技术领域Technical field
本发明涉及微生物技术领域，特别涉及一种植物乳杆菌、植物乳杆菌的培养方法及其应用。The present invention relates to the technical field of microorganisms, and in particular to a method for culturing Lactobacillus plantarum and Lactobacillus plantarum and its application.
背景技术Background technique
肠道菌群与人类宿主及其祖先共同进化了数百万年，在提供营养、抵御病原体和预防疾病等方面对人体的做出了重大贡献。目前，健康、无癌、长寿人群的肠道菌群尤其吸引广大研究者的关注。The intestinal flora has co-evolved with the human host and its ancestors for millions of years and has made significant contributions to the human body in providing nutrition, resisting pathogens, and preventing disease. At present, the intestinal flora of healthy, cancer-free, and long-lived people is particularly attracting the attention of researchers.
研究指出，由于百岁老人肠道菌群结构明显不同于年轻健康人群，其中的肠道菌群研究更具价值。Studies have pointed out that because the structure of the intestinal flora of centenarians is significantly different from that of young healthy people, the study of the intestinal flora among them is more valuable.
目前，提取自健康、无癌、长寿人群来源的益生菌的活菌数的生物量较低。Currently, probiotics extracted from sources of healthy, cancer-free, long-lived people have low biomass of viable bacteria.
发明内容Contents of the invention
基于此，本发明的目的是提供一种植物乳杆菌、植物乳杆菌的培养方法及其应用，旨在解决背景技术中提出的现有技术中的肠道菌群活菌数的生物量较低的问题。Based on this, the purpose of the present invention is to provide a culture method and application of Lactobacillus plantarum and Lactobacillus plantarum, aiming to solve the problem of low biomass of viable intestinal flora in the prior art proposed in the background art. The problem.
本发明实施例是这样实现的：The embodiment of the present invention is implemented as follows:
一种植物乳杆菌MH-301，已保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.23397。Lactobacillus plantarum MH-301 has been deposited in the General Microbiology Center of the China Microbial Culture Collection Committee, with the deposit number CGMCC No. 23397.
一种植物乳杆菌MH-301的培养方法，用于培养上述的植物乳杆菌MH-301，所述培养方法包括：A culture method for Lactobacillus plantarum MH-301, used to culture the above-mentioned Lactobacillus plantarum MH-301, the culture method includes:
制备高密度发酵培养基，将活化好的所述植物乳杆菌MH-301接种于高密度发酵培养基中进行发酵培养；Prepare a high-density fermentation medium, and inoculate the activated Lactobacillus plantarum MH-301 into the high-density fermentation medium for fermentation culture;
通过控温控氧和补料分批发酵技术提高所述植物乳杆菌MH-301活菌数，从而获得高密度的菌液。The number of viable bacteria of Lactobacillus plantarum MH-301 is increased through temperature control, oxygen control and fed-batch fermentation technology, thereby obtaining a high-density bacterial liquid.
进一步的，上述植物乳杆菌MH-301的培养方法，其中，所述高密度发酵培养基包含：Further, the above-mentioned cultivation method of Lactobacillus plantarum MH-301, wherein the high-density fermentation medium contains:
胰蛋白胨、大豆蛋白胨、葡萄糖、牛肉粉、酵母浸出粉、乳糖、KHPO2·7H2O 、甘油磷酸钠，抗坏血酸钠、甲酸、吐温、MgSO2、醋酸钠、柠檬酸三铵和MnSO2。Tryptone, soy peptone, glucose, beef meal, yeast extract powder, lactose, KHPO2 ·7H2 O, sodium glycerophosphate, sodium ascorbate, formic acid, Tween, MgSO2 , sodium acetate, triammonium citrate and MnSO2 .
进一步的，上述植物乳杆菌MH-301的培养方法，其中，按质量百分比计所述高密度发酵培养基包含：Further, the above-mentioned cultivation method of Lactobacillus plantarum MH-301, wherein the high-density fermentation medium contains in mass percentage:
0.5-1%胰蛋白胨、0.4-1%大豆蛋白胨、0.5-15%葡萄糖、0.3-0.5%牛肉粉、0.3-0.5%酵母浸出粉、0.2-0.5%乳糖、0.1-0.2% KHPO2·7H2O 、1-1.5%甘油磷酸钠，余量为抗坏血酸钠、甲酸、吐温、MgSO2、醋酸钠、柠檬酸三铵和MnSO2。0.5-1% tryptone, 0.4-1% soy peptone, 0.5-15% glucose, 0.3-0.5% beef meal, 0.3-0.5% yeast extract powder, 0.2-0.5% lactose, 0.1-0.2% KHPO2 ·7H2O, 1-1.5% sodium glycerophosphate, the balance is sodium ascorbate, formic acid, Tween, MgSO2, sodium acetate, triammonium citrate andMnSO2 .
本发明的另一个目的在于提供一种微生物临床制剂，所述微生物临床制剂是以上述的植物乳杆菌MH-301为活性成分的微生物制剂。Another object of the present invention is to provide a microbial clinical preparation, which contains the above-mentioned Lactobacillus plantarum MH-301 as an active ingredient.
本发明的另一个目的在于提供一种微生物临床制剂的制备方法，用于制备上述的微生物临床制剂，所述制备方法包括：Another object of the present invention is to provide a method for preparing a microbial clinical preparation, which is used to prepare the above-mentioned microbial clinical preparation. The preparation method includes:
将所述植物乳杆菌MH-301通过高密度发酵技术进行发酵培养，使发酵液中活菌量达到至少1.5×109 CFU/mL；The Lactobacillus plantarum MH-301 is fermented and cultured through high-density fermentation technology, so that the amount of viable bacteria in the fermentation broth reaches at least 1.5×109 CFU/mL;
采用冷冻干燥技术进行冷冻干燥，制成具有内层包埋层的活性微生物菌剂，后进行外层包埋以得到所述微生物临床制剂。The freeze-drying technology is used for freeze-drying to prepare an active microbial agent with an inner embedding layer, and then the outer layer is embedded to obtain the microbial clinical preparation.
本发明的另一个目的在于提供一种可食性益生菌，所述可食性益生菌是以上述的植物乳杆菌MH-301固化得到。Another object of the present invention is to provide an edible probiotic, which is obtained by solidifying the above-mentioned Lactobacillus plantarum MH-301.
本发明的另一个目的在于提供一种可食性益生菌的制备方法，用于制备上述的可食性益生菌，所述制备方法包括：Another object of the present invention is to provide a preparation method of edible probiotics for preparing the above-mentioned edible probiotics. The preparation method includes:
将培养成熟的植物乳杆菌MH-301种子按比例加入到含有适合益生菌生长繁殖的固定化载体溶液中；Add the cultured and mature Lactobacillus plantarum MH-301 seeds in proportion to an immobilized carrier solution suitable for the growth and reproduction of probiotics;
在所述固定化载体溶液滴入一种固化液；或将所述固化液加入固定化载体中使之凝固；Drop a solidifying liquid into the immobilized carrier solution; or add the solidifying liquid to the immobilized carrier to solidify it;
将凝固后的含有植物乳杆菌MH-301的固定化载体用无菌水洗涤，后置于预设温度下密闭培养预设时间。The coagulated immobilized carrier containing Lactobacillus plantarum MH-301 is washed with sterile water, and then placed in an airtight culture at a preset temperature for a preset time.
进一步的，上述可食性益生菌的制备方法，其中，所述预设温度为10℃- 45℃、预设时间为8-72小时。Further, in the above preparation method of edible probiotics, the preset temperature is 10°C-45°C and the preset time is 8-72 hours.
与现有技术相比：本发明首次提取出健康、无癌、长寿人群来源的益生菌，借助高密度液体发酵技术，显著增加了益生菌制剂的活菌数（不低于1.0×109CFU/mL）,与传统筛菌所得益生菌活菌数（1.0×106CFU/mL～1.0×107CFU/mL）相比较，菌体生物量提高了至少2个数量级。Compared with the existing technology: this invention extracts probiotics from healthy, cancer-free and longevity people for the first time. With the help of high-density liquid fermentation technology, the number of viable bacteria in the probiotic preparation is significantly increased (not less than 1.0×109 CFU /mL), compared with the number of viable probiotic bacteria (1.0×106 CFU/mL ~ 1.0×107 CFU/mL) obtained by traditional screening bacteria, the bacterial biomass has increased by at least 2 orders of magnitude.
另外，本发明至少还具有如下的有益效果：In addition, the present invention at least also has the following beneficial effects:
1、将活化好的益生菌菌种接种于高密度发酵培养基中进行发酵培养，通过控温控氧和补料分批发酵技术等提高益生菌的活菌数，从而获得高密度的菌液。并将废弃发酵液进行灭菌处理，再与原发酵液对半稀释使用，也可以进行益生菌的高密度培养，实现了发酵培养基的二次利用；1. Inoculate the activated probiotic strains into a high-density fermentation medium for fermentation culture, and increase the number of viable probiotics through temperature and oxygen control and fed-batch fermentation technology to obtain a high-density bacterial liquid. . The waste fermentation liquid is sterilized and then diluted in half with the original fermentation liquid for use. High-density cultivation of probiotics can also be carried out, realizing the secondary utilization of the fermentation medium;
2、将肠道益生菌通过高密度发酵技术进行发酵培养，使发酵液中活菌量达到至少1.5×109CFU/mL。采用冷冻干燥技术进行冷冻干燥，制成具有内层包埋层的活性微生物菌剂。后进行外层包埋。有效地增加益生菌的抗逆性，延长益生菌保存期的活性，保护益生菌不受胃酸的侵蚀而使其安全到达小肠、大肠后靶向定植，发挥益生菌的生物学功效。经多层包埋技术包埋的益生菌，抗逆性、稳定性和存活率比一般未经包埋的益生菌效果要好的多。2. Ferment and culture intestinal probiotics through high-density fermentation technology, so that the amount of viable bacteria in the fermentation broth reaches at least 1.5×109 CFU/mL. Freeze-drying technology is used to freeze-dry the active microbial agent with an inner embedded layer. Then carry out outer layer embedding. It effectively increases the stress resistance of probiotics, prolongs the shelf life of probiotics, and protects probiotics from being eroded by gastric acid so that they can safely reach the small intestine and large intestine for targeted colonization and exert the biological effects of probiotics. The stress resistance, stability and survival rate of probiotics embedded through multi-layer encapsulation technology are much better than those of ordinary probiotics that are not embedded.
3、将培养成熟的益生菌种子加入到含有适合益生菌生长繁殖的固定化载体溶液中，然后将益生菌种子的固定化载体溶液滴入一种固化液中，或将固化液加入固定化载体中使之凝固。凝固后的含有益生菌的固定化载体用无菌水洗涤，然后置于10℃- 45℃密闭培养8-72小时。该技术生产的固定化益生菌不需要分离冻干，可直接作为益生菌服用，也可放入其他食品中；常温下可长期密闭保存，菌数仍可不断生长。3. Add the mature probiotic seeds to an immobilized carrier solution suitable for the growth and reproduction of probiotics, and then drop the immobilized carrier solution of the probiotic seeds into a solidifying solution, or add the solidifying solution to the immobilized carrier. Make it solidify. The coagulated immobilized carrier containing probiotic bacteria is washed with sterile water, and then placed in a closed culture at 10°C-45°C for 8-72 hours. The immobilized probiotics produced by this technology do not need to be separated and freeze-dried. They can be taken directly as probiotics or put into other foods. They can be stored airtightly at room temperature for a long time, and the number of bacteria can still continue to grow.
附图说明Description of the drawings
图1为本发明一实施例当中提出的植物乳杆菌的培养方法中的不同发酵pH对活菌数的影响的示意图；Figure 1 is a schematic diagram illustrating the impact of different fermentation pH on the number of viable bacteria in the culture method of Lactobacillus plantarum proposed in one embodiment of the present invention;
图2为本发明一实施例当中提出的植物乳杆菌的培养方法中的不同胆盐浓度对活菌数的影响的示意图；Figure 2 is a schematic diagram illustrating the impact of different bile salt concentrations on the number of viable bacteria in the culture method of Lactobacillus plantarum proposed in one embodiment of the present invention;
图3为本发明一实施例当中提出的植物乳杆菌的培养方法中的分离株抗氧化性测定的示意图；Figure 3 is a schematic diagram of the determination of antioxidant properties of isolates in the culture method of Lactobacillus plantarum proposed in one embodiment of the present invention;
图4为本发明一实施例当中提出的植物乳杆菌的培养方法中的抑制致病菌能力实验结果的示意图。Figure 4 is a schematic diagram illustrating the experimental results of the ability to inhibit pathogenic bacteria in the culture method of Lactobacillus plantarum proposed in one embodiment of the present invention.
如下具体实施方式将结合上述附图进一步说明本发明。The following specific embodiments will further illustrate the present invention in conjunction with the above-mentioned drawings.
具体实施方式Detailed ways
为了便于理解本发明，下面将对本发明进行更全面的描述。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容更加透彻全面。In order to facilitate an understanding of the invention, the invention will be described more fully below. However, the invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“及／或”包括一个或多个相关的所列项目的任意的和所有的组合。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the invention belongs. The terminology used herein in the description of the invention is for the purpose of describing specific embodiments only and is not intended to limit the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
实施例一Embodiment 1
植物乳杆菌MH-301的获取Acquisition of Lactobacillus plantarum MH-301
1）菌株分离1) Strain isolation
采集江西省上饶市婺源县赋春镇源头村健康长寿志愿者的早晨第一次粪样（三个月内未服用抗生素，非素食者），立即转入密封厌氧塑料操作袋内（85％ N2，5％ H2，10％CO2）放置一小时，之后称取10 g粪便样品到内置灭菌甘油的 10 mL 的离心管内，甘油含量为样品的 10％，用封口胶密封离心管口（以上操作在厌氧袋内完成）。将样品保存在4℃保温盒中，迅速送实验室进行下一步处理。Collect the first morning stool samples from healthy and longevity volunteers in Yuanyuan Village, Fuchun Town, Wuyuan County, Shangrao City, Jiangxi Province (people who have not taken antibiotics within three months, non-vegetarians), and immediately transfer them into sealed anaerobic plastic operating bags (85% N2 , 5% H2 , 10% CO2 ) for one hour, then weigh 10 g of stool sample into a 10 mL centrifuge tube with built-in sterilized glycerol. The glycerin content is 10% of the sample. Seal the centrifuge tube with sealing tape. mouth (the above operations are completed in an anaerobic bag). Store the samples in a 4°C incubator and quickly send them to the laboratory for further processing.
无菌采取粪便0.5-1.0 g，加入试管中，添加无菌玻璃珠对收集的粪便进行充分振荡，混匀后进行10倍比例稀释，选择合适浓度在商品化MRS琼脂培养基上培养计数。待菌落长出后，挑选平皿中底为黄色、菌落中等大小，凸起，微白色，湿润，边缘整齐，菌落呈圆形，直径为3.0 mm±1 mm的菌落。同时对菌落进行革兰染色，革兰染色为阳性的菌落即为乳酸杆菌。Aseptically collect 0.5-1.0 g of feces, add it to a test tube, add sterile glass beads, shake the collected feces thoroughly, mix and dilute 10 times, select the appropriate concentration and culture and count on commercial MRS agar medium. After the colonies grow, select colonies with a yellow bottom in the flat dish, medium-sized colonies, convex, slightly white, moist, neat edges, round colonies, and a diameter of 3.0 mm ± 1 mm. At the same time, Gram stain was performed on the colonies. The colonies with positive Gram stain were Lactobacilli.
2）鉴定与保藏2) Identification and preservation
利用 DNA 提取试剂盒分离单菌基因组 DNA，进行16S rRNA测序。测序结果去掉载体序列，可得到目的序列。在 NCBI 上运行 BLAST 程序，在 Gene Bank 中对克隆的目的基因进行同源性搜索，寻找具有较高同源性的16SrRNA 序列，取相似度大于 99％以上的序列做参考，做出鉴定。最终筛选得到植物乳杆菌(Lactobacillus plantarum)并已进行保藏，命名为植物乳杆菌MH-301，保藏号为CGMCC No.23397，保藏时间为2021年09月13日，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。Use a DNA extraction kit to isolate single bacterial genomic DNA and perform 16S rRNA sequencing. The target sequence can be obtained by removing the vector sequence from the sequencing results. Run the BLAST program on NCBI, perform a homology search on the cloned target gene in Gene Bank, look for 16SrRNA sequences with higher homology, and use sequences with a similarity greater than 99% as reference to make identifications. Lactobacillus plantarum was finally screened and preserved, named Lactobacillus plantarum MH-301, with the preservation number CGMCC No. 23397, and the preservation date is September 13, 2021. It was deposited in the China Microbial Culture Collection Management General Microbiology Center of the Committee, the depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.
实施例二Embodiment 2
耐酸实验、耐胆盐实验、分离株抗氧化性的测定、抑制致病菌能力实验、分离株细胞粘附能力评价Acid resistance test, bile salt resistance test, determination of antioxidant properties of isolates, ability test to inhibit pathogenic bacteria, evaluation of cell adhesion ability of isolates
耐酸实验流程：Acid resistance test process:
用 1mol/L 的盐酸将 MRS 液体培养基的 pH 调节为 2.0， 3.0， 4.0， 5.0， 6.0和 7.0，将 18 h 培养的益生菌菌液以 1％的比例分别接入常规液体培养基和 pH 为2.0， 3.0， 4.0， 5.0， 6.0 和 7.0 液体培养基中，检测培养 3 h 的益生菌生长状况，采取倍量稀释的方法将菌液点于平板培养基上，计算活菌数，每一计数均重复三次取平均值，以各候选菌株常规液体发酵液作对照，按公式计其存活率。存活率（％） =候选菌株不同 pH值培养基发酵液中活菌数/侯选菌株常规培养基发酵液中活菌数× 100％。Use 1 mol/L hydrochloric acid to adjust the pH of the MRS liquid culture medium to 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0. Add the probiotic liquid cultured for 18 hours into the conventional liquid culture medium and pH at a ratio of 1%. In 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0 liquid culture media, detect the growth status of probiotics after 3 h of culture. Use the method of doubling dilution to spot the bacterial solution on the plate culture medium and calculate the number of viable bacteria. The counts were repeated three times and the average value was taken. The conventional liquid fermentation broth of each candidate strain was used as a control, and the survival rate was calculated according to the formula. Survival rate (%) = number of viable bacteria in the fermentation broth of different pH media for the candidate strain/number of viable bacteria in the fermentation broth of the candidate strain's conventional culture media × 100%.
其中，如图1所示，试验结果表明经过培养出的植物乳杆菌的耐酸性尚佳，能很好的应对现实当中的酸性条件。Among them, as shown in Figure 1, the test results show that the cultured Lactobacillus plantarum has good acid resistance and can well cope with the acidic conditions in reality.
耐胆盐实验流程：Bile salt resistance test procedure:
取耐酸实验筛选后具有良好耐酸性能的菌株，培养 18 h，将菌液以 1％的比例接入常规液体培养基和分别含有0.1％，0.3％和0.5％牛胆盐的液体培养基中，检测培养 18h 的益生菌生长状况，采取倍量稀释的方法将益生菌液点于平板培养基上，计算活菌数，每一计数均重复三次取平均值，以各候选菌株常规液体发酵液作对照， 计算其存活率。存活率（％） =候选菌株不同胆盐浓度培养基发酵液中活菌数/候选菌株常规培养基发酵液中活菌数× 100％。Take the strains with good acid resistance after screening by acid resistance experiment, culture them for 18 hours, and add the bacterial liquid into the conventional liquid culture medium at a ratio of 1% and the liquid culture medium containing 0.1%, 0.3% and 0.5% ox bile salts respectively. Detect the growth status of probiotics after culturing for 18 hours. Use the method of doubling dilution to spot the probiotic liquid on the plate culture medium and calculate the number of viable bacteria. Each count is repeated three times and the average value is taken. Use the conventional liquid fermentation broth of each candidate strain as the Control and calculate the survival rate. Survival rate (%) = the number of viable bacteria in the fermentation broth of the candidate strain's culture medium with different bile salt concentrations/the number of viable bacteria in the conventional culture medium and fermentation broth of the candidate strain × 100%.
其中，如图2所示，试验结果表明经过培养出的植物乳杆菌的耐胆盐能力尚佳，能很好的应对现实当中的胆盐浓度条件。Among them, as shown in Figure 2, the test results show that the cultured Lactobacillus plantarum has good bile salt tolerance and can well cope with the actual bile salt concentration conditions.
分离株抗氧化性的测定：Determination of antioxidant properties of isolates:
将分离鉴定后得到的细菌用对应（不加 L-半胱氨酸）液体培养基培养1-2 d，待长到1010CFU/mL 左右时，离心取上清，4℃保存备用。用于DPPH自由基清除能力、清除超氧自由基（O2-）能力、清除羟自由基（HO·）能力、对Fe2+螯合能力及还原活性的测定。其中，测试结果如图3所示，The bacteria obtained after isolation and identification were cultured in the corresponding (without L-cysteine) liquid medium for 1-2 d. When the bacteria grew to about 1010 CFU/mL, centrifuge to take the supernatant and store it at 4°C for later use. Used for the determination of DPPH free radical scavenging ability, superoxide radical scavenging ability (O2- ), hydroxyl radical scavenging ability (HO·), Fe2+ chelating ability and reducing activity. Among them, the test results are shown in Figure 3,
抑制致病菌能力实验：Experiment on the ability to inhibit pathogenic bacteria:
通过酸面团分离筛选得到测试细菌拮抗的8株常见食源性致病菌，包括E.coliO157、S.aureus、S.hemolytic-β、S.castellani、C.albicans、S.enteritidis、S.typhimurium，将7株致病菌用LB平板培养基活化后，并用LB液体培养基扩大培养；将8株致病菌的菌体浓度调至108CFU/mL 左右，分别取200μL菌液涂布于LB固体培养基（5%琼脂），每株致病菌涂布3个平板；待菌液凝固后，在每个平板中放置无菌牛津杯，向牛津杯中加入200 μL益生菌的原倍培养上清液37℃培养6-8 h。观察抑菌圈形状并拍照，同时用游标卡尺计量抑菌圈直径。Through sourdough isolation and screening, 8 strains of common foodborne pathogenic bacteria were tested for bacterial antagonism, includingE.coliO157 ,S.aureus ,S.hemolytic-β ,S.castellani ,C.albicans ,S.enteritidis , andS.typhimurium , after activating 7 strains of pathogenic bacteria with LB plate culture medium, and expanding the culture with LB liquid culture medium; adjust the cell concentration of 8 strains of pathogenic bacteria to about 108 CFU/mL, and apply 200 μL of bacterial liquid on LB solid medium (5% agar), apply each pathogenic bacteria to 3 plates; after the bacterial liquid solidifies, place a sterile Oxford cup in each plate, and add 200 μL of the original times of the probiotics to the Oxford cup Culture supernatant was cultured at 37°C for 6-8 h. Observe the shape of the inhibition zone and take photos, and use a vernier caliper to measure the diameter of the inhibition zone.
其中，如图4所示，试验结果表明经过培养出的植物乳杆菌的抑菌尚佳，能很好的应对现实当中的菌体环境。Among them, as shown in Figure 4, the test results show that the cultured Lactobacillus plantarum has good antibacterial properties and can well cope with the actual bacterial environment.
分离株细胞粘附能力评价：Evaluation of cell adhesion ability of isolates:
将培养好的HT29细胞进行消化，用DMEM溶液稀释细胞，使浓度为5×105cell/mL，从中吸出1 mL细胞悬液于12孔细胞培养板中，5％CO2培养箱内于37℃下培养细胞长至单层。用灭菌的PBS溶液洗三次，然后加入1 mL cFDA-SE标记的益生菌悬液，静置培养，灭菌溶液洗三次，除去未粘附的益生菌。每个孔中加入0.7 mL胰酶消化，待细胞与培养板底部完全脱落，加入0.3 mL DMEM培养液终止消化，收集孔内的菌体悬液，使用荧光分光光度计测定益生菌悬浮液的荧光强度。荧光检测条件：激发波长492 nm，发射波长517 nm，狭缝宽2.5nm。粘附率按如下公式计算：粘附率（％）=粘附益生菌荧光强度/初始益生菌荧光强度×100％。发明人进行植物乳杆菌MH-301的细胞黏附率测定，黏附率达50%±14%。Digest the cultured HT29 cells, dilute the cells with DMEM solution to a concentration of 5×105 cell/mL, and suck out 1 mL of cell suspension into a 12-well cell culture plate, incubate in a 5% CO2 incubator at 37 Cells were cultured at ℃ until they grew into a monolayer. Wash three times with sterile PBS solution, then add 1 mL cFDA-SE labeled probiotic suspension, let it stand for culture, wash three times with sterile solution, and remove non-adherent probiotics. Add 0.7 mL trypsin to each well for digestion. When the cells are completely detached from the bottom of the culture plate, add 0.3 mL DMEM culture solution to terminate digestion. Collect the bacterial suspension in the well and use a fluorescence spectrophotometer to measure the fluorescence of the probiotic suspension. strength. Fluorescence detection conditions: excitation wavelength 492 nm, emission wavelength 517 nm, slit width 2.5nm. The adhesion rate was calculated according to the following formula: adhesion rate (%) = fluorescence intensity of adherent probiotics/initial probiotic fluorescence intensity × 100%. The inventor measured the cell adhesion rate of Lactobacillus plantarum MH-301 and found that the adhesion rate reached 50% ± 14%.
实施例三Embodiment 3
植物乳杆菌MH-301高密度培养Lactobacillus plantarum MH-301 high-density culture
实施例提供一种植物乳杆菌MH-301高密度培养方法，具体包括以下步骤：The embodiment provides a high-density culture method for Lactobacillus plantarum MH-301, which specifically includes the following steps:
1）制备高密度发酵培养基1) Preparation of high-density fermentation medium
高密度培养基的组成为：胰蛋白胨5-10g/L、大豆蛋白胨4-10g/L、葡萄糖5-15 g/L、牛肉粉3-5 g/L、酵母浸出粉3-5 g/L、乳糖2-5 g/L、KHPO2·7H2O 1-2 g/L、甘油磷酸钠10-15 g/L，抗坏血酸钠0.1-0.5 g/L、甲酸0.1-0.3 g/L、吐温0.5-2.0 g/L、MgSO20.58-0.6g/L、醋酸钠 1-10 g/L、柠檬酸三铵1.0-2.0 g/L和MnSO20.28-0.3 g/L。将上述原料用蒸馏水定容至1000mL，调节pH至6.8±0.3，搅拌溶解均匀后，0 .08-0 .10MPa灭菌15-20min，冷却即可。The composition of high-density culture medium is: tryptone 5-10g/L, soy peptone 4-10g/L, glucose 5-15 g/L, beef meal 3-5 g/L, yeast extract powder 3-5 g/L , lactose 2-5 g/L, KHPO2 ·7H2 O 1-2 g/L, sodium glycerophosphate 10-15 g/L, sodium ascorbate 0.1-0.5 g/L, formic acid 0.1-0.3 g/L, vomiting Temperature 0.5-2.0 g/L, MgSO2 0.58-0.6g/L, sodium acetate 1-10 g/L, triammonium citrate 1.0-2.0 g/L and MnSO2 0.28-0.3 g/L. Dilute the above raw materials to 1000mL with distilled water, adjust the pH to 6.8±0.3, stir and dissolve evenly, sterilize at 0.08-0.10MPa for 15-20 minutes, and cool.
2）活化接种2) Activation vaccination
将活化好的植物乳杆菌MH-301接种于高密度发酵培养基中进行培养。The activated Lactobacillus plantarum MH-301 was inoculated into a high-density fermentation medium for culture.
3）发酵3) Fermentation
通过对温度，初始 pH、摇床转速等条件的研究得到菌体在此培养基中适宜的试管培养条件为：初始 pH6.5，36.2℃，保温静止发酵。Through the study of temperature, initial pH, shaker speed and other conditions, we found that the suitable test tube culture conditions for the bacteria in this medium are: initial pH 6.5, 36.2°C, insulated static fermentation.
分批补料：Feeding in batches:
发酵罐的pH探头能够实时监测发酵液的pH变化，因此采取恒流补碱的方法，滴加15%的NaOH溶液，使发酵液的pH保持在6.5±0.1。葡萄糖的补加应根据实际发酵情况而定，当葡萄糖浓度降至5g·L-1时开始第1次补糖，初次应补充10%的灭菌葡萄糖170 m L，使糖浓度升至7 g·L-1。当葡萄糖浓度降至5 g·L-1左右时重复补加葡萄糖，整个过程保持葡萄糖浓度在5~7 g·L-1范围内。The pH probe of the fermentation tank can monitor the pH changes of the fermentation broth in real time. Therefore, a constant flow alkali supplement method is adopted, and 15% NaOH solution is added dropwise to maintain the pH of the fermentation broth at 6.5±0.1. The supplementation of glucose should be based on the actual fermentation conditions. When the glucose concentration drops to 5g·L-1 , the first sugar supplementation should be started. For the first time, 170 mL of 10% sterilized glucose should be added to increase the sugar concentration to 7 g. ·L-1 . When the glucose concentration drops to about 5 g·L-1 , glucose is added repeatedly to keep the glucose concentration within the range of 5 to 7 g·L-1 during the entire process.
分别对不同参数下的培养基的组成制备的高密度发酵培养基培养出的活菌数进行测定，如下表一所示：The number of viable bacteria cultured in the high-density fermentation medium prepared with the composition of the medium under different parameters was measured, as shown in Table 1 below:
表一Table I
从表一可以明显看出，借助独创的原有的高密度液体发酵技术，显著增加了益生菌制剂的活菌数（不低于1.0×109CFU/mL）,与传统筛菌所得益生菌活菌数（1.0×106CFU/mL～1.0×107CFU/mL）相比较，菌体生物量提高了至少2个数量级。It can be clearly seen from Table 1 that with the help of the original high-density liquid fermentation technology, the number of viable bacteria in the probiotic preparation has been significantly increased (not less than 1.0×109 CFU/mL), which is comparable to the probiotics obtained by traditional screening of bacteria. Compared with the number of viable bacteria (1.0×106 CFU/mL ~ 1.0×107 CFU/mL), the bacterial biomass increased by at least 2 orders of magnitude.
实施例四Embodiment 4
本实施例提供一种微生物临床制剂及其制备方法，具体包括以下步骤：This embodiment provides a microbial clinical preparation and a preparation method thereof, which specifically includes the following steps:
1）菌群的扩大培养1) Expansion of culture of bacterial flora
将植物乳杆菌MH-301通过高密度发酵技术（例如上述实施例1当中的高密度培养）进行发酵培养，使发酵液中活菌量达到至少1.5×109CFU/mL。Lactobacillus plantarum MH-301 is fermented and cultured through high-density fermentation technology (such as the high-density culture in Example 1 above), so that the amount of viable bacteria in the fermentation broth reaches at least 1.5×109 CFU/mL.
2）冷冻干燥2) Freeze drying
进行冷冻干燥前将单株益生菌用MRS培养基进行发酵36 h- 48 h，并进行活菌计数，至1.5×109CFU/mL，达到相应剂量后3500 r/min离心15 min，弃上清，生理盐水洗涤，离心弃上清。用等体积脱脂乳吹打混匀，存放于-80 ℃，48 h后用冻干机继续冻干，上机约48h后取出，碾碎，加入红薯粉等辅料，混匀，称取1 g混匀的粉剂进行活菌计数，根据活菌计数情况计算剂量，加入多孔淀粉、乳清粉和海藻糖混合后形成的包埋层进行包埋，制作胶囊，之后随机挑选几粒胶囊进行活菌计数，达到要求后低温保存。Before freeze-drying, ferment the single strain of probiotics in MRS culture medium for 36 h-48 h, and count the viable bacteria to 1.5×109 CFU/mL. After reaching the corresponding dose, centrifuge at 3500 r/min for 15 min and discard. Clear, wash with physiological saline, centrifuge and discard the supernatant. Use an equal volume of skim milk to mix evenly, store it at -80°C, and continue freeze-drying with a freeze dryer after 48 hours. Take it out after about 48 hours in the machine, crush it, add sweet potato powder and other accessories, mix well, and weigh 1 g of the mixture. Use a uniform powder to count viable bacteria. Calculate the dose based on the viable bacteria count. Add porous starch, whey powder and trehalose to form an embedding layer and embed it into capsules. Then randomly select a few capsules to count viable bacteria. , stored at low temperature after meeting the requirements.
实施例五Embodiment 5
本实施例提供一种可食性益生菌及其制备方法，具体包括以下步骤：This embodiment provides an edible probiotic and a preparation method thereof, which specifically includes the following steps:
1）固化1) Curing
将培养成熟的植物乳杆菌MH-301种子按比例加入到含有适合益生菌生长繁殖的固定化载体溶液中，在固定化载体溶液滴入固化液；或将固化液加入固定化载体中使之凝固。Add the mature Lactobacillus plantarum MH-301 seeds in proportion to an immobilized carrier solution suitable for the growth and reproduction of probiotics, and drop the solidifying solution into the immobilized carrier solution; or add the solidifying solution to the immobilized carrier to solidify it. .
2）固化洗涤培养2) Solidification washing culture
将凝固后的含有植物乳杆菌MH-301的固定化载体用无菌水洗涤，后置于10℃- 45℃下密闭培养8-72h。The coagulated immobilized carrier containing Lactobacillus plantarum MH-301 is washed with sterile water, and then placed in a closed culture at 10°C-45°C for 8-72 hours.
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。The above-mentioned embodiments only express several implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the patent scope of the present invention. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the scope of protection of the patent of the present invention should be determined by the appended claims.
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