Application of saccharomyces cerevisiae in preparation of anti-reproductive aging product
By screening specific gene mutant yeasts in the Saccharomyces cerevisiae gene knockout strain library, the side effects and instability problems caused by the improvement of reproductive capacity in the existing technology were solved, and the effect of significantly improving reproductive capacity and delaying reproductive aging was achieved. Extend lifespan and improve exercise capacity.
Patent Information
- Application Number
- CN202411681511.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-22
- Publication Date
- 2025-01-07
AI Technical Summary
The prior art often has side effects and instability in improving host reproductive capacity, and it is difficult to take into account both prolonging life and improving long-term health.
By screening the Saccharomyces cerevisiae gene knockout strain library, select Saccharomyces cerevisiae strains that lack specific genes, and are used to prepare anti-reproductive aging products, which significantly improves reproductive ability, delays reproductive aging and improves physiological functions.
Significantly improve the reproductive ability and lifespan of the host, delay reproductive aging, reduce adverse health effects, improve exercise ability, and achieve standardized production.
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Figure CN119258103A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technology, and in particular to the application of brewer's yeast in the preparation of anti-reproductive aging products. Background Art
[0002] Currently, existing technologies for improving host reproductive capacity mainly rely on exogenous drugs or hormone regulation. Although these methods are effective in the short term, they are usually accompanied by side effects, such as hormone imbalance and physiological burden. In addition, the effects of these technologies are easily affected by environmental and individual differences, resulting in instability, and excessive focus on improving reproductive capacity often comes at the expense of the overall health of the host, failing to simultaneously extend lifespan and improve long-term health. Summary of the invention
[0003] The purpose of the present invention is to provide the use of brewer's yeast in the preparation of anti-reproductive aging products, wherein the brewer's yeast can significantly improve reproductive capacity, delay reproductive aging and improve physiological function; in addition, the strain can also achieve standardized production.
[0004] To achieve the above object, the present invention provides the use of Saccharomyces cerevisiae in the preparation of anti-reproductive aging products, wherein the Saccharomyces cerevisiae is screened from a Saccharomyces cerevisiae gene-knockout strain library, and the Saccharomyces cerevisiae gene-knockout strain library is purchased from Invitrogen. The Saccharomyces cerevisiae gene-knockout collection (YKOC) is a resource library containing a large number of gene knockout mutants, which covers 6000 genes in the Saccharomyces cerevisiae genome. The present invention can significantly improve the aging degree of the host and prolong life by screening 13 mutant genes from the gene knockout library.
[0005] The brewer's yeast lacks one gene among YAL044C, YJR044C, YDR176W, YPL257W, YCL051W, YAL053W, YER164W, YPL253C, YER169W, YBR041W, YNL125C, YER174C, and YLL015W; the nucleotide sequences of YAL044C, YJR044C, YDR176W, YPL257W, YCL051W, YAL053W, YER164W, YPL253C, YER169W, YBR041W, YNL125C, YER174C, and YLL015W are shown in SEQ ID NOs.1-13.
[0006] Preferably, the brewer's yeast lacks YAL044C, and the product is used to significantly improve the host's reproductive capacity and reproductive cycle and delay the host's reproductive aging.
[0007] Preferably, the brewer's yeast lacks one gene among YER164W, YER174C, YNL125C, YLL015W, YCL051W, YPL257W, and YPL253C; and the product is used to increase the total number of eggs laid by the host and improve reproductive capacity.
[0008] Preferably, the product is used to increase the life span of the host and maintain and improve the host's motility.
[0009] Preferably, the product is one of fermented food, health food, daily chemical products, feed additives, probiotic preparations, and medicines.
[0010] Compared with the prior art, the present invention has the following beneficial effects:
[0011] (1) Improving reproductive capacity: The gene mutant yeast screened by the present invention can significantly improve the reproductive capacity of the host (fruit fly); specifically, it increases the total number of eggs laid by the fruit fly and prolongs its reproductive cycle, allowing it to maintain a high reproductive activity for a longer period of time; this advantage is achieved without the need for additional drug intervention, thereby reducing adverse effects on the host's health;
[0012] (2) Delaying reproductive aging: The genetically engineered yeast of the present invention delays the aging process of the reproductive system, slows down the rate of decline in reproductive capacity, and prolongs the peak reproductive period without damaging the healthy life span of the host;
[0013] (3) Improving physiological functions: The genetically engineered yeast selected can significantly extend the healthy lifespan of Drosophila melanogaster and improve the athletic ability of Drosophila, including the climbing ability of young and middle-aged Drosophila. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 This is a schematic diagram of the application effect process of an embodiment of the present invention;
[0015] Figure 2 Schematic diagram of the effect of Saccharomyces cerevisiae gene mutation on the reproductive ability of Drosophila, where (A) 1 male and 1 female per tube, (B) 5 males and 5 females per tube;
[0016] Figure 3 Schematic diagram of the effect of Saccharomyces cerevisiae gene mutation on the reproductive cycle of Drosophila, including (A) 1 male and 1 female / tube (12 days), (B) 5 males and 5 females / tube (12 days), (C) 5 males and 5 females / tube (18 days);
[0017] Figure 4Schematic diagram of the effect of Saccharomyces cerevisiae gene mutation on the healthy lifespan of Drosophila; (A) is the effect of the mutant strain on the host lifespan, and (B) is the effect of YAL044C on the locomotion of the host fruit fly in its young and old stages. DETAILED DESCRIPTION
[0018] The present invention is further described in detail below in conjunction with specific embodiments.
[0019] Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0020] Example
[0021] This example uses the biological model Drosophila melanogaster to screen for Saccharomyces cerevisiae mutants that improve reproductive capacity from a library of 2,600 single-gene mutant strains of Saccharomyces cerevisiae; and measures the effects of anti-reproductive aging yeast on the reproductive capacity and health status of the host. Figure 1 As shown, the details are as follows:
[0022] 1. Experimental process
[0023] 1. Main reagent formula
[0024] (1) Methyl p-hydroxybenzoate solvent: 0.8 g of methyl p-hydroxybenzoate was dissolved in 4 mL of anhydrous ethanol.
[0025] (2) 0.6% sodium hypochlorite solution: 0.6 g sodium hypochlorite was dissolved in 100 mL sterile water.
[0026] (3) Saccharomyces cerevisiae culture medium (YPD medium): dissolve 10 g yeast extract, 20 g peptone, and 20 g glucose in 1 L deionized water and sterilize by high pressure for 30 min. For solid YPD, add 20 g agar powder to the above.
[0027] 2. Cultivation of Drosophila
[0028] (1) Preparation of basic Drosophila culture medium
[0029] 1) Mix 350 ml of deionized water, 16 g of corn flour and 16 g of soybean flour, and boil to obtain a corn and soybean flour mixture for later use.
[0030] 2) Bring 350 ml of deionized water, 12 g of agar, and 16 g of yeast extract (CAS number 8013-01-2) to a boil and then slowly add the corn and soybean flour mixture.
[0031] 3) Add methyl paraben solvent and continue boiling for 3 minutes.
[0032] 4) Pour the above solution into a bottle and sterilize at 121°C for 20 minutes.
[0033] 5) Pour 100 ml of deionized water, 24 g of sucrose, and 12 g of glucose into another bottle and sterilize.
[0034] 6) After sterilization, mix in a clean bench and add 4 mL of propionic acid and mix well.
[0035] 7) After the bottles have cooled, plug them and place them in a 4°C refrigerator for later use.
[0036] (2) Preparation of Drosophila culture medium for experiments
[0037] The yeast extract was removed from step 2) of the preparation of the basal Drosophila culture medium, and the remaining steps were the same as those in the above-mentioned "(1) Preparation of basal Drosophila culture medium".
[0038] (3) Drosophila synchronization
[0039] Transfer young and energetic wild-type Drosophila melanogaster (WT) to a bottle containing a basic Drosophila culture medium and adapt for 1-2 days as spare Drosophila. Transfer the male and female Drosophila in the spare bottle to a wide-mouth bottle containing a basic Drosophila culture medium. Collect the Drosophila eggs after 6-8 hours and put them in a 25°C incubator for hatching. Feed them with yeast powder (i.e. Angel yeast powder sold in the market) to supplement nutrition during the 2nd to 5th day of Drosophila larvae. After about 10 days, a large number of Drosophila born at the same time can be hatched.
[0040] (4) Determining the sex of fruit flies
[0041] Take the fruit flies on the first day of eclosion after synchronization and place them on a fly sorting plate (fruit fly flight plate). Anesthetize the fruit flies by passing CO2, and use a fly sorting pen under a stereo microscope to pick out male or female fruit flies. Male fruit flies are smaller than female fruit flies, and the three black stripes behind their tails are connected into one piece. Male fruit flies have a pair of umbrella-shaped genitals at the end of their abdomen, while female fruit flies have a pair of reproductive tract openings. Most importantly, male fruit flies have sexual sparseness while females do not. Confirm the sex of the fruit flies based on these phenotypes. Ensure that the fly sorting equipment is relatively clean. Wipe it with 75% ethanol before and after each use, and sterilize it by high pressure once a week.
[0042] (5) Cultivation of sterile fruit flies
[0043] 1) Synchronize the flies in the jars according to the normal synchronization procedure and then transfer out the adult flies.
[0044] 2) Add double distilled water to rinse, gently brush off the eggs with a brush, and use a pipette to transfer the eggs into a centrifuge tube.
[0045] 3) Rinse with 0.6% sodium hypochlorite 5 times, disinfect with 75% ethanol 2 times, and clean with double distilled water 3 times.
[0046] 4) Transfer the eggs into new experimental culture medium and place them in a 25°C incubator to wait for them to emerge as adults.
[0047] 3. Cultivation of strains
[0048] (1) Strain recovery
[0049] 1) Take out some yeast mutants (i.e. selected from the single gene mutant strain library of Saccharomyces cerevisiae) from the -80 degree refrigerator. In the clean bench, use a pipette to take a certain amount of bacterial liquid ice crystals and add 1mL YPD culture base to the EP tube, mix, mark, and place in a shaker at 200rpm and 30℃ for one night.
[0050] 2) On the second day, remove the EP tube from the shaker, take a portion of the bacterial solution and place it on a slide, and observe under a microscope to see if there is any contamination; transfer the uncontaminated bacterial solution to a centrifuge tube containing 35 mL of YPD culture medium and place it in a shaker for shaking culture overnight.
[0051] 3) On the second day, take out the centrifuge tube, centrifuge at 7000 r / min for 5 min, then remove the centrifuge tube and discard the supernatant for later use.
[0052] (2) Bacteria spotting
[0053] Take several tubes of revived yeast and dilute them with 400 μL sterile PBS buffer. Use a pipette to transfer 200 μL of suspension drops into the fruit fly tubes containing fruit fly experimental culture medium, four tubes in a group. After the bacterial solution solidifies, plug it with cotton plugs and refrigerate for later use. The above operations are performed on a clean bench.
[0054] 4. Drosophila Reproduction Experiment
[0055] Freshly eclosing fruit flies (day 0) were separated by sex on the flight plate, and 5 male and 5 female fruit flies were placed in fruit fly tubes, 3 tubes per group (or one male and one female were placed in each tube, 10 tubes per group), marked with the date and strain, and stored in a 25°C incubator. The culture medium and strain were replaced every 5-7 days. The fruit flies were transferred to new fruit fly tubes at the same time every 2 days, and the previous fruit fly tubes were retained. After 10 days, the number of fruit flies in the old fruit fly tubes was counted to assess the total egg laying on that day.
[0056] 5. Drosophila lifespan experiment
[0057] The one-day-old male fruit flies to be tested were transferred into the fruit fly tubes containing the experiments after inoculation, with four tubes in each group and 20 flies in each tube. The tubes were marked with the date and strain, and stored in an incubator at 25°C. The culture medium and strain were replaced every 5-7 days, and the number of fruit flies that died in each group was recorded at a fixed time every day until all the fruit flies died. The survival indicators were recorded: the average number of days that all the fruit flies in each group survived was the average lifespan of the group; based on the data obtained, a survival curve was constructed to observe the effects of different mutant strains of brewer's yeast on the survival rate of fruit flies. The algorithm for the average lifespan of fruit flies is as follows: let the number of days that the fruit flies survive be d, and the number of fruit flies that died on the same day be xd. Then the average lifespan of fruit flies in each tube is
[0058] L=∑d*xd / 20
[0059] All data of repeated life span experiments were expressed as Mean ± SEM, and independent sample t-test was used to compare the experimental group and the control group, with P < 0.05 as statistically significant.
[0060] 6. Fruit fly climbing experiment
[0061] In each experiment, a camera was used to record the climbing process of fruit flies for observation. The day when fruit flies were transferred to the experimental tube was recorded as day 1. At 18:00 every day, the experimental tubes of day 10 and day 15 were taken horizontally and inverted. The fruit flies were gently shaken to the bottom of the experimental tube with the same force. The number of fruit flies that reached the top of the experimental tube within 10 seconds was tested, and the climbing index was calculated (the number of fruit flies that reached the top of the experimental tube within 10 seconds / the total number of surviving fruit flies in the tube). Each tube of fruit flies was tested at least 3 times, with at least 1 minute interval between each test. The average climbing index of each tube was recorded for each group.
[0062] Figure 4 The statistical results in B are expressed as the degree of change compared with the control bacteria (%), and the specific calculation method is: (the average climbing value of fruit flies fed with mutant bacteria-the average climbing value of fruit flies fed with wild control BY4743 strain) / the average climbing value of fruit flies fed with wild control BY4743 strain)*100%).
[0063] 7. Data statistics and analysis
[0064] The experimental data were analyzed using GraphPad Prism 8 software. The experimental results were expressed as "Mean ± SEM or SD" (SD was used for the mean within the group, and SEM was used for the mean between groups). The significance of the difference between the groups was analyzed using the Unpaired t test. If the variance was not uniform, a non-parametric test method was used. The significance level was P < 0.05 (*).
[0065] 2. Experimental Results Analysis
[0066] 1. Detect the effect of Saccharomyces cerevisiae gene mutation on the reproductive ability of Drosophila
[0067] Using male Drosophila melanogaster as an animal model and 1,600 strains of single-gene knockout Saccharomyces cerevisiae as research subjects, the effects of different mutant strains on the lifespan and motor ability of fruit flies were tested, and mutant strains with significantly increased lifespan were selected for reproductive experiments.
[0068] The study found that compared with the wild control BY4743 strain, the reproductive capacity of fruit flies fed with the YAL044C mutant bacteria changed significantly. Specifically, after 12 days of feeding different mutant bacteria, the total number of egg production of fruit flies increased compared with the control strain, and the total number of egg production of YAL044C increased most significantly (P<0.05) ( Figure 2 A and 2B).
[0069] 2. Detecting the effects of Saccharomyces cerevisiae gene mutations on the reproductive cycle of Drosophila
[0070] The reproductive cycle of the mutant bacteria that can increase the total number of host reproduction is detected. The peak reproductive period of fruit flies is 3-7 days after birth, and it will be significantly reduced after 10-14 days. The present invention detects and counts the egg production at each stage.
[0071] The study found that mutant bacteria that can increase the total number of host reproduction also have a significant effect on the number of eggs laid by the host at different stages. Among them, YAL044C has a positive effect on the host's reproduction at different time points and delays reproductive aging ( Figure 3 A, 3B). In addition, the present invention extended the time to 18 days and found that the YAL044C mutant bacteria can delay the reproductive aging cycle ( Figure 3 C).
[0072] 3. Detect the effects of mutant strains that enhance reproductive capacity on the healthy lifespan of Drosophila
[0073] The fruit flies were fed with YAL044C mutant bacteria until they died, and their lifespan and health status were observed and recorded.
[0074] The anti-reproductive aging mutant YAL044C can significantly increase the lifespan of Drosophila ( Figure 4 A), and YAL044C improved the athletic ability of the host fruit flies both in the young and old stages ( Figure 4 B).
Claims
1. Use of Saccharomyces cerevisiae in preparing an anti-reproductive aging product, wherein the Saccharomyces cerevisiae is screened out from a Saccharomyces cerevisiae gene knockout strain library, and the Saccharomyces cerevisiae lacks one of the genes YAL044C, YJR044C, YDR176W, YPL257W, YCL051W, YAL053W, YER164W, YPL253C, YER169W, YBR041W, YNL125C, YER174C, and YLL015W; the nucleotide sequences of the YAL044C, YJR044C, YDR176W, YPL257W, YCL051W, YAL053W, YER164W, YPL253C, YER169W, YBR041W, YNL125C, YER174C, and YLL015W are as shown in SEQ ID NOs.1-13.
2. The use of the brewer's yeast in the preparation of anti-reproductive aging products according to claim 1, characterized in that: The brewer's yeast lacks YAL044C, and the product is used to significantly improve the host's reproductive capacity and reproductive cycle and delay the host's reproductive aging.
3. The use of the brewer's yeast according to claim 1 or 2 in the preparation of an anti-reproductive aging product, characterized in that: The brewer's yeast lacks one gene among YER164W, YER174C, YNL125C, YLL015W, YCL051W, YPL257W and YPL253C; the product is used to increase the total number of eggs laid by the host and improve the reproductive capacity.
4. The use of the brewer's yeast according to claim 1 or 2 in the preparation of an anti-reproductive aging product, characterized in that: The product is used to increase the host's lifespan and maintain and improve the host's motor ability.
5. The use of the brewer's yeast according to claim 1 or 2 in the preparation of anti-reproductive aging products, characterized in that: The product is one of fermented food, health food, daily chemical products, feed additives, probiotic preparations and medicines.