration of 10 9 About CFU/ml, washing with PBS buffer solution for 2 times, and then re-suspending with 1ml DMEM in a 5ml centrifuge tube;
digesting the cultured Caco-2 cells with pancreatin, and adjusting the concentration of the cell suspension to 5×10 with DMEM complete medium 5 The cells with the adjusted concentration are spread in a 24-well plate and placed in 37 ℃ (5% CO) 2 ) In the incubator of (2), until the cells are plated, the intestinal epithelial tissue is simulated;
after sucking out the cell culture solution in the 24-hole plate, flushing the cell culture solution once by using PBS buffer solution, adding 1ml of bacterial suspension into each hole, repeating the concentration of each bacterial suspension for 2 times, counting the total number of bacterial colonies of the initial bacterial suspension, and placing the cell culture solution into a carbon dioxide incubator at 37 ℃ for incubation for 3 hours;
after incubation was completed, the cells were washed 4 times with PBS buffer, lysed with 1ml of sterilized 1% (v/v) Triton X100, and the number of strains adhering to the cells was measured by performing gradient dilution (dilution method: 300. Mu.L to 2.7ml saline), and incubated at 37℃for 3 days to count the total number of colonies, and the results are shown in Table 2:
TABLE 2 results of intestinal epithelial cell adhesion test of strains
As can be seen from table 2, the bifidobacterium animalis subspecies i797 has good intestinal epithelial cell adhesion capability, and the good gastrointestinal transit rate and the good intestinal epithelial cell adhesion capability ensure that the bifidobacterium animalis subspecies i797 can exist in the intestinal tract of a subject for a long time, so as to play a probiotic role.
3. Relieving asthenopia
1. Experimental materials
Probiotic mother liquor:
inoculating activated Lactobacillus bifidus subspecies i797 strain into a high-density culture medium with an inoculum size of 2%, fermenting and culturing for 18h, stopping fermentation, centrifuging to obtain bacterial mud, mixing bacterial mud and a freeze-drying protective agent at a weight ratio of 1:2 for 30min, immediately cooling to-35 ℃ to-40 ℃ at a cooling rate of 4 ℃/min, maintaining for 24min, freeze-drying, pulverizing the freeze-dried bacterial cells in a pulverizer, sieving the pulverized product with a 40-mesh sieve, and storing in an environment at-80 ℃ to obtain the bacterial count of 2×10 6 -5×10 10 A bifidobacterium animalis subspecies i797 probiotic in the CFU/g range;
the probiotic preparation is prepared by the same method for Bifidobacterium longum subspecies i772, bifidobacterium breve i1088 and Bifidobacterium bifidum i 771;
the preparation method comprises the steps of selecting a long subspecies i772, a short bifidobacterium i1088, a bifidobacterium bifidum i771 and an animal bifidobacterium lactis subspecies i797 probiotics with viable bacteria as shown in table 3, and preparing 2.00mg/mL probiotics mother liquor by using standard dilution water respectively for preparation at present;
experimental animals:
the zebra fish is cultured in water at 28deg.C (water quality: 200mg instant sea salt per 1L reverse osmosis water, conductivity 450-550 μS/cm, pH 6.5-8.5, and hardness 50-100 mg/LCaCO) 3 ) The experimental animal use license number is provided by the fish culture propagation center of the company: SYXK (Zhe) 2022-0004, feeding management meets the requirements of International AAALAC authentication (authentication number: 001458), IACUC ethical examination number: IACUC-2023-6993-01;
wild type AB strain zebra fish is bred in a natural pairing mating breeding mode.
2. Experimental method
Randomly selecting 120 wild AB strain zebra fish with the age of 1dpf, placing the wild AB strain zebra fish in a 6-hole plate, treating 30 zebra fish in each hole, setting the experiment group, and respectively water-dissolving and giving 3mL of probiotic liquid (respectively diluting the probiotic mother liquid according to different dilution factors to obtain the probiotic liquid, so that the viable bacteria amount of the probiotic liquid in the probiotic liquid is the same, and the viable bacteria amount of the probiotic liquid is shown in Table 3); meanwhile, 30 wild AB strain zebra fish with the age of 1dpf are randomly selected and placed in a 6-hole plate to be set as a control group, and 3mL of water is added. After each group is treated for 24 hours at 28 ℃, acridine Orange (AO) is used for dyeing, 10 zebra fishes selected randomly after the dyeing is finished are placed under a fluorescence microscope for photographing, NIS-Elements D3.20 advanced image processing software is used for analyzing and collecting data, the fluorescence intensity of eye apoptosis cells is analyzed, the effect of bacterial liquid for relieving asthenopia is evaluated according to the statistical analysis result of the index, the statistical analysis is carried out according to SPSS26.0 software, p <0.05 shows that the difference has statistical significance, meanwhile, the dose of the zebra fishes used for evaluating the effect is converted into the human dose according to the method disclosed in the invention patent with publication number of CN 113496071A, and the experimental result is shown in table 3:
TABLE 3 bacterial liquid dosage and visual fatigue relieving efficacy experiment results for zebra fish
Note that: * Represents p <0.01 compared to model control group
Research shows that the main physiological mechanisms of asthenopia include oxidative stress injury, inflammatory injury, cell aging and excessive visual cell loss, when the eyeball is in a state of long-time work, high concentration, high oxygen pressure and the like, the metabolism rate of the extraocular muscles and ciliary muscles is increased, and a large amount of peroxide, ROS and other metabolites are accumulated, so that the oxidative stress injury is caused to the eyes; in addition, ROS readily oxidize polyunsaturated fatty acids in the retina, produce large amounts of lipid peroxidation, and further produce large amounts of reactive aldehydes, which, when combined with intracellular proteins and DNA, can cause cellular inflammation and apoptosis, thereby inducing asthenopia. The asthenopia improving effect of the sample can be evaluated by detecting the apoptotic cell condition.
The zebra fish eye structure development is the same as that of a human body, three different embryo tissues are used for developing the zebra fish eye structure, the zebra fish has visual response at 72hpf, the retina is similar to the shape of an adult retina at the time, the retina is similar to the human body in terms of anatomy (the structural characteristics of different retina layers and the similarity of cell arrangement) and functions, meanwhile, the eye vascular structure and the retina structure of the zebra fish are highly similar to those of the human body, the zebra fish eye cell apoptosis can be induced by the mycophenolate mofetil, the eye disease of the human can be simulated, and therefore, the effect of improving the visual fatigue of a sample can be represented by the level of the zebra fish eye cell apoptosis.
As can be seen from table 3, under this experimental condition, the probiotic powder of bifidobacterium animalis subspecies i797 has a significant effect of relieving asthenopia, whereas the effects of bifidobacterium longum subspecies i772, bifidobacterium breve i1088 and bifidobacterium bifidum i771 are not significant.
The bifidobacterium animalis subspecies i797 can also be used for preparing yoghurt or milk powder with the effect of relieving visual fatigue, or other foods or medicines.
Claims (4)
1. Use of bifidobacterium animalis subspecies i797 in the manufacture of an eye care product.
2. The use according to claim 1, wherein the eye-care product is a food or a pharmaceutical product having an effect of relieving asthenopia.
3. The use according to claim 1, wherein the eye care product comprises milk powder, yogurt and probiotics having asthenopia relieving effect.
4. The use according to claim 1, wherein the eye-protection product contains bifidobacterium animalis subspecies i797 with a viable count of 2 x 10 6 -5×10 10 CFU/g or CFU/mL.
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