CN117384990B - A method for extracting peptidoglycan from lactobacillus based on spatiotemporal variables - Google Patents

Abstract

The invention provides a method for extracting peptidoglycan by lactobacillus based on space-time variable. The method is characterized in that the yield of the peptidoglycan is high and the immunocompetence is strong by increasing the culture temperature of the lactobacillus and increasing the osmotic pressure of a culture medium to induce the lactobacillus to generate more cell walls and purifying and extracting the peptidoglycan.

Description

A method for extracting peptidoglycan from lactobacillus based on spatiotemporal variables

Technical Field

The invention relates to a method for extracting peptidoglycan by lactobacillus, in particular to a method for extracting peptidoglycan by lactobacillus based on time-space variables.

Background technique

Peptidoglycan is a unique component of bacterial cell walls. Its basic structure is composed of N-acetylglucosamine and N-acetylmuramic acid connected by β-1,4 glycosidic bonds, and the sugar chains are cross-linked by peptide chains to form a stable network structure. In recent years, it has been found that peptidoglycan plays an important role in improving the body's immunity and resisting bacterial infections. Due to the complex and diverse structure of peptidoglycan, its specific mechanism of improving the body's immunity has not yet been fully explored.

At present, the preparation method of peptidoglycan mainly involves extracting the cell wall of Gram-positive bacteria, such as lactic acid bacteria, by physical or chemical methods after fermentation and purifying to obtain peptidoglycan. Therefore, it is of great significance to further improve the yield of peptidoglycan preparation and further improve the immunological activity of peptidoglycan.

In view of this, the present invention is proposed.

Summary of the invention

In order to solve the above problems, the present invention provides a method for extracting peptidoglycan from lactobacillus based on spatiotemporal variables. The peptidoglycan prepared by the method has the characteristics of high yield and strong immune activity.

The present application provides a method for extracting peptidoglycan from lactobacillus based on spatiotemporal variables, comprising the following steps:

S1: adjusting the MRS medium to a level of 600-1000 mOsmol/kg in osmotic pressure, inoculating Lactobacillus plantarum at a volume percentage of 1.0-4.0%, and culturing at 37-42° C. for 24-30 hours;

S2: After culturing, centrifuge at 8000-10000 rpm for 10-15 minutes to collect the cells; wash the collected cells with physiological saline, centrifuge and collect the cells, repeat the above washing and centrifugation 1-2 times; wash with ion exchange water, centrifuge and collect the cells;

S3: Take the collected bacteria, mix 1-1.2 g of wet bacteria with 10-12 ml of 10% mass volume ratio trichloroacetic acid solution, boil it in a boiling water bath for 20-30 minutes, centrifuge it at 8000-10000 rpm for 10-15 minutes to collect the precipitate, and then wash the precipitate with sterile distilled water for 2-3 times to obtain precipitate A;

S4: 1-1.2 g of precipitate A was uniformly mixed with 10-12 ml of 40-50 g/L SDS solution, and then the mixture was boiled in a water bath for 20-30 minutes, and the precipitate was collected by centrifugation at 8000-10000 rpm for 10-15 minutes, and then the precipitate was washed 2-3 times with sterile distilled water to obtain precipitate B;

S5: 1-1.2 g of precipitate B was mixed with 0.5-0.7 ml of 1-3 mg/ml trypsin buffer, digested at 30-37°C for 3-5 hours, centrifuged at 4-8°C, 2000-3000 rpm for 2-5 minutes, and the supernatant was collected. The supernatant was centrifuged at 8000-10000 rpm for 10-15 minutes to obtain precipitate C;

S6: suspend the precipitate C in ether, let it stand at 20-30°C for 20-30 minutes, and then centrifuge it at 8000-10000 rpm for 10-15 minutes to obtain the precipitate D; dehydrate the precipitate D with anhydrous ethanol, and obtain the peptidoglycan product after drying at 60-70°C.

Through the above technical scheme, the culture conditions, that is, the temporal and spatial variables of the culture, are controlled to induce the growth and structural changes of the lactic acid bacteria cell wall, increase the production and yield of peptidoglycan, and enhance the immune activity of peptidoglycan.

The above-mentioned lactic acid bacteria and peptidoglycan can be added to food as food excipients to make functional food; peptidoglycan can also be used as a medicine to enhance the body's immune activity.

Detailed ways

During the long-term research and practice, the applicant unexpectedly discovered that by increasing the culture temperature of lactic acid bacteria and increasing the osmotic pressure of the culture medium, the production of peptidoglycan can be increased and the immune activity of peptidoglycan can be enhanced. On this basis, the applicant carried out a systematic study, and the specific research process is as follows:

1. Lactic acid bacteria culture temperature test

This study uses Lactobacillus plantarum as an example. The instruction manual recommends a culture temperature of 30°C for Lactobacillus plantarum (strain number ATCC14917).

1.1 Lactic acid bacteria culture

Plant Lactobacillus (strain number ATCC14917) was cultured independently in MRS medium at 25°C, 30°C, 37°C, 40°C, and 42°C for 24 hours. After culture, the bacteria were collected by centrifugation at 8000rpm for 10 minutes; washed with physiological saline and centrifuged, and the above washing and centrifugation were repeated 2 times; washed with ion exchange water and centrifuged. Samples were taken at this step, and the bacteria were suspended in ion exchange water to prepare a lactic acid bacteria suspension, and the bacterial concentration (bacterial cell number/mL) was measured, and the diaminopimelate content of the bacteria was detected.

1.2 Extraction of peptidoglycan

1.2.1 Removal of teichoic acid

Take the collected bacteria, mix 1 gram of wet bacteria with 10 ml of 10% trichloroacetic acid solution, and place in a boiling water bath for 20 minutes while stirring; centrifuge at 4000 r/min for 10 minutes, and discard the supernatant; finally wash the precipitate with sterile distilled water for 3 times to obtain a precipitate I without teichoic acid;

1.2.2 Protein removal

After uniformly mixing 1 gram of precipitate I with 10 ml of 8g/dl SDS solution, the mixture was placed in a boiling water bath with stirring for 30 minutes; the mixture was centrifuged at 4000 r/min for 30 minutes and the supernatant was discarded; the precipitate was washed 4 times with sterile distilled water; the washed precipitate was mixed with 0.5 ml of 1mg/ml trypsin buffer at 1 gram of weight and shaken at 37°C for 3 hours; after 3 hours, the mixture was centrifuged at 4°C and 3000 r/min for 5 minutes; the supernatant was collected, and the supernatant was centrifuged at 12000 r/min for 15 minutes, and then the precipitate II was collected.

1.2.3 Removal of lipids

The precipitate II was suspended in ether, allowed to stand at room temperature for 30 minutes, and then centrifuged at 12000r/min for 15 minutes to collect the precipitate; dehydrated with anhydrous ethanol, and dried in an oven at 70°C to obtain the peptidoglycan product. The peptidoglycan product was taken for interleukin-12 induction activity determination.

1.3 Detection Method

1.3.1 Diaminopimelic acid measurement method

Diaminopimelic acid is an amino acid that constitutes peptides and cross-links cell wall sugar chains and is an index that reflects the composition of the cell wall.

6 mol/L hydrochloric acid was added to the cells and hydrolyzed at 100°C for 20 hours. The resulting product was then evaporated to dryness using a centrifugal concentrator (produced by Thermo SCIENTIFIC Co.). 0.05 mol/L hydrochloric acid was added to the dried product to a cell concentration of 1 mg dry cells/mL. The resulting product was filtered through a disc filter with a pore size of 0.2 μm and then analyzed by HPLC using a L-8800 Hitachi high-speed amino acid analyzer.

1.3.2 Interleukin-12 induction activity assay

2 mL of 4.05% thioglycolate was intraperitoneally administered to BALB/c mice (8 weeks old, female). Four days later, intraperitoneal macrophages were collected using PBS and adjusted to 2×10 ⁶ cells/mL in RPMI1640 medium containing 10% FBS, and then seeded in a 48-well plate at 0.5 mL per well.

Each well was added with peptidoglycan at 100 μg/mL, and after 24 hours of culture, the interleukin-12 concentration in the culture medium was measured. The active form of interleukin-12 is p70, in which the p35 subunit binds the p40 subunit, so this test measures interleukin-12 (p70). The instrument used to measure interleukin-12 is the OptEIA mouse interleukin-12 measurement kit (produced by BD Pharmingen Co.).

1.4 Results and Analysis

1.4.1 Bacterial concentration

After the culture was completed, the results of the lactobacillus cell concentration were shown in Table 1. The bacterial concentration was the highest at 30°C, which was consistent with the recommended culture temperature.

Table 1 Results of bacterial concentrations cultured at different temperatures

1.4.2 Diaminopimelate content

The results of diaminopimelate measurement are shown in Table 2. When cultured at 30°C, the concentration of diaminopimelate increased to about twice that of 25°C. As the culture temperature increased, the concentration of diaminopimelate increased. Culture at 42°C produced the maximum concentration.

Table 2 Results of diaminopimelate content when cultured at different temperatures

1.4.3 Results of interleukin-12 induction activity assay

The in vitro immunostimulatory activity was evaluated by interleukin-12 induction activity. The results are shown in Table 3. When the culture temperature was 30°C to 42°C, the temperature increased and the induced IL-12 increased.

Table 3 Results of interleukin-12 induction activity assay at different temperatures

1.5 Test conclusion

1.5.1 The optimum culture temperature for bacterial growth in this experiment is 30°C, the optimum culture temperature for producing specific cell wall peptidoglycan is 40°C, and the optimum culture temperature for producing immune activity is 40°C. When the bacteria are at a temperature higher than the optimum production temperature, a stress response is generated, inducing cell wall synthesis, thereby producing more peptidoglycan, and peptidoglycan has a higher immune induction response.

2. Culture test with different osmotic pressures

At the aforementioned culture temperature of 40° C., the culture medium was adjusted to different osmotic pressures, and the culture was carried out to examine the effect of osmotic pressure on the induction of lactic acid bacteria peptidoglycan.

2.1 Lactic acid bacteria culture

Use sorbitol to adjust the MRS medium so that its osmotic pressure reaches 500mOsmol/kg, 600mOsmol/kg, 800mOsmol/kg, and 1000mOsmol/kg, respectively. Together with the unadjusted MRS medium (osmotic pressure of 430mOsmol/kg), add plant lactobacillus (strain number ATCC14917), and culture them independently at 40°C for 24 hours. After cultivation, collect the bacteria by centrifugation at 8000rpm for 10 minutes; wash and centrifuge with physiological saline, and repeat the above washing and centrifugation twice; wash and centrifuge with ion exchange water. Take samples at this step, suspend the bacteria in ion exchange water to prepare a lactic acid bacteria suspension, measure the bacterial concentration (bacterial cell number/mL), and detect the diaminopimelic acid content of the bacteria.

2.2 Extraction of peptidoglycan

2.2.1 Removal of teichoic acid

Take the collected bacteria, mix 1 gram of wet bacteria with 10 ml of 10% trichloroacetic acid solution, and boil it in boiling water for 20 minutes while stirring; centrifuge at 4000 r/min for 10 minutes, discard the supernatant; finally, wash the precipitate with sterile distilled water for 3 times to obtain precipitate I without teichoic acid.

2.2.2 Protein removal

After uniformly mixing 1 gram of precipitate I with 10 ml of 8g/dl SDS solution, the mixture was placed in a boiling water bath with stirring for 30 minutes; the mixture was centrifuged at 4000 r/min for 30 minutes and the supernatant was discarded; the precipitate was washed 4 times with sterile distilled water; the washed precipitate was mixed with 0.5 ml of 1mg/ml trypsin buffer at 1 gram of weight and shaken at 37°C for 3 hours; after 3 hours, the mixture was centrifuged at 4°C and 3000 r/min for 5 minutes; the supernatant was collected, and the supernatant was centrifuged at 12000 r/min for 15 minutes, and then the precipitate II was collected.

2.2.3 Removal of lipids

The precipitate II was suspended in ether, allowed to stand at room temperature for 30 minutes, and then centrifuged at 12000r/min for 15 minutes to collect the precipitate; dehydrated with anhydrous ethanol, and dried in an oven at 70°C to obtain the peptidoglycan product. The peptidoglycan product was taken for interleukin-12 induction activity determination.

2.3 Detection methods

The measurement method of diaminopimelane acid and the determination of interleukin-12 induction activity are the same as those of the “Lactic acid bacteria culture temperature test”.

2.4 Results and Analysis

2.4.1 Bacterial concentration

After the culture was completed, the results of the lactobacillus cell concentration were shown in Table 4. When the osmotic pressure of the culture medium was 500 mOsmol/kg, the bacterial concentration was the highest.

Table 4 Results of bacterial concentration when cultured at different osmotic pressures

2.4.2 Diaminopimelate content

The measurement results of diaminopimelic acid are shown in Table 5. When the osmotic pressure of the culture medium was 750 mOsmol/kg, diaminopimelic acid produced the maximum concentration, which was 1.22 times that of 430 mOsmol/kg.

Table 5 Results of diaminopimelate content during culture at different osmotic pressures

2.4.3 Results of interleukin-12 induction activity assay

The in vitro immunostimulatory activity was evaluated by interleukin-12 induction activity, and the results are shown in Table 6. When the osmotic pressure of the culture medium was 750 mOsmol/kg, the induced production of IL-12 was the highest.

Table 6 Results of interleukin-12 induction activity assay at different temperatures

2.5 Experimental conclusion

In this experiment, the optimal osmotic pressure for producing cell wall peptidoglycan was 800mOsmol/kg, and the optimal osmotic pressure for producing immune activity was 800mOsmol/kg. When the bacteria are in a higher osmotic pressure environment, a stress response is produced, inducing cell wall synthesis, thereby producing more peptidoglycan, and peptidoglycan has a higher immune induction response.

Based on the above research results, the applicant verified the technical solution according to the research results. The verification results are shown in Examples 1 to 3, and compared and analyzed with Comparative Examples 1 to 3.

Example 1

S1: Adjust the MRS medium to a level of 600 mOsmol/kg in osmotic pressure, inoculate Lactobacillus plantarum at a volume percentage of 1.0% on the MRS medium, and culture at 37° C. for 24 hours;

S2: After culturing, the cells were collected by centrifugation at 8000 rpm for 10 minutes; the collected cells were washed with physiological saline, centrifuged and collected, and the above washing and centrifugation were repeated twice; the cells X were washed with ion exchange water and centrifuged;

S3: Take the collected bacteria, mix 1 gram of wet bacteria with 10 ml of 10% mass volume ratio trichloroacetic acid solution, boil it in a water bath for 20 minutes, centrifuge it at 8000 rpm for 10 minutes to collect the precipitate, and then wash the precipitate twice with sterile distilled water to obtain precipitate A;

S4: After 1 gram of precipitate A was uniformly mixed with 10 ml of 40 g/L SDS solution, the mixture was placed in a boiling water bath for 20 minutes, and the precipitate was collected by centrifugation at 8000 rpm for 10 minutes. The precipitate was then washed twice with sterile distilled water to obtain precipitate B.

S5: 1 g of precipitate B was mixed with 0.5 ml of 1 mg/ml trypsin buffer, digested at 30°C for 3 hours, centrifuged at 2000 rpm for 2 minutes at 4°C, and the supernatant was collected. The supernatant was centrifuged at 8000 rpm for 10 minutes to obtain precipitate C;

S6: The precipitate C was suspended in ether, allowed to stand at 20°C for 20 minutes, and then centrifuged at 8000 rpm for 10 minutes to obtain the precipitate D; the precipitate D was dehydrated with anhydrous ethanol, and the peptidoglycan product Y was obtained after drying at 60°C.

According to the peptidoglycan production, the peptidoglycan yield per 100 g of bacteria was calculated. The peptidoglycan yield calculation method was: weight of peptidoglycan X obtained in step S6 (g) × 100 g/weight of bacteria Y obtained in step S2 (g).

The peptidoglycan product was taken and the interleukin-12 induction activity was determined by the same method as that used in the research process, which will not be described again.

The results of the peptidoglycan yield and interleukin-12 induction activity assay are shown in Table 7.

Example 2

S1: Adjust the MRS medium to a level where the osmotic pressure reaches 1000 mOsmol/kg, inoculate Lactobacillus plantarum at a volume percentage of 4.0% on the MRS medium, and culture at 42° C. for 30 hours;

S2: After culturing, the cells were collected by centrifugation at 10,000 rpm for 15 minutes; the collected cells were washed with physiological saline, centrifuged and collected, and the above washing and centrifugation were repeated twice; the cells X were washed with ion exchange water and centrifuged;

S3: Take the collected bacteria, mix 1.2 g of wet bacteria with 12 ml of 10% mass volume ratio trichloroacetic acid solution, boil in a water bath for 30 minutes, centrifuge at 10000 rpm for 15 minutes to collect the precipitate, and then wash the precipitate with sterile distilled water for 3 times to obtain precipitate A;

S4: 1.2 g of precipitate A was uniformly mixed with 12 ml of 50 g/L SDS solution, and then the mixture was placed in a boiling water bath for 30 minutes. The precipitate was collected by centrifugation at 10,000 rpm for 15 minutes, and then washed three times with sterile distilled water to obtain precipitate B.

S5: 1.2 g of precipitate B was mixed with 0.7 ml of 3 mg/ml trypsin buffer, digested at 37°C for 5 hours, centrifuged at 8°C, 3000 rpm for 5 minutes, and the supernatant was collected. The supernatant was centrifuged at 10000 rpm for 15 minutes to obtain precipitate C;

S6: The precipitate C was suspended in ether, allowed to stand at 30°C for 30 minutes, and then centrifuged at 10,000 rpm for 15 minutes to obtain the precipitate D; the precipitate D was dehydrated with anhydrous ethanol, and the peptidoglycan product Y was obtained after drying at 70°C.

According to the peptidoglycan production, the peptidoglycan yield per 100 g of bacteria was calculated. The peptidoglycan yield calculation method was: weight of peptidoglycan X obtained in step S6 (g) × 100 g/weight of bacteria Y obtained in step S2 (g).

The peptidoglycan product was taken and the interleukin-12 induction activity was determined by the same method as that used in the research process, which will not be described in detail here.

The results of the peptidoglycan yield and interleukin-12 induction activity assay are shown in Table 7.

Example 3

S1: Adjust the MRS medium to a level of 800 mOsmol/kg in osmotic pressure, inoculate Lactobacillus plantarum at a volume percentage of 3.0% on the MRS medium, and culture at 40° C. for 28 hours;

S2: After culturing, the cells were collected by centrifugation at 9000 rpm for 13 minutes; the collected cells were washed with physiological saline, centrifuged and collected, and the above washing and centrifugation were repeated twice; the cells X were washed with ion exchange water and centrifuged;

S3: Take the collected bacteria, mix 1.1 g of wet bacteria with 11 ml of 10% mass volume ratio trichloroacetic acid solution, boil in a boiling water bath for 25 minutes, centrifuge at 9000 rpm for 12 minutes to collect the precipitate, and then wash the precipitate with sterile distilled water for 3 times to obtain precipitate A;

S4: 1.1 g of precipitate A was uniformly mixed with 11 ml of 45 g/L SDS solution, and then the mixture was boiled in a water bath for 25 minutes, and the precipitate was collected by centrifugation at 9000 rpm for 13 minutes, and then washed with sterile distilled water for 3 times to obtain precipitate B;

S5: 1.1 g of precipitate B was mixed with 0.6 ml of 2 mg/ml trypsin buffer, digested at 35°C for 4 hours, centrifuged at 6°C, 2500 rpm for 3 minutes, and the supernatant was collected. The supernatant was centrifuged at 9000 rpm for 12 minutes to obtain precipitate C;

S6: The precipitate C was suspended in ether, allowed to stand at 25°C for 25 minutes, and then centrifuged at 9000 rpm for 13 minutes to obtain the precipitate D; the precipitate D was dehydrated with anhydrous ethanol, and the peptidoglycan product Y was obtained after drying at 65°C.

According to the peptidoglycan production, the peptidoglycan yield per 100 g of bacteria was calculated. The peptidoglycan yield calculation method was: weight of peptidoglycan X obtained in step S6 (g) × 100 g/weight of bacteria Y obtained in step S2 (g).

The peptidoglycan product was taken and the interleukin-12 induction activity was determined by the same method as that used in the research process, which will not be described again.

The results of the peptidoglycan yield and interleukin-12 induction activity assay are shown in Table 7.

Comparative Example 1

S1: using MRS medium, inoculating Lactobacillus plantarum at a volume percentage of 1.0% on the MRS medium, and culturing at 30°C for 24 hours;

S2: After culturing, the cells were collected by centrifugation at 8000 rpm for 10 minutes; the collected cells were washed with physiological saline, centrifuged and collected, and the above washing and centrifugation were repeated twice; the cells X were washed with ion exchange water and centrifuged;

S3: Take the collected bacteria, mix 1 gram of wet bacteria with 10 ml of 10% mass volume ratio trichloroacetic acid solution, boil it in a water bath for 20 minutes, centrifuge it at 8000 rpm for 10 minutes to collect the precipitate, and then wash the precipitate twice with sterile distilled water to obtain precipitate A;

S4: After 1 gram of precipitate A was uniformly mixed with 10 ml of 40 g/L SDS solution, the mixture was placed in a boiling water bath for 20 minutes, and the precipitate was collected by centrifugation at 8000 rpm for 10 minutes. The precipitate was then washed twice with sterile distilled water to obtain precipitate B.

S5: 1 g of precipitate B was mixed with 0.5 ml of 1 mg/ml trypsin buffer, digested at 30°C for 3 hours, centrifuged at 2000 rpm for 2 minutes at 4°C, and the supernatant was collected. The supernatant was centrifuged at 8000 rpm for 10 minutes to obtain precipitate C;

S6: The precipitate C was suspended in ether, allowed to stand at 20°C for 20 minutes, and then centrifuged at 8000 rpm for 10 minutes to obtain the precipitate D; the precipitate D was dehydrated with anhydrous ethanol, and the peptidoglycan product Y was obtained after drying at 60°C.

According to the peptidoglycan production, the peptidoglycan yield per 100 g of bacteria was calculated. The peptidoglycan yield calculation method was: weight of peptidoglycan X obtained in step S6 (g) × 100 g/weight of bacteria Y obtained in step S2 (g).

The peptidoglycan product was taken and the interleukin-12 induction activity was determined by the same method as that used in the research process, which will not be described again.

The results of the peptidoglycan yield and interleukin-12 induction activity assay are shown in Table 7.

Comparative Example 2

S1: using MRS medium, inoculating Lactobacillus plantarum at a volume percentage of 4.0% on the MRS medium, and culturing at 30°C for 30 hours;

S2: After culturing, the cells were collected by centrifugation at 10,000 rpm for 15 minutes; the collected cells were washed with physiological saline, centrifuged and collected, and the above washing and centrifugation were repeated twice; the cells X were washed with ion exchange water and centrifuged;

S3: Take the collected bacteria, mix 1.2 g of wet bacteria with 12 ml of 10% mass volume ratio trichloroacetic acid solution, boil in a water bath for 30 minutes, centrifuge at 10000 rpm for 15 minutes to collect the precipitate, and then wash the precipitate with sterile distilled water for 3 times to obtain precipitate A;

S4: 1.2 g of precipitate A was uniformly mixed with 12 ml of 50 g/L SDS solution, and then the mixture was placed in a boiling water bath for 30 minutes. The precipitate was collected by centrifugation at 10,000 rpm for 15 minutes, and then washed three times with sterile distilled water to obtain precipitate B.

S5: 1.2 g of precipitate B was mixed with 0.7 ml of 3 mg/ml trypsin buffer, digested at 37°C for 5 hours, centrifuged at 8°C, 3000 rpm for 5 minutes, and the supernatant was collected. The supernatant was centrifuged at 10000 rpm for 15 minutes to obtain precipitate C;

S6: The precipitate C was suspended in ether, allowed to stand at 30°C for 30 minutes, and then centrifuged at 10,000 rpm for 15 minutes to obtain the precipitate D; the precipitate D was dehydrated with anhydrous ethanol, and the peptidoglycan product Y was obtained after drying at 70°C.

According to the peptidoglycan production, the peptidoglycan yield per 100 g of bacteria was calculated. The peptidoglycan yield calculation method was: weight of peptidoglycan X obtained in step S6 (g) × 100 g/weight of bacteria Y obtained in step S2 (g).

The peptidoglycan product was taken and the interleukin-12 induction activity was determined by the same method as that used in the research process, which will not be described again here.

The results of the peptidoglycan yield and interleukin-12 induction activity assay are shown in Table 7.

Comparative Example 3

S1: using MRS medium, inoculating Lactobacillus plantarum at an inoculum rate of 3.0% by volume onto the MRS medium, and culturing at 30° C. for 28 hours;

S2: After culturing, the cells were collected by centrifugation at 9000 rpm for 13 minutes; the collected cells were washed with physiological saline, centrifuged and collected, and the above washing and centrifugation were repeated twice; the cells X were washed with ion exchange water and centrifuged;

S3: Take the collected bacteria, mix 1.1 g of wet bacteria with 11 ml of 10% mass volume ratio trichloroacetic acid solution, boil in a boiling water bath for 25 minutes, centrifuge at 9000 rpm for 12 minutes to collect the precipitate, and then wash the precipitate with sterile distilled water for 3 times to obtain precipitate A;

S4: 1.1 g of precipitate A was uniformly mixed with 11 ml of 45 g/L SDS solution, and then the mixture was placed in a boiling water bath for 25 minutes, and the precipitate was collected by centrifugation at 9000 rpm for 13 minutes. The precipitate was then washed three times with sterile distilled water to obtain precipitate B.

S5: 1.1 g of precipitate B was mixed with 0.6 ml of 2 mg/ml trypsin buffer, digested at 35°C for 4 hours, centrifuged at 6°C, 2500 rpm for 3 minutes, and the supernatant was collected. The supernatant was centrifuged at 9000 rpm for 12 minutes to obtain precipitate C;

S6: The precipitate C was suspended in ether, allowed to stand at 25°C for 25 minutes, and then centrifuged at 9000 rpm for 13 minutes to obtain the precipitate D; the precipitate D was dehydrated with anhydrous ethanol, and the peptidoglycan product Y was obtained after drying at 65°C.

According to the peptidoglycan production, the peptidoglycan yield per 100 g of bacteria was calculated. The peptidoglycan yield calculation method was: weight of peptidoglycan X obtained in step S6 (g) × 100 g/weight of bacteria Y obtained in step S2 (g).

The peptidoglycan product was taken and the interleukin-12 induction activity was determined by the same method as that used in the research process, which will not be described again here.

The results of the peptidoglycan yield and interleukin-12 induction activity assay are shown in Table 7.

The results of the embodiments and comparative examples were statistically analyzed using the one-way analysis of variance method in the EXCEL document. The results showed that the p-values of the peptidoglycan yield (g) per 100g of bacterial cells and the interleukin-12 induction activity were both <0.05, which were significantly different, further confirming that the technical scheme of the present invention can significantly improve the peptidoglycan yield and the immune activity of peptidoglycan.

Table 7 Statistical analysis of the results of the embodiments and comparative examples

Claims (8)

1. A method for extracting peptidoglycan from lactobacillus based on spatiotemporal variables, comprising the steps of: S1: using sorbitol to adjust the MRS medium to a level of 600-1000 mOsmol/kg, inoculating Lactobacillus plantarum ATCC14917 at a volume percentage of 1.0-4.0% on the MRS medium, with a culture temperature of 40-42°C and a culture time of 24-30 hours; S2: After culturing, the cells are collected at a centrifugal speed of 8000-10000 rpm and a centrifugal time of 10-15 minutes; the collected cells are washed with physiological saline, centrifuged and collected, and the above washing and centrifugation are repeated 1-2 times; the cells are washed with ion exchange water and centrifuged to collect; S3: Take the collected bacteria, mix 1-1.2 g of wet bacteria with 10-12 ml of 10% mass volume ratio trichloroacetic acid solution, boil it in a boiling water bath for 20-30 minutes, centrifuge it at 8000-10000 rpm for 10-15 minutes to collect the precipitate, and then wash the precipitate with sterile distilled water for 2-3 times to obtain precipitate A; S4: 1-1.2 g of precipitate A was uniformly mixed with 10-12 ml of 40-50 g/L SDS solution, and then the mixture was boiled in a water bath for 20-30 minutes. The precipitate was collected by centrifugation at 8000-10000 rpm for 10-15 minutes, and then washed with sterile distilled water for 2-3 times to obtain precipitate B. S5: Mix the precipitate B with trypsin buffer in a ratio of 1-1.2 g precipitate B: 0.5-0.7 ml trypsin buffer, the concentration of the trypsin buffer is 1-3 mg/ml, the reaction temperature is 30-37° C., the reaction time is 3-5 hours, centrifuge at 4-8° C., 2000-3000 rpm for 2-5 minutes, collect the supernatant, centrifuge the supernatant at 8000-10000 rpm for 10-15 minutes, and obtain precipitate C; S6: suspend the precipitate C in ether, let it stand at 20-30°C for 20-30 minutes, and then centrifuge it at 8000-10000 rpm for 10-15 minutes to obtain the precipitate D; dehydrate the precipitate D with anhydrous ethanol, and obtain the peptidoglycan product after drying at 60-70°C. 2. The method for extracting peptidoglycan from lactobacillus according to claim 1, characterized in that the culture temperature in step S1 is 40°C. 3. the method for extracting peptidoglycan from lactobacillus according to claim 2 is characterized in that, the MRS culture medium in adjustment step S1 is adjusted so that its osmotic pressure reaches 800mOsmol/kg. 4. The method for extracting peptidoglycan from lactobacillus according to claim 3, wherein the incubation time in step S1 is 28 hours. 5. The method for extracting peptidoglycan from lactobacillus according to claim 4, characterized in that the inoculum amount in step S1 is 3.0% by volume. 6. The method for extracting peptidoglycan from lactobacillus according to claim 5, wherein the centrifugal speed for collecting the thalline in step S2 is 9000 rpm and the thalline is centrifuged for 13 minutes. 7. The method for extracting peptidoglycan from lactobacillus according to claim 6, characterized in that the mixing ratio in step S5 is 1.1 g of precipitate B: 0.6 ml of trypsin buffer, and the concentration of the trypsin buffer is 2 mg/ml. 8. The method for extracting peptidoglycan from lactobacillus according to claim 7, characterized in that the reaction temperature in step S5 is 35°C and the reaction time is 4 hours.