Lactobacillus gasseri capable of improving memory and promoting growth and metagen of lactobacillus gasseri

Abstract

The invention discloses a lactobacillus gasseri strain capable of improving memory and promoting growth and a metagen thereof, and belongs to the technical field of microbial fermentation. A strain of lactobacillus gasseri is obtained through screening, thalli and metabiotics of the thalli are obtained through culture, the metabiotics and the reed rhizome extract form the compound, and the compound shows a synergistic interaction effect which is specifically shown as follows: in a water maze test, the incubation period of a mouse is shortened; in a rat growth test, the body length, the body width and the testis weight of the rat are all improved. The lactobacillus gasseri and the preparation method thereof have practical value on comprehensive utilization of lactobacillus gasseri and the metaplasts thereof, and also have important value in the aspects of improving memory of an organism and promoting growth.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and particularly relates to a Lactobacillus gasseri and its postbiotics for improving memory and promoting growth. Background Art

[0002] Lactobacillus gasseri, also known as Lactobacillus gasseri, is a homofermentative lactic acid bacterium that naturally exists in the gastrointestinal tract, vagina, or breast milk. It is defined by the US FDA as a generally recognized as safe (GRAS) status microorganism and can be used in the food industry to maintain the balance of the intestinal microecosystem.

[0003] Phragmitis rhizoma is the fresh or dried rhizome of the grass Phragmites australis (Cav.) Trin. ex Steud. Phragmites australis is distributed throughout China and is commonly found along the banks of rivers, lakes, ponds, ditches, and low wetlands. Phragmites australis is a perennial herb with a well-developed creeping rhizome, namely phragmitis rhizoma. Phragmitis rhizoma can be dug throughout the year and can be used as food for people or as medicine, with the effects of clearing heat and promoting fluid production, treating gum bleeding, stopping vomiting, and promoting diuresis.

[0004] Although there is a certain understanding of the effects of Lactobacillus gasseri and phragmitis rhizoma in various fields, there has been no report on the combined use of the above two substances for a long time, and the effects of adding Lactobacillus gasseri and its postbiotics on improving memory and promoting growth are also unknown. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a Lactobacillus gasseri and its postbiotics for improving memory and promoting growth.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, a Lactobacillus gasseri for improving memory and promoting growth is provided. The Lactobacillus gasseri is Lactobacillus gasseri WZA-LG12, which is preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: M 20241508.

[0008] In the second aspect of the present invention, the application of the above-mentioned Lactobacillus gasseri in the preparation of a product for improving memory and promoting growth is provided. In the product, the viable count of Lactobacillus gasseri is greater than 1×10 8 cfu / mL.

[0009] In the third aspect of the present invention, a postbiotic of Lactobacillus gasseri is provided, and the postbiotic of Lactobacillus gasseri is prepared by the following method:

[0010] After activating the Lactobacillus gasseri, inoculate it into a fermentation medium for fermentation. The fermentation temperature is 20 - 50 °C, and the fermentation time is 5 - 60 h; centrifuge and separate the fermented product, and freeze-dry the supernatant to obtain the postbiotic of Lactobacillus gasseri.

[0011] Furthermore, the composition of the fermentation medium is as follows: peptone 5 - 20 parts, beef extract 5 - 20 parts, yeast extract 2 - 10 parts, glucose 10 - 40 parts, magnesium sulfate 0.1 - 1 part, sodium acetate 2 - 10 parts, diammonium hydrogen citrate 1 - 10 parts, dipotassium hydrogen phosphate 1 - 10 parts, manganese sulfate 0.01 - 0.1 part, Tween - 80 0.5 - 5 parts, distilled water 800 - 1200 parts.

[0012] In the fourth aspect of the present invention, the application of the postbiotic of Lactobacillus gasseri in the preparation of a product for improving memory and promoting growth is provided. The product for improving memory and promoting growth is composed of the postbiotic of Lactobacillus gasseri and reed root extract in a mass ratio of (0.5 - 5):(0.5 - 5).

[0013] Furthermore, the reed root extract is prepared by the following method:

[0014] Take fresh reed roots, wash them clean, remove the buds, fibrous roots and membranous leaves, cut them into pieces, dry them in an oven at 60 °C, and crush them through a 500 - mesh sieve to obtain reed root powder; perform normal-temperature extraction of the reed root powder in 70% v / v ethanol at a mass - volume ratio of 1 g∶3 ml, with each extraction time of 9 h, and extract 3 times in total; combine the extraction solutions obtained each time, perform vacuum concentration at 60 °C, and then dry them in an oven at 75 °C to obtain the reed root extract.

[0015] Advantages of the present invention:

[0016] (1) In the present invention, Lactobacillus gasseri is obtained through screening. This bacterium has the characteristics of acid and bile salt tolerance and has the effects of improving memory and promoting growth.

[0017] (2) In the present invention, Lactobacillus gasseri is fermented, and the postbiotic of Lactobacillus gasseri is obtained by freeze - drying the supernatant. The postbiotic and reed root extract are combined to form a complex, namely the Lactobacillus gasseri postbiotic complex.

[0018] (3) The present invention uses the probiotic complex of Lactobacillus gasseri for experiments. From the experimental results, it can be seen that the complex group shows the best performance: in the water maze test, the latency of the mice in the complex group is (23.8 ± 2.3) s, the health status of the mice is improved, and the memory of the mice is improved; in the rat growth test, the body length of the rats in the complex group is (23.6 ± 0.19) cm, the body width is (9.09 ± 0.05) cm, and the testis weight is (2.28 ± 0.01) g. The rats are improved in terms of body length and body width, which are basic growth indicators, and testis weight, which is a development indicator, and the growth of the rats is accelerated. It can be seen that when the probiotic is combined with the extract of Phragmites australis root to form a complex, the complex exhibits a synergistic effect, demonstrating the effects of improving memory and promoting growth. Detailed implementation mode

[0019] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0020] 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 combination with specific embodiments.

[0021] The test materials not specifically described in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels. SAM P8 aging mice were purchased from Beijing Zhongke Zesheng Biotechnology Co., Ltd., and SD rats were purchased from Vital River Laboratories. Animal welfare was ensured throughout the experiment.

[0022] The preservation information of Lactobacillus gasseri used in the present invention is as follows:

[0023] Chinese name: Lactobacillus gasseri WZA-LG12;

[0024] Latin name: Lactobacillus gasseri WZA-LG12;

[0025] Preservation number: CCTCC NO: M 20241508;

[0026] Preservation date: July 8, 2024;

[0027] Preservation institution: China Center for Type Culture Collection (CCTCC);

[0028] Preservation location: Wuhan University, Wuhan, China.

[0029] Example 1: Screening of Lactobacillus gasseri

[0030] 1. Isolation and primary screening of strains:

[0031] Collect 10g of fecal samples from newborns, take 1g of each sample and put it into a 50mL centrifuge tube containing 9mL of sterile saline, shake for 15min to mix thoroughly, let it stand for 25s, take 2mL of suspension from each sample and place it in an 80℃ water bath for 10min, take 1mL of each sample for 10-fold gradient dilution, and then select the appropriate gradient suspension to apply on a 0.3% CaCO 3 The strain was cultured on MRS agar medium (prepared by yourself) at 37°C incubator for 48 hours. The single colonies with calcium-dissolving rings were streaked and purified, and the colonies were numbered and stored after their morphology was stable.

[0032] 2. Strain screening: acid resistance and bile resistance

[0033] The strains were treated in simulated gastric fluid at pH = 3 for 0h, 1h, 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%, 0.75%, and 1%, and the survival rate was recorded. The strains with higher survival rates were selected for subsequent identification.

[0034] 3. Strain identification:

[0035] The strain was sent to Henan Industrial Microbial Strain Engineering Technology Research Center for 16S rDNA gene sequence identification, the sequence is shown in SEQ ID NO: 1, and the identification result showed that the strain belonged to Lactobacillus gasseri. The strain WZA-LG12 was biologically preserved.

[0036] Example 2: Preparation of postbiotics of Lactobacillus gasseri

[0037] The preparation method is as follows:

[0038] (1) Activate the Lactobacillus gasseri preserved in Example 1 to obtain an activated bacterial solution of Lactobacillus gasseri. The activation temperature is 37° C. and the activation time is 24 h.

[0039] (2) the activated Lactobacillus gasseri bacterial liquid obtained in step (1) is inoculated into a fermentation medium according to the inoculum amount of 3wt% (g / g) of the fermentation medium to obtain a Lactobacillus gasseri fermentation liquid. The fermentation temperature is 37°C, 5% carbon dioxide, the fermentation time is 48h, and the fermentation medium used is an MRS liquid medium, which is composed of: 10g / L peptone, 8g / L beef extract, 4g / L yeast extract, 20g / L glucose, 0.2g / L magnesium sulfate, 5g / L sodium acetate, 2g / L diammonium hydrogen citrate, 2g / L dipotassium hydrogen phosphate, 0.05g / L manganese sulfate, 1g / L Tween-80, 1.0L distilled water, pH=5.7.

[0040] (3) Take the Lactobacillus gasseri fermentation broth obtained in step (2), centrifuge it at 30 °C, and lyophilize the supernatant to obtain the postbiotics of Lactobacillus gasseri.

[0041] Use spectrophotometry to detect the content of Lactobacillus gasseri in the precipitate, and store it in a 4 °C refrigerator at a concentration of 5×10 8 cfu / ml.

[0042] Example 3: Preparation of Lactobacillus gasseri postbiotic complex

[0043] Preparation of reed root extract

[0044] Take fresh reed roots, wash them clean, remove the buds, fibrous roots and membranous leaves, cut them into pieces, dry them in an oven at 60 °C, crush them to pass through a 500-mesh sieve to obtain reed root powder; soak the reed root powder in 70% v / v ethanol at room temperature according to a mass-volume ratio of 1 g∶3 ml, with each extraction time of 9 h, and extract 3 times in total; combine the extraction liquids obtained each time, carry out vacuum concentration at 60 °C, and then dry them in an oven at 75 °C to obtain reed root extract.

[0045] Preparation of the complex

[0046] Mix the postbiotics of Lactobacillus gasseri prepared in Example 2 and the reed root extract prepared in Example 3 according to a mass ratio of 1:1 to obtain the Lactobacillus gasseri postbiotic complex.

[0047] Experimental example 1: Animal experiment

[0048] 1. Morris water maze test

[0049] Take 3-month-old rapidly aging model mice SAMP8, with equal numbers of males and females, randomly divide them into groups, with 6 mice in each group, set three parallels, and the grouping is as follows: the postbiotic group, with the additive being the postbiotics of Lactobacillus gasseri prepared in Example 2; the asparagus group, with the additive being the asparagus extract prepared in Example 3; the complex group, with the additive being the Lactobacillus gasseri postbiotic complex prepared in Example 3; each additive group is gavage-fed according to the dose of 2 mg / kg based on the animal body weight, and the control group is given an equal amount of normal saline. Except for using the same normal feed for feeding, each group of mice is gavaged once a day until the end of the experiment. After 6 months of conventional breeding, the surviving mice are subjected to the Morris water maze test, specifically as follows:

[0050] Put the mice into a circular pool containing a circular platform, with the water depth of 30 cm. If the mice cannot find the platform within 90 s, the experimenter will put the mice on the platform to familiarize them with the environment for 10 s. After 3 consecutive days of training, record the time for each mouse to find the platform, that is, the latency. The length of the latency represents the quality of the animal's spatial learning and memory ability. A short latency indicates good learning and memory ability of the animal, which can be used as an index to improve memory. The results are shown in Table 1.

[0051] Table 1. Water maze test

[0052]

[0053] As can be seen from Table 1, compared with the control group, the latency of mice in each experimental group was shortened. Among them, the complex group showed the best performance, and the latency of mice in the complex group was (23.8 ± 2.3) s. It can be seen that after the treatment of the complex group, the health status of mice was improved, the treatment of the complex group showed a synergistic effect, and the memory of mice was improved.

[0054] 2. Rat growth test

[0055] Three-week-old male SD rat pups were selected and randomly divided into groups of 5 each. Three parallels were set in the experimental groups, and the grouping was as follows: the postbiotic group, with the additive being the postbiotic of Lactobacillus gasseri prepared in Example 2; the Phragmites communis group, with the additive being the Phragmites communis extract prepared in Example 3; the probiotic group, with the additive being the preserved Lactobacillus gasseri prepared in Example 2 (1 ml is regarded as 1 g); the complex group, with the additive being the Lactobacillus gasseri postbiotic complex prepared in Example 3. Each additive group was gavaged and fed according to the animal body weight at a dose of 3 mg / kg, and the control group was given an equal amount of normal saline. In addition to using the same normal feeding for each group, the rats were gavaged once a day until the end of the experiment. After 3 weeks of conventional rearing, the surviving rats were tested.

[0056] During the rearing period, the reactions, activities, mental states, and hair changes of the rats were observed regularly, and the morphological changes of the rats were photographed and recorded. The feces were collected 24 hours after the last gavage, and then the rats were fasted overnight. The body length, body width, and testis weight were measured the next day to obtain the growth test data, and the results are shown in Table 2.

[0057] Table 2. Growth test

[0058]

[0059] As can be seen from Table 2, compared with the control group, the body length, body width, and testis weight of rats in each experimental group increased. Among them, the complex group showed the best performance. The body length of rats in the complex group was (23.6 ± 0.19) cm, the body width was (9.09 ± 0.05) cm, and the testis weight was (2.28 ± 0.01) g. It can be seen that after the treatment of the complex group, the rats were improved in terms of body length and body width, which are basic growth indicators, and testis weight, which is a development indicator. The treatment of the complex group showed a synergistic effect, and the growth of rats was accelerated.

[0060] In summary, the complex group showed the best performance: in the water maze test, the latency of the mice in the complex group was (23.8 ± 2.3) s, the health of the mice was improved, and the memory of the mice was enhanced; in the rat growth test, the body length of the rats in the complex group was (23.6 ± 0.19) cm, the body width was (9.09 ± 0.05) cm, and the testis weight was (2.28 ± 0.01) g. The rats were improved in terms of body length and body width as basic growth indicators and testis weight as a development indicator, and the growth of the rats was accelerated. It can be seen that when the postbiogen is combined with the reed root extract to form a complex, the complex exhibits a synergistic effect, demonstrating the effects of improving memory and promoting growth.

[0061] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A strain of Lactobacillus gasseri for improving memory and promoting growth, characterized in that: The Lactobacillus gasseri is Lactobacillus gasseri WZA-LG12, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241508.

2. The use of Lactobacillus gasseri according to claim 1 in preparing a product for improving memory and promoting growth, characterized in that: The number of viable Lactobacillus gasseri in the product is greater than 1×10 8 cfu / mL.

3. A postbiotic of Lactobacillus gasseri, characterized in that The postbiotics of Lactobacillus gasseri are prepared by the following method: The activated Lactobacillus gasseri of claim 1 is inoculated into a fermentation medium for fermentation at a fermentation temperature of 20-50° C. for 5-60 hours; the fermented product is centrifuged and the supernatant is freeze-dried to obtain the postbiotics of Lactobacillus gasseri.

4. The postbiotic of Lactobacillus gasseri according to claim 3, characterized in that The composition of the fermentation medium is as follows: 5-20 parts of peptone, 5-20 parts of beef extract, 2-10 parts of yeast extract, 10-40 parts of glucose, 0.1-1 parts of magnesium sulfate, 2-10 parts of sodium acetate, 1-10 parts of diammonium hydrogen citrate, 1-10 parts of dipotassium hydrogen phosphate, 0.01-0.1 parts of manganese sulfate, 0.5-5 parts of Tween-80, and 800-1200 parts of distilled water.

5. Use of the postbiotics of Lactobacillus gasseri according to claim 3 or 4 in the preparation of products for improving memory and promoting growth, characterized in that: The product for improving memory and promoting growth is prepared by mixing the postbiotics of Lactobacillus gasseri and the extract of Phragmites australis in a mass ratio of (0.5-5):(0.5-5).

6. The use of the postbiotics of Lactobacillus gasseri in preparing products for improving memory and promoting growth according to claim 5, characterized in that: The rhizoma reedii extract is prepared by the following method: Take fresh reed root, clean it, remove buds, fibrous roots and membranous leaves, cut it into pieces and dry it in a 60℃ oven, crush it into 500 mesh and sieve it to obtain reed root powder; extract the reed root powder in 70% v / v ethanol at room temperature with a mass volume ratio of 1g:3ml, each extraction time is 9h, and the extraction is performed 3 times in total; the extracts obtained each time are combined, concentrated under reduced pressure at 60℃, and then dried in a 75℃ oven to obtain reed root extract.

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