A strain of Weizmannella coagulans with anti-aging and antioxidant effects and its postbiotics and applications

Abstract

The present invention discloses a strain of Weizmannella coagulans with anti-aging and antioxidant effects, its postbiotics and applications, and belongs to the field of microbial fermentation technology. The present invention screens and obtains a strain of Weizmannella coagulans, obtains the bacterial body and its postbiotics by culture, and forms a complex with asparagus extract and NAD. The complex exhibits a synergistic effect, which is specifically manifested in reducing cell mortality, increasing the activity of antioxidant substances such as cell CAT, SOD, GSH‑Px, and maintaining the health of mice for a long time, and having significant anti-aging and antioxidant effects on mice. The present invention not only has practical value for the comprehensive utilization of Weizmannella coagulans and its postbiotics, but also has important social value for delaying aging and maintaining body activity.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a strain of Weizmannella coagulans with anti-aging and antioxidant effects, and a postbiotic and application thereof. Background Art

[0002] Aging refers to the phenomenon that the body's physiological and psychological adaptability to the environment is progressively reduced and gradually tends to death. Aging can be divided into two categories: physiological aging and pathological aging. The former refers to the physiological degeneration process that occurs after maturity, and the latter is the senile changes caused by various external factors (including various diseases). Aging is inevitable, but delaying aging is possible. Delaying aging means postponing or reducing the occurrence of diseases, thereby improving the quality of life of individuals. Antioxidation refers to antioxidant free radicals. The human body continuously produces free radicals in the body due to continuous contact with the outside world, including breathing, external pollution, radiation exposure and other factors. Aging or other diseases are mostly related to the production of excessive free radicals, so removing free radicals can delay the aging process.

[0003] Weizmannella coagulans Weizmannia coagulans ) is a type of Gram-positive, facultative anaerobic to microaerobic bacteria. This strain has been certified GRAS (Generally Recognized as Safe) by the U.S. Food and Drug Administration. In 2016, my country also approved Weizmannella coagulans as a food-usable strain, which can be used in various types of probiotic foods. Currently, Weizmannella coagulans is used in feed, food, fermented products and other industries because of its ability to inhibit harmful bacteria, regulate intestinal flora and enhance host immunity.

[0004] Nicotinamide Adenine Dinucleotide (NAD or NAD + ), also known as oxidized coenzyme I, is a biological substance that transfers electrons. + As a coenzyme in many dehydrogenases in the body, its function is to connect the tricarboxylic acid cycle and the respiratory chain, and to transfer hydrogen removed during metabolism to flavoproteins. The paper "Progress in the Synthesis and Function of Nicotinamide Adenine Dinucleotide (Yang Min et al., Biochemical Engineering)" believes that with the increase of age, NAD in the body + The content of NAD decreases, which causes a series of metabolic disorders. Therefore, exogenous supplementation of NAD + It is very necessary.

[0005] Asparagus Asparagus officinalis L..) is the seedling of the perennial herb Asparagus of the Asparagaceae family. It can be eaten as a vegetable. my country is the largest producer of asparagus. Asparagus is rich in vitamin B, vitamin A, trace elements such as folic acid, selenium, iron, manganese, zinc, and various amino acids necessary for the human body, and has high nutritional value. The paper "Progress in Research on the Nutritional Function of Asparagus and the Effect of Selenium Application on Asparagus Quality (Shen Lecheng et al., China Inspection and Testing)" believes that asparagus is rich in nutrients and contains a variety of bioactive ingredients such as polysaccharides, saponins and flavonoids. It has a variety of health functions such as anti-cancer, improving immunity, lowering blood lipids, anti-fatigue, and anti-inflammation.

[0006] Although people in all walks of life have a certain understanding of the effects of Weizmannella coagulans, NAD and asparagus, there has been no report on the combined use of the above three substances for a long time, and the effects of adding Weizmannella coagulans and its postbiotics to them are even more unknown. Summary of the invention

[0007] In view of the above-mentioned prior art, the purpose of the present invention is to provide a strain of Weizmannella coagulans having anti-aging and antioxidant effects, and its postbiotics and applications.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] In a first aspect of the present invention, a strain of Weizmannella coagulans having anti-aging and antioxidant effects is provided. The Weizmannella coagulans is Weizmannella coagulans WZA-WE70, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241411.

[0010] The second aspect of the present invention provides the use of the coagulant Weizmannella in the preparation of anti-aging and antioxidant products, wherein the total viable count of the coagulant Weizmannella is (0.01-9)×10 10 cfu / mL.

[0011] The third aspect of the present invention provides a postbiotic for coagulating Weizmannella, wherein the postbiotic for coagulating Weizmannella is prepared by the following method:

[0012] (1) activating the Weizmannella coagulans to obtain an activated Weizmannella coagulans bacterial solution;

[0013] (2) inoculating the activated Weizmannella coagulans bacterial solution obtained in step (1) into a fermentation medium for fermentation to obtain a Weizmannella coagulans fermentation solution;

[0014] (3) Centrifuging the fermentation broth of Weizmannella coagulans obtained in step (2), and freeze-drying the separated supernatant to obtain postbiotics of Weizmannella coagulans.

[0015] Furthermore, in step (2), the composition of the fermentation medium is as follows: 1-10 parts of peptone, 10-30 parts of yeast extract, 5-15 parts of glucose, 1-10 parts of corn starch, 1-10 parts of dipotassium hydrogen phosphate, 0.1-1 parts of magnesium sulfate, 0.01-0.1 parts of manganese sulfate, and 500-1200 parts of distilled water; the fermentation temperature is 30-50°C, the shaking speed is 100-500rpm, and the fermentation time is 10-30h.

[0016] The fourth aspect of the present invention provides the use of the postbiotics of Weizmannella coagulans in the preparation of anti-aging antioxidant products, wherein the anti-aging antioxidant product is prepared by mixing the postbiotics of Weizmannella coagulans, asparagus extract and NAD in a mass ratio of (0.5-5):(0.5-5):(0.5-5).

[0017] Furthermore, the asparagus extract is prepared by the following method:

[0018] Fresh green asparagus aerial stems were selected, washed and dried at 85°C, crushed to pass through a 200-mesh sieve, and 50 g of the sieved powder was added to a 50% ethanol aqueous solution at a mass volume ratio of 1 g: 10 mL. The mixture was extracted three times at 70°C, the obtained extracts were combined and concentrated to 100 mL, centrifuged at 5000 r / min for 20 min, and the precipitate was freeze-dried to obtain asparagus extract.

[0019] Beneficial effects of the present invention:

[0020] (1) The present invention obtains Weizmannella coagulans through screening. The bacterium has the characteristics of acid resistance and bile salt resistance. Cell experiments show that the Weizmannella coagulans has the effect of enhancing the anti-aging and antioxidant functions of cells.

[0021] (2) The present invention ferments Weizmannella coagulans, freeze-dries the supernatant, obtains the postbiotics of Weizmannella coagulans, and forms a complex with asparagus extract and NAD, namely, the Weizmannella coagulans postbiotic complex. The Weizmannella coagulans postbiotic complex was used in the experiment. Among the experimental groups, the mortality rate of the complex group was the lowest, which was 31.2±0.2%, indicating that the Weizmannella coagulans postbiotic complex has an anti-aging effect on cells. The activities of CAT, SOD, and GSH-Px were further determined. The complex group was able to more significantly improve the CAT, SOD, and GSH-Px activities of the corresponding components, with CAT being (2.67±0.77) KU / L, SOD being (24.45±3.65) KU / L, and GSH-Px being (20.48±2.36) KU / L. The substances in the complex group showed a synergistic effect, indicating that the Weizmannella coagulans postbiotic complex improved the cellular antioxidant function.

[0022] (3) The present invention uses the Weizmannella coagulans postbiotic complex for animal experiments. In the water maze test, the mouse latency was (24.0±3.4) s, and the thymus index and spleen index were (2.8±0.4) mg / g and (5.5±0.9) mg / g, respectively. The results were significantly improved compared with the postbiotic group, asparagus group, and NAD group used alone, reflecting a synergistic effect, which is specifically manifested in the long-term maintenance of the health of mice and the significant anti-aging and antioxidant effects on mice. The present invention not only has practical value for the comprehensive utilization of Weizmannella coagulans and its postbiotics, but also has important social value in delaying aging and maintaining body activity. DETAILED DESCRIPTION

[0023] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0024] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0025] The experimental materials used in the embodiments of the present invention that are not specifically described are all conventional experimental materials in the field and can be purchased through commercial channels. Nicotinamide adenine dinucleotide (CAS: 53-84-9) used in this application was purchased from Merck, the human normal colon epithelial cells (NCM-460) and DMEM-H complete medium (90% DMEM-H + 10% FBS) used were purchased from Henan Industrial Microbial Strain Engineering Technology Research Center, SOD, CAT and GSH-Px kits were purchased from Biyuntian Biotechnology Company; SAM P8 aged mice were purchased from Beijing Zhongke Zesheng Biotechnology Co., Ltd., and animal welfare was guaranteed throughout the experiment.

[0026] The deposit information of Weizmannella coagulans used in the present invention is as follows:

[0027] Chinese name: Weizmannella coagulans WZA-WE70;

[0028] Latin name: Weizmannia coagulans WZA-WE70;

[0029] Deposit number: CCTCC NO:M 20241411;

[0030] Deposit date: July 1, 2024;

[0031] Depository: China Center for Type Culture Collection (CCTCC);

[0032] Deposit: Wuhan University, Wuhan, China.

[0033] Example 1: Screening of Weizmannella coagulans

[0034] 1. Isolation and initial screening of strains:

[0035] 10 g of fecal samples from healthy adults were collected, and 1 g of each sample was placed in a 50 mL centrifuge tube containing 9 mL of sterile saline, shaken for 15 min to mix thoroughly, and allowed to stand for 25 s. 2 mL of suspension was taken from each sample and placed in an 80 ℃ water bath for 10 min. 1 mL was taken for 10-fold gradient dilution, and then the appropriate gradient suspension was selected and spread on MRS agar medium (self-prepared) containing 0.25% calcium carbonate, and placed in a 37 ℃ incubator for constant temperature culture for 48 h. Single colonies with different morphologies on the initial screening plate were repeatedly streaked and purified on PCA medium (self-prepared) plates. After the single colony morphology was stable, it was numbered and stored.

[0036] 2. Strain screening: acid resistance and bile resistance

[0037] The strains were treated in simulated gastric fluid at pH = 3 for 0h, 2h, 3h, and 4h, and the survival rate was recorded; the strains were treated at bile salt concentrations of 0.1%, 0.3%, 0.5%, and 1%, and the survival rate was recorded. The strains with higher survival rates were selected for subsequent identification.

[0038] 3. Identification of strains:

[0039] The strain was sent to Henan Industrial Microbial Engineering Technology Research Center for 16S rDNA gene sequence identification (SEQ ID NO: 1), and the identification results showed that the strain belonged to Weizmannella coagulans ( Weizmannia coagulans ). The strain WZA-WE70 was biologically deposited.

[0040] Example 2: Preparation of postbiotics for coagulating Weizmannella

[0041] The preparation method is as follows:

[0042] (1) Activate the Weizmannella coagulans preserved in Example 1 to obtain an activated bacterial solution of Weizmannella coagulans. The activation temperature is 39°C, the shaking speed is 250 rpm, and the activation time is 10 h. The composition of the activation medium is as follows: 9 g of peptone, 15 g of yeast extract, 11 g of glucose, 3 g of dipotassium hydrogen phosphate, 0.3 g of magnesium sulfate, 0.01 g of manganese sulfate, and distilled water is added to 1 L.

[0043] (2) The activated bacterial liquid of Weizmannella coagulans obtained in step (1) is inoculated into a fermentation medium at an inoculum amount of 4 wt% of the fermentation medium to obtain a fermentation liquid of Weizmannella coagulans. The fermentation temperature is 42° C., the shaking speed is 300 rpm, the fermentation time is 24 h, and the composition of the fermentation medium used is as follows: 8 g of peptone, 24 g of yeast extract, 10 g of glucose, 8 g of corn starch, 6 g of dipotassium hydrogen phosphate, 0.5 g of magnesium sulfate, 0.03 g of manganese sulfate, and distilled water is added to 1 L.

[0044] (3) Take the fermentation liquid of Weizmannella coagulans obtained in step (2), centrifuge at 30°C, and freeze-dry the supernatant to obtain the postbiotics of Weizmannella coagulans. The content of Weizmannella coagulans in the precipitate was detected by spectrophotometry. The number of viable bacteria was 7×10 10 The cfu / ml concentration was stored in a refrigerator at 4°C.

[0045] Example 3: Preparation of coagulated Weizmannella postbiotic complex

[0046] Preparation of Asparagus Extract

[0047] Fresh green asparagus aerial stems were selected, washed and dried at 85°C, crushed to pass through a 200-mesh sieve, and 50 g of the sieved powder was added to a 50% ethanol aqueous solution at a mass volume ratio of 1 g: 10 mL. The mixture was extracted three times at 70°C, the obtained extracts were combined and concentrated to 100 mL, centrifuged at 5000 r / min for 20 min, and the precipitate was freeze-dried to obtain asparagus extract.

[0048] Preparation of complexes

[0049] The postbiotics of Weizmannella coagulans prepared in Example 1, the asparagus extract prepared in Example 2, and NAD were mixed in a mass ratio of 1:1:1 to obtain a Weizmannella coagulans postbiotic complex.

[0050] Test Example 1: Anti-aging Cell Experiment

[0051] Here are the steps:

[0052] (1) Human normal colon epithelial cells were subcultured, and the third generation cells were selected for the experiment. They were then inoculated in 6-well plates, with a total of seven groups, three parallels in each group. After the cells adhered to the wall, the control group and the aging group were cultured in DMEM+10% FBS medium; the probiotic group, postbiotic group, asparagus group, NAD group and complex group were cultured in DMEM+10% FBS medium containing 10 mg / ml of the corresponding additives.

[0053] The corresponding additives of each experimental group were set as follows: the probiotic group was added with the preserved Weizmannella coagulans prepared in Example 2 (1 ml was regarded as 1 g); the postbiotic group was added with the postbiotics of Weizmannella coagulans prepared in Example 2; the asparagus group was added with the asparagus extract prepared in Example 3; the NAD group was added with NAD; the complex group was added with the Weizmannella coagulans postbiotic complex prepared in Example 3; the control group and the aging group were added with an equal amount of PBS.

[0054] (2) After 24 h of culture, the control group was cultured with new DMEM + 10% FBS medium, while the other groups were cultured with DMEM + 10% FBS medium containing 30 mmol / L glucose.

[0055] (3) Continue culturing for 3 days, and replace the culture medium with fresh medium according to the method in step (2) within 3 days. Then perform SA-β-Gal staining, observe under a microscope and take pictures. The cell death rate results are shown in Table 1.

[0056] Table 1. Cell death rate results (%)

[0057]

[0058] As can be seen from Table 1, compared with the cells in the control group, the mortality rate of the experimental group cells treated with high sugar increased significantly; among the experimental groups, the mortality rate of the complex group was the lowest, which was 31.2±0.2%, indicating that the Weizmannia coagulans postbiotic complex has an anti-aging effect on cells.

[0059] Test Example 2: Antioxidant Cell Experiment

[0060] CAT, SOD, GSH-Px activity assay

[0061] Normal human colon epithelial cells were subcultured, and the third generation cells were selected for the experiment. The groups were set up as follows: probiotic group, added with the coagulant Weizmannella prepared and preserved in Example 2 (1 ml was regarded as 1 g); postbiotic group, added with the postbiotics of coagulant Weizmannella prepared in Example 2; asparagus group, added with the asparagus extract prepared in Example 3; NAD group, added with NAD; complex group, added with the coagulant Weizmannella postbiotic complex prepared in Example 3; control group, added with PBS as a control; in the cell culture medium, the final concentration of the above additives was 15 mg / L, and 3 parallels were set for each treatment. The antioxidant enzyme activity of cells cultured for 72 hours was determined: the cells of each group were collected and ultrasonically disrupted. After centrifugation, the supernatant was collected, and the activities of CAT, SOD, and GSH-Px were determined according to the method in the kit instructions. The results are shown in Table 2.

[0062] Table 2. CAT, SOD and GSH-Px test results

[0063]

[0064] From the results in Table 2, it can be seen that the probiotic group, postbiotic group, asparagus group, NAD group, and complex group can increase the activities of CAT, SOD, and GSH-Px compared with the control group. Compared with the control group, the complex group can more significantly increase the activities of CAT, SOD, and GSH-Px of the corresponding components, with CAT being (2.67±0.77) KU / L, SOD being (24.45±3.65) KU / L, and GSH-Px being (20.48±2.36) KU / L. The substances in the complex group showed a synergistic effect, indicating that the Weizmannella postbiotic complex enhanced the cellular antioxidant function.

[0065] Experimental Example 3: Aging Mouse Model Experiment

[0066] Take 3-month-old rapid aging model mice SAMP8, half male and half female, randomly divided into groups, 18 in each group, set up three parallel groups, and the group settings are as follows: postbiotic group, the additive is the postbiotic of Weizmannella coagulans prepared in Example 1; asparagus group, the additive is asparagus extract prepared in Example 2; NAD group, the additive is NAD; complex group, the additive is the Weizmannella coagulans postbiotic complex prepared in Example 3; each additive group is gavage-fed at a dose of 2 mg / kg according to the weight of the animal, and the control group is given an equal amount of normal saline. In addition to feeding with the same normal feed, each group is gavaged once a day until the end of the experiment. After 6 months of conventional feeding, the surviving mice are subjected to water maze test and thymus index and spleen index determination, as follows:

[0067] Water maze test: Place mice in a circular pool with a circular platform, 30 cm deep. If the mice cannot find the platform for more than 90 seconds, the experimenter will place the mice on the platform to familiarize them with the environment for 10 seconds. After 3 consecutive days of training, the time it takes for each mouse to find the platform, i.e., the latency period, is recorded. Its length represents the quality of the animal's spatial learning and memory ability. A short latency period indicates that the animal has a good learning and memory ability, which can be used as an indicator of the aging process. The results are shown in Table 3.

[0068] Table 3. Water maze test

[0069]

[0070] As shown in Table 3, compared with the control group, the incubation period of mice in each experimental group was shortened. Among them, the compound group performed best, with a latency of (24.0±3.4) s. It can be seen that after the compound group was treated, the health of the mice improved, and the compound group treatment showed a synergistic effect, the health of the mice could be maintained for a long time, and the aging process was delayed.

[0071] Determination of thymus index and spleen index: The thymus and spleen of the experimental mice were taken and weighed, and the ratio of the thymus and spleen weight to the mouse body weight was calculated. The results are shown in Table 4.

[0072] Table 4. Thymus index and spleen index

[0073]

[0074] As shown in Table 4, compared with the control group, the spleen index and thymus index of mice in each experimental group increased. The thymus and spleen indexes depend on the degree of lymphocyte proliferation, which can estimate the strength of immune function. Among them, the complex group performed best, with the thymus index and spleen index of mice in the complex group being (2.8±0.4) mg / g and (5.5±0.9) mg / g, respectively. It can be seen that after treatment with the complex group, the mice had stronger immune function, maintained better health, and delayed aging.

[0075] In summary, the complex group performed best, with CAT of (2.67±0.77) KU / L, SOD of (24.45±3.65) KU / L, GSH-Px of (20.48±2.36) KU / L, latency of (24.0±3.4) s in the water maze test, thymus index and spleen index of (2.8±0.4) mg / g and (5.5±0.9) mg / g, respectively. This result not only had significant advantages over the control group, but also had a significant improvement over the postbiotic group, asparagus group, and NAD group used alone, reflecting a synergistic effect, which was specifically manifested in the long-term maintenance of the health status of mice and significant anti-aging and antioxidant effects on mice.

[0076] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A strain of Weizmannella coagulans having anti-aging and antioxidant effects, characterized in that: The coagulant Weizmannella is coagulant Weizmannella WZA-WE70 ( Weizmannia coagulans WZA-WE70), deposited in China Center for Type Culture Collection with the deposit number of CCTCC NO:M 20241411.

2. The use of the Weizmannella coagulans according to claim 1 in the preparation of anti-aging and antioxidant products, characterized in that: The total number of viable cells of Weizmannella coagulans is (0.01-9)×10 10 cfu / mL.

3. A postbiotic for coagulating Weizmannella, characterized in that The postbiotics of coagulating Weizmannella are prepared by the following method: (1) activating the Weizmannella coagulans described in claim 1 to obtain an activated Weizmannella coagulans bacterial solution; (2) inoculating the activated Weizmannella coagulans bacterial solution obtained in step (1) into a fermentation medium for fermentation to obtain a Weizmannella coagulans fermentation solution; (3) Centrifuging the fermentation broth of Weizmannella coagulans obtained in step (2), and freeze-drying the separated supernatant to obtain postbiotics of Weizmannella coagulans.

4. The postbiotic of Weizmannella coagulans according to claim 3, characterized in that In step (2), the composition of the fermentation medium is as follows: 1-10 parts of peptone, 10-30 parts of yeast extract, 5-15 parts of glucose, 1-10 parts of corn starch, 1-10 parts of dipotassium hydrogen phosphate, 0.1-1 parts of magnesium sulfate, 0.01-0.1 parts of manganese sulfate, and 500-1200 parts of distilled water; the fermentation temperature is 30-50°C, the shaking speed is 100-500rpm, and the fermentation time is 10-30h.

5. The use of the postbiotics of Weizmannella coagulans according to claim 3 or 4 in the preparation of anti-aging and antioxidant products, characterized in that: The anti-aging antioxidant product is prepared by mixing postbiotics of Weizmannella coagulans, asparagus extract and NAD in a mass ratio of (0.5-5):(0.5-5):(0.5-5); The asparagus extract is prepared by the following method: Fresh green asparagus aerial stems were selected, washed and dried at 85°C, crushed to pass through a 200-mesh sieve, and 50 g of the sieved powder was added to a 50% ethanol aqueous solution at a mass volume ratio of 1 g: 10 mL. The mixture was extracted three times at 70°C, the obtained extracts were combined and concentrated to 100 mL, centrifuged at 5000 r / min for 20 min, and the precipitate was freeze-dried to obtain asparagus extract.

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