CN117384990B - Method for extracting peptidoglycan by lactobacillus based on space-time variable - Google Patents
Method for extracting peptidoglycan by lactobacillus based on space-time variable Download PDFInfo
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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
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 space-time variable.
Background
Peptidoglycans are characteristic components of bacterial cell walls, the basic structure of which is formed by connecting N-Acetylglucosamine (N-Acetylglucosamine) and N-acetylmuramic acid (N-acetylmuramic acid) through beta-1, 4 glycosidic bonds, and the sugar chains are crosslinked by peptide chains to form a stable network structure. In recent years, peptidoglycans have been found to play an important role in enhancing the immunity of the body and combating bacterial infections. Because of the complex and diverse structures of peptidoglycans, the specific mechanisms of enhancing the immunity of the organism are not completely explored.
At present, the preparation method of the peptidoglycan mainly comprises the steps of fermenting and culturing gram positive bacteria such as lactobacillus, extracting cell walls of the bacteria by a physical or chemical method, and purifying to obtain the peptidoglycan. Therefore, the preparation yield of the peptidoglycan is further improved, and the immunocompetence of the peptidoglycan is further improved.
In view of this, the present invention has been made.
Disclosure of Invention
In order to solve the problems, the invention provides a method for extracting peptidoglycan by lactobacillus based on space-time variable, and the peptidoglycan prepared by the method has the characteristics of high yield and strong immunocompetence.
The application provides a method for extracting peptidoglycan by lactobacillus based on space-time variables, which comprises the following steps:
s1: regulating MRS culture medium to make its osmotic pressure reach 600-1000 mOsmol/kg, inoculating lactobacillus plantarum according to the inoculum size of 1.0-4.0% by volume percentage to MRS culture medium, culturing at 37-42 deg.C for 24-30 hr;
s2: centrifuging at 8000-10000 rpm for 10-15 min after culturing to collect thalli; washing the collected thalli with physiological saline, centrifuging and collecting thalli, and repeating the washing and centrifuging for 1-2 times; washing with ion exchange water, and centrifugally collecting thalli;
s3: uniformly mixing 1-1.2 g of collected thalli with 10-12 ml of trichloroacetic acid solution with the mass volume ratio of 10%, carrying out boiling water bath for 20-30 minutes, centrifuging at 8000-10000 rpm for 10-15 minutes, collecting precipitate, and washing the precipitate with sterilized distilled water for 2-3 times to obtain precipitate A;
s4: uniformly mixing 1-1.2 g of precipitate A with 10-12 ml of 40-50 g/L SDS solution, carrying out boiling water bath for 20-30 minutes, centrifuging at 8000-10000 rpm for 10-15 minutes, collecting precipitate, and washing the precipitate with sterilized distilled water for 2-3 times to obtain precipitate B;
s5: mixing 1-1.2 g of precipitate B with 0.5-0.7 ml of 1-3 mg/ml trypsin buffer solution uniformly, digesting for 3-5 hours at 30-37 ℃, centrifuging at 4-8 ℃ and 2000-3000 rpm for 2-5 minutes, collecting supernatant, and centrifuging the supernatant at 8000-10000 rpm for 10-15 minutes to obtain precipitate C;
s6: suspending the precipitate C in diethyl ether, standing for 20-30 min at 20-30 ℃, and centrifuging at 8000-10000 rpm for 10-15 min to obtain precipitate D; and dehydrating the precipitate D by using absolute ethyl alcohol, and drying at 60-70 ℃ to obtain a peptidoglycan product.
By the technical scheme, culture conditions, namely culture space-time variables, are controlled, the cell wall growth and structural change of the lactic acid bacteria are induced, the yield and the yield of the peptidoglycan are improved, and the immunocompetence of the peptidoglycan is enhanced.
The lactobacillus and peptidoglycan can be used as food auxiliary materials to be added into food to prepare functional food; the peptidoglycan can also be used as a medicine to improve the immune activity of the organism.
Detailed Description
In the long-term research and practice process, the applicant unexpectedly found that the yield of peptidoglycan can be improved and the immunocompetence of peptidoglycan can be enhanced by increasing the culture temperature of lactic acid bacteria and increasing the osmotic pressure of a culture medium. On this basis, the applicant has conducted systematic studies, the specific course of which is as follows:
1. lactic acid bacteria culture temperature test
The present study was conducted using lactobacillus plantarum as an example. Lactobacillus plantarum (strain number ATCC 14917), the specification recommends a culture temperature of 30 ℃.
1.1 lactic acid bacteria culture
Lactobacillus plantarum (strain No. ATCC 14917) was independently cultured in MRS medium at 25℃at 30℃at 37℃at 40℃at 42℃for 24 hours. After incubation, bacteria were collected by centrifugation at 8000rpm for 10 minutes; washing with physiological saline, centrifuging, and repeating the washing and centrifuging for 2 times; washing with ion-exchanged water, and centrifuging. In this step, a sample was taken, and bacteria were suspended in ion-exchanged water to prepare a lactic acid bacteria suspension, and the concentration of bacteria (number of bacterial cells/mL) was measured to measure the diaminopimelic acid content of the cells.
1.2 extraction of peptidoglycans
1.2.1 removal of teichoic acid
Uniformly mixing 1 g of collected thallus and 10 ml of 10% trichloroacetic acid solution by volume, and stirring while boiling water for 20min; centrifuging at 4000r/min for 10min, and discarding the supernatant; finally, washing the precipitate with sterilized distilled water for 3 times to obtain a precipitate I without teichoic acid;
1.2.2 removal of protein
Uniformly mixing 1 g of precipitate I with 10 ml of 8g/dl SDS solution by volume, and stirring while boiling water for 30min; centrifuging at 4000r/min for 30min, and discarding the supernatant; washing the precipitate with sterilized distilled water for 4 times; uniformly mixing the washed precipitate with 0.5 milliliter volume of 1mg/ml trypsin buffer solution according to 1 gram weight part, and oscillating for 3 hours at 37 ℃; centrifuging at 4deg.C and 3000r/min for 5min after 3 hr; the supernatant was collected and after centrifugation at 12000r/min for 15min, precipitate II was collected.
1.2.3 removal of lipid
Suspending the precipitate II in diethyl ether, standing at room temperature for 30min, centrifuging at 12000r/min for 15min, and collecting precipitate; dehydrating with absolute ethanol, and drying in oven at 70deg.C to obtain peptidoglycan product. And taking the peptidoglycan product, and measuring the interleukin-12 induction activity.
1.3 detection method
1.3.1 diaminopimelic acid measurement method
Diaminopimelic acid is an amino acid that constitutes a peptide and crosslinks the cell wall sugar chain, and belongs to an index reflecting the cell wall composition.
6mol/L hydrochloric acid was added to the cells, and the cells were hydrolyzed at 100℃for 20 hours. The resulting product was then evaporated to dryness using a centrifugal concentrator (manufactured by Thermo SCIENTIFIC co.). To the dried product, 0.05mol/L hydrochloric acid was added to bring the cell concentration to 1mg of dried cells/mL. The resulting product was filtered through a disc filter having a pore size of 0.2 μm and then analyzed by HPLC treatment using an L-8800 Hitachi high-speed amino acid analyzer.
1.3.2 determination of Interleukin-12 Induction Activity
BALB/c mice (8 week old, female) were given 2mL 4.05% thioglycolate intraperitoneally. Four days later, intraperitoneal macrophages were collected with PBS and conditioned to 2X 10 in RPMI1640 medium containing 10% FBS 6 Individual cells/mL, then seeded in 48-well plates at 0.5mL per well.
The concentration of interleukin-12 in the medium was measured after culturing for 24 hours by adding peptidoglycan per well at 100. Mu.g/mL. The active form of interleukin-12 is p70, where the p35 subunit binds to the p40 subunit, so this assay measures interleukin-12 (p 70). The instrument used for measuring interleukin-12 is an OptEIA mouse interleukin-12 measuring kit (manufactured by BD Pharmingen co.).
1.4 results and analysis
1.4.1 cell concentration
After the completion of the culture, the results of the concentration of Lactobacillus cells are shown in Table 1. The highest concentration of bacteria at 30℃was consistent with the recommended incubation temperature.
TABLE 1 results of cell concentration in different temperature cultures
1.4.2 diaminopimelic acid content
The diaminopimelic acid measurements are shown in Table 2. When incubated at 30℃the concentration of diaminopimelic acid increased to about twice that at 25 ℃. As the culture temperature increases, the concentration of diaminopimelic acid increases. Incubation at 42℃produced the maximum concentration.
TABLE 2 results of diaminopimelic acid content in cultures at different temperatures
1.4.3 results of Interleukin-12 Induction Activity assay
In vitro immunostimulatory activity was assessed by interleukin-12 induction and the results are shown in Table 3, with elevated temperatures at 30℃to 42℃in culture, inducing an increase in IL-12 produced.
TABLE 3 results of measurement of interleukin-12 Induction Activity at different temperature culture
1.5 test conclusion
1.5.1 the culture temperature optimum for the growth of the cells in this test was 30℃and for the production of a specific cell wall peptidoglycan was 40℃and for the production of an immunocompetence was 40 ℃. When the temperature of the thalli is higher than the optimal production temperature of the thalli, stress reaction is generated, and cell wall synthesis is induced, so that more peptidoglycan is generated, and the peptidoglycan has higher immune induction reaction.
2. Differential osmotic culture assays
At the culture temperature of 40 ℃, the culture medium is regulated to different osmotic pressures, and the culture is carried out, so that the induction condition of the osmotic pressure on the lactic acid bacteria peptidoglycan is examined.
2.1 lactic acid bacteria culture
The MRS medium was adjusted with sorbitol so that the osmotic pressure thereof became 500mOsmol/kg, 600mOsmol/kg, 800mOsmol/kg, 1000mOsmol/kg, respectively, together with the unregulated MRS medium (osmotic pressure: 430 mOsmol/kg), and Lactobacillus plantarum (strain No. ATCC 14917) was added thereto, and then cultured independently at 40℃for 24 hours, respectively. After incubation, bacteria were collected by centrifugation at 8000rpm for 10 minutes; washing with physiological saline, centrifuging, and repeating the washing and centrifuging for 2 times; washing with ion-exchanged water, and centrifuging. In this step, a sample was taken, and bacteria were suspended in ion-exchanged water to prepare a lactic acid bacteria suspension, and the concentration of bacteria (number of bacterial cells/mL) was measured to measure the diaminopimelic acid content of the cells.
2.2 extraction of peptidoglycans
2.2.1 removal of teichoic acid
Uniformly mixing 1 g of collected thallus and 10 ml of 10% trichloroacetic acid solution by volume, and stirring while boiling water for 20min; centrifuging at 4000r/min for 10min, and discarding the supernatant; finally, washing the precipitate with sterilized distilled water for 3 times to obtain a precipitate I without teichoic acid.
2.2.2 removal of protein
Uniformly mixing 1 g of precipitate I with 10 ml of 8g/dl SDS solution by volume, and stirring while boiling water for 30min; centrifuging at 4000r/min for 30min, and discarding the supernatant; washing the precipitate with sterilized distilled water for 4 times; uniformly mixing the washed precipitate with 0.5 milliliter volume of 1mg/ml trypsin buffer solution according to 1 gram weight part, and oscillating for 3 hours at 37 ℃; centrifuging at 4deg.C and 3000r/min for 5min after 3 hr; the supernatant was collected and after centrifugation at 12000r/min for 15min, precipitate II was collected.
2.2.3 removal of lipid
Suspending the precipitate II in diethyl ether, standing at room temperature for 30min, centrifuging at 12000r/min for 15min, and collecting precipitate; dehydrating with absolute ethanol, and drying in oven at 70deg.C to obtain peptidoglycan product. And taking the peptidoglycan product, and measuring the interleukin-12 induction activity.
2.3 detection method
The diaminopimelic acid measurement method and the interleukin-12 induction activity measurement are the same as those of the "lactobacillus culture temperature test".
2.4 results and analysis
2.4.1 cell concentration
After the completion of the culture, the results of the lactobacillus cell concentration are shown in Table 4. The bacteria concentration was highest when the medium osmotic pressure was 500 mOsmol/kg.
TABLE 4 results of cell concentration at different osmotic pressure cultures
2.4.2 diaminopimelic acid content
The diaminopimelic acid measurement results are shown in Table 5. When the osmotic pressure of the culture medium is 750mOsmol/kg, the maximum concentration of diaminopimelic acid is generated, and the concentration is 1.22 times that of 430 mOsmol/kg.
TABLE 5 results of diaminopimelic acid content in different osmotically cultured cells
2.4.3 results of Interleukin-12 Induction Activity assay
The in vitro immunostimulatory activity was evaluated by interleukin-12 induction and the results are shown in Table 6. IL-12 induction was greatest when the medium osmotic pressure was 750 mOsmol/kg.
TABLE 6 results of measurement of interleukin-12 Induction Activity at different temperature cultures
2.5 test conclusion
In this test, the optimum osmotic pressure for producing cell wall peptidoglycan was 800mOsmol/kg, and the optimum osmotic pressure for producing immunological activity was 800mOsmol/kg. When the thalli are in a higher osmotic pressure environment, stress reaction is generated, and cell wall synthesis is induced, so that more peptidoglycan is generated, and the peptidoglycan has higher immune induction reaction.
Based on the above-mentioned results, the applicant has verified the technical scheme according to the results of the study, and the verification results are shown in examples 1 to 3, and comparative analysis is performed with comparative examples 1 to 3.
Example 1
S1: regulating MRS culture medium to make osmotic pressure reach 600mOsmol/kg, inoculating lactobacillus plantarum with 1.0% of inoculation amount by volume percent to MRS culture medium, and culturing at 37deg.C for 24 hr;
s2: after the culture, the cells were collected by centrifugation at 8000rpm for 10 minutes; washing the collected thalli with physiological saline, centrifuging and collecting thalli, and repeating the washing and centrifuging for 2 times; washing with ion exchange water, and centrifugally collecting thalli X;
s3: uniformly mixing 1 g of the collected thalli with 10 ml of trichloroacetic acid solution with the mass volume ratio of 10%, carrying out boiling water bath for 20 minutes, centrifuging at 8000rpm for 10 minutes, collecting precipitate, and washing the precipitate with sterilized distilled water for 2 times to obtain precipitate A;
s4: uniformly mixing 1 g of precipitate A with 10 ml of 40g/L SDS solution, carrying out boiling water bath for 20 minutes, centrifuging at 8000rpm for 10 minutes, collecting precipitate, and washing the precipitate with sterilized distilled water for 2 times to obtain precipitate B;
s5: mixing 1 g of precipitate B with 0.5ml of 1mg/ml trypsin buffer, digesting for 3 hours at 30 ℃, centrifuging at 4 ℃ and 2000rpm for 2 minutes, collecting supernatant, centrifuging at 8000rpm for 10 minutes, and obtaining precipitate C;
s6: suspending the precipitate C in diethyl ether, standing at 20deg.C for 20min, and centrifuging at 8000rpm for 10min to obtain precipitate D; the precipitate D was dehydrated with absolute ethanol and dried at 60℃to obtain the peptidoglycan product Y.
According to the yield of the peptidoglycan, the yield of the peptidoglycan of each 100g of thalli is calculated, and the method for calculating the yield of the peptidoglycan comprises the following steps: the weight (g) of peptidoglycan X obtained in step S6. Times.100 g/weight (g) of cell Y obtained in step S2.
The peptidoglycan product was assayed for interleukin-12 induction activity in the same manner as in the study, and no further description is given here.
The results of the peptidoglycan yield and interleukin-12 induction activity measurements are shown in Table 7.
Example 2
S1: regulating MRS culture medium to make osmotic pressure reach 1000mOsmol/kg, inoculating lactobacillus plantarum with inoculum size of 4.0% by volume percent to MRS culture medium, and culturing at 42 deg.C for 30 hr;
s2: after culturing, the cells were collected by centrifugation at 10000rpm for 15 minutes; washing the collected thalli with physiological saline, centrifuging and collecting thalli, and repeating the washing and centrifuging for 2 times; washing with ion exchange water, and centrifugally collecting thalli X;
s3: uniformly mixing 1.2 g of the collected thalli with 12 ml of trichloroacetic acid solution with the mass volume ratio of 10%, carrying out boiling water bath for 30 minutes, centrifuging at 10000rpm for 15 minutes, collecting precipitate, and washing the precipitate 3 times with sterilized distilled water to obtain precipitate A;
s4: after 1.2 g of precipitate A was uniformly mixed with 12 ml of 50g/L SDS solution, the mixture was subjected to boiling water bath for 30 minutes, centrifuged at 10000rpm for 15 minutes, and the precipitate was collected and washed 3 times with sterilized distilled water to obtain precipitate B;
s5: mixing 1.2 g of precipitate B with 0.7 ml of trypsin buffer of 3mg/ml, digesting for 5 hours at 37 ℃, centrifuging at 8 ℃ and 3000rpm for 5 minutes, collecting supernatant, and centrifuging the supernatant at 10000rpm for 15 minutes to obtain precipitate C;
s6: suspending the precipitate C in diethyl ether, standing at 30deg.C for 30min, and centrifuging at 10000rpm for 15min to obtain precipitate D; the precipitate D was dehydrated with absolute ethanol and dried at 70℃to obtain the peptidoglycan product Y.
According to the yield of the peptidoglycan, the yield of the peptidoglycan of each 100g of thalli is calculated, and the method for calculating the yield of the peptidoglycan comprises the following steps: the weight (g) of peptidoglycan X obtained in step S6. Times.100 g/weight (g) of cell Y obtained in step S2.
The peptidoglycan product was assayed for interleukin-12 induction activity in the same manner as in the study, and no further description is given here.
The results of the peptidoglycan yield and interleukin-12 induction activity measurements are shown in Table 7.
Example 3
S1: regulating MRS culture medium to make osmotic pressure reach 800mOsmol/kg, inoculating lactobacillus plantarum with an inoculum size of 3.0% by volume percent to MRS culture medium, and culturing at 40deg.C for 28 hr;
s2: after culturing, the cells were collected by centrifugation at 9000rpm for 13 minutes; washing the collected thalli with physiological saline, centrifuging and collecting thalli, and repeating the washing and centrifuging for 2 times; washing with ion exchange water, and centrifugally collecting thalli X;
s3: uniformly mixing 1.1 g of the collected thalli with 11 ml of trichloroacetic acid solution with the mass volume ratio of 10%, carrying out boiling water bath for 25 minutes, centrifuging at 9000rpm for 12 minutes, collecting precipitate, and washing the precipitate 3 times with sterilized distilled water to obtain precipitate A;
s4: after 1.1 g of precipitate A was uniformly mixed with 11 ml of 45g/L SDS solution, the mixture was subjected to boiling water bath for 25 minutes, centrifuged at 9000rpm for 13 minutes to collect the precipitate, and the precipitate was washed 3 times with sterilized distilled water to obtain precipitate B;
s5: 1.1 g of precipitate B was mixed with 0.6 ml of 2mg/ml trypsin buffer, digested for 4 hours at 35℃and centrifuged at 6℃and 2500rpm for 3 minutes, and the supernatant was collected and centrifuged at 9000rpm for 12 minutes to obtain precipitate C;
s6: suspending the precipitate C in diethyl ether, standing at 25deg.C for 25 min, and centrifuging at 9000rpm for 13 min to obtain precipitate D; the precipitate D was dehydrated with absolute ethanol and dried at 65℃to give the peptidoglycan product Y.
According to the yield of the peptidoglycan, the yield of the peptidoglycan of each 100g of thalli is calculated, and the method for calculating the yield of the peptidoglycan comprises the following steps: the weight (g) of peptidoglycan X obtained in step S6. Times.100 g/weight (g) of cell Y obtained in step S2.
The peptidoglycan product was assayed for interleukin-12 induction activity in the same manner as in the study, and no further description is given here.
The results of the peptidoglycan yield and interleukin-12 induction activity measurements are shown in Table 7.
Comparative example 1
S1: inoculating lactobacillus plantarum on an MRS culture medium according to an inoculum size of 1.0 percent by volume, and culturing at 30 ℃ for 24 hours;
s2: after the culture, the cells were collected by centrifugation at 8000rpm for 10 minutes; washing the collected thalli with physiological saline, centrifuging and collecting thalli, and repeating the washing and centrifuging for 2 times; washing with ion exchange water, and centrifugally collecting thalli X;
s3: uniformly mixing 1 g of the collected thalli with 10 ml of trichloroacetic acid solution with the mass volume ratio of 10%, carrying out boiling water bath for 20 minutes, centrifuging at 8000rpm for 10 minutes, collecting precipitate, and washing the precipitate with sterilized distilled water for 2 times to obtain precipitate A;
s4: uniformly mixing 1 g of precipitate A with 10 ml of 40g/L SDS solution, carrying out boiling water bath for 20 minutes, centrifuging at 8000rpm for 10 minutes, collecting precipitate, and washing the precipitate with sterilized distilled water for 2 times to obtain precipitate B;
s5: mixing 1 g of precipitate B with 0.5ml of 1mg/ml trypsin buffer, digesting for 3 hours at 30 ℃, centrifuging at 4 ℃ and 2000rpm for 2 minutes, collecting supernatant, centrifuging at 8000rpm for 10 minutes, and obtaining precipitate C;
s6: suspending the precipitate C in diethyl ether, standing at 20deg.C for 20min, and centrifuging at 8000rpm for 10min to obtain precipitate D; the precipitate D was dehydrated with absolute ethanol and dried at 60℃to obtain the peptidoglycan product Y.
According to the yield of the peptidoglycan, the yield of the peptidoglycan of each 100g of thalli is calculated, and the method for calculating the yield of the peptidoglycan comprises the following steps: the weight (g) of peptidoglycan X obtained in step S6. Times.100 g/weight (g) of cell Y obtained in step S2.
The peptidoglycan product was assayed for interleukin-12 induction activity in the same manner as in the study, and no further description is given here.
The results of the peptidoglycan yield and interleukin-12 induction activity measurements are shown in Table 7.
Comparative example 2
S1: inoculating lactobacillus plantarum on an MRS culture medium according to an inoculum size of 4.0 percent by volume, and culturing at 30 ℃ for 30 hours;
s2: after culturing, the cells were collected by centrifugation at 10000rpm for 15 minutes; washing the collected thalli with physiological saline, centrifuging and collecting thalli, and repeating the washing and centrifuging for 2 times; washing with ion exchange water, and centrifugally collecting thalli X;
s3: uniformly mixing 1.2 g of the collected thalli with 12 ml of trichloroacetic acid solution with the mass volume ratio of 10%, carrying out boiling water bath for 30 minutes, centrifuging at 10000rpm for 15 minutes, collecting precipitate, and washing the precipitate 3 times with sterilized distilled water to obtain precipitate A;
s4: after 1.2 g of precipitate A was uniformly mixed with 12 ml of 50g/L SDS solution, the mixture was subjected to boiling water bath for 30 minutes, centrifuged at 10000rpm for 15 minutes, and the precipitate was collected and washed 3 times with sterilized distilled water to obtain precipitate B;
s5: mixing 1.2 g of precipitate B with 0.7 ml of trypsin buffer of 3mg/ml, digesting for 5 hours at 37 ℃, centrifuging at 8 ℃ and 3000rpm for 5 minutes, collecting supernatant, and centrifuging the supernatant at 10000rpm for 15 minutes to obtain precipitate C;
s6: suspending the precipitate C in diethyl ether, standing at 30deg.C for 30min, and centrifuging at 10000rpm for 15min to obtain precipitate D; the precipitate D was dehydrated with absolute ethanol and dried at 70℃to obtain the peptidoglycan product Y.
According to the yield of the peptidoglycan, the yield of the peptidoglycan of each 100g of thalli is calculated, and the method for calculating the yield of the peptidoglycan comprises the following steps: the weight (g) of peptidoglycan X obtained in step S6. Times.100 g/weight (g) of cell Y obtained in step S2.
The peptidoglycan product was assayed for interleukin-12 induction activity in the same manner as in the study, and no further description is given here.
The results of the peptidoglycan yield and interleukin-12 induction activity measurements are shown in Table 7.
Comparative example 3
S1: inoculating lactobacillus plantarum on an MRS culture medium according to an inoculum size of 3.0 percent by volume, and culturing at 30 ℃ for 28 hours;
s2: after culturing, the cells were collected by centrifugation at 9000rpm for 13 minutes; washing the collected thalli with physiological saline, centrifuging and collecting thalli, and repeating the washing and centrifuging for 2 times; washing with ion exchange water, and centrifugally collecting thalli X;
s3: uniformly mixing 1.1 g of the collected thalli with 11 ml of trichloroacetic acid solution with the mass volume ratio of 10%, carrying out boiling water bath for 25 minutes, centrifuging at 9000rpm for 12 minutes, collecting precipitate, and washing the precipitate 3 times with sterilized distilled water to obtain precipitate A;
s4: after 1.1 g of precipitate A was uniformly mixed with 11 ml of 45g/L SDS solution, the mixture was subjected to boiling water bath for 25 minutes, centrifuged at 9000rpm for 13 minutes to collect the precipitate, and the precipitate was washed 3 times with sterilized distilled water to obtain precipitate B;
s5: 1.1 g of precipitate B was mixed with 0.6 ml of 2mg/ml trypsin buffer, digested for 4 hours at 35℃and centrifuged at 6℃and 2500rpm for 3 minutes, and the supernatant was collected and centrifuged at 9000rpm for 12 minutes to obtain precipitate C;
s6: suspending the precipitate C in diethyl ether, standing at 25deg.C for 25 min, and centrifuging at 9000rpm for 13 min to obtain precipitate D; the precipitate D was dehydrated with absolute ethanol and dried at 65℃to give the peptidoglycan product Y.
According to the yield of the peptidoglycan, the yield of the peptidoglycan of each 100g of thalli is calculated, and the method for calculating the yield of the peptidoglycan comprises the following steps: the weight (g) of peptidoglycan X obtained in step S6. Times.100 g/weight (g) of cell Y obtained in step S2.
The peptidoglycan product was assayed for interleukin-12 induction activity in the same manner as in the study, and no further description is given here.
The results of the peptidoglycan yield and interleukin-12 induction activity measurements are shown in Table 7.
The results of the statistical analysis of the example and the comparative example by using the single-factor analysis of variance method in the EXCEL document show that the yield (g) of peptidoglycan and the induction activity p value of interleukin-12 are less than 0.05 per 100g of thalli, and the significant difference exists, thereby further proving that the technical scheme of the invention can significantly improve the yield of peptidoglycan and the immunocompetence of peptidoglycan.
Table 7 statistical analysis of results for examples and comparative examples
Claims (8)
1. A method for extracting peptidoglycan by lactobacillus based on space-time variables, which is characterized by comprising the following steps:
s1: regulating an MRS culture medium by using sorbitol to ensure that the osmotic pressure reaches 600-1000 mOsmol/kg, inoculating lactobacillus plantarum ATCC14917 to the MRS culture medium according to the inoculum size of 1.0-4.0% by volume percent, wherein the culture temperature is 40-42 ℃ and the culture time is 24-30 hours;
s2: culturing and collecting thalli according to the centrifugal rotation speed of 8000-10000 rpm and the centrifugal time of 10-15 minutes; washing the collected thalli with physiological saline, centrifuging and collecting thalli, and repeating the washing and centrifuging for 1-2 times; washing with ion exchange water, and centrifugally collecting thalli;
s3: uniformly mixing 1-1.2 g of collected thalli with 10-12 ml of trichloroacetic acid solution with the mass volume ratio of 10%, carrying out boiling water bath for 20-30 minutes, centrifuging at 8000-10000 rpm for 10-15 minutes, collecting precipitate, and washing the precipitate with sterilized distilled water for 2-3 times to obtain precipitate A;
s4: uniformly mixing 1-1.2 g of precipitate A with 10-12 ml of 40-50 g/L SDS solution, carrying out boiling water bath for 20-30 minutes, centrifuging at 8000-10000 rpm for 10-15 minutes, collecting precipitate, and washing the precipitate with sterilized distilled water for 2-3 times to obtain precipitate B;
s5: mixing the precipitate B with trypsin buffer solution in a mixing ratio of 1-1.2 g of precipitate B: 0.5-0.7 ml trypsin buffer solution, wherein the concentration of the trypsin buffer solution is 1-3 mg/ml, the reaction temperature is 30-37 ℃, the reaction time is 3-5 hours, the centrifugation is carried out at 4-8 ℃ and 2000-3000 rpm for 2-5 minutes, the supernatant is collected, and the supernatant is centrifuged at 8000-10000 rpm for 10-15 minutes, thus obtaining a precipitate C;
s6: suspending the precipitate C in diethyl ether, standing for 20-30 min at 20-30 ℃, and centrifuging at 8000-10000 rpm for 10-15 min to obtain precipitate D; and dehydrating the precipitate D by using absolute ethyl alcohol, and drying at 60-70 ℃ to obtain a peptidoglycan product.
2. The method for extracting peptidoglycan from lactobacillus according to claim 1, wherein the culture temperature in step S1 is 40 ℃.
3. The method for extracting peptidoglycan from lactobacillus according to claim 2, wherein the MRS medium in step S1 is adjusted to have an osmotic pressure of 800mOsmol/kg.
4. A 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, wherein the inoculation amount in the step S1 is 3.0% by volume.
6. The method for extracting peptidoglycan from lactobacillus according to claim 5, wherein the centrifugation speed of the collected cells in step S2 is 9000rpm and the centrifugation is performed for 13 minutes.
7. The method for extracting peptidoglycan from lactobacillus according to claim 6, wherein the mixing ratio in step S5 is 1.1 g of precipitate B:0.6 ml trypsin buffer with a concentration of 2mg/ml trypsin buffer.
8. The method for extracting peptidoglycan from lactobacillus according to claim 7, wherein the reaction temperature in step S5 is 35 ℃ and the reaction time is 4 hours.
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| CN101717806A (en) * | 2009-12-03 | 2010-06-02 | 内蒙古双奇药业股份有限公司 | Integrated peptidoglycan and preparation method thereof |
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| CN101717806A (en) * | 2009-12-03 | 2010-06-02 | 内蒙古双奇药业股份有限公司 | Integrated peptidoglycan and preparation method thereof |
| CN101947213A (en) * | 2010-09-29 | 2011-01-19 | 黑龙江大学 | Method for preparing microcapsules from peptidoglycan in lactic acid bacterial cell walls |
| CN113024678A (en) * | 2019-12-25 | 2021-06-25 | 南京农业大学 | Method for extracting peptidoglycan from lactobacillus |
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