技术领域technical field
本发明涉及生物技术领域，尤其是涉及一种乳酸片球菌GLP06及其应用和产品。The invention relates to the field of biotechnology, in particular to Pediococcus lactis GLP06 and its application and products.
背景技术Background technique
乳酸片球菌(Pediococcus acidilactici，P.acidilactici)分类于片球菌属(Pediococcus)。研究表明，乳酸片球菌具有良好的益生作用，如能够通过调节小鼠肠道菌群来改善小鼠便秘症状；降低患糖尿病大鼠的血糖水平并增强胰腺β细胞功能；还可以提高断奶仔猪营养物质消化率，调节其血清生化指标，提高其抗氧化能力；另有研究表明乳酸片球菌会产生片球菌素，片球菌素具有耐热、耐酸碱、易被蛋白酶降解，具有相对宽的抑菌谱，对一些病原菌(如：李斯特菌)有较强的抑制作用，因此乳酸片球菌及其代谢产物已被作为生物防腐剂添加到饲料和发酵食品及肉制品的保存中。Pediococcus acidilactici (P. acidilactici) is classified in the genus Pediococcus. Studies have shown that Pediococcus lactis has good probiotic effects, such as being able to improve the symptoms of constipation in mice by regulating the intestinal flora of mice; reducing blood sugar levels in diabetic rats and enhancing the function of pancreatic β cells; it can also improve the nutrition of weaned piglets material digestibility, adjust its serum biochemical indicators, and improve its antioxidant capacity; other studies have shown that Pediococcus lactis can produce pediocin, which is heat-resistant, acid-alkali-resistant, and easily degraded by proteases, and has a relatively wide inhibitory effect. Spectrum of bacteria, has a strong inhibitory effect on some pathogenic bacteria (such as: Listeria), so Pediococcus lactis and its metabolites have been added as biological preservatives to the preservation of feed, fermented food and meat products.
随着犬、猫角色由“看家护院”到“情感陪伴”的转变，其社会角色也转变成伴侣动物或家庭成员，越来越多的宠物主愿意为宠物的健康和改善宠物福利付出更多的时间和金钱。伴侣动物饮食已经从自己猎食和简单的喂养转变成追求科学化、精细化、健康化的饮食模式，伴侣动物的食品研发已经向营养均衡和保证动物健康的方向发展。无毒副作用、无残留的益生菌制剂在伴侣动物疾病治疗和预防保健中的应用越来越广泛。有研究人员提出使用来自宿主物种肠道的益生菌才是最理想的，然而，对于目前市场上绝大多数伴侣动物的益生菌产品来说都是非宿主源的，宿主源菌种的研究和应用仍较少。使用从动物自身分离得到的宿主源能更好的适应胃肠道环境，从而能更好地发挥其益生特性，取得最大化的治疗和保健效果。As the roles of dogs and cats change from "home care" to "emotional companionship", their social roles are also transformed into companion animals or family members. More and more pet owners are willing to pay for the health of their pets and the improvement of pet welfare. More time and money. The diet of companion animals has changed from self-hunting and simple feeding to a scientific, refined and healthy diet pattern. The research and development of food for companion animals has developed towards the direction of nutritional balance and ensuring animal health. Probiotic preparations without toxic side effects and residues are more and more widely used in the treatment and preventive health care of companion animal diseases. Some researchers have proposed that the use of probiotics from the gut of the host species is the most ideal. However, the vast majority of probiotic products for companion animals on the market are non-host-derived. The research and application of host-derived strains Still less. Using the host source isolated from the animal itself can better adapt to the gastrointestinal tract environment, so that it can better exert its probiotic properties and achieve maximum therapeutic and health care effects.
目前关于犬源益生菌的报道逐渐增加，周雨等将犬源益生菌(罗伊氏乳酸菌)与马齿苋联合用来防治番泻叶导致的犬腹泻。王明艳等将犬源植物乳杆菌和枯草芽孢杆制成二联菌粉可一定程度调节肠道菌群，改善犬腹泻程度。但犬源乳酸片球菌的报道较少，市场响应产品更少。At present, the reports on canine probiotics are gradually increasing. Zhou Yu et al. combined canine probiotics (Lactobacillus reuteri) and purslane to prevent and treat dog diarrhea caused by senna. Wang Mingyan et al. made canine-derived Lactobacillus plantarum and Bacillus subtilis into a dual bacterial powder, which can regulate intestinal flora to a certain extent and improve the degree of dog diarrhea. However, there are fewer reports on Pediococcus lactis from canine origin, and fewer products responding to the market.
有鉴于此，特提出本发明。In view of this, the present invention is proposed.
发明内容Contents of the invention
本发明的第一目的在于提供一种乳酸片球菌(Pediococcus acidilactici)GLP06，以解决上述问题中的至少一种。The first object of the present invention is to provide a Pediococcus acidilactici GLP06 to solve at least one of the above problems.
本发明的第二目的在于提供一种菌剂。The second object of the present invention is to provide a bacterial agent.
本发明的第三目的在于提供上述乳酸片球菌(Pediococcus acidilactici)GLP06或者菌剂在制备抑菌产品中的应用。The third object of the present invention is to provide the application of the above-mentioned Pediococcus acidilactici GLP06 or bacterial agent in the preparation of bacteriostatic products.
本发明的第四目的在于提供上述乳酸片球菌(Pediococcus acidilactici)GLP06或者菌剂在制备提高免疫力的产品中的应用。The fourth object of the present invention is to provide the application of the above-mentioned Pediococcus acidilactici GLP06 or bacterial agent in the preparation of products for improving immunity.
本发明的第五目的在于提供上述乳酸片球菌(Pediococcus acidilactici)GLP06或者菌剂在制备缓解动物氧化应激的产品中的应用。The fifth object of the present invention is to provide the application of the above-mentioned Pediococcus acidilactici GLP06 or bacterial agent in the preparation of products for alleviating oxidative stress in animals.
本发明的第六目的在于提供上述乳酸片球菌(Pediococcus acidilactici)GLP06或者菌剂在制备提高动物肠道菌群物种多样性的产品中的应用。The sixth object of the present invention is to provide the application of the above-mentioned Pediococcus acidilactici GLP06 or bacterial agent in the preparation of products for increasing species diversity of animal intestinal flora.
本发明的第七目的在于提供一种饲料。The seventh object of the present invention is to provide a feed.
本发明的第八目的在于提供一种药物。The eighth object of the present invention is to provide a medicine.
第一方面，本发明提供了一种乳酸片球菌(Pediococcus acidilactici)GLP06，所述乳酸片球菌(Pediococcus acidilactici)GLP06保藏于中国典型培养物保藏中心，保藏编号为：CCTCC NO:M 2023200。In a first aspect, the present invention provides a Pediococcus acidilactici GLP06, which is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: M 2023200.
作为进一步技术方案，所述乳酸片球菌(Pediococcus acidilactici)GLP06来源于犬。As a further technical solution, the Pediococcus acidilactici GLP06 is derived from dogs.
第二方面，本发明提供了一种菌剂，包括所述的乳酸片球菌(Pediococcusacidilactici)GLP06。In a second aspect, the present invention provides a bacterial agent, including the Pediococcus acidilactici GLP06.
第三方面，本发明提供了所述的乳酸片球菌(Pediococcus acidilactici)GLP06或者菌剂在制备抑菌产品中的应用。In a third aspect, the present invention provides the application of the Pediococcus acidilactici GLP06 or bacterial agent in the preparation of bacteriostatic products.
第四方面，本发明提供了所述的乳酸片球菌(Pediococcus acidilactici)GLP06或者菌剂在制备提高免疫力的产品中的应用。In a fourth aspect, the present invention provides the application of the Pediococcus acidilactici GLP06 or bacterial agent in the preparation of immunity-enhancing products.
第五方面，本发明提供了所述的乳酸片球菌(Pediococcus acidilactici)GLP06或者菌剂在制备缓解动物氧化应激的产品中的应用。In the fifth aspect, the present invention provides the application of the Pediococcus acidilactici GLP06 or bacterial agent in the preparation of products for alleviating oxidative stress in animals.
第六方面，本发明提供了所述的乳酸片球菌(Pediococcus acidilactici)GLP06或者菌剂在制备提高动物肠道菌群物种多样性的产品中的应用。In the sixth aspect, the present invention provides the application of the Pediococcus acidilactici GLP06 or bacterial agent in the preparation of products for increasing species diversity of animal intestinal flora.
第七方面，本发明提供了一种饲料，包括所述的乳酸片球菌(Pediococcusacidilactici)GLP06或者菌剂。In the seventh aspect, the present invention provides a feed, including the Pediococcus acidilactici (Pediococcus acidilactici) GLP06 or bacterial agent.
第八方面，本发明提供了一种药物，包括所述的乳酸片球菌(Pediococcusacidilactici)GLP06或者菌剂。In the eighth aspect, the present invention provides a medicament comprising the Pediococcus acidilactici GLP06 or bacterial agent.
作为进一步技术方案，所述药物还包括辅料。As a further technical solution, the medicine also includes auxiliary materials.
与现有技术相比，本发明提供的乳酸片球菌GLP06具有如下有益效果：Compared with the prior art, Pediococcus lactis GLP06 provided by the present invention has the following beneficial effects:
1.来自于健康犬的粪便中，作为宠物益生菌更适合宠物胃肠道，可缓解宠物市场上宠物益生菌来源不明确的棘手问题。1. From the feces of healthy dogs, as a pet probiotic, it is more suitable for the gastrointestinal tract of pets, and can alleviate the thorny problem of unclear sources of pet probiotics in the pet market.
2.抗菌效果很好，可预防犬腹泻。2. The antibacterial effect is very good, which can prevent dog diarrhea.
3.耐高温性强，超过70℃存活率仍然较高，可解决益生菌无法高温喷涂的技术壁垒。3. High temperature resistance, the survival rate is still high above 70°C, which can solve the technical barrier that probiotics cannot be sprayed at high temperature.
4.耐酸，耐胆盐，耐人工肠胃液，粘附性强，都说明其具有很好的益生潜力，在肠道中定值能力好，具有优秀的益生潜力。4. Acid resistance, bile salt resistance, artificial gastrointestinal juice resistance, and strong adhesion all indicate that it has good probiotic potential. It has good value-setting ability in the intestinal tract and has excellent probiotic potential.
5.抗氧化能力好，能显著降低灌服小鼠血液中的MDA和BUN含量，提高SOD活性，可缓解宠物应激导致过氧化反应，延缓衰老，延长宠物寿命。5. Good anti-oxidation ability, can significantly reduce the content of MDA and BUN in the blood of fed mice, increase the activity of SOD, relieve the peroxidation reaction caused by pet stress, delay aging, and prolong the life of pets.
6.饲喂小鼠后可显著提高小鼠胸腺器官指数，说明其可以促进宿主免疫系统的发育，提高免疫力，具有预防疾病的潜力。6. After feeding mice, the thymus organ index of mice can be significantly increased, indicating that it can promote the development of the host immune system, improve immunity, and have the potential to prevent diseases.
7.饲喂小鼠后可显著提高小鼠肠道的α多样性，说明饲喂其可以提高肠道菌群的物种多样性。7. After feeding the mice, the α-diversity of the mouse intestine can be significantly increased, indicating that feeding it can increase the species diversity of the intestinal flora.
8.饲喂小鼠后可提高小鼠肠道内Muribaculaceae丰度，降低与其处于相同生态位的致病菌Clostridium丰度，阻碍致病菌在肠道的定居，说明其可通过调节宿主胃肠菌群来改善动物健康。8. Feeding mice can increase the abundance of Muribaculaceae in the intestinal tract of mice, reduce the abundance of pathogenic bacteria Clostridium in the same ecological niche, and hinder the colonization of pathogenic bacteria in the intestinal tract, indicating that it can regulate the host gastrointestinal bacteria flocks to improve animal health.
附图说明Description of drawings
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the specific implementation or description of the prior art. Obviously, the accompanying drawings in the following description The drawings show some implementations of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.
图1为乳酸片球菌GLP06菌落与形态；Figure 1 is the colony and morphology of Pediococcus lactis GLP06;
图2为乳酸片球菌GLP06进化树；Fig. 2 is the phylogenetic tree of Pediococcus lactis GLP06;
图3为乳酸片球菌GLP06生长及产酸速率曲线；Fig. 3 is the growth and acid production rate curve of Pediococcus lactis GLP06;
图4为乳酸片球菌GLP06对高温的耐受性；Figure 4 shows the tolerance of Pediococcus lactis GLP06 to high temperature;
图5为乳酸片球菌GLP06对酸度的耐受性；Figure 5 is the tolerance of Pediococcus lactis GLP06 to acidity;
图6为乳酸片球菌GLP06在人工胃肠液中的存活率；Fig. 6 is the survival rate of Pediococcus lactis GLP06 in artificial gastrointestinal fluid;
图7为乳酸片球菌GLP06在酸和不同胆盐浓度中的存活率；Figure 7 is the survival rate of Pediococcus lactis GLP06 in acid and different bile salt concentrations;
图8为乳酸片球菌GLP06粘附细胞结果；Figure 8 is the result of adherent cells of Pediococcus lactis GLP06;
图9为乳酸片球菌GLP06的抑菌活性；Fig. 9 is the antibacterial activity of Pediococcus lactis GLP06;
图10为乳酸片球菌GLP06的溶血结果；Figure 10 is the hemolysis result of Pediococcus lactis GLP06;
图11为饲喂乳酸片球菌GLP06小鼠生长及采食情况；Figure 11 is the growth and food intake of mice fed Pediococcus lactis GLP06;
图12为饲喂乳酸片球菌GLP06小鼠器官指数；Figure 12 is the organ index of feeding Pediococcus lactis GLP06 mice;
图13为饲喂乳酸片球菌GLP06小鼠血清生化指标；Figure 13 is the serum biochemical index of mice fed Pediococcus lactis GLP06;
图14为饲喂乳酸片球菌GLP06小鼠血清抗氧化指标；Figure 14 is the serum antioxidant index of mice fed Pediococcus lactis GLP06;
图15为饲喂乳酸片球菌GLP06小鼠肠道菌群α多样性；Figure 15 is the α-diversity of intestinal flora of mice fed Pediococcus lactis GLP06;
图16为饲喂乳酸片球菌GLP06对小鼠肠道菌群的影响。Figure 16 shows the effect of feeding Pediococcus lactis GLP06 on the intestinal flora of mice.
具体实施方式Detailed ways
下面将结合实施方式和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施方式和实施例仅用于说明本发明，而不应视为限制本发明的范围。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only for illustrating the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention. If the specific conditions are not specified, follow the general conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.
第一方面，本发明提供了一种乳酸片球菌GLP06，该菌株的分类命名为：乳酸片球菌GLP06，拉丁文学名：Pediococcus acidilactici GLP06，保藏于中国典型培养物保藏中心，保藏地址：湖北省武汉市武昌区八一路299号，保藏日期：2023年2月27日，保藏编号：CCTCC NO:M 2023200。In the first aspect, the present invention provides a Pediococcus acidilactici GLP06, the classification of the strain is named: Pediococcus acidilactici GLP06, Latin literary name: Pediococcus acidilactici GLP06, preserved in the China Type Culture Collection Center, preservation address: Wuhan, Hubei Province No. 299, Bayi Road, Wuchang District, City, date of deposit: February 27, 2023, deposit number: CCTCC NO:M 2023200.
本发明提供的乳酸片球菌(Pediococcus acidilactici)GLP06来源于健康比格犬的粪便，该菌株培养24h后，菌落中间凸起，边缘整齐，表面光滑；兼性厌氧，革兰氏阳性，呈球状。Pediococcus acidilactici (Pediococcus acidilactici) GLP06 provided by the present invention is derived from the feces of healthy beagle dogs. After the bacterial strain is cultivated for 24 hours, the colony is raised in the middle, with neat edges and smooth surface; facultative anaerobic, Gram-positive, spherical .
经发明人研究发现，本发明提供的乳酸片球菌GLP06的抗菌效果好，耐高温性强，耐酸，耐胆盐，粘附性强，具有良好的益生潜力；抗氧化能力好，可缓解宠物应激导致过氧化反应，延缓衰老，延长宠物寿命；能够提高动物胸腺器官指数和肠道的α多样性，降低致病菌丰度，进而提高动物免疫力，改善动物健康。The inventors have found through research that the Pediococcus lactis GLP06 provided by the present invention has good antibacterial effect, strong high temperature resistance, acid resistance, bile salt resistance, strong adhesion, and good probiotic potential; good antioxidant capacity, which can relieve pet stress. Stimulate the peroxidation reaction, delay aging, and prolong the life of pets; it can increase the thymus organ index and the α-diversity of the intestinal tract, reduce the abundance of pathogenic bacteria, and then improve animal immunity and animal health.
第二方面，本发明提供了一种菌剂，包括所述的乳酸片球菌(Pediococcusacidilactici)GLP06。In a second aspect, the present invention provides a bacterial agent, including the Pediococcus acidilactici GLP06.
本发明提供的菌剂，包括乳酸片球菌GLP06，或者乳酸片球菌GLP06与辅料或者其他菌的混合，例如，可以是本发明的乳酸片球菌GLP06与其他的菌混合制备的菌剂，也可以是本发明的乳酸片球菌GLP06与辅料制备得到的菌剂。该菌剂具有本发明乳酸片球菌GLP06的所有有益效果。The microbial preparation provided by the present invention includes Pediococcus lactis GLP06, or a mixture of Pediococcus lactis GLP06 and auxiliary materials or other bacteria, for example, it can be a bacterial preparation prepared by mixing Pediococcus lactis GLP06 with other bacteria of the present invention, or it can be The bacterium preparation prepared from Pediococcus lactis GLP06 and auxiliary materials of the present invention. The bacterial agent has all the beneficial effects of the Pediococcus lactis GLP06 of the present invention.
第三方面，本发明提供了所述的乳酸片球菌(Pediococcus acidilactici)GLP06或者菌剂在制备抑菌产品中的应用。In a third aspect, the present invention provides the application of the Pediococcus acidilactici GLP06 or bacterial agent in the preparation of bacteriostatic products.
经发明人研究发现，本发明提供的乳酸片球菌GLP06的发酵产物对于大肠杆菌、沙门氏菌、金黄色葡萄球菌、铜绿假单胞菌和单核细胞增生李斯特菌均有较好的抑制作用，能够用于制备抑菌产品。The inventors found that the fermentation product of Pediococcus lactis GLP06 provided by the present invention has a good inhibitory effect on Escherichia coli, Salmonella, Staphylococcus aureus, Pseudomonas aeruginosa and Listeria monocytogenes, and can For the preparation of antibacterial products.
第四方面，本发明提供了所述的乳酸片球菌(Pediococcus acidilactici)GLP06或者菌剂在制备提高免疫力的产品中的应用。In a fourth aspect, the present invention provides the application of the Pediococcus acidilactici GLP06 or bacterial agent in the preparation of immunity-enhancing products.
经发明人研究发现，饲喂本发明的乳酸片球菌GLP06可显著提高小鼠胸腺器官指数，说明本发明菌株可以促进宿主免疫系统的发育，提高免疫力，具有预防疾病的潜力。The inventors found that feeding the Pediococcus lactis GLP06 of the present invention can significantly increase the thymus organ index of mice, indicating that the strain of the present invention can promote the development of the host's immune system, improve immunity, and have the potential to prevent diseases.
第五方面，本发明提供了所述的乳酸片球菌(Pediococcus acidilactici)GLP06或者菌剂在制备缓解动物氧化应激的产品中的应用。In the fifth aspect, the present invention provides the application of the Pediococcus acidilactici GLP06 or bacterial agent in the preparation of products for alleviating oxidative stress in animals.
经发明人研究发现，本发明提供的乳酸片球菌GLP06抗氧化能力好，能显著降低灌服小鼠血液中的MDA和BUN含量，提高SOD活性，可缓解宠物应激导致过氧化反应，延缓衰老，延长宠物寿命。The inventors found that the Pediococcus lactis GLP06 provided by the present invention has good antioxidant capacity, can significantly reduce the MDA and BUN content in the blood of fed mice, increase the activity of SOD, relieve the peroxidation reaction caused by pet stress, and delay aging , Extend the life of pets.
第六方面，本发明提供了所述的乳酸片球菌(Pediococcus acidilactici)GLP06或者菌剂在制备提高动物肠道菌群物种多样性的产品中的应用。In the sixth aspect, the present invention provides the application of the Pediococcus acidilactici GLP06 or bacterial agent in the preparation of products for increasing species diversity of animal intestinal flora.
经发明人研究发现，饲喂本发明的乳酸片球菌GLP06可显著提高小鼠肠道的α多样性，说明饲喂本发明菌株可以提高肠道菌群的物种多样性。The inventors found that feeding the Pediococcus lactis GLP06 of the present invention can significantly increase the α-diversity of the intestinal tract of mice, indicating that feeding the strain of the present invention can increase the species diversity of the intestinal flora.
作为进一步技术方案，所述产品包括饲料或药物。As a further technical solution, the product includes feed or medicine.
第七方面，本发明提供了一种饲料，包括所述的乳酸片球菌(Pediococcusacidilactici)GLP06或者菌剂。In the seventh aspect, the present invention provides a feed, including the Pediococcus acidilactici (Pediococcus acidilactici) GLP06 or bacterial agent.
第八方面，本发明提供了一种药物，包括所述的乳酸片球菌(Pediococcusacidilactici)GLP06或者菌剂。In the eighth aspect, the present invention provides a medicament comprising the Pediococcus acidilactici GLP06 or bacterial agent.
本发明提供的饲料和药物包含本发明的乳酸片球菌GLP06，因此具有该乳酸片球菌GLP06所有的有益效果。The feed and medicine provided by the present invention contain the Pediococcus lactis GLP06 of the present invention, and therefore have all the beneficial effects of the Pediococcus lactis GLP06.
在一些优选的实施方式中，所述药物还包括辅料。本发明中对于辅料的选择不作具体限制，采用本领域技术人员所熟知的药物辅料即可。In some preferred embodiments, the medicament further includes excipients. The selection of auxiliary materials in the present invention is not specifically limited, and the pharmaceutical auxiliary materials well known to those skilled in the art can be used.
下面通过具体的实施例进一步说明本发明，但是，应当理解为，这些实施例仅仅是用于更详细地说明之用，而不应理解为用于以任何形式限制本发明。The present invention will be further described below through specific examples, however, it should be understood that these examples are only used for more detailed description, and should not be construed as limiting the present invention in any form.
实施例Example
1.犬源乳酸片球菌的分离鉴定1. Isolation and identification of canine-derived Pediococcus lactis
1.1分离用培养基1.1 Culture medium for isolation
MRS培养基，培养基成分如表1。MRS medium, the medium components are shown in Table 1.
表1培养基成分表Table 1 Medium composition list
pH 6.2～6.5，121℃灭菌15min。pH 6.2-6.5, sterilized at 121°C for 15 minutes.
1.2分离方法1.2 Separation method
(1)样品的采集与微生物分离：粪便样本采集于中国青岛即墨畜牧实验场的3只成年的雌性比格犬。通过无菌拭子直肠取样，收集粪便样本，放置到含有生理盐水的50mL灭菌螺口盖试管中。在冷藏条件下运输至青岛农业大学特种经济动物营养实验室，立即进行梯度稀释，每梯度三个平板，倾注法分离培养，于37℃倒置培养。(1) Sample collection and microbial isolation: Fecal samples were collected from 3 adult female Beagle dogs at the Jimo Animal Husbandry Experimental Farm in Qingdao, China. Rectal sampling was performed by sterile swab, and fecal samples were collected and placed into sterile 50-mL screw-cap tubes containing normal saline. It was transported to the Special Economic Animal Nutrition Laboratory of Qingdao Agricultural University under refrigerated conditions, and immediately carried out gradient dilution, with three plates for each gradient, separated and cultured by pouring method, and cultured upside down at 37°C.
(2)分离纯化：取以上长有各种细菌的平板，选取乳酸菌单菌落，采用连续划线法进行分离纯化。将有不同菌落形态的单菌落均进行纯化。重复3-4次，镜检，直至获得纯种。(2) Separation and purification: Take the above plate with various bacteria, select a single colony of lactic acid bacteria, and use continuous streaking method to carry out separation and purification. Single colonies with different colony morphology were all purified. Repeat 3-4 times, and microscopically check until pure species are obtained.
(3)观察菌落特征，利用革兰氏染色、镜检，观察细菌形态。(3) Observe the characteristics of the colonies, and use Gram staining and microscopic examination to observe the bacterial morphology.
(4)按照北京天根生物公司细菌基因组提取试剂盒说明书提取细菌DNA，利用细菌的通用引物：27F，1492R对所获得乳酸菌保守序列进行扩增并测序，引物合成及测序送检北京擎科生物科技有限公司完成，对16S rRNA序列进行同源性比较。16S rDNA的扩增体系如下，扩增体系的总体积为50μl：(4) Extract bacterial DNA according to the instructions of Beijing Tiangen Biological Company’s Bacterial Genome Extraction Kit, use the general primers of bacteria: 27F, 1492R to amplify and sequence the conserved sequence of lactic acid bacteria obtained, and send the primer synthesis and sequencing to Beijing Qingke Bio for inspection Completed by Technology Co., Ltd., homology comparison of 16S rRNA sequences. The 16S rDNA amplification system is as follows, and the total volume of the amplification system is 50 μl:
PCR反应程序：94℃预变性5min，94℃变性45s，56℃退火30s，72℃延伸45s，共35个循环，最后72℃延伸10min，4℃保存。PCR reaction program: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 45 s, annealing at 56°C for 30 s, extension at 72°C for 45 s, a total of 35 cycles, a final extension at 72°C for 10 min, and storage at 4°C.
1.3乳酸片球菌的分离筛选鉴定1.3 Isolation, screening and identification of Pediococcus lactis
经形态学观察，显微镜检及碳水化合物利用实验，结合16S rRNA测序比对，鉴定为乳酸片球菌，命名为GLP06，16S rRNA基因序列如SEQ ID NO.1所示。菌株培养24h后，菌落中间凸起，边缘整齐，表面光滑；兼性厌氧，革兰氏阳性，呈球状。GLP06的菌落形态如图1中的A所示，显微镜检形态如图1中的B所示。After morphological observation, microscopic examination and carbohydrate utilization experiment, combined with 16S rRNA sequencing comparison, it was identified as Pediococcus lactis and named GLP06. The 16S rRNA gene sequence is shown in SEQ ID NO.1. After the strain was cultured for 24 hours, the center of the colony was raised, the edges were neat, and the surface was smooth; it was facultatively anaerobic, Gram-positive, and spherical. The colony morphology of GLP06 is shown in A in Figure 1, and the microscopic morphology is shown in B in Figure 1.
对GLP06的16S rRNA基因进行系统发育分析(如图2所示)，确定它们与乳酸片球菌的亲缘关系密切。Phylogenetic analysis of the 16S rRNA genes of GLP06 (as shown in Figure 2) determined that they were closely related to Pediococcus lactis.
1)16S rRNA基因序列1) 16S rRNA gene sequence
TATAATGCAAGTCGAACGAACTTCCGTTAATTGATCATGACGTGCTTGCACTGAATGAGATTTTAATATGAAGTGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCAGAAGCAGGGGATAACACCTGGAAACAGATGCTAATACCGTATAACAGAGAAAACCGCCTGGTTTTCTTTTAAAAGATGGCTCTGCTATCACTTCTGGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGATGATGCGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACGTGGGTGAGAGTAACTGTTCACCCAGTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGARCGCAGGCGGTCTTTTAAGTCTAATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGATTACTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGTAATCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAAGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGCCAACCTAAGAGATTAGGCGTTCCCTTCGGGGACAGAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTACTAGTTGCCAGCATTCAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTCGCGAAACCGCGAGGTTTAGCTAATCTCTTAAAACCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGGGGTAACCTTTTAGGAGCTAGCCGT(SEQ ID NO.1)。TATAATGCAAGTCGAACGAACTTCCGTTAATTGATCATGACGTGCTTGCACTGAATGAGATTTTAATATGAAGTGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCAGAAGCAGGGGATAACACCTGGAAACAGATGCTAATACCGTAATAACAGAGAAAACCGCCTGGTTTTCTTTTAAAAGATGGCTCTGCTATCACTTCTGGATG GACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGATGATGCGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTA AAGAAGAACGTGGGTGAGAGTAACTGTTCACCCAGTGACGGTATTTAACCAGAAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGARCGCAGGCGGTCTTTTAAGTCTAATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATTGGAAACTGGGAGACTTGAGTGCAGAAGAG GACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGATTACTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGTAATCCGC CTGGGGAGTACGACCGCAAGGTTGAAACTCAAAAGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGCCAACCTAAGAGATTAGGCGTTCCCTTCGGGGACAGAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCC GCAACGAGCGCAACCCTTATTTACTAGTTGCCAGCATTCAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTCGCGAAACCGCGAGGTTTAGCTAATCTCTTAAAACTCTCTCAGTGGACTGTAGGCTG CAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGGGGTAACCTTTTAGGAGCTAGCCGT (SEQ ID NO. 1).
2.生长特性2. Growth characteristics
2.1生理生化特性2.1 Physiological and biochemical characteristics
取活化培养18h后的分离菌菌液，参照《伯杰氏细菌鉴定手册》、《常见细菌系统鉴定手册》和《乳酸细菌分类鉴定及实验方法》进行乳酸菌生理生化特性鉴定，明胶试验、V-P试验、甲基红(MR)试验、糖发酵试验等生化特性进行鉴定。Take the isolated bacteria liquid after 18 hours of activation culture, and refer to the "Bergey's Bacteria Identification Manual", "Common Bacteria System Identification Manual" and "Lactic Acid Bacteria Classification Identification and Experimental Methods" to carry out the identification of the physiological and biochemical characteristics of lactic acid bacteria, gelatin test, V-P test , methyl red (MR) test, sugar fermentation test and other biochemical characteristics were identified.
2.2生长性能及产酸性能测定2.2 Determination of growth performance and acid production performance
取活化培养18h后的分离菌菌液以2.0％的接种量接种于MRS培养基中，37℃恒温厌氧培养24h，对照组为未接菌种液的液体培养基。分别于1、2、3、5、7、9、12、15、18、21、24、27、30、33、36、48h取样测定培养液600nm处吸光度OD值，分别于1、2、3、5、7、9、12、15、18、21、24、27、30、33、36、48h取样测定pH。以时间为横坐标，以培养液的pH和OD值为纵坐标，绘制其生长曲线和产酸曲线。The isolated bacteria liquid after 18 hours of activation culture was inoculated in MRS medium with an inoculum amount of 2.0%, anaerobic culture at a constant temperature of 37°C for 24 hours, and the control group was a liquid medium without seed liquid. Samples were taken at 1, 2, 3, 5, 7, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, and 48 hours to measure the absorbance OD value at 600 nm of the culture solution. , 5, 7, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 48h sampling and determination of pH. Taking time as the abscissa and pH and OD values of the culture medium as the ordinate, draw the growth curve and acid production curve.
2.3耐酸特性试验2.3 Acid resistance test
取活化培养18h后的分离菌菌液以2.0％的接种量接种于初始pH分别为1.0、2.0、3.0、4.0、5.0、6.0、7.0的MRS液体培养基中，37℃恒温厌氧培养。分别于3、6、9、12、18、24、30、36、42h测定其OD600值，每组3个平行。测定完成后取3组平行试验结果计算平均值，根据计算结果绘制曲线。The isolated bacteria liquid after 18 hours of activation culture was inoculated with 2.0% inoculum in MRS liquid medium with initial pHs of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0, and anaerobic culture was carried out at 37°C. The OD600 values were measured at 3, 6, 9, 12, 18, 24, 30, 36, and 42 hours respectively, with 3 parallels in each group. After the measurement is completed, the results of 3 parallel experiments are taken to calculate the average value, and the curve is drawn according to the calculation results.
2.4耐高温特性试验2.4 High temperature resistance test
取活化培养18h后的分离菌菌液，分别于50℃、60℃、70℃、80℃和90℃的水浴锅中处理5min，处理后立即放在冰盒中。取100μL培养液梯度稀释后涂布，用平板计数法计算活菌数，以0h的活菌数为对照计算存活率。Take the isolated bacteria liquid after 18 hours of activation culture, and treat them in water baths at 50°C, 60°C, 70°C, 80°C and 90°C for 5 minutes respectively, and place them in an ice box immediately after treatment. Take 100 μL of the culture solution and apply it after gradient dilution. The number of viable bacteria is calculated by the plate counting method, and the survival rate is calculated using the number of viable bacteria at 0 h as a control.
存活率Survivability(％)＝Ttreatment1/Tinitial1×100％(1)；Survivability (%) = Ttreatment1 /Tinitial1 × 100% (1);
其中Tinitial1和Ttreatment1分别是0h和5min存活细菌的数量(log CFU/mL)。Where Tinitial1 and Ttreatment1 are the number of surviving bacteria (log CFU/mL) at 0h and 5min, respectively.
2.5胃肠道定植能力2.5 Gastrointestinal colonization ability
2.5.1耐酸和耐胆盐试验2.5.1 Acid resistance and bile salt resistance test
取活化培养18h后的分离菌菌液，于4℃，10000rpm离心10min，弃去上清液后，用无菌中性PBS缓冲液(pH 7.0)洗涤两次，然后在无菌PBS缓冲液(pH 2.5)中重新悬浮。在37℃培养0和3h后，连续稀释的培养物涂布于MRS琼脂平板上，然后在37℃下孵育24h，通过活菌落来评估分离菌株对酸的耐受性，重复3次试验。Get the isolated bacterial liquid after 18 h of activation culture, centrifuge at 10000 rpm for 10 min at 4 °C, discard the supernatant, wash twice with sterile neutral PBS buffer (pH 7.0), and then wash in sterile PBS buffer (pH 7.0) pH 2.5) to resuspend. After incubation at 37°C for 0 and 3h, serially diluted cultures were plated on MRS agar plates and then incubated at 37°C for 24h. The acid tolerance of isolated strains was assessed by viable colonies, and the test was repeated 3 times.
取活化培养18h后的分离菌菌液，于4℃，10000rpm离心10min，弃去上清液后，用无菌中性PBS缓冲液(pH 7.0)洗涤两次，重新悬浮在含有0.3％和0.5％(w/v)猪胆汁盐(Solarbio，China)的PBS缓冲液中。在37℃培养0和4h后，将10×连续稀释的培养物涂布于MRS琼脂平板上，然后在37℃下孵育24h，通过活菌菌落来评估分离菌株对胆盐的耐受性，重复3次试验。Take the isolated bacterial liquid after 18 h of activation culture, centrifuge at 10000 rpm for 10 min at 4 °C, discard the supernatant, wash twice with sterile neutral PBS buffer (pH 7.0), and resuspend in a solution containing 0.3% and 0.5 % (w/v) pig bile salts (Solarbio, China) in PBS buffer. After incubation at 37°C for 0 and 4h, spread 10× serially diluted cultures on MRS agar plates, and then incubate at 37°C for 24h, evaluate the tolerance of isolated strains to bile salts by viable colonies, and repeat 3 trials.
使用以下公式计算存活率：Survival was calculated using the following formula:
存活率Survivability(％)＝Ttreatment2/Tinitial2×100％(2)；Survivability (%) = Ttreatment2 /Tinitial2 × 100% (2);
其中Tinitial2和Ttreatment2分别是在温育前(0h)以及在温育后(3h/4h)存活细菌的数量(log CFU/mL)。Where Tinitial2 and Ttreatment2 are the number of surviving bacteria (log CFU/mL) before incubation (0h) and after incubation (3h/4h), respectively.
2.5.2人工胃肠液耐受性试验2.5.2 Artificial gastrointestinal fluid tolerance test
人工胃液和人工肠液的配置参考2010年版《中国药典》。The configuration of artificial gastric juice and artificial intestinal juice refers to the 2010 edition of "Chinese Pharmacopoeia".
取活化培养18h后的分离菌菌液，于4℃，10000rpm离心10min，弃去上清液后，用无菌中性PBS缓冲液(pH 7.0)洗涤两次，重悬于人造胃液中，并在37℃，200rpm搅拌孵育1.5h。孵育后，用无菌中性PBS缓冲液(pH 7.0)洗涤细菌沉淀两次，在10000rpm和4℃下离心10min，然后重悬于人工肠液中，并在37℃，200rpm搅拌孵育2h。在人工胃液和肠液中孵育前后，通过平板计数法测定活菌数量。重复3次试验。使用以下公式计算存活率：Take the isolated bacterial liquid after 18 hours of activation culture, centrifuge at 10,000 rpm for 10 minutes at 4°C, discard the supernatant, wash twice with sterile neutral PBS buffer (pH 7.0), resuspend in artificial gastric juice, and Incubate at 37° C. with stirring at 200 rpm for 1.5 h. After incubation, the bacterial pellet was washed twice with sterile neutral PBS buffer (pH 7.0), centrifuged at 10,000 rpm and 4°C for 10 min, then resuspended in artificial intestinal fluid, and incubated at 37°C for 2 h with stirring at 200 rpm. Before and after incubation in artificial gastric juice and intestinal juice, the number of viable bacteria was determined by plate counting. Repeat the test 3 times. Survival was calculated using the following formula:
存活率Survivability(％)＝Ttreatment3/Tinitial3×100％(3)；Survivability (%) = Ttreatment3 /Tinitial3 × 100% (3);
其中Tinitial3和Ttreatment3分别是在人工胃液或肠液中孵育之前以及在人工胃液或肠液中孵育后存活细菌的数量(log CFU/mL)。Where Tinitial3 and Ttreatment3 are the number of surviving bacteria (log CFU/mL) before incubation in artificial gastric juice or intestinal juice and after incubation in artificial gastric juice or intestinal juice, respectively.
2.5.3表面疏水性测定2.5.3 Determination of Surface Hydrophobicity
采用细菌碳烃化合物黏着法(bacterium adhesion to hydrocarbons，BATH)，通过乳酸菌对碳烃化合物的亲和力来反应菌株表面的疏水性能。取活化培养18h后的分离菌菌液(10000×g，10分钟，4℃)用无菌中性PBS缓冲液(pH 7.0)洗涤3次后，调节菌液OD600值为0.25±0.05(A1)备用。然后，以1∶1的比例加入二甲苯和氯仿，涡旋振荡3min，然后在37℃下孵育3h。最后，小心地吸收水相，测量OD600值(A2)。重复3次试验。疏水率计算公式如下：The bacteria adhesion to hydrocarbons (BATH) method was used to reflect the hydrophobic properties of the strain surface through the affinity of lactic acid bacteria to hydrocarbons. Take the isolated bacteria liquid (10000×g, 10 minutes, 4°C) after 18 hours of activation culture and wash with sterile neutral PBS buffer (pH 7.0) for 3 times, adjust theOD600 value of the bacterial liquid to 0.25±0.05 (A1 ) Spare. Then, xylene and chloroform were added at a ratio of 1:1, vortexed for 3 min, and then incubated at 37° C. for 3 h. Finally, carefully absorb the aqueous phase and measure theOD600 value (A2 ). Repeat the test 3 times. The calculation formula of hydrophobic rate is as follows:
疏水率CSH(％)＝(1-A2/A1)×100％(4)；Hydrophobicity CSH(%)=(1-A2 /A1 )×100%(4);
其中A1和A2分别是孵育3小时之前和之后的吸光度。whereA1 andA2 are the absorbance before and after incubation for 3 h, respectively.
2.5.4自凝聚性测定2.5.4 Determination of self-aggregation
取培养过夜(18h)的分离菌株菌液(10000×g，10分钟，4℃)用无菌中性PBS缓冲液(pH 7.0)洗涤2次，然后重新悬浮在相同体积的无菌中性PBS缓冲液(pH 7.0)中，测量OD600值(A3)，然后涡旋振荡10s，37℃下，孵育8h，避免搅拌，仔细吸收上部，并在600nm处测量吸光度(A4)，重复3次试验。Take the isolated strain cultured overnight (18h) (10000×g, 10 minutes, 4°C), wash twice with sterile neutral PBS buffer (pH 7.0), and then resuspend in the same volume of sterile neutral PBS In the buffer (pH 7.0), measure the OD600 value (A3 ), then vortex for 10 s, incubate at 37°C for 8 h, avoid stirring, carefully absorb the upper part, and measure the absorbance at 600 nm (A4 ), repeat 3 times trials.
自动聚集率计算如下：The automatic aggregation rate is calculated as follows:
Autoaggregation ability(％)＝(1-A4/A3)×100％ (5)；Autoaggregation ability(%)=(1-A4 /A3 )×100% (5);
其中A3和A4分别是孵育8小时之前和之后的吸光度。whereA3 andA4 are the absorbance before and after 8 h incubation, respectively.
2.5.5细胞粘附试验2.5.5 Cell adhesion test
2.5.5.1细胞爬片2.5.5.1 Cell slide
Caco-2细胞在含有1.0％(w/v)非必需氨基酸和20％(v/v)胎牛血清(Gibco，USA)的MEM培养液(HyClone，USA)的细胞培养瓶中培养，并添加100μg青霉素和100μg链霉素(Sigma，USA)。当细胞铺满细胞瓶90％左右的空间，加入1mL 0.25％胰蛋白酶-EDTA消化液(Solarbio，China)37℃消化5min，加培养基吹打均匀制成细胞悬液(5×104CFU/mL)，均匀铺在已放置玻璃爬片的24孔板中，培养至致密单层后弃去培养液，用无菌中性PBS缓冲液(pH 7.0)清洗3次后每孔加入1mL活化培养24h后的分离菌液(菌液浓度调整为1×108CFU/mL)和1mL细胞培养液，37℃厌氧培养2h。吸出孔内的混合液体，用无菌中性PBS缓冲液(pH7.0)清洗3次后每孔加入适量的多聚甲醛固定30min。吸出孔内的多聚甲醛，用无菌中性PBS缓冲液(pH 7.0)清洗3次后进行革兰染色，显微镜下观察。Caco-2 cells were cultured in cell culture flasks in MEM medium (HyClone, USA) containing 1.0% (w/v) non-essential amino acids and 20% (v/v) fetal calf serum (Gibco, USA), and added 100 μg penicillin and 100 μg streptomycin (Sigma, USA). When the cells covered about 90% of the space of the cell bottle, add 1 mL of 0.25% trypsin-EDTA digestion solution (Solarbio, China) to digest at 37°C for 5 min, add medium and pipette evenly to make a cell suspension (5×104 CFU/mL ), spread evenly in a 24-well plate with glass slides, discard the culture medium after culturing to a dense monolayer, wash with sterile neutral PBS buffer (pH 7.0) for 3 times, add 1 mL per well for activation and culture for 24 hours The isolated bacteria liquid (the concentration of the bacteria liquid was adjusted to 1×108 CFU/mL) and 1 mL of cell culture liquid were cultured anaerobically at 37° C. for 2 h. The mixed liquid in the wells was sucked out, washed with sterile neutral PBS buffer (pH 7.0) for 3 times, and fixed by adding an appropriate amount of paraformaldehyde to each well for 30 min. The paraformaldehyde in the hole was sucked out, washed with sterile neutral PBS buffer (pH 7.0) for 3 times, then Gram stained and observed under a microscope.
2.5.5.2粘附性2.5.5.2 Adhesion
将瓶中培养好的Caco-2细胞消化下来制备成5×105/mL的细胞悬液，接入到24孔细胞培养板中培养至长成单层细胞。将已长成单层的细胞用MEM清洗2次后，每孔中加入100uL的菌悬液(1×108CFU/mL)和900uL MEM培养液(不含抗生素)，置37℃、5％CO2/RH90％的二氧化碳培养箱中培养90min。培养结束后用PBS清洗单层细胞5次，以除去未粘附的细菌。加入0.5mL含有0.05％(v/w)曲拉通X-100的PBS溶液，于37℃裂解细胞20min，处理后的含有菌体的细胞裂解液经适当稀释后涂布于MRS琼脂平板，37℃培养48h后计数。每个实验做3个平行，根据以下公式计算粘附力：The Caco-2 cells cultured in the flask were digested to prepare a 5×105 /mL cell suspension, which was inserted into a 24-well cell culture plate and cultured until it grew into a single layer of cells. After the cells that have grown into a monolayer were washed twice with MEM, 100uL of bacterial suspension (1×108 CFU/mL) and 900uL of MEM culture solution (without antibiotics) were added to each well, and placed at 37°C, 5% Cultivate in a CO2 /RH90% carbon dioxide incubator for 90 minutes. After incubation, the monolayer cells were washed 5 times with PBS to remove non-adherent bacteria. Add 0.5 mL of PBS solution containing 0.05% (v/w) Triton X-100, lyse the cells at 37°C for 20 min, and spread the treated cell lysate containing bacteria on the MRS agar plate after appropriate dilution, 37 Count after 48 hours of cultivation at ℃. Three parallel experiments were done for each experiment, and the adhesion force was calculated according to the following formula:
粘附率(CFU/100cells)＝粘附的菌数(CFU)×100％/粘附的Caco-2细胞数(6)。Adhesion rate (CFU/100cells) = number of adhered bacteria (CFU) × 100%/number of adhered Caco-2 cells (6).
2.6抑菌试验2.6 Antibacterial test
菌株的抑菌性能采用牛津杯琼脂扩散法进行测定，取活化培养18h后的分离菌菌液(10000×g，10分钟，4℃)，去除无细胞上清液(CFS)，将细菌沉淀(BP)重悬于等体积无菌中性PBS缓冲液(pH 7.0)中。将无菌牛津杯放置在LB琼脂平板上，LB平板含有蛋白胨(10.0g/L)、牛肉提取物(1.0g/L)、酵母提取物(5.0g/L)、氯化钠(5.0g/L)和琼脂(20.0g/L)，取200uL浓度为1×107CFU/mL的致病菌(大肠杆菌ATCC25922；沙门氏菌ATCC14028；金黄色葡萄球菌ATCC25923；铜绿假单胞菌ATCC27853；单核细胞增生李斯特菌ATCC19115)涂布于LB琼脂平板上，待菌液吸收完全后，每个孔接种100uL分离菌株的BS，CFS，BP和CFSpH7.0，平板于37℃恒温培养箱正置放置培养，48h后观察是否有抑菌圈形成，并精确量取抑菌圈直径。The antibacterial performance of the bacterial strain was determined by the Oxford cup agar diffusion method. The isolated bacterial liquid (10000 × g, 10 minutes, 4 °C) after 18 hours of activation culture was taken, the cell-free supernatant (CFS) was removed, and the bacterial precipitate ( BP) were resuspended in an equal volume of sterile neutral PBS buffer (pH 7.0). Place a sterile Oxford cup on an LB agar plate containing peptone (10.0g/L), beef extract (1.0g/L), yeast extract (5.0g/L), sodium chloride (5.0g/L) L) and agar (20.0g /L), take 200uL concentration of pathogenic bacteria (Escherichia coli ATCC25922; Salmonella ATCC14028; Staphylococcus aureus ATCC25923; Pseudomonas aeruginosa ATCC27853; Listeria monocytogenes ATCC19115) was spread on the LB agar plate, after the bacterial solution was completely absorbed, each well was inoculated with 100uL of BS, CFS, BP and CFSpH7.0 of the isolated strain, and the plate was placed upright in a 37°C constant temperature incubator for culture After 48 hours, observe whether there is an inhibition zone, and accurately measure the diameter of the inhibition zone.
2.7抗生素敏感性试验2.7 Antibiotic susceptibility test
菌株的抗生素敏感性采用纸片扩散法进行测定。选择21种抗生素以6mm纸片(BIO-KONT，China)的形式进行测试：青霉素、苯唑西林、氨苄西林、哌拉西林、亚胺培南、万古霉素、链霉素、庆大霉素、阿米卡星、卡那霉素、四环素、氯霉素、米诺环素、强力霉素、复方新诺明、阿奇霉素、红霉素、克林霉素、诺氟沙星、环丙沙星和左氧氟沙星。The antibiotic susceptibility of the strains was determined by disc diffusion method. 21 antibiotics were selected for testing in the form of 6mm discs (BIO-KONT, China): penicillin, oxacillin, ampicillin, piperacillin, imipenem, vancomycin, streptomycin, gentamicin , amikacin, kanamycin, tetracycline, chloramphenicol, minocycline, doxycycline, co-trimoxazole, azithromycin, erythromycin, clindamycin, norfloxacin, ciprofloxacin star and levofloxacin.
取活化培养18h后的分离菌菌液(10000×g，10分钟，4℃)，用无菌中性PBS缓冲液(pH 7.0)洗涤2次后，然后在无菌中性PBS缓冲液(pH7.0)中重悬以获得对数生长期的细菌溶液(0.5McFarland悬浮液)。取100uL分离菌株的McFarland悬浮液涂布于MRS琼脂平板上。最后，将抗生素纸片在15min内贴于MRS琼脂平板表面，将平板在37℃恒温培养48h，观察是否有抑菌圈形成，并用游标卡尺测定抑菌圈直径。Take the isolated bacteria liquid (10000×g, 10 minutes, 4°C) after activating and culturing for 18 hours, wash it twice with sterile neutral PBS buffer (pH 7.0), and then wash it twice in sterile neutral PBS buffer (pH 7.0). .0) to obtain a bacterial solution in logarithmic growth phase (0.5 McFarland suspension). Take 100uL of the McFarland suspension of the isolated strain and spread it on the MRS agar plate. Finally, paste the antibiotic disc on the surface of the MRS agar plate within 15 minutes, incubate the plate at a constant temperature of 37°C for 48 hours, observe whether there is an inhibition zone, and measure the diameter of the inhibition zone with a vernier caliper.
2.8自由基清除试验2.8 Free radical scavenging test
用索莱宝DPPH检测试剂盒来测定分离菌株的DPPH清除能力，按照说明书操作，简而言之，吸取100μL培养过夜(18h)的分离菌株菌液加入900μL提取液，旋涡振荡混匀，室温10000rpm离心10min，取25μL上清液加入到975μL DPPH工作溶液中，涡旋混匀，室温避光静止30min。于515nm处记录吸光度。Use the Solebao DPPH detection kit to measure the DPPH removal ability of the isolated strain, and operate according to the instructions. In short, draw 100 μL of the isolated strain cultured overnight (18h) and add 900 μL of the extract, vortex and shake, room temperature 10000rpm After centrifugation for 10 min, 25 μL of the supernatant was added to 975 μL of DPPH working solution, vortexed to mix, and kept at room temperature for 30 min in the dark. Absorbance was recorded at 515 nm.
DPPH自由基清除率的计算公式如下：The calculation formula of DPPH free radical scavenging rate is as follows:
其中：Aassay1为待测样品的吸光度；Acontrol1为分离菌株处理上清液和无水乙醇混合液的吸光度；Ablank1为提取液和工作液混合的吸光度。Among them: Aassay1 is the absorbance of the sample to be tested; Acontrol1 is the absorbance of the mixture of the isolated strain treated supernatant and absolute ethanol; Ablank1 is the absorbance of the mixture of the extract and the working solution.
2.9安全性试验2.9 Safety test
2.9.1溶血实验2.9.1 Hemolysis test
取活化培养18h后的分离菌菌液在胰蛋白酶大豆琼脂(TSA)上划线，其中含有5.0％(w/v)的羊血(Oxoid,Germany)，将平板在37℃下培养48h，并检查血琼脂平板是否存在β-溶血(菌落周围的清晰区域)、α-溶血(菌落周围的绿色区域)或γ-溶血(菌落周围无区域)迹象。用金黄色葡萄球菌(ATCC25923)菌株作为阳性对照。Get the isolated bacterial liquid after activating culture for 18h and streak on tryptic soy agar (TSA), which contains 5.0% (w/v) sheep blood (Oxoid, Germany), culture the plate at 37°C for 48h, and Examine blood agar plates for signs of beta-hemolysis (clear areas around colonies), alpha-hemolysis (green areas around colonies), or gamma-hemolysis (no areas around colonies). A strain of Staphylococcus aureus (ATCC25923) was used as a positive control.
2.9.2小鼠安全性试验2.9.2 Mice Safety Test
为了进行体内安全性评价，将生长健康，体重接近的昆明系小白鼠80只随机分为4组，每组20只。对照组(CK)小鼠灌服0.2mL无菌生理盐水，而其他三组的小鼠分别灌服0.2mL分离菌液，浓度分别为1×109(LG06)，1×1010(MG06)和1×1011(HG06)CFU/mL。每3天测量一次体重和采食量，并记录小鼠的健康状况。持续28天后，将小鼠禁食12小时，然后用1％戊巴比妥钠(50mg/kg)麻醉。腹主动脉采血收集血液，并通过离心(4000rpm，4℃，10min)收集血清以进行进一步分析。处死小鼠后，解剖观察各组小鼠的内脏中毒情况，并收集肝脏、肾脏、脾脏和胸腺等器官并称重，器官指数计算为器官重量/体重×100。In order to evaluate the safety in vivo, 80 Kunming mice with healthy growth and similar body weight were randomly divided into 4 groups, 20 in each group. The mice in the control group (CK) were fed with 0.2mL sterile normal saline, while the mice in the other three groups were fed with 0.2mL of isolated bacteria solution, the concentrations were 1×109 (LG06), 1×1010 (MG06) and 1×1011 (HG06) CFU/mL. Body weight and feed intake were measured every 3 days, and the health status of the mice was recorded. After 28 days, the mice were fasted for 12 hours and then anesthetized with 1% sodium pentobarbital (50 mg/kg). Blood was collected by abdominal aortic bleeding, and serum was collected by centrifugation (4000rpm, 4°C, 10min) for further analysis. After the mice were sacrificed, the visceral poisoning of the mice in each group was observed by dissection, and organs such as liver, kidney, spleen and thymus were collected and weighed. The organ index was calculated as organ weight/body weight×100.
使用商用ELISA试剂盒(南京建成)测定小鼠血清中的天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、总胆红素(TBIL)、直接胆红素(DBIL)、血尿素氮(BUN)、丙二醛(MDA)和超氧化物歧化酶(SOD)。Aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), direct bilirubin (DBIL) in mouse serum were determined using a commercial ELISA kit (Nanjing Jiancheng). ), blood urea nitrogen (BUN), malondialdehyde (MDA) and superoxide dismutase (SOD).
从粪便样品中提取DNA用Illumina NovaSeq平台对V3-V4区进行测序。序列以97％的相似性聚类在OTU中。所有分析从聚类到确定α和β多样性使用QIIME进行。DNA extracted from stool samples was sequenced on the V3 -V4 region using the Illumina NovaSeq platform. Sequences clustered in OTUs with 97% similarity. All analyzes from clustering to determination of alpha and beta diversity were performed using QIIME.
3结果与分析3 Results and Analysis
3.1生长特性3.1 Growth characteristics
3.1.1生理生化特性3.1.1 Physiological and biochemical characteristics
菌株GLP06的部分生理生化鉴定结果如表2所示。乳酸片球菌GLP06可利用葡萄糖、果糖、半乳糖等单糖，还可利用纤维二糖、麦芽糖、阿拉伯糖和木糖。MR试验结果为阳性，其余为阴性。Some physiological and biochemical identification results of strain GLP06 are shown in Table 2. Pediococcus lactis GLP06 can utilize monosaccharides such as glucose, fructose, and galactose, as well as cellobiose, maltose, arabinose, and xylose. MR test results were positive, and the rest were negative.
表2乳酸片球菌GLP06生理生化特性Table 2 Physiological and biochemical characteristics of Pediococcus lactis GLP06
注：+：阳性；–：阴性。Note: +: Positive; –: Negative.
3.1.2生长性能及产酸性能测定3.1.2 Determination of growth performance and acid production performance
乳酸片球菌GLP06生长及产酸速率曲线如图3所示，由图3可知，接种后0-3h生长缓慢，处于生长滞缓区；3h后菌液吸光度值迅速升高，进入对数生长期；18h后菌液吸光度趋于平缓，菌株达到生长稳定期，该时期菌株活力强，活菌数量达到高峰。培养液的pH变化趋势与生长曲线相一致，乳酸片球菌GLP06接种后3h内pH下降缓慢；3h后进入对数生长期，pH下降速率加快，18h后菌液pH变化趋于平缓，稳定在3.76左右。The growth and acid production rate curves of Pediococcus lactis GLP06 are shown in Figure 3. From Figure 3, it can be seen that the growth rate of Pediococcus lactis GLP06 is slow at 0-3 hours after inoculation, and it is in the growth retardation zone; after 3 hours, the absorbance value of the bacteria solution increases rapidly, and enters the logarithmic growth phase ; After 18 hours, the absorbance of the bacterial solution tends to be flat, and the strain reaches a stable growth period. During this period, the strain has strong vitality and the number of viable bacteria reaches a peak. The pH change trend of the culture solution was consistent with the growth curve. The pH of Pediococcus lactis GLP06 decreased slowly within 3 hours after inoculation; after 3 hours, it entered the logarithmic growth phase, and the pH decrease rate accelerated. about.
3.1.3菌株对高温的耐受性3.1.3 Tolerance of strains to high temperature
乳酸片球菌GLP06对高温的耐受性的结果如图4所示。由图4可知，60℃以下存活率可超过70％，70℃存活率接近50％，80℃存活率超过40％，说明乳酸片球菌GLP06具有较好的耐高温能力。The results of high temperature tolerance of Pediococcus lactis GLP06 are shown in FIG. 4 . It can be seen from Fig. 4 that the survival rate below 60°C can exceed 70%, the survival rate at 70°C is close to 50%, and the survival rate at 80°C exceeds 40%, indicating that Pediococcus lactis GLP06 has better high temperature resistance.
3.1.4菌株对酸的耐受性3.1.4 Tolerance of strains to acid
乳酸片球菌GLP06对酸度的耐受性结果如图5所示。由图5可知，菌株GLP06在pH≤3.0时生长完全被抑制，随培养时间延长，OD值未见变化。在pH＝4.0时，菌株能缓慢生长，但OD值明显低于pH＝5.0、6.0、7.0时。在pH≥5.0时，菌株GLP06均能正常生长，但在到达稳定期时OD值存在一定差别，其中OD值pH＝7.0>pH＝6.0>pH＝5.0，活菌数与OD值变化趋势相一致。The tolerance results of Pediococcus lactis GLP06 to acidity are shown in Fig. 5 . It can be seen from Figure 5 that the growth of strain GLP06 was completely inhibited at pH ≤ 3.0, and the OD value did not change with the extension of culture time. At pH=4.0, the strain can grow slowly, but the OD value is obviously lower than that at pH=5.0, 6.0, 7.0. When pH ≥ 5.0, the strain GLP06 can grow normally, but there is a certain difference in OD value when it reaches the stable stage, among which OD value pH=7.0>pH=6.0>pH=5.0, the number of viable bacteria is consistent with the change trend of OD value .
3.2菌株在胃肠道内的定植能力3.2 Colonization ability of the strains in the gastrointestinal tract
3.2.1菌株对人工胃肠液的耐受性3.2.1 Tolerance of strains to artificial gastrointestinal fluid
乳酸片球菌GLP06在人工胃肠液中的存活率如图6所示。由图6可知，乳酸片球菌GLP06在人工胃肠液中具有很强的存活能力。接种于人工胃液1.5h后菌株的存活率为61.94％，活菌数约为9.73×104CFU/mL；接种于人工肠液2h后菌株的存活率为90.43％，活菌数约为4.67×107CFU/mL，人工肠胃液共同消化3.5h后，存活率为54.83％，活菌数约为9.40×104CFU/mL。The survival rate of Pediococcus lactis GLP06 in artificial gastrointestinal fluid is shown in Figure 6. It can be seen from Figure 6 that Pediococcus lactis GLP06 has a strong ability to survive in artificial gastrointestinal fluid. After being inoculated in artificial gastric juice for 1.5 hours, the survival rate of the strain was 61.94%, and the number of viable bacteria was about 9.73×104 CFU/mL; after being inoculated in artificial intestinal juice for 2 hours, the survival rate of the strain was 90.43%, and the number of viable bacteria was about 4.67×107 CFU/mL, after co-digestion with artificial gastrointestinal juice for 3.5 hours, the survival rate was 54.83%, and the number of viable bacteria was about 9.40×104 CFU/mL.
3.2.2菌株对酸和胆盐的耐受性3.2.2 Tolerance of strains to acids and bile salts
乳酸片球菌GLP06在酸和不同胆盐浓度中的存活率如图7所示。由图7可知，菌株GLP06对胆盐和酸具有较强的耐受性。接种于pH＝2.5的MRS液体培养基中3h后菌株的存活率为72.17％，活菌数约为2.80×106CFU/mL。接种于胆盐浓度为0.3％的MRS液体培养基中4h后菌株的存活率为95.70％，活菌数约为1.08×108CFU/mL；接种于胆盐浓度为0.5％的MRS液体培养基中4h后菌株的存活率为90.22％，活菌数约为1.30×107CFU/mL。The survival rate of P. lactis GLP06 in acid and different bile salt concentrations is shown in FIG. 7 . It can be seen from Figure 7 that the strain GLP06 has strong tolerance to bile salts and acids. The survival rate of the strain was 72.17% after being inoculated in MRS liquid medium with pH=2.5 for 3 hours, and the number of viable bacteria was about 2.80×106 CFU/mL. After being inoculated in MRS liquid medium with a bile salt concentration of 0.3% for 4 hours, the survival rate of the strain was 95.70%, and the number of viable bacteria was about 1.08×108 CFU/mL; inoculated in an MRS liquid medium with a bile salt concentration of 0.5% After 4 hours of incubation, the survival rate of the strain was 90.22%, and the number of viable bacteria was about 1.30×107 CFU/mL.
3.2.3菌株的表面疏水性能及自凝聚性能3.2.3 Surface hydrophobic properties and self-aggregation properties of the strains
细菌根据表面疏水性定义标准，一般设定CSH％>50％为高度疏水，CSH％介于20％和50％为中度疏水，CSH％<20％为非疏水。自凝聚能力一般分为：低(16％-35％)，中等(35％-50％)和高(50％以上)三类。由表3可知，乳酸片球菌GLP06具有高度疏水性，且自凝聚性能较高。According to the definition standard of surface hydrophobicity, bacteria generally set CSH%>50% as highly hydrophobic, CSH% between 20% and 50% as moderately hydrophobic, and CSH%<20% as non-hydrophobic. The self-aggregation ability is generally divided into three categories: low (16%-35%), medium (35%-50%) and high (above 50%). It can be seen from Table 3 that Pediococcus lactis GLP06 is highly hydrophobic and has high self-aggregation performance.
表3乳酸片球菌GLP06的表面疏水率及自凝聚率Table 3 Surface hydrophobicity and self-aggregation rate of Pediococcus lactis GLP06
3.2.4菌株对肠上皮细胞的粘附能力3.2.4 Adhesion ability of strains to intestinal epithelial cells
乳酸片球菌GLP06粘附能力结果如图8所示。图8中的A是细胞爬片革兰染色镜检(1000×)结果图，可见乳酸片球菌GLP06对细胞粘附性较好；图8中的B是涂板法粘附性结果，可见乳酸片球菌GLP06对Caco2细胞粘附性为74.63个/细胞。The results of the adhesion ability of Pediococcus lactis GLP06 are shown in Fig. 8 . A in Fig. 8 is the results of Gram staining microscopic examination (1000 ×) of the cell slides. It can be seen that Pediococcus lactis GLP06 has better adhesion to cells; B in Fig. 8 is the adhesion result of the plate method, and lactic acid can be seen The adhesion of Pediococcus GLP06 to Caco2 cells was 74.63/cell.
3.3菌株的抑菌性能3.3 Bacteriostatic properties of the strains
乳酸片球菌GLP06的抑菌活性如图9所示。图9可知，菌体蛋白抑菌性很弱，发酵上清液和菌悬液对大肠杆菌，沙门氏菌，金黄色葡萄球菌，铜绿假单胞菌，单核细胞增生李斯特菌均有较好的抑制作用，二者均对沙门氏菌的抑菌活性最强，抑菌圈直径大于17mm；其次是单核细胞增生李斯特菌，抑菌圈直径大于16mm；再次是铜绿假单胞菌、大肠杆菌和金黄色葡萄球菌。但将上清液pH调整到7.0，无抑菌作用，猜想是菌株代谢产物有机酸起到了抑菌作用。The antibacterial activity of Pediococcus lactis GLP06 is shown in Figure 9. It can be seen from Fig. 9 that the bacteriostasis of thalline protein is very weak, and the fermentation supernatant and bacterial suspension all have good effect on Escherichia coli, Salmonella, Staphylococcus aureus, Pseudomonas aeruginosa, and Listeria monocytogenes. Inhibitory effect, both of them have the strongest antibacterial activity against Salmonella, and the diameter of the inhibition zone is greater than 17mm; followed by Listeria monocytogenes, the diameter of the inhibition zone is greater than 16mm; thirdly, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus. However, the pH of the supernatant was adjusted to 7.0, and there was no antibacterial effect. It is guessed that the organic acid of the metabolites of the strain played an antibacterial effect.
3.4菌株的抗生素敏感性3.4 Antibiotic susceptibility of strains
药敏片直径为6mm，故设抑菌圈直径大于7mm的为具有抑菌性，小于等于7mm视为无抑菌性。抗生素敏感性结果如表4所示。由表4可知，菌株GLP06对各种常见抗生素的敏感性不同。对青霉素、氯霉素和亚胺培南敏感性强，抑菌圈直径均大于23mm；对苯唑西林、氨苄西林、头孢氨苄、头孢唑林、头孢呋辛钠、头孢哌酮、红霉素、克林霉素、强力霉素和氟苯尼考中度敏感，抑菌圈直径均大于15mm；头孢曲松、米诺环素和阿奇霉素敏感性弱，抑菌圈直径在7～14mm范围内；对哌拉西林、头孢他啶、阿米卡星、庆大霉素、卡那霉素、链霉素、四环素、林可霉素、万古霉素、多粘菌素B、复方新诺明、头孢他啶、诺氟沙星、环丙沙星和左氧氟沙星则不敏感。The diameter of the drug-sensitive tablet is 6 mm, so the antibacterial zone with a diameter greater than 7 mm is regarded as having antibacterial activity, and the diameter less than or equal to 7 mm is regarded as having no antibacterial activity. The antibiotic susceptibility results are shown in Table 4. It can be seen from Table 4 that strain GLP06 has different sensitivities to various common antibiotics. Strong sensitivity to penicillin, chloramphenicol and imipenem, and the diameter of the inhibition zone is greater than 23mm; to oxacillin, ampicillin, cephalexin, cefazolin, cefuroxime sodium, cefoperazone, erythromycin , clindamycin, doxycycline, and florfenicol are moderately sensitive, and the diameter of the inhibition zone is greater than 15 mm; ceftriaxone, minocycline, and azithromycin are weakly sensitive, and the diameter of the inhibition zone is within the range of 7 to 14 mm ;Piperacillin, ceftazidime, amikacin, gentamicin, kanamycin, streptomycin, tetracycline, lincomycin, vancomycin, polymyxin B, cotrimoxazole, ceftazidime , norfloxacin, ciprofloxacin and levofloxacin are not sensitive.
表4乳酸片球菌GLP06对常用抗生素的敏感性Table 4 Sensitivity of Pediococcus lactis GLP06 to commonly used antibiotics
注：抑菌圈直径(mm)：+++：23-30mm；++：15-22mm；+：7-14mm；-：没有抑菌圈。Note: Diameter of the inhibition zone (mm): +++: 23-30mm; ++: 15-22mm; +: 7-14mm; -: no inhibition zone.
3.5菌株的自由基清除能力3.5 Free radical scavenging ability of strains
本实验对菌株GLP06菌体的DPPH清除能力进行了测定，根据测定结果计算得到菌株GLP06的DPPH自由基清除率为55.87％，说明GLP06具有较强的自由基清除力。In this experiment, the DPPH scavenging ability of the bacterial strain GLP06 was measured. According to the measurement results, the DPPH free radical scavenging rate of the strain GLP06 was calculated to be 55.87%, indicating that GLP06 has a strong free radical scavenging ability.
3.6安全性试验结果3.6 Safety test results
3.6.1溶血实验结果3.6.1 Hemolysis test results
乳酸片球菌GLP06的溶血结果如图10所示，以金黄色葡萄球菌作阳性对照(图10中的A)，可见明显β-溶血(菌落周围的清晰区域)，但GLP06为γ-溶血(菌落周围无区域)，可初步判断菌种GLP06安全无致病性。The hemolysis results of Pediococcus lactis GLP06 are shown in Figure 10, with Staphylococcus aureus as a positive control (A in Figure 10), it can be seen that obvious β-hemolysis (clear area around the colony), but GLP06 is γ-hemolysis (colonies There is no area around), it can be preliminarily judged that the strain GLP06 is safe and non-pathogenic.
3.6.2小鼠实验结果3.6.2 Mouse experiment results
临床观察所有小鼠精神状况良好，均能正常采食及饮水，粪便未见异常，试验期间未有试验小鼠死亡。试验结束后剖检观察其内脏均未见异常病理组织学变化及细菌易位，说明菌株针对小鼠临床应用无毒副作用。According to the clinical observation, all the mice were in good spirits and could eat and drink normally. There was no abnormality in the feces, and no test mice died during the experiment. No abnormal histopathological changes and bacterial translocation were found in the viscera after the autopsy, indicating that the strain has no toxic and side effects for clinical application in mice.
体重、食物摄入量和器官指数是评价动物健康状况的常用指标，也用来评估分离菌株的安全性。饲喂不同剂量GLP06小鼠生长及采食情况如图11所示。补充不同剂量GLP06的小鼠体重与CK组之间无显著差异(图11中的A-B)。而MG06组小鼠的平均日增重(ADG)和平均日采食量(ADFI)较CK组显著提高(P<0.01或P<0.05，图11中的C-D)。以上结果表明，添加GLP06对小鼠的体重和摄食量均无不良影响。Body weight, food intake, and organ indices are commonly used indicators to evaluate the health status of animals and are also used to assess the safety of isolated strains. The growth and food intake of mice fed with different doses of GLP06 are shown in Figure 11. There was no significant difference between the body weight of mice supplemented with different doses of GLP06 and the CK group (A-B in Figure 11). The average daily gain (ADG) and average daily feed intake (ADFI) of the mice in the MG06 group were significantly higher than those in the CK group (P<0.01 or P<0.05, C-D in Figure 11). The above results showed that the addition of GLP06 had no adverse effects on the body weight and food intake of mice.
我们还研究了饲喂不同剂量的GLP06如何影响小鼠的器官指数，结果如图12所示。除胸腺外，各组间心脏、肝脏、脾脏和肾脏脏器重均无显著性差异。LG06组和MG06组胸腺指数均显著高于CK组(P<0.01)。胸腺指数是免疫系统发育的标志物，在LG06和MG06组中显著增加。这表明菌株GLP06可能会促进免疫系统的发育，进而对动物具有健康和益生作用。We also investigated how feeding different doses of GLP06 affects the organ index of mice, and the results are shown in Figure 12. Except for the thymus, there were no significant differences in the organ weights of the heart, liver, spleen and kidney among the groups. The thymus index of LG06 group and MG06 group were significantly higher than that of CK group (P<0.01). Thymus index, a marker of immune system development, was significantly increased in the LG06 and MG06 groups. This suggests that strain GLP06 may promote the development of the immune system, which in turn has health and probiotic effects on animals.
我们还研究了饲喂不同剂量的GLP06如何影响小鼠的血液生化指标，见图13。除BUN外，各组间AST、ALT、T-BIL、I-BIL、D-BIL含量无显著差异。MG06组BUN含量明显低于CK组(P<0.01)和LG06组(P<0.05，图13中的C)。抗氧化方面，如图14所示，HG06组和MG06组小鼠血清SOD水平显著高于LG06组和CK组(P<0.01，图14中的A)。此外，MG06组小鼠血清MDA水平显著低于CK组(P<0.05，图14中的B)。这些结果显示，补充GLP06不但对小鼠没有毒性作用，而且对促进免疫系统的发育和减少氧化应激对器官的损害亦有重要作用。We also studied how feeding different doses of GLP06 affects the blood biochemical indicators of mice, see Figure 13. Except for BUN, there were no significant differences in the contents of AST, ALT, T-BIL, I-BIL, and D-BIL among the groups. The BUN content of MG06 group was significantly lower than that of CK group (P<0.01) and LG06 group (P<0.05, C in Figure 13). In terms of anti-oxidation, as shown in Figure 14, the serum SOD levels of the mice in the HG06 group and the MG06 group were significantly higher than those in the LG06 group and the CK group (P<0.01, A in Figure 14). In addition, the serum MDA level of mice in the MG06 group was significantly lower than that in the CK group (P<0.05, B in FIG. 14 ). These results show that GLP06 supplementation not only has no toxic effect on mice, but also plays an important role in promoting the development of the immune system and reducing the damage of organs caused by oxidative stress.
α多样性可以反映宿主微生物群落的丰度和多样性。饲喂GLP06小鼠肠道菌群α多样性结果如图15所示。HG06组小鼠肠道微生物Richness，Chao1和ACE指数显著高于CK组(P<0.05，图15中的A-B和F)，补充不同剂量GLP06的小鼠肠道微生物的Shannon和Simpson指数无显著差异(图15中的C-D)，HG06组和MG06组小鼠肠道微生物的PD whole tree显著高于CK组(P<0.05，图15中的E)。Alpha diversity can reflect the abundance and diversity of the host microbial community. The results of α-diversity of intestinal flora of mice fed with GLP06 are shown in Figure 15. The Richness, Chao1 and ACE indexes of intestinal microorganisms in HG06 group were significantly higher than those in CK group (P<0.05, A-B and F in Figure 15), and the Shannon and Simpson indexes of intestinal microorganisms in mice supplemented with different doses of GLP06 had no significant difference (C-D in Figure 15), the PD whole tree of intestinal microorganisms in HG06 group and MG06 group was significantly higher than that in CK group (P<0.05, E in Figure 15).
饲喂GLP06小鼠肠道菌群影响结果如图16所示。在Veen图(图16中的A)中计算了每个组常见和独特的扩增序列变体(amplicon sequence variants，ASV)的数量，可以看出每个组独特的ASV的数量随着GLP06饲喂量的增加而增加，主成分分析未出现明显聚集(图16中的B-C)。在物种组成方面，Firmicutes，Bacteroidota，Deferribacterota，Desulfobacterota，Campilobacterota和Actinobacteriota是门水平主要菌群(图16中的D)。图16中的F展示了肠道微生物属水平的丰度。The results of feeding the intestinal flora of GLP06 mice are shown in Figure 16. The number of common and unique amplified sequence variants (amplicon sequence variants, ASV) in each group was calculated in the Veen diagram (A in Figure 16), and it can be seen that the number of unique ASVs in each group increased with GLP06 feeding The increase of the feeding amount increased, and the principal component analysis did not show obvious aggregation (B-C in Fig. 16). In terms of species composition, Firmicutes, Bacteroidota, Deferribacterota, Desulfobacterota, Campilobacterota and Actinobacteriota were the main bacterial groups at the phylum level (D in Figure 16). F in Figure 16 shows the abundance at the genus level of gut microbes.
以上实验结果表明，新分离的犬源乳酸片球菌GLP06具有良好的体外益生菌活性，安全性好，具有改善小鼠抗氧化能力，预防腹泻，提高肠道微生物多样性，提高肠道内Muribaculaceae丰度，降低与其处于相同生态位的致病菌Clostridium丰度，阻碍致病菌在肠道的定居，调节宿主胃肠道健康。表明乳酸片球菌GLP06可成为预防和治疗宠物胃肠道疾病的潜在益生菌。The above experimental results show that the newly isolated Pediococcus lactis GLP06 has good in vitro probiotic activity, good safety, can improve the antioxidant capacity of mice, prevent diarrhea, increase the diversity of intestinal microorganisms, and increase the abundance of Muribaculaceae in the intestinal tract , reduce the abundance of pathogenic bacteria Clostridium in the same ecological niche, hinder the colonization of pathogenic bacteria in the intestinal tract, and regulate the health of the host gastrointestinal tract. It indicated that Pediococcus lactis GLP06 could be a potential probiotic for the prevention and treatment of gastrointestinal diseases in pets.
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. scope.
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