CN117384990B - A method for extracting peptidoglycan from lactobacillus based on spatiotemporal variables - Google Patents
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Abstract
Description
技术领域Technical Field
本发明涉及一种乳杆菌提取肽聚糖的方法,具体而言,涉及一种基于时空变量的乳杆菌提取肽聚糖的方法。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.
背景技术Background technique
肽聚糖是细菌细胞壁的特有组成部分,其基本结构是由N-乙酰葡萄糖胺(N-Acetylglucosamine)和N-乙酰胞壁酸(N-AcetylmuramicAcid)通过β-1,4糖苷键连接,糖链间由肽链交联,构成稳定的网状结构。近年来发现,肽聚糖在提高机体免疫力、抗细菌感染方面发挥重要作用。由于肽聚糖结构复杂多样,其提高机体免疫力的具体机制尚未完全探索清楚。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.
目前,肽聚糖的制备方法主要是对革兰氏阳性细菌,例如乳酸菌,发酵培养后,采用物理或化学方法提取细菌的细胞壁,纯化获得肽聚糖。因此,进一步提高肽聚糖制备收率,进一步提高肽聚糖免疫活性具有重要意义。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.
有鉴于此,特提出本发明。In view of this, the present invention is proposed.
发明内容Summary of the invention
为了解决上述问题,本发明提供了一种基于时空变量的乳杆菌提取肽聚糖的方法,以该方法制备的肽聚糖具有收率高、免疫活性强的特点。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.
本申请提供一种基于时空变量的乳杆菌提取肽聚糖的方法,包括如下步骤:The present application provides a method for extracting peptidoglycan from lactobacillus based on spatiotemporal variables, comprising the following steps:
S1:调整MRS培养基,使其渗透压达到600~1000mOsmol/kg,将植物乳杆菌按体积百分比1.0~4.0%的接种量,接种至MRS培养基上,于37~42℃培养24~30小时;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:培养后以8000~10000rpm离心10~15分钟收集菌体;用生理盐水洗涤收集的菌体,离心并收集菌体,上述洗涤、离心重复进行1~2次;用离子交换水洗涤、离心收集菌体;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:取收集到的菌体,按1~1.2克湿菌体与10~12毫升10%质量体积比的三氯乙酸溶液均匀混合后,沸水浴20~30分钟,8000~10000rpm离心10~15分钟收集沉淀,再用灭菌蒸馏水洗涤沉淀2~3次,获得沉淀物A;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:按1~1.2克沉淀物A与10~12毫升40~50g/L的SDS溶液均匀混合后,沸水浴20~30分钟,8000~10000rpm离心10~15分钟收集沉淀,再用灭菌蒸馏水洗涤沉淀2~3次,获得沉淀物B;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:按1~1.2克沉淀物B与0.5~0.7毫升的1~3mg/ml的胰蛋白酶缓冲液混匀,30~37℃消化3~5小时,在4~8℃,2000~3000rpm离心2~5分钟,收集上清液,该上清液8000~10000rpm离心10~15分钟,获得沉淀物C;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:将沉淀物C悬浮于乙醚中,20~30℃下静置20~30分钟,再于8000~10000rpm离心10~15分钟,获得沉淀物D;用无水乙醇对沉淀物D脱水,于60~70℃干燥后获得肽聚糖产物。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.
通过上述技术方案,控制培养条件,即培养的时空变量,诱导乳酸菌细胞壁生长和结构改变,提高肽聚糖的产量和收率,增强肽聚糖免疫活性。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.
上述乳酸菌、肽聚糖均可作为食品辅料添加至食品中,制成功能食品;肽聚糖也可作为药品提高机体免疫活性。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.
具体实施方式Detailed ways
申请人在长期的研究、实践过程中,意外的发现通过提高乳酸菌的培养温度,增加培养基的渗透压,可以提高肽聚糖的产量,并增强肽聚糖免疫活性。在此基础上,申请人开展了系统的研究,具体研究过程如下: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、乳酸菌培养温度试验1. Lactic acid bacteria culture temperature test
本研究以植物乳杆菌为例开展研究。植物乳杆菌(菌种编号ATCC14917),说明书推荐培养温度为30℃。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乳酸菌培养1.1 Lactic acid bacteria culture
将植物乳杆菌(菌种编号ATCC14917)于25℃、30℃、37℃、40℃、42℃下,在MRS培养基中独立培养24小时。培养后,通过以8000rpm离心10分钟来收集细菌;用生理盐水洗涤、离心,上述洗涤、离心再重复进行2次;用离子交换水洗涤、离心。在此步骤取样,将细菌悬浮在离子交换水中制备乳酸菌悬浮液,测量细菌浓度(细菌细胞数/mL),检测菌体二氨基庚二酸含量。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肽聚糖的提取1.2 Extraction of peptidoglycan
1.2.1去除磷壁酸1.2.1 Removal of teichoic acid
取收集到的菌体,按1克重量份湿菌体与10毫升体积份10%的三氯乙酸溶液均匀混合后,边搅拌边沸水浴20min;4000r/min离心10min,弃去上清;最后用灭菌蒸馏水3次洗涤沉淀,即获得不含磷壁酸的沉淀物I;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去除蛋白质1.2.2 Protein removal
按1克重量份沉淀物I与10毫升体积份8g/dl的SDS溶液均匀混合后,边搅拌边沸水浴30min;通过4000r/min离心30min,弃去上清;再用灭菌蒸馏水4次洗涤沉淀;将洗涤后的沉淀按1克重量份与0.5毫升体积份的1mg/ml的胰蛋白酶缓冲液混匀,37℃振荡3h;3h后在4℃,3000r/min离心5min;收集上清液,该上清液在12000r/min离心15min后收集沉淀物II。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去除脂类1.2.3 Removal of lipids
将沉淀物II悬浮于乙醚中,室温下静置30min,再于12000r/min离心15min后收集沉淀;用无水乙醇脱水,于70℃烘箱内干燥后即可得到肽聚糖产物。取肽聚糖产物,进行白细胞介素-12诱导活性测定。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检测方法1.3 Detection Method
1.3.1二氨基庚二酸测量方法1.3.1 Diaminopimelic acid measurement method
二氨基庚二酸是构成肽并交联细胞壁糖链的氨基酸,属于反映细胞壁成分的指数。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.
向菌体中加入6mol/L的盐酸,在100℃下水解20小时。然后,使用离心浓缩机(由Thermo SCIENTIFIC Co.生产)将所得产物蒸发干燥。向干燥的产物中加入0.05mol/L的盐酸,使细胞浓度到达1mg干燥细胞/mL。所得产物经孔径为0.2μm的盘式过滤器过滤,然后用L-8800日立高速氨基酸分析仪进行HPLC处理分析。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白细胞介素-12诱导活性测定1.3.2 Interleukin-12 induction activity assay
向BALB/c小鼠(8周龄,雌性)腹膜内给予2mL 4.05%巯基乙酸盐。四天后,使用PBS收集腹膜内巨噬细胞,并在含有10%FBS的RPMI1640培养基中调节至2×106个细胞/mL,然后以每孔0.5mL的量接种在48孔板中。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.
每个孔按照100μg/mL添加肽聚糖,培养24小时后,测量培养基中的白细胞介素-12浓度。白细胞介素-12的活性形式是p70,其中p35亚基结合p40亚基,因此本次试验测量白细胞介素-12(p70)。用于测量白细胞介素-12的仪器是OptEIA小鼠白细胞介素-12测量试剂盒(由BD Pharmingen Co.生产)。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结果与分析1.4 Results and Analysis
1.4.1菌体浓度1.4.1 Bacterial concentration
培养结束后,乳杆菌菌体浓度结果见表1。在30℃时细菌浓度最高,与推荐培养温度一致。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.
表1不同温度培养菌体浓度结果Table 1 Results of bacterial concentrations cultured at different temperatures
1.4.2二氨基庚二酸含量1.4.2 Diaminopimelate content
二氨基庚二酸测量结果见表2。在30℃下培养时,二氨基庚二酸浓度增加到25℃时的约两倍。随着培养温度的升高,二氨基庚二酸浓度增加。在42℃培养产生最大浓度。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.
表2不同温度培养时二氨基庚二酸含量结果Table 2 Results of diaminopimelate content when cultured at different temperatures
1.4.3白细胞介素-12诱导活性测定结果1.4.3 Results of interleukin-12 induction activity assay
以白细胞介素-12诱导活性评估体外免疫刺激活性,结果见表3,培养温度30℃至42℃时,温度升高,诱导产生的IL-12增加。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.
表3不同温度培养时白细胞介素-12诱导活性测定结果Table 3 Results of interleukin-12 induction activity assay at different temperatures
1.5试验结论1.5 Test conclusion
1.5.1本次试验菌体生长最适的培养温度为30℃,产生具体细胞壁肽聚糖的最适的培养温度为40℃,产生免疫活性的最适的培养温度为40℃。当菌体在高于菌体最适生产温度时,产生应激反应,诱导细胞壁合成,从而产生了更多的肽聚糖,且肽聚糖具有更高的免疫诱导反应。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、不同渗透压培养试验2. Culture test with different osmotic pressures
在前述40℃培养温度下,调节培养基至不同的渗透压,进行培养,考察渗透压对乳酸菌肽聚糖的诱导情况。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乳酸菌培养2.1 Lactic acid bacteria culture
使用山梨糖醇调整MRS培养基,使其渗透压分别达到500mOsmol/kg、600mOsmol/kg、800mOsmol/kg、1000mOsmol/kg,连同未调整的MRS培养基(渗透压为430mOsmol/kg),添加植物乳杆菌(菌种编号ATCC14917)后,于40℃下,分别独立培养24小时。培养后,通过以8000rpm离心10分钟来收集细菌;用生理盐水洗涤、离心,上述洗涤、离心再重复进行2次;用离子交换水洗涤、离心。在此步骤取样,将细菌悬浮在离子交换水中制备乳酸菌悬浮液,测量细菌浓度(细菌细胞数/mL),检测菌体二氨基庚二酸含量。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肽聚糖的提取2.2 Extraction of peptidoglycan
2.2.1去除磷壁酸2.2.1 Removal of teichoic acid
取收集到的菌体,按1克重量份湿菌体与10毫升体积份10%的三氯乙酸溶液均匀混合后,边搅拌边沸水浴20min;4000r/min离心10min,弃去上清;最后用灭菌蒸馏水3次洗涤沉淀,即获得不含磷壁酸的沉淀物I。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去除蛋白质2.2.2 Protein removal
按1克重量份沉淀物I与10毫升体积份8g/dl的SDS溶液均匀混合后,边搅拌边沸水浴30min;通过4000r/min离心30min,弃去上清;再用灭菌蒸馏水4次洗涤沉淀;将洗涤后的沉淀按1克重量份与0.5毫升体积份的1mg/ml的胰蛋白酶缓冲液混匀,37℃振荡3h;3h后在4℃,3000r/min离心5min;收集上清液,该上清液在12000r/min离心15min后收集沉淀物II。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去除脂类2.2.3 Removal of lipids
将沉淀物II悬浮于乙醚中,室温下静置30min,再于12000r/min离心15min后收集沉淀;用无水乙醇脱水,于70℃烘箱内干燥后即可得到肽聚糖产物。取肽聚糖产物,进行白细胞介素-12诱导活性测定。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检测方法2.3 Detection methods
二氨基庚二酸测量方法、白细胞介素-12诱导活性测定同“乳酸菌培养温度试验”。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结果与分析2.4 Results and Analysis
2.4.1菌体浓度2.4.1 Bacterial concentration
培养结束后,乳杆菌菌体浓度结果见表4。当培养基渗透压为500mOsmol/kg时,细菌浓度最高。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.
表4不同渗透压培养时菌体浓度结果Table 4 Results of bacterial concentration when cultured at different osmotic pressures
2.4.2二氨基庚二酸含量2.4.2 Diaminopimelate content
二氨基庚二酸测量结果见表5。当培养基渗透压为750mOsmol/kg时,二氨基庚二酸产生最大浓度,浓度达到430mOsmol/kg时的1.22倍。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.
表5不同渗透压培养时二氨基庚二酸含量结果Table 5 Results of diaminopimelate content during culture at different osmotic pressures
2.4.3白细胞介素-12诱导活性测定结果2.4.3 Results of interleukin-12 induction activity assay
以白细胞介素-12诱导活性评估体外免疫刺激活性,结果见表6。当培养基渗透压为750mOsmol/kg时,诱导产生的IL-12最多。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.
表6不同温度培养时白细胞介素-12诱导活性测定结果Table 6 Results of interleukin-12 induction activity assay at different temperatures
2.5试验结论2.5 Experimental conclusion
本次试验,产生细胞壁肽聚糖的最适的渗透压为800mOsmol/kg,产生免疫活性的最适的的渗透压为800mOsmol/kg。当菌体在较高渗透压环境时,产生应激反应,诱导细胞壁合成,从而产生了更多的肽聚糖,且肽聚糖具有更高的免疫诱导反应。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.
基于上述研究结果,申请人根据研究结果对技术方案进行了验证,验证结果见实施例1~实施例3,并与对比例1~对比例3进行了对比分析。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.
实施例1Example 1
S1:调整MRS培养基,使其渗透压达到600mOsmol/kg,将植物乳杆菌按体积百分比1.0%的接种量,接种至MRS培养基上,于37℃培养24小时;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:培养后以8000rpm离心10分钟收集菌体;用生理盐水洗涤收集的菌体,离心并收集菌体,上述洗涤、离心重复进行2次;用离子交换水洗涤、离心收集菌体X;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:取收集到的菌体,按1克湿菌体与10毫升10%质量体积比的三氯乙酸溶液均匀混合后,沸水浴20分钟,8000rpm离心10分钟收集沉淀,再用灭菌蒸馏水洗涤沉淀2次,获得沉淀物A;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:按1克沉淀物A与10毫升40g/L的SDS溶液均匀混合后,沸水浴20分钟,8000rpm离心10分钟收集沉淀,再用灭菌蒸馏水洗涤沉淀2次,获得沉淀物B;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:按1克沉淀物B与0.5毫升的1mg/ml的胰蛋白酶缓冲液混匀,30℃消化3小时,在4℃,2000rpm离心2分钟,收集上清液,该上清液8000rpm离心10分钟,获得沉淀物C;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:将沉淀物C悬浮于乙醚中,20℃下静置20分钟,再于8000rpm离心10分钟,获得沉淀物D;用无水乙醇对沉淀物D脱水,于60℃干燥后获得肽聚糖产物Y。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.
根据肽聚糖产量,计算每100g菌体的肽聚糖收率,肽聚糖收率计算方法为:步骤S6获得的肽聚糖X重量(g)×100g/步骤S2获得的菌体Y重量(g)。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).
取肽聚糖产物,进行白细胞介素-12诱导活性测定,测定方法与研究过程中白细胞介素-12诱导活性测定方法相同,在此不再赘述。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.
肽聚糖收率及白细胞介素-12诱导活性测定结果见表7。The results of the peptidoglycan yield and interleukin-12 induction activity assay are shown in Table 7.
实施例2Example 2
S1:调整MRS培养基,使其渗透压达到1000mOsmol/kg,将植物乳杆菌按体积百分比4.0%的接种量,接种至MRS培养基上,于42℃培养30小时;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:培养后以10000rpm离心15分钟收集菌体;用生理盐水洗涤收集的菌体,离心并收集菌体,上述洗涤、离心重复进行2次;用离子交换水洗涤、离心收集菌体X;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:取收集到的菌体,按1.2克湿菌体与12毫升10%质量体积比的三氯乙酸溶液均匀混合后,沸水浴30分钟,10000rpm离心15分钟收集沉淀,再用灭菌蒸馏水洗涤沉淀3次,获得沉淀物A;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:按1.2克沉淀物A与12毫升50g/L的SDS溶液均匀混合后,沸水浴30分钟,10000rpm离心15分钟收集沉淀,再用灭菌蒸馏水洗涤沉淀3次,获得沉淀物B;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:按1.2克沉淀物B与0.7毫升的3mg/ml的胰蛋白酶缓冲液混匀,37℃消化5小时,在8℃,3000rpm离心5分钟,收集上清液,该上清液10000rpm离心15分钟,获得沉淀物C;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:将沉淀物C悬浮于乙醚中,30℃下静置30分钟,再于10000rpm离心15分钟,获得沉淀物D;用无水乙醇对沉淀物D脱水,于70℃干燥后获得肽聚糖产物Y。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.
根据肽聚糖产量,计算每100g菌体的肽聚糖收率,肽聚糖收率计算方法为:步骤S6获得的肽聚糖X重量(g)×100g/步骤S2获得的菌体Y重量(g)。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).
取肽聚糖产物,进行白细胞介素-12诱导活性测定,测定方法与研究过程中白细胞介素-12诱导活性测定方法相同,在此不再赘述。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.
肽聚糖收率及白细胞介素-12诱导活性测定结果见表7。The results of the peptidoglycan yield and interleukin-12 induction activity assay are shown in Table 7.
实施例3Example 3
S1:调整MRS培养基,使其渗透压达到800mOsmol/kg,将植物乳杆菌按体积百分比3.0%的接种量,接种至MRS培养基上,于40℃培养28小时;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:培养后以9000rpm离心13分钟收集菌体;用生理盐水洗涤收集的菌体,离心并收集菌体,上述洗涤、离心重复进行2次;用离子交换水洗涤、离心收集菌体X;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:取收集到的菌体,按1.1克湿菌体与11毫升10%质量体积比的三氯乙酸溶液均匀混合后,沸水浴25分钟,9000rpm离心12分钟收集沉淀,再用灭菌蒸馏水洗涤沉淀3次,获得沉淀物A;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:按1.1克沉淀物A与11毫升45g/L的SDS溶液均匀混合后,沸水浴25分钟,9000rpm离心13分钟收集沉淀,再用灭菌蒸馏水洗涤沉淀3次,获得沉淀物B;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:按1.1克沉淀物B与0.6毫升的2mg/ml的胰蛋白酶缓冲液混匀,35℃消化4小时,在6℃,2500rpm离心3分钟,收集上清液,该上清液9000rpm离心12分钟,获得沉淀物C;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:将沉淀物C悬浮于乙醚中,25℃下静置25分钟,再于9000rpm离心13分钟,获得沉淀物D;用无水乙醇对沉淀物D脱水,于65℃干燥后获得肽聚糖产物Y。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.
根据肽聚糖产量,计算每100g菌体的肽聚糖收率,肽聚糖收率计算方法为:步骤S6获得的肽聚糖X重量(g)×100g/步骤S2获得的菌体Y重量(g)。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).
取肽聚糖产物,进行白细胞介素-12诱导活性测定,测定方法与研究过程中白细胞介素-12诱导活性测定方法相同,在此不再赘述。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.
肽聚糖收率及白细胞介素-12诱导活性测定结果见表7。The results of the peptidoglycan yield and interleukin-12 induction activity assay are shown in Table 7.
对比例1Comparative Example 1
S1:使用MRS培养基,将植物乳杆菌按体积百分比1.0%的接种量,接种至MRS培养基上,于30℃培养24小时;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:培养后以8000rpm离心10分钟收集菌体;用生理盐水洗涤收集的菌体,离心并收集菌体,上述洗涤、离心重复进行2次;用离子交换水洗涤、离心收集菌体X;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:取收集到的菌体,按1克湿菌体与10毫升10%质量体积比的三氯乙酸溶液均匀混合后,沸水浴20分钟,8000rpm离心10分钟收集沉淀,再用灭菌蒸馏水洗涤沉淀2次,获得沉淀物A;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:按1克沉淀物A与10毫升40g/L的SDS溶液均匀混合后,沸水浴20分钟,8000rpm离心10分钟收集沉淀,再用灭菌蒸馏水洗涤沉淀2次,获得沉淀物B;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:按1克沉淀物B与0.5毫升的1mg/ml的胰蛋白酶缓冲液混匀,30℃消化3小时,在4℃,2000rpm离心2分钟,收集上清液,该上清液8000rpm离心10分钟,获得沉淀物C;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:将沉淀物C悬浮于乙醚中,20℃下静置20分钟,再于8000rpm离心10分钟,获得沉淀物D;用无水乙醇对沉淀物D脱水,于60℃干燥后获得肽聚糖产物Y。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.
根据肽聚糖产量,计算每100g菌体的肽聚糖收率,肽聚糖收率计算方法为:步骤S6获得的肽聚糖X重量(g)×100g/步骤S2获得的菌体Y重量(g)。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).
取肽聚糖产物,进行白细胞介素-12诱导活性测定,测定方法与研究过程中白细胞介素-12诱导活性测定方法相同,在此不再赘述。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.
肽聚糖收率及白细胞介素-12诱导活性测定结果见表7。The results of the peptidoglycan yield and interleukin-12 induction activity assay are shown in Table 7.
对比例2Comparative Example 2
S1:使用MRS培养基,将植物乳杆菌按体积百分比4.0%的接种量,接种至MRS培养基上,于30℃培养30小时;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:培养后以10000rpm离心15分钟收集菌体;用生理盐水洗涤收集的菌体,离心并收集菌体,上述洗涤、离心重复进行2次;用离子交换水洗涤、离心收集菌体X;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:取收集到的菌体,按1.2克湿菌体与12毫升10%质量体积比的三氯乙酸溶液均匀混合后,沸水浴30分钟,10000rpm离心15分钟收集沉淀,再用灭菌蒸馏水洗涤沉淀3次,获得沉淀物A;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:按1.2克沉淀物A与12毫升50g/L的SDS溶液均匀混合后,沸水浴30分钟,10000rpm离心15分钟收集沉淀,再用灭菌蒸馏水洗涤沉淀3次,获得沉淀物B;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:按1.2克沉淀物B与0.7毫升的3mg/ml的胰蛋白酶缓冲液混匀,37℃消化5小时,在8℃,3000rpm离心5分钟,收集上清液,该上清液10000rpm离心15分钟,获得沉淀物C;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:将沉淀物C悬浮于乙醚中,30℃下静置30分钟,再于10000rpm离心15分钟,获得沉淀物D;用无水乙醇对沉淀物D脱水,于70℃干燥后获得肽聚糖产物Y。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.
根据肽聚糖产量,计算每100g菌体的肽聚糖收率,肽聚糖收率计算方法为:步骤S6获得的肽聚糖X重量(g)×100g/步骤S2获得的菌体Y重量(g)。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).
取肽聚糖产物,进行白细胞介素-12诱导活性测定,测定方法与研究过程中白细胞介素-12诱导活性测定方法相同,在此不再赘述。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.
肽聚糖收率及白细胞介素-12诱导活性测定结果见表7。The results of the peptidoglycan yield and interleukin-12 induction activity assay are shown in Table 7.
对比例3Comparative Example 3
S1:使用MRS培养基,将植物乳杆菌按体积百分比3.0%的接种量,接种至MRS培养基上,于30℃培养28小时;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:培养后以9000rpm离心13分钟收集菌体;用生理盐水洗涤收集的菌体,离心并收集菌体,上述洗涤、离心重复进行2次;用离子交换水洗涤、离心收集菌体X;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:取收集到的菌体,按1.1克湿菌体与11毫升10%质量体积比的三氯乙酸溶液均匀混合后,沸水浴25分钟,9000rpm离心12分钟收集沉淀,再用灭菌蒸馏水洗涤沉淀3次,获得沉淀物A;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:按1.1克沉淀物A与11毫升45g/L的SDS溶液均匀混合后,沸水浴25分钟,9000rpm离心13分钟收集沉淀,再用灭菌蒸馏水洗涤沉淀3次,获得沉淀物B;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:按1.1克沉淀物B与0.6毫升的2mg/ml的胰蛋白酶缓冲液混匀,35℃消化4小时,在6℃,2500rpm离心3分钟,收集上清液,该上清液9000rpm离心12分钟,获得沉淀物C;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:将沉淀物C悬浮于乙醚中,25℃下静置25分钟,再于9000rpm离心13分钟,获得沉淀物D;用无水乙醇对沉淀物D脱水,于65℃干燥后获得肽聚糖产物Y。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.
根据肽聚糖产量,计算每100g菌体的肽聚糖收率,肽聚糖收率计算方法为:步骤S6获得的肽聚糖X重量(g)×100g/步骤S2获得的菌体Y重量(g)。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).
取肽聚糖产物,进行白细胞介素-12诱导活性测定,测定方法与研究过程中白细胞介素-12诱导活性测定方法相同,在此不再赘述。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.
肽聚糖收率及白细胞介素-12诱导活性测定结果见表7。The results of the peptidoglycan yield and interleukin-12 induction activity assay are shown in Table 7.
使用EXCEL文档中的单因素方差分析法统计分析实施例和对比例结果,结果表明,每100g菌体肽聚糖收率(g)和白细胞介素-12诱导活性p值均<0.05,有显著性差异,进一步证实了本发明的技术方案可以显著提高肽聚糖收率以及肽聚糖的免疫活性。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.
表7实施例和对比例结果统计分析Table 7 Statistical analysis of the results of the embodiments and comparative examples
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