技术领域Technical Field
本申请涉及微生物制剂领域，特别是涉及一种微生物或微生物相关的组合物及其应用。The present application relates to the field of microbial preparations, and in particular to a microorganism or a microorganism-related composition and applications thereof.
背景技术Background Art
炎症性肠道疾病(inflammatory bowel disease，IBD)是一种病因不明的慢性炎症性肠道疾病，该疾病易反复，严重影响患者的生活质量。现代医学认为，引发炎症性肠道疾病(IBD)的因素有遗传、饮食、感染、自身免疫、心理因素以及环境等。炎症性肠道疾病包括溃疡性肠炎(UC)和克罗恩病(CD)，均属于炎症相关疾病。Inflammatory bowel disease (IBD) is a chronic inflammatory bowel disease with unknown etiology. The disease is prone to recurrence and seriously affects the patient's quality of life. Modern medicine believes that the factors that cause inflammatory bowel disease (IBD) include genetics, diet, infection, autoimmunity, psychological factors, and environment. Inflammatory bowel disease includes ulcerative colitis (UC) and Crohn's disease (CD), both of which are inflammation-related diseases.
溃疡性肠炎(ulcerative colitis，UC)是炎症性肠道疾病(inflammatory boweldisease，IBD)一种重要的疾病类型，其发病原因不明，主要病变部位是在结肠粘膜的粘膜下层，属于一种慢性的肠道疾病。基于目前的研究，主要认为引发UC的病因有宿主遗传易感性、肠道菌群以及肠粘膜的免疫反应，临床病理表现为持续腹痛、腹泻和黏液血便，且病情反复，近年来我国UC的患病人数呈明显上升趋势。Ulcerative colitis (UC) is an important type of inflammatory bowel disease (IBD). The cause of the disease is unknown. The main lesion site is the submucosal layer of the colon mucosa. It is a chronic intestinal disease. Based on current research, the main causes of UC are host genetic susceptibility, intestinal flora, and immune response of the intestinal mucosa. Clinical pathology manifests as persistent abdominal pain, diarrhea, and mucus and blood in the stool, and the disease recurs. In recent years, the number of UC patients in my country has shown a clear upward trend.
目前，临床上针对UC的用药主要有水杨酸类药物、肾上腺糖皮质激素类药物和免疫抑制剂。这三类药物均可以一定程度上对UC进行缓解，但是也都存在不足。水杨酸类药物可以比较好的抑制前列腺素合成，清除氧自由基从而达到缓解炎症反应的目的，但也只能短期缓解，无法实现根治，临床上治疗UC常见的水杨酸类西药主要是柳氮磺胺吡啶(SASP)，主要针对轻度、中度以及慢性UC患者。并且，水杨酸类药物还具有很多副作用，例如引发消化道反应、头痛、网织红细胞增多、精子减少，以及过敏反应引起的皮疹、肝毒性、白细胞减少、贫血等，这类药物同时也具有抗菌作用，容易引起菌群紊乱以及耐药性增强。肾上腺糖皮质激素类药物是重症或者爆发性UC患者的首选用药，典型的药物比如倍他米松；但是，肾上腺糖皮质激素类药物会导致机体代谢紊乱，水潴留等副作用，仅可作为应急用药，不能长期服用。免疫抑制剂，例如环孢素，可以通过抑制T细胞IL-2的产生，影响免疫反应的进展，从而对UC进行抑制；但是，免疫抑制剂治疗对药物依赖性较大，治疗周期长，容易引起肾毒性及二次感染，只能作为一种辅助治疗的手段。因此，目前来说尚没有安全有效的炎症及其相关疾病，尤其是溃疡性肠炎的治疗药物。At present, the main drugs used clinically for UC are salicylic acid drugs, adrenal glucocorticoid drugs and immunosuppressants. All three types of drugs can relieve UC to a certain extent, but they also have shortcomings. Salicylic acid drugs can better inhibit prostaglandin synthesis and scavenge oxygen free radicals to achieve the purpose of alleviating inflammatory reactions, but they can only provide short-term relief and cannot achieve a radical cure. The common salicylic acid Western medicine for the treatment of UC in clinical practice is mainly sulfasalazine (SASP), which is mainly used for patients with mild, moderate and chronic UC. In addition, salicylic acid drugs also have many side effects, such as gastrointestinal reactions, headaches, reticulocytosis, sperm reduction, and rashes caused by allergic reactions, hepatotoxicity, leukopenia, anemia, etc. This type of drug also has antibacterial effects, which can easily cause flora disorders and enhance drug resistance. Adrenal glucocorticoids are the first choice for patients with severe or fulminant UC, and a typical drug is betamethasone; however, adrenal glucocorticoids can cause metabolic disorders, water retention and other side effects, and can only be used as emergency medications and cannot be taken for a long time. Immunosuppressants, such as cyclosporine, can inhibit the production of T cell IL-2 and affect the progression of the immune response, thereby inhibiting UC; however, immunosuppressant treatment is highly drug-dependent, has a long treatment cycle, and is prone to cause nephrotoxicity and secondary infection, so it can only be used as an auxiliary treatment. Therefore, there is currently no safe and effective treatment for inflammation and its related diseases, especially ulcerative enteritis.
发明内容Summary of the invention
本申请的目的是提供一种组合物及其应用。The purpose of the present application is to provide a composition and its application.
本申请采用了以下技术方案：This application adopts the following technical solutions:
本申请的一方面公开了一种组合物，该组合物包括加氏乳杆菌和/或其代谢产物、嗜酸乳杆菌和/或其代谢产物，以及假小链状双歧杆菌和/或其代谢产物。One aspect of the present application discloses a composition comprising Lactobacillus gasseri and/or its metabolites, Lactobacillus acidophilus and/or its metabolites, and Bifidobacterium pseudocatenulatus and/or its metabolites.
需要说明的是，本申请的关键在于，研究发现加氏乳杆菌、嗜酸乳杆菌和假小链状双歧杆菌三者联合使用能够预防和治疗炎症及炎症相关疾病，尤其是能够有效预防和治疗溃疡性肠炎；研究显示，其预防或治疗预防作用主要由两方面组成，一方面，是加氏乳杆菌、嗜酸乳杆菌和假小链状双歧杆菌对体内肠道微生态进行改善，形成由有益菌构成的生态保护屏障，从而起到炎症或炎症相关疾病的预防和治疗效果；另一方面，是加氏乳杆菌、嗜酸乳杆菌和假小链状双歧杆菌的代谢产物作为益生材料，起到炎症或炎症相关疾病的预防和治疗作用。因此，本申请的组合物，其重要的用途就是用于预防或治疗炎症或炎症相关疾病，尤其是用于预防或治疗溃疡性肠炎或其相关疾病。It should be noted that the key to the present application is that studies have found that the combined use of Lactobacillus gasseri, Lactobacillus acidophilus and Bifidobacterium pseudocatinellae can prevent and treat inflammation and inflammation-related diseases, especially can effectively prevent and treat ulcerative enteritis; Studies show that its prevention or treatment preventive effect is mainly composed of two aspects, on the one hand, Lactobacillus gasseri, Lactobacillus acidophilus and Bifidobacterium pseudocatinellae improve the intestinal microecology in vivo, forming an ecological protection barrier composed of beneficial bacteria, thereby playing the prevention and treatment effect of inflammation or inflammation-related diseases; on the other hand, the metabolites of Lactobacillus gasseri, Lactobacillus acidophilus and Bifidobacterium pseudocatinellae are used as prebiotic materials to play the prevention and treatment effect of inflammation or inflammation-related diseases. Therefore, the composition of the present application, its important use is to prevent or treat inflammation or inflammation-related diseases, especially to prevent or treat ulcerative enteritis or its related diseases.
还需要说明的是，本申请的一种实现方式中，由加氏乳杆菌、嗜酸乳杆菌和假小链状双歧杆菌组成的组合物，通过三个菌对体内肠道微生态进行改善，形成由有益菌构成的生态保护屏障，起到溃疡性肠炎预防和治疗效果。可以理解，这种微生态的改善，不仅对溃疡性肠炎有预防和治疗效果，对于其它与微生态相关疾病，例如普通肠炎或胃炎等，同样具有效果；因此，本申请的组合物可以用于预防或治疗炎症或炎症相关疾病。It should also be noted that in one implementation of the present application, the composition composed of Lactobacillus gasseri, Lactobacillus acidophilus and Bifidobacterium pseudocatinellae improves the intestinal microecology in the body through three bacteria, forming an ecological protection barrier composed of beneficial bacteria, and plays a role in the prevention and treatment of ulcerative enteritis. It can be understood that this improvement in microecology not only has a preventive and therapeutic effect on ulcerative enteritis, but also has an effect on other diseases related to microecology, such as common enteritis or gastritis; therefore, the composition of the present application can be used to prevent or treat inflammation or inflammation-related diseases.
优选的，加氏乳杆菌为保藏号GDMCC 60092的加氏乳杆菌TF08-1，嗜酸乳杆菌为保藏号GDMCC 60091的嗜酸乳杆菌AM13-1，假小链状双歧杆菌为保藏号GDMCC 60089的假小链状双歧杆菌TM12-14。Preferably, Lactobacillus gasseri is Lactobacillus gasseri TF08-1 with a preservation number of GDMCC 60092, Lactobacillus acidophilus is Lactobacillus acidophilus AM13-1 with a preservation number of GDMCC 60091, and Bifidobacterium pseudocatenulatum is Bifidobacterium pseudocatenulatum TM12-14 with a preservation number of GDMCC 60089.
需要说明的是，本申请的关键在于研究发现加氏乳杆菌、嗜酸乳杆菌和假小链状双歧杆菌三者联合使用能够预防和治疗炎症及炎症相关疾病，而加氏乳杆菌TF08-1、嗜酸乳杆菌AM13-1和假小链状双歧杆菌TM12-14，这三个菌株是本申请研究过程中发现的联合使用效果较好的三个菌株，因此分别对三个菌株进行了保藏。可以理解，一方面，不排除在本申请的发明思路下，还可以采用其它菌株的加氏乳杆菌、嗜酸乳杆菌和假小链状双歧杆菌，达到甚至超过本申请三个菌株的效果；另一方面，在对炎症或炎症相关疾病预防或治疗效果要求较低的情况下，也可以采用其它菌株的加氏乳杆菌、嗜酸乳杆菌和假小链状双歧杆菌。It should be noted that the key to this application is that the study found that the combined use of Lactobacillus gasseri, Lactobacillus acidophilus and Bifidobacterium pseudocatinellae can prevent and treat inflammation and inflammation-related diseases, and Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1 and Bifidobacterium pseudocatinellae TM12-14, these three strains are three strains with good combined use effects found in the research process of this application, so the three strains are preserved respectively. It can be understood that, on the one hand, it is not excluded that under the inventive thinking of this application, other strains of Lactobacillus gasseri, Lactobacillus acidophilus and Bifidobacterium pseudocatinellae can also be used to achieve or even exceed the effects of the three strains of this application; on the other hand, in the case of lower requirements for the prevention or treatment effect of inflammation or inflammation-related diseases, other strains of Lactobacillus gasseri, Lactobacillus acidophilus and Bifidobacterium pseudocatinellae can also be used.
优选的，本申请的组合物还含有其它益生菌和/或益生元。Preferably, the composition of the present application further contains other probiotics and/or prebiotics.
需要说明的是，本申请的组合物，其关键在于加氏乳杆菌、嗜酸乳杆菌和假小链状双歧杆菌三者联合使用能够预防和治疗炎症及炎症相关疾病，可以理解，在不影响加氏乳杆菌、嗜酸乳杆菌和假小链状双歧杆菌三者联合作用效果的情况下，还可以添加其它益生菌或者益生元，使得本申请的组合物具备更多的功能，或者对其原本的功效进行加强，这些益生菌或益生元可以采用现有研究报道过的益生菌或益生元，在此不做具体限定。It should be noted that the key to the composition of the present application is that the combined use of Lactobacillus gasseri, Lactobacillus acidophilus and Bifidobacterium pseudocatenulatum can prevent and treat inflammation and inflammation-related diseases. It can be understood that other probiotics or prebiotics can be added without affecting the combined effect of Lactobacillus gasseri, Lactobacillus acidophilus and Bifidobacterium pseudocatenulatum, so that the composition of the present application has more functions or its original efficacy is enhanced. These probiotics or prebiotics can be probiotics or prebiotics reported in existing studies, and are not specifically limited here.
优选的，本申请的一种实现方式中，本申请组合物中的益生元，选自低聚果糖(FOS)、低聚半乳糖(GOS)、低聚木糖(XOS)、低聚乳果糖(LACT)、大豆低聚糖(SOS)、菊粉(Inulin)和寡聚糖中的至少一种。Preferably, in one implementation of the present application, the prebiotics in the composition of the present application are selected from at least one of fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), xylooligosaccharides (XOS), lactofructooligosaccharides (LACT), soy oligosaccharides (SOS), inulin and oligosaccharides.
优选的，本申请的组合物还含有有助于保持加氏乳杆菌、嗜酸乳杆菌和假小链状双歧杆菌中的至少一种的活力的物质。Preferably, the composition of the present application further contains a substance that helps maintain the vitality of at least one of Lactobacillus gasseri, Lactobacillus acidophilus and Bifidobacterium pseudocatenulatus.
可以理解，为了保持组合物中加氏乳杆菌、嗜酸乳杆菌和假小链状双歧杆菌的活力，保障其功效，还可以在组合物中添加各种维持菌株活力的物质，这些活力物质可以是现有研究报道过的活力物质，在此不做具体限定。It is understandable that in order to maintain the vitality of Lactobacillus gasseri, Lactobacillus acidophilus and Bifidobacterium pseudocatenulatus in the composition and ensure its efficacy, various substances that maintain the vitality of the strains can also be added to the composition. These vitality substances can be vitality substances reported in existing studies and are not specifically limited here.
优选的，本申请的一种实现方式中，有助于保持加氏乳杆菌、嗜酸乳杆菌和假小链状双歧杆菌中的至少一种的活力的物质，选自半胱氨酸、谷胱甘肽、丁基羟基茴香醚、二丁基甲基甲苯、生育酚、竹叶抗氧化物、D-异抗坏血酸或其钠盐、抗坏血酸钠、抗坏血酸钙、磷脂、维生素C和维生素E中的至少一种。Preferably, in one implementation of the present application, the substance that helps to maintain the vitality of at least one of Lactobacillus gasseri, Lactobacillus acidophilus and Bifidobacterium pseudocatenulatus is selected from at least one of cysteine, glutathione, butylated hydroxyanisole, dibutylmethyltoluene, tocopherol, bamboo leaf antioxidants, D-isoascorbic acid or its sodium salt, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C and vitamin E.
优选的，本申请的组合物还包括药学上或食品上可接受的载体或辅料。Preferably, the composition of the present application further comprises a pharmaceutically or food acceptable carrier or auxiliary material.
需要说明的是，本申请的组合物具有预防和治疗炎症及炎症相关疾病的效果，本申请的一种实现方式中，主要通过食用本申请的组合物，达到预防和治疗效果。因此，组合物中还可以包括药学上或食品上可接受的载体或辅料，以方便使用。It should be noted that the composition of the present application has the effect of preventing and treating inflammation and inflammation-related diseases. In one implementation of the present application, the prevention and treatment effects are achieved mainly by consuming the composition of the present application. Therefore, the composition may also include a pharmaceutically or food-acceptable carrier or excipient for easy use.
优选的，药学上或食品上可接受的载体或辅料，选自葡萄糖、乳糖、蔗糖、淀粉、甘露醇、糊精、脂肪酸甘油酯、聚乙二醇、羟乙基淀粉、乙二醇、聚氧乙烯山梨糖醇酐脂肪酸酯、氨基酸、明胶、白蛋白、水和生理食盐水中的至少一种。Preferably, the pharmaceutically or food acceptable carrier or excipient is selected from at least one of glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin, water and physiological saline.
本申请的另一面公开了本申请的组合物在制备用于治疗或预防炎症或炎症相关疾病的食品、保健品、食品添加剂或药品中的应用。Another aspect of the present application discloses the use of the composition of the present application in the preparation of food, health products, food additives or medicines for treating or preventing inflammation or inflammation-related diseases.
可以理解，本申请的组合物具有预防和治疗炎症及炎症相关疾病的效果，为了方便使用，可以将本申请的组合物制成各种食品、保健品、食品添加剂或药品。It can be understood that the composition of the present application has the effect of preventing and treating inflammation and inflammation-related diseases. For the convenience of use, the composition of the present application can be made into various foods, health products, food additives or medicines.
优选的，本申请的组合物尤其可以用于制备治疗或预防溃疡性肠炎或其相关疾病的食品、保健品、食品添加剂或药品。Preferably, the composition of the present application can be used in particular for preparing food, health products, food additives or medicines for treating or preventing ulcerative enteritis or diseases related thereto.
本申请的再一面公开了本申请的组合物在制备用于控制哺乳动物体重下降的食品、保健品、食品添加剂或药品中的应用。Another aspect of the present application discloses the use of the composition of the present application in the preparation of food, health products, food additives or medicines for controlling weight loss in mammals.
其中，哺乳动物体重下降，尤其指哺乳动物因炎症导致的体重下降。Among them, weight loss in mammals, particularly refers to weight loss in mammals caused by inflammation.
优选的，炎症为溃疡性肠炎，即控制哺乳动物因溃疡性肠炎导致的体重下降。Preferably, the inflammation is ulcerative colitis, ie, controlling the weight loss of a mammal caused by ulcerative colitis.
本申请的再一面公开了本申请的组合物在制备用于降低哺乳动物的疾病活动指数的食品、保健品、食品添加剂或药品中的应用。Another aspect of the present application discloses the use of the composition of the present application in the preparation of food, health products, food additives or medicines for reducing the disease activity index of mammals.
本申请的再一面公开了本申请的组合物在制备用于改善哺乳动物肠道病变的食品、保健品、食品添加剂或药品中的应用。Another aspect of the present application discloses the use of the composition of the present application in the preparation of food, health products, food additives or medicines for improving intestinal lesions in mammals.
需要说明的是，本申请的组合物能够预防和治疗炎症及炎症相关疾病，其关键就体现在能够对炎症及炎症相关疾病引起的体重下降进行控制、降低炎症及炎症相关疾病引起的疾病活动指数，并改善哺乳动物肠道病变；因此，本申请的组合物也可以单独的用于制备控制哺乳动物体重下降、降低哺乳动物疾病活动指数或改善哺乳动物肠道病变的食品、保健品、食品添加剂或药品。It should be noted that the composition of the present application can prevent and treat inflammation and inflammation-related diseases. The key lies in its ability to control weight loss caused by inflammation and inflammation-related diseases, reduce the disease activity index caused by inflammation and inflammation-related diseases, and improve intestinal lesions in mammals; therefore, the composition of the present application can also be used alone to prepare foods, health products, food additives or medicines that control weight loss in mammals, reduce the disease activity index in mammals or improve intestinal lesions in mammals.
本申请的再一面公开了采用本申请的组合物进行炎症或炎症相关疾病的治疗或预防的方法。Another aspect of the present application discloses a method for treating or preventing inflammation or inflammation-related diseases using the composition of the present application.
本申请的再一面公开了采用本申请的组合物控制哺乳动物体重下降的方法。Another aspect of the present application discloses a method for controlling weight loss in mammals using the composition of the present application.
本申请的再一面公开了采用本申请的组合物降低哺乳动物的疾病活动指数的方法。Another aspect of the present application discloses a method for reducing the disease activity index of a mammal using the composition of the present application.
本申请的再一面公开了采用本申请的组合物改善哺乳动物肠道病变的方法。Another aspect of the present application discloses a method for improving intestinal lesions in mammals using the composition of the present application.
需要说明的是，以上各种方法中，主要是通过食用本申请的组合物，以起到治疗或预防炎症或炎症相关疾病、控制哺乳动物体重下降、降低哺乳动物疾病活动指数或改善哺乳动物肠道病变的效果。It should be noted that among the above methods, the composition of the present application is mainly consumed to treat or prevent inflammation or inflammation-related diseases, control weight loss in mammals, reduce the disease activity index of mammals, or improve intestinal lesions in mammals.
本申请的再一面公开了一种食品，该食品中含有本申请的组合物。Another aspect of the present application discloses a food, wherein the food contains the composition of the present application.
优选的，食品为乳酸饮品或豆乳饮品。本申请的食品，是广义上的以任何形态存在的可食用物品，并不仅限于乳酸饮品或豆乳饮品，例如还可以是发酵食品，也可以是动物饲料等。Preferably, the food is a lactic acid drink or a soy milk drink. The food of the present application is a broadly defined edible item in any form, and is not limited to lactic acid drinks or soy milk drinks, and may also be fermented food, animal feed, etc.
需要说明的是，本申请的食品由于含有本申请的组合物，因此同样具有治疗或预防炎症或炎症相关疾病、控制哺乳动物体重下降、降低哺乳动物疾病活动指数和改善哺乳动物肠道病变的效果。可以理解，本申请的食品，其关键在于其中含有本申请的组合物，至于食品的具体形态，如固体、液体等，可以根据不同的食品产品或使用需求而定，在此不做具体限定。本申请的一种实现方式中，主要是将本申请的组合物制成常见的乳酸饮品或豆乳饮品，以方便饮用；当然，也可以制成例如奶片、奶酪条等固体食品，在此不做具体限定。It should be noted that the food of the present application, because it contains the composition of the present application, also has the effect of treating or preventing inflammation or inflammation-related diseases, controlling weight loss in mammals, reducing the disease activity index of mammals, and improving intestinal lesions in mammals. It can be understood that the key to the food of the present application is that it contains the composition of the present application. As for the specific form of the food, such as solid, liquid, etc., it can be determined according to different food products or usage requirements, and is not specifically limited here. In one implementation of the present application, the composition of the present application is mainly made into a common lactic acid drink or soy milk drink for easy drinking; of course, it can also be made into solid foods such as milk slices and cheese strips, which are not specifically limited here.
还需要说明的是，本申请的食品中加氏乳杆菌、嗜酸乳杆菌和假小链状双歧杆菌的活性菌量或者摄取量不做具体限定，实际应用中，可以根据实际状况灵活选择。以加氏乳杆菌TF08-1、嗜酸乳杆菌AM13-1和假小链状双歧杆菌TM12-14为例，本申请的研究显示，每天摄入加氏乳杆菌TF08-1、嗜酸乳杆菌AM13-1和假小链状双歧杆菌TM12-14的浓度都是109cfu/mL的组合物0.2mL，对溃疡性肠炎有良好的治疗效果，该活性菌剂量可以作为食品、保健品、食品添加剂或药品的参考用量或参考摄取量。It should also be noted that the amount of active bacteria or intake of Lactobacillus gasseri, Lactobacillus acidophilus and Bifidobacterium pseudocatinellae in the food of the present application is not specifically limited, and in practical applications, it can be flexibly selected according to actual conditions. Taking Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1 and Bifidobacterium pseudocatinellae TM12-14 as examples, the research of the present application shows that daily intake of 0.2 mL of a composition with a concentration of 109 cfu/mL of Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1 and Bifidobacterium pseudocatinellae TM12-14 has a good therapeutic effect on ulcerative enteritis, and the active bacteria dosage can be used as a reference dosage or reference intake of food, health products, food additives or medicines.
本申请的再一面公开了一种保健品，该保健品中含有本申请的组合物。Another aspect of the present application discloses a health product, which contains the composition of the present application.
需要说明的是，本申请的保健品由于含有本申请的组合物，因此同样具有治疗或预防炎症或炎症相关疾病、控制哺乳动物体重下降、降低哺乳动物疾病活动指数和改善哺乳动物肠道病变的效果。It should be noted that the health care product of the present application, since it contains the composition of the present application, also has the effect of treating or preventing inflammation or inflammation-related diseases, controlling weight loss in mammals, reducing the disease activity index of mammals and improving intestinal lesions in mammals.
本申请的再一面公开了一种食品添加剂，该食品添加剂中含有本申请的组合物。Another aspect of the present application discloses a food additive, wherein the food additive contains the composition of the present application.
需要说明的是，本申请的组合物可以与通常的食品材料配合食用。例如，谷类及薯类，谷类包括米、面、杂粮，薯类包括马铃薯、红薯等；动物性食物，包括肉、禽、鱼、奶、蛋等；豆类及其制品，包括大豆及其它干豆类；蔬菜水果类，包括鲜豆、根茎、叶菜、茄果等；纯热能食物，包括动植物油、淀粉、食用糖和酒类等；因此，本申请的组合物可以单独的作为食品添加剂或者调制剂加入各种食材中直接食用，起到炎症及其相关疾病的治疗或预防效果。It should be noted that the composition of the present application can be eaten with common food materials. For example, cereals and potatoes, cereals include rice, noodles, and grains, potatoes include potatoes, sweet potatoes, etc.; animal foods include meat, poultry, fish, milk, eggs, etc.; beans and their products, including soybeans and other dried beans; vegetables and fruits, including fresh beans, rhizomes, leafy vegetables, eggplants, etc.; pure energy foods, including animal and vegetable oils, starch, edible sugar, and alcohol, etc.; therefore, the composition of the present application can be added to various food materials as a food additive or modulator and eaten directly, to achieve the therapeutic or preventive effect of inflammation and related diseases.
本申请的再一面公开了一种药品，该药品中含有本申请的组合物。Another aspect of the present application discloses a medicine, which contains the composition of the present application.
优选的，药品为片剂、颗粒剂、散剂、肠溶剂、溶液剂或悬浮剂。Preferably, the medicine is a tablet, granule, powder, enteric solvent, solution or suspension.
需要说明的是，本申请的药品由于含有本申请的组合物，因此具有炎症及其相关疾病的治疗或预防效果；本申请的药品，可以是单独的本申请的组合物，也可以与其它炎症药物配合使用，只要相互之间不影响活性即可。可以理解，本申请的药品，只要不影响组合物中各菌株的活性，药品可以采用现有的各种剂型。而本申请的药品中，进一步的还可以包括药品或剂型中常用的辅料，例如稳定剂、湿润剂、乳化剂、粘合剂、等渗剂等。It should be noted that the medicine of the present application has the therapeutic or preventive effect of inflammation and related diseases because it contains the composition of the present application; the medicine of the present application can be a single composition of the present application, or it can be used in combination with other inflammatory drugs, as long as they do not affect the activity of each other. It can be understood that the medicine of the present application can adopt various existing dosage forms as long as it does not affect the activity of each strain in the composition. The medicine of the present application can further include excipients commonly used in medicines or dosage forms, such as stabilizers, wetting agents, emulsifiers, adhesives, isotonic agents, etc.
本申请的药品可以以口服液、片剂、针剂、口崩片、冻干粉制剂或肠溶剂型的任一种形式给药。优选肠溶剂型，如胶囊或肠溶片，以便于药品的活性成分即微生物能顺利通过胃而不被胃酸所破坏。更优选的，本申请的药品可制成肠溶片供口服使用。The drug of the present application can be administered in any form of oral liquid, tablet, injection, orodisintegrating tablet, lyophilized powder preparation or enteric coating formulation. Enteric coating formulations, such as capsules or enteric-coated tablets, are preferred, so that the active ingredient of the drug, i.e., the microorganism, can pass through the stomach smoothly without being destroyed by gastric acid. More preferably, the drug of the present application can be made into enteric-coated tablets for oral use.
本申请的肠溶剂型是指在胃液中不崩解，而在肠液中能够崩解和吸收的一种药品剂型，肠溶剂型包括胶囊和肠溶片。其中，胶囊由粉末状的药品封装在的常规药物允许使用的胶囊壳中形成；肠溶片则是在普通的片剂药品外面包裹一层肠溶包衣而成。本申请的“肠溶包衣”简称“肠衣”，包括所有常规药物允许使用的包衣，这些包衣不被胃酸降解，但在小肠中能充分分解并快速释放出本申请的药品。例如，本申请的肠衣能在合成胃酸如pH＝1的HCl溶液中在36-38℃维持2小时以上，并优选在合成肠液如pH＝7.0的缓冲液中在1.0小时内分解。The enteric-coated dosage form of the present application refers to a drug dosage form that does not disintegrate in gastric juice but can disintegrate and be absorbed in intestinal juice. The enteric-coated dosage form includes capsules and enteric-coated tablets. Among them, capsules are formed by encapsulating powdered drugs in capsule shells that are allowed to be used in conventional drugs; enteric-coated tablets are formed by wrapping a layer of enteric coating on the outside of ordinary tablet drugs. The "enteric coating" of the present application is referred to as "enteric coating", which includes all coatings allowed to be used in conventional drugs. These coatings are not degraded by gastric acid, but can be fully decomposed in the small intestine and quickly release the drugs of the present application. For example, the enteric coating of the present application can be maintained at 36-38°C for more than 2 hours in synthetic gastric acid such as HCl solution with pH=1, and preferably decomposed within 1.0 hour in synthetic intestinal juice such as buffer solution with pH=7.0.
优选的，本申请的肠溶片中，其肠衣厚度为5-100μm，理想的厚度为20-80μm。肠衣成分选自已公开知晓的常规材料。Preferably, in the enteric-coated tablets of the present application, the thickness of the enteric coating is 5-100 μm, and the ideal thickness is 20-80 μm. The enteric coating components are selected from conventional materials that are publicly known.
本申请的药品中益生菌组合物的各菌株的活性菌含量，或者药剂量不受特别限制，实际应用中，可以根据给药对象的健康状况灵活选择。但是，本申请的研究显示，每天摄入加氏乳杆菌TF08-1、嗜酸乳杆菌AM13-1和假小链状双歧杆菌TM12-14的浓度都是109cfu/mL的益生菌组合物0.2mL，对溃疡性肠炎有良好的治疗效果，此用量可以作为药品中活性菌含量或者给药剂量参考。The active bacterial content of each strain of the probiotic composition in the medicine of the present application, or the dosage of the medicine is not particularly limited. In practical applications, it can be flexibly selected according to the health status of the subject. However, the research of the present application shows that daily intake of 0.2 mL of the probiotic composition with a concentration of 109 cfu/mL of Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1 and Bifidobacterium pseudocatenulatus TM12-14 has a good therapeutic effect on ulcerative enteritis. This dosage can be used as a reference for the active bacterial content or dosage in the medicine.
本申请的有益效果在于：The beneficial effects of this application are:
本申请的组合物，通过加氏乳杆菌、嗜酸乳杆菌和假小链状双歧杆菌三者联合使用，能够对炎症，尤其是溃疡性肠炎，及其相关疾病有良好的治疗和预防效果，为炎症及其相关疾病的治疗和预防提供了一种新的安全、有效、毒副作用小，且不易产生抗性的组合物。The composition of the present application, through the combined use of Lactobacillus gasseri, Lactobacillus acidophilus and Bifidobacterium pseudocatenulatus, can have a good therapeutic and preventive effect on inflammation, especially ulcerative enteritis, and related diseases, and provides a new safe, effective, low-toxic and non-resistant composition for the treatment and prevention of inflammation and related diseases.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1是本申请实施例中Control组、模型组、VSL#3组和益生菌组合物治疗组小鼠的体重的变化曲线；FIG1 is a curve showing changes in body weight of mice in the Control group, the model group, the VSL# 3 group, and the probiotic composition treatment group in the examples of the present application;
图2是本申请实施例中Control组、模型组、VSL#3组和益生菌组合物治疗组小鼠的DAI指数的变化曲线。FIG. 2 is a curve showing changes in the DAI index of mice in the Control group, the model group, the VSL# 3 group, and the probiotic composition treatment group in the examples of the present application.
本申请的加氏乳杆菌TF08-1于2016年10月13日保藏于广东省微生物菌种保藏中心，保藏单位的地址为中国广东省广州市先烈中路100号省微生物所实验楼五楼，保藏号为GDMCC 60092。The Lactobacillus gasseri TF08-1 of the present application was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 13, 2016. The address of the depository is the fifth floor of the laboratory building of the Provincial Microbiology Institute, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, China, and the deposit number is GDMCC 60092.
嗜酸乳杆菌AM13-1于2016年10月13日保藏于广东省微生物菌种保藏中心，保藏单位的地址为中国广东省广州市先烈中路100号省微生物所实验楼五楼，保藏号为GDMCC60091。Lactobacillus acidophilus AM13-1 was deposited in Guangdong Microbial Culture Collection Center on October 13, 2016. The address of the depository is 5th Floor, Laboratory Building, Provincial Institute of Microbiology, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, China. The deposit number is GDMCC60091.
假小链状双歧杆菌TM12-14于2016年10月13日保藏于广东省微生物菌种保藏中心，保藏单位的地址为中国广东省广州市先烈中路100号省微生物所实验楼五楼，保藏号为GDMCC 60089。Bifidobacterium pseudocatenulatus TM12-14 was deposited in Guangdong Microbial Culture Collection Center on October 13, 2016. The address of the depository is 5th Floor, Laboratory Building, Provincial Institute of Microbiology, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, China, and the deposit number is GDMCC 60089.
具体实施方式DETAILED DESCRIPTION
随着肠道微生态的深入研究，发现溃疡性肠炎的发病与肠道微生物的组成密切相关，肠道细菌的失衡与肠粘膜炎症反应有着密切的关系，其中有害菌的过度增殖可以引发炎症反应，进而诱发溃疡性肠炎的发病。健康的人体肠道中存在着大量的有益菌，这些有益菌构成肠道的第一层生物屏障。With the in-depth study of intestinal microecology, it is found that the onset of ulcerative enteritis is closely related to the composition of intestinal microorganisms, and the imbalance of intestinal bacteria is closely related to the inflammatory response of the intestinal mucosa. The excessive proliferation of harmful bacteria can trigger an inflammatory response, which in turn induces the onset of ulcerative enteritis. There are a large number of beneficial bacteria in the healthy human intestine, which constitute the first biological barrier of the intestine.
基于以上研究和认识，本申请研发并提出了一种新的组合物，该组合物包括加氏乳杆菌和/或其代谢产物、嗜酸乳杆菌和/或其代谢产物，以及假小链状双歧杆菌和/或其代谢产物。本申请的组合物不仅具有溃疡性肠炎治疗和预防功能，而且，对于其它与微生态相关疾病，例如普通肠炎或胃炎等，同样具有效果；因此，本申请的组合物能够用于预防或治疗炎症或炎症相关疾病。Based on the above research and understanding, the present application has developed and proposed a new composition, which includes Lactobacillus gasseri and/or its metabolites, Lactobacillus acidophilus and/or its metabolites, and Bifidobacterium pseudocatenulatum and/or its metabolites. The composition of the present application not only has the function of treating and preventing ulcerative enteritis, but also has the same effect on other diseases related to microecology, such as common enteritis or gastritis; therefore, the composition of the present application can be used to prevent or treat inflammation or inflammation-related diseases.
本申请的一种实现方式中，由保藏号为GDMCC 60092的加氏乳杆菌TF08-1、保藏号为GDMCC 60091的嗜酸乳杆菌AM13-1和保藏号为GDMCC60089的假小链状双歧杆菌TM12-14组成的组合物，对溃疡性肠炎的治疗效果优于美国Alfasigma公司生产的VSL#3复合益生菌剂，能够用于制备治疗或预防炎症及其相关疾病的食品、保健品、食品添加剂或药品。In one implementation of the present application, a composition consisting of Lactobacillus gasseri TF08-1 with a preservation number of GDMCC 60092, Lactobacillus acidophilus AM13-1 with a preservation number of GDMCC 60091, and Bifidobacterium pseudocatenulatus TM12-14 with a preservation number of GDMCC60089 has a therapeutic effect on ulcerative enteritis that is better than the VSL# 3 composite probiotic produced by Alfasigma, Inc. of the United States, and can be used to prepare foods, health products, food additives or medicines for the treatment or prevention of inflammation and related diseases.
下面通过具体实施例对本申请作进一步详细说明。以下实施例仅对本申请进行进一步说明，不应理解为对本申请的限制。The present application is further described in detail below through specific examples. The following examples are only used to further illustrate the present application and should not be construed as limiting the present application.
实施例一Embodiment 1
本例以分子量36000-50000的硫酸葡聚糖钠(缩写DSS)诱导的溃疡性肠炎小鼠模型为研究对象，研究加氏乳杆菌(Lactobacillus gasseri)TF08-1、嗜酸乳杆菌(Lactobacillus acidophilus)AM13-1和假小链状双歧杆菌(Bifidobacteriumpseudocatenulatum)TM12-14益生菌组合物对溃疡性肠炎的治疗效果。具体如下：In this case, the ulcerative colitis mouse model induced by dextran sulfate sodium (DSS) with a molecular weight of 36000-50000 was used as the research object to study the therapeutic effect of the probiotic composition of Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1 and Bifidobacteriumpseudocatenulatum TM12-14 on ulcerative colitis. The details are as follows:
一、材料和方法1. Materials and Methods
1.菌株培养和鉴定1. Strain culture and identification
1.1加氏乳杆菌TF08-11.1 Lactobacillus gasseri TF08-1
本例的加氏乳杆菌TF08-1采用PYG培养基进行分离，分离条件为37℃厌氧条件。TF08-1在PYG培养基培养2天的菌落为白色、低凸起、近圆形、边缘波状，菌落直径约1-2mm，菌体的显微形态为杆状，革兰氏阳性、不产芽孢和鞭毛。菌株保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC 60092。In this example, Lactobacillus gasseri TF08-1 was isolated using PYG medium under anaerobic conditions at 37°C. The colonies of TF08-1 cultured in PYG medium for 2 days were white, low-raised, nearly round, with wavy edges, and a colony diameter of about 1-2 mm. The microscopic morphology of the bacteria was rod-shaped, Gram-positive, and did not produce spores and flagella. The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC 60092.
加氏乳杆菌TF08-1的具体分离鉴定步骤如下：The specific isolation and identification steps of Lactobacillus gasseri TF08-1 are as follows:
1.1.1样品收集1.1.1 Sample collection
分离的样品来自于一位16岁健康女性志愿者的粪便样品，志愿者居住于广东省深圳市。并详细记录该志愿者的饮食情况和身体情况。The isolated sample came from a fecal sample of a 16-year-old healthy female volunteer who lived in Shenzhen, Guangdong Province. The volunteer's diet and physical condition were recorded in detail.
1.1.2菌株的分离培养1.1.2 Isolation and culture of strains
提前制备好分离培养基，培养基选用PYG培养基，购自环凯微生物科技公司，具体成分为：蛋白胨5g，胰化酪蛋白5g，酵母粉10g，牛肉膏5g，葡萄糖5g，K2HPO4 2g，Tween 801mL，Cysteine-HCl·H2O 0.5g，硫化钠0.25g，血红素5mg，维生素K1 1μL，无机盐溶液40mL，刃天青1mg，蒸馏水950mL，pH6.8～7.0，115℃灭菌25min。固体培养基加入1.5％的琼脂，在厌氧操作箱中倾倒。每1L无机盐溶液含有CaCl2·2H2O 0.25g，MgSO4·7H2O 0.5g，K2HPO41g，KH2PO4 1g，NaHCO3 10g，NaCl 2g。Prepare the separation medium in advance. The medium is PYG medium purchased from Huankai Microbiological Technology Co., Ltd. The specific ingredients are:5g peptone, 5g tryptic casein, 10g yeast powder, 5g beef extract, 5g glucose, 2gK2HPO4 , 1mL Tween80 , 0.5g Cysteine-HCl·H2O, 0.25g sodium sulfide, 5mg heme,1μL vitamin K1, 40mL inorganic salt solution, 1mg resazurin, 950mL distilled water, pH 6.8-7.0, sterilized at 115℃ for 25min. Add 1.5% agar to the solid medium and pour it in the anaerobic operation box. Each 1L of the inorganic salt solution contains 0.25g of CaCl2 ·2H2 O, 0.5g of MgSO4 ·7H2 O, 1g of K2 HPO4 , 1g of KH2 PO4 , 10g of NaHCO3 , and 2g of NaCl.
收集的新鲜粪便样品转移至厌氧箱，取0.2g粪便悬浮于1mL无菌磷酸盐缓冲液(缩写PBS)中，充分混匀，然后进行梯度稀释，取100μL稀释液进行平板涂布，37℃厌氧培养3-4天，厌氧的气体组分为N2∶CO2∶H2＝90∶5∶5。待平板长出菌落选取单个菌落进行划线分纯，获得纯培养菌株，然后进行鉴定和功能验证。The collected fresh feces samples were transferred to an anaerobic box, 0.2 g of feces was suspended in 1 mL of sterile phosphate buffered saline (PBS), mixed thoroughly, and then graded diluted. 100 μL of the dilution was spread on a plate and cultured anaerobically at 37°C for 3-4 days. The anaerobic gas composition was N2 ∶CO2 ∶H2 ＝90∶5∶5. When colonies grew on the plate, a single colony was selected for streaking and purification to obtain a pure culture strain, which was then identified and functionally verified.
1.1.3菌株的16S rDNA鉴定1.1.3 16S rDNA Identification of Strains
分离得到的菌株进行16S rDNA鉴定，以确定菌株的物种分类信息。将获得的分离菌株在液体PYG培养基中培养24h，取1mL菌液进行10000r/min离心5min，收集菌体，提取菌株的基因组DNA，以基因组DNA作为模板进行16S rDNA的扩增，使用16S rDNA的通用引物。The isolated strains were identified by 16S rDNA to determine the species classification information of the strains. The isolated strains were cultured in liquid PYG medium for 24 hours, 1 mL of the bacterial solution was centrifuged at 10,000 r/min for 5 minutes, the bacteria were collected, the genomic DNA of the strains was extracted, and the genomic DNA was used as a template for 16S rDNA amplification using universal primers for 16S rDNA.
16S rDNA的PCR扩增体系为：10×PCR缓冲液3μL、dNTP 2.5μL、上游引物27F 0.5μL、下游引物1492R 0.5μL、Taq酶0.3μL、模板1μL、ddH2O 18.2μL。The PCR amplification system of 16S rDNA was: 3 μL of 10×PCR buffer, 2.5 μL of dNTP, 0.5 μL of upstream primer 27F, 0.5 μL of downstream primer 1492R, 0.3 μL of Taq enzyme, 1 μL of template, and 18.2 μL of ddH2 O.
16S rDNA的扩增条件为：95℃预变性4min，然后进入30个循环：95℃变性30s、57℃退火40s、72℃延伸1min 30s。The amplification conditions of 16S rDNA were as follows: pre-denaturation at 95°C for 4 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 57°C for 40 s, and extension at 72°C for 1 min 30 s.
对16S rDNA的PCR扩增产物进行纯化，3730测序，获得菌株的16S rDNA序列，然后进行NCBI的数据库的比对。The 16S rDNA PCR amplification product was purified and sequenced by 3730 to obtain the 16S rDNA sequence of the strain, which was then compared with the NCBI database.
本试验的16S rDNA通用引物的上下游引物分别为SEQ ID NO.1和SEQ ID NO.2所示序列。分离获得的菌株TF08-1的16S rDNA序列结果为SEQ ID NO.3所示序列。NCBI blast比对结果显示，本例分离的TF08-1菌株与Lactobacillus gasseri同源性最高，相似度为99.9％，因此，判断TF08-1为加氏乳杆菌，命名为加氏乳杆菌TF08-1，并对其进行保藏。The upstream and downstream primers of the 16S rDNA universal primer in this experiment are the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 respectively. The 16S rDNA sequence of the isolated strain TF08-1 is the sequence shown in SEQ ID NO.3. The NCBI blast comparison results show that the isolated TF08-1 strain in this example has the highest homology with Lactobacillus gasseri, with a similarity of 99.9%. Therefore, TF08-1 is judged to be Lactobacillus gasseri, named Lactobacillus gasseri TF08-1, and preserved.
SEQ ID NO.1：5’-AGAGTTTGATCATGGCTCAG-3’SEQ ID NO.1: 5’-AGAGTTTGATCATGGCTCAG-3’
SEQ ID NO.2：5’-TAGGGTTACCTTGTTACGACTT-3’SEQ ID NO.2: 5’-TAGGGTTACCTTGTTACGACTT-3’
1.2嗜酸乳杆菌AM13-11.2 Lactobacillus acidophilus AM13-1
嗜酸乳杆菌AM13-1采用PYG培养基进行分离，分离条件为37℃厌氧条件。AM13-1在PYG培养基培养2天的菌落为白色，凸起，较粘稠，不透明，圆形，边缘整齐，直径约2-3mm，菌体的显微形态为杆状，革兰氏阳性、不产芽孢和鞭毛。菌株保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC 60091。Lactobacillus acidophilus AM13-1 was isolated using PYG medium under anaerobic conditions at 37°C. The colonies of AM13-1 cultured in PYG medium for 2 days were white, convex, viscous, opaque, round, with neat edges and a diameter of about 2-3 mm. The microscopic morphology of the bacteria was rod-shaped, Gram-positive, and did not produce spores or flagella. The strain was deposited in the Guangdong Microbiological Culture Collection Center with the deposit number GDMCC 60091.
嗜酸乳杆菌AM13-1的具体分离鉴定步骤如下：The specific isolation and identification steps of Lactobacillus acidophilus AM13-1 are as follows:
1.2.1分离培养1.2.1 Isolation and culture
分离的样品来自于深圳市一位健康男性的粪便样品，嗜酸乳杆菌AM13-1的分离过程如下：The isolated sample came from a fecal sample of a healthy male in Shenzhen. The isolation process of Lactobacillus acidophilus AM13-1 is as follows:
(1)将样品转移至厌氧箱中，取约0.2g样品悬浮于1mL无菌PBS中，充分混匀，然后进行梯度稀释；(1) Transfer the sample to an anaerobic chamber, suspend about 0.2 g of the sample in 1 mL of sterile PBS, mix thoroughly, and then perform gradient dilutions;
(2)取100μL稀释液于PYG培养基平板上，然后进行涂布，涂布均匀后放置在37℃厌氧环境中进行培养，厌氧的气体组成为：氮气∶氢气∶二氧化碳＝90∶5∶5；PYG培养基与“1.1.2菌株的分离培养”相同；(2) Take 100 μL of the dilution on a PYG medium plate, then spread it, and place it in an anaerobic environment at 37°C for culture. The anaerobic gas composition is: nitrogen: hydrogen: carbon dioxide = 90:5:5; the PYG medium is the same as that in "1.1.2 Isolation and culture of strains";
(3)培养4天，待平板上长出菌落之后，挑选单菌落进行划线分纯，37℃厌氧培养；(3) After 4 days of incubation, when colonies grow on the plate, single colonies are selected for streaking and purification, and then incubated anaerobically at 37°C;
(4)对分纯的单菌进行甘油保藏和真空冷冻干燥保藏。(4) The purified single bacteria are stored in glycerol or vacuum freeze-dried.
1.2.2 AM13-1的16S rDNA鉴定1.2.2 16S rDNA identification of AM13-1
提取基因组DNA，以DNA作为模板进行16S rDNA扩增，采用16S rDNA的通用引物，扩增条件是95℃预变性4min，然后进入30个循环：95℃变性30s、57℃退火40s、72℃延伸1min30s。扩增的PCR产物进行纯化，3730测序，获得AM13-1的16S rDNA全长序列，将AF13-1的16SrDNA序列在NCBI的数据库比对。Genomic DNA was extracted and used as a template for 16S rDNA amplification. The universal primers of 16S rDNA were used. The amplification conditions were 95℃ pre-denaturation for 4min, followed by 30 cycles of 95℃ denaturation for 30s, 57℃ annealing for 40s, and 72℃ extension for 1min30s. The amplified PCR product was purified and sequenced by 3730 to obtain the full-length sequence of 16S rDNA of AM13-1. The 16S rDNA sequence of AF13-1 was compared with the NCBI database.
本试验的16S rDNA通用引物和PCR扩增体系与“1.1.3菌株的16S rDNA鉴定”相同。分离获得的菌株AM13-1的16S rDNA序列结果为SEQ ID NO.4所示序列。NCBI blast比对结果显示，本例分离的AM13-1菌株与Lactobacillus acidophilus同源性最高，相似度为100％，因此，判断AM13-1为嗜酸乳杆菌，命名为嗜酸乳杆菌AM13-1，并对其进行保藏。The 16S rDNA universal primers and PCR amplification system of this test are the same as those of "1.1.3 Identification of 16S rDNA of strains". The 16S rDNA sequence of the isolated strain AM13-1 is the sequence shown in SEQ ID NO.4. The NCBI blast comparison results show that the AM13-1 strain isolated in this example has the highest homology with Lactobacillus acidophilus, with a similarity of 100%. Therefore, AM13-1 is judged to be Lactobacillus acidophilus, named Lactobacillus acidophilus AM13-1, and preserved.
1.3假小链状双歧杆菌TM12-141.3 Bifidobacterium pseudocatenulatum TM12-14
假小链状双歧杆菌TM12-14采用PYG培养基进行分离，分离条件为37℃厌氧条件。TM12-14在PYG培养基培养2天的菌落为白色、凸起、圆形、边缘整齐，菌落直径约1-2mm，菌体显微形态呈现分歧杆状，革兰氏染色为阳性，没有芽孢和鞭毛产生。菌株保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC 60089。Bifidobacterium pseudocatinum TM12-14 was isolated using PYG medium under anaerobic conditions at 37°C. The colonies of TM12-14 cultured in PYG medium for 2 days were white, convex, round, with neat edges, and a colony diameter of about 1-2 mm. The microscopic morphology of the bacteria was bifurcated rod-shaped, Gram staining was positive, and no spores and flagella were produced. The strain was deposited in the Guangdong Microbiological Culture Collection Center with the deposit number GDMCC 60089.
假小链状双歧杆菌TM12-14的具体分离鉴定步骤如下：The specific isolation and identification steps of Bifidobacterium pseudocatenulatum TM12-14 are as follows:
1.3.1样品收集1.3.1 Sample collection
分离样品来自于一位14岁健康的男性粪便，将粪便采集至无菌的样品管中，1h内带回实验室进行分选。The separation sample came from the feces of a healthy 14-year-old male. The feces was collected into a sterile sample tube and brought back to the laboratory for sorting within 1 hour.
1.3.2假小链状双歧杆菌的分离纯化1.3.2 Isolation and purification of Bifidobacterium pseudocatenulatus
收集的新鲜样品立刻转移至厌氧操作箱中，取0.2g样品于1mL无菌的PBS中，充分震荡混匀，然后进行梯度稀释涂布，培养基采用PYG培养基平板，于37℃厌氧培养，厌氧的气体组分为N2∶CO2∶H2＝90∶5∶5。培养3天后，挑取单菌落进行划线分纯，获得每株单菌的纯培养。其中，PYG培养基与“1.1.2菌株的分离培养”相同。The collected fresh samples were immediately transferred to the anaerobic operation box, 0.2 g of the sample was taken in 1 mL of sterile PBS, and the mixture was thoroughly shaken and mixed, and then gradient dilution was applied. The culture medium was PYG medium plate, and the culture was carried out anaerobically at 37°C. The anaerobic gas composition was N2 ∶CO2 ∶H2 ＝90∶5∶5. After 3 days of culture, a single colony was picked for streaking and purification to obtain a pure culture of each single strain. Among them, the PYG culture medium was the same as that of "1.1.2 Isolation and Culture of Strains".
1.3.3菌种保藏1.3.3 Strain preservation
对获得的纯培养菌株进行培养，至浓度约为109cfu/mL，取400μL菌液添加40％甘油400μL，使其甘油浓度达到20％，然后进行-80℃超低温保藏。The obtained pure culture strain was cultured to a concentration of about 109 cfu/mL, 400 μL of the bacterial solution was added with 400 μL of 40% glycerol to make the glycerol concentration reach 20%, and then ultra-low temperature storage was performed at -80°C.
1.3.4 16S rDNA鉴定1.3.4 16S rDNA identification
将获得的分离菌株在液体PYG培养基中培养24h，取1mL菌液进行10000r/min离心5min，收集菌体，提取基因组DNA。以基因组DNA作为模板，使用16S rDNA通用引物进行PCR扩增，PCR扩增引物、体系和条件与“1.1.3菌株的16S rDNA鉴定”相同。The isolated strain was cultured in liquid PYG medium for 24 hours, 1 mL of bacterial solution was centrifuged at 10,000 r/min for 5 minutes, the bacteria were collected, and genomic DNA was extracted. The genomic DNA was used as a template and 16S rDNA universal primers were used for PCR amplification. The PCR amplification primers, system and conditions were the same as those in "1.1.3 Identification of 16S rDNA of strains".
将获得的16S rDNA扩增产物进行电泳检测、纯化、3730测序，获得菌株的16S rDNA序列，然后进行NCBI的数据库的比对。The obtained 16S rDNA amplification product was subjected to electrophoresis detection, purification, and 3730 sequencing to obtain the 16S rDNA sequence of the strain, which was then compared with the NCBI database.
测序结果显示，菌株TM12-14的16S rDNA长度为1400bp，序列如SEQ ID NO.5所示。NCBI blast比对结果显示，菌株TM12-14与Bifidobacterium pseudocatenulatum同源性最高，鉴定其为假小链状双歧杆菌Bifidobacterium pseudocatenulatum，命名为假小链状双歧杆菌TM12-14，并对其进行保藏。The sequencing results showed that the 16S rDNA of strain TM12-14 was 1400 bp in length, and the sequence was shown in SEQ ID NO. 5. The NCBI blast comparison results showed that strain TM12-14 had the highest homology with Bifidobacterium pseudocatenulatum, and was identified as Bifidobacterium pseudocatenulatum, named Bifidobacterium pseudocatenulatum TM12-14, and preserved.
2.小鼠模型2. Mouse Model
本例所选取的小鼠模型为：DSS(硫酸葡聚糖钠Dextran Sulfate，Na，分子量36000-50000)诱导的溃疡性肠炎小鼠模型。The mouse model selected in this example is: DSS (Dextran Sulfate, Na, molecular weight 36000-50000) induced ulcerative colitis mouse model.
具体的，采用购自湖北医学实验动物中心的小鼠品系为C57bl/6小鼠48只，所有小鼠8周龄，体重20g±2g，在SPF级别的鼠房环境中饲养。48只小鼠随机分为4组，每组12只进行后续试验。Specifically, 48 C57bl/6 mice were purchased from Hubei Medical Experimental Animal Center. All mice were 8 weeks old, weighed 20 g ± 2 g, and were raised in a SPF-level mouse house environment. The 48 mice were randomly divided into 4 groups, with 12 mice in each group for subsequent experiments.
DSS造模：续给小鼠饮用0.15％的DSS七天，即获得溃疡性肠炎小鼠模型。DSS modeling: The mice were fed 0.15% DSS for seven consecutive days to obtain an ulcerative colitis mouse model.
3.试验方法3. Test methods
48只小鼠随机分为4组，每组12只，4组分别为正常组(即control组)、模型组、益生菌组合物治疗组和VSL#3治疗组，各组的具体处理方式如下：48 mice were randomly divided into 4 groups, 12 mice in each group, the 4 groups were normal group (i.e. control group), model group, probiotic composition treatment group and VSL# 3 treatment group, the specific treatment methods of each group were as follows:
正常组：采用普通饲料进行饲喂，每只小鼠每天灌胃0.2mL的PBS缓冲液。Normal group: The mice were fed with ordinary feed, and each mouse was gavaged with 0.2 mL of PBS buffer every day.
模型组：采用相同的饲料饲喂，并进行DSS造模：将DSS添加到小鼠饮水中，DSS添加量为终浓度0.15％，饲喂七天，每只小鼠每天灌胃0.2mL的PBS缓冲液。Model group: The mice were fed with the same feed and DSS modeling was performed: DSS was added to the drinking water of the mice with a final concentration of 0.15%. The mice were fed for seven days and each mouse was gavaged with 0.2 mL of PBS buffer per day.
益生菌组合物治疗组：采用相同的饲料饲喂，在进行DSS造模前3天，每天给每只小鼠灌胃0.2mL的益生菌组合物菌液，然后再进行DSS造模：将DSS添加到小鼠饮水中，DSS添加量为终浓度0.15％，饲喂七天，每只小鼠每天灌胃0.2mL的益生菌组合物菌液。Probiotic composition treatment group: The same feed was used for feeding. Three days before DSS modeling, each mouse was gavaged with 0.2 mL of the probiotic composition solution every day. Then DSS modeling was performed: DSS was added to the drinking water of the mice. The final concentration of DSS addition was 0.15%. The mice were fed for seven days, and each mouse was gavaged with 0.2 mL of the probiotic composition solution every day.
VSL#3治疗组：采用相同的饲料饲喂，在进行DSS造模前3天，每天给每只小鼠灌胃0.2mL的VSL#3菌液，然后再进行DSS造模：将DSS添加到小鼠饮水中，DSS添加量为终浓度0.15％，饲喂七天，每只小鼠每天灌胃0.2mL的VSL#3菌液。VSL# 3 treatment group: The mice were fed with the same feed. Three days before DSS modeling, each mouse was gavaged with 0.2 mL of VSL# 3 bacterial solution every day. Then DSS modeling was performed: DSS was added to the drinking water of the mice. The final concentration of DSS was 0.15%. The mice were fed for seven days. Each mouse was gavaged with 0.2 mL of VSL# 3 bacterial solution every day.
其中，益生菌组合物菌液采用如下方法配制：The probiotic composition bacterial liquid is prepared by the following method:
将加氏乳杆菌TF08-1、嗜酸乳杆菌AM13-1和假小链状双歧杆菌TM12-14分别培养24h，离心收集菌体，用PBS进行悬浮，调整菌浓度至109cfu/mL，三株菌按照1∶1∶1进行混合，制备成益生菌组合物菌液。Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1 and Bifidobacterium pseudocatenulatus TM12-14 were cultured for 24 hours respectively, the cells were collected by centrifugation, suspended with PBS, the bacterial concentration was adjusted to 109 cfu/mL, and the three strains were mixed at a ratio of 1:1:1 to prepare a probiotic composition bacterial liquid.
VSL#3菌液采用如下方法配制：VSL# 3 bacterial solution was prepared as follows:
VSL#3购自美国Alfasigma公司，是一种包含干酪乳杆菌、植物乳杆菌、嗜酸乳杆菌、德式乳杆菌保加利亚亚种、嗜热链球菌、长双歧杆菌、短双歧杆菌、婴儿双岐杆菌等8种有益菌的复合益生菌剂；同样，采用PBS对VSL#3进行悬浮，调节浓度至109cfu/mL，即获得VSL#3菌液。VSL# 3 was purchased from Alfasigma, USA. It is a composite probiotic containing 8 kinds of beneficial bacteria, including Lactobacillus casei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium infantis. Similarly, VSL# 3 was suspended in PBS and the concentration was adjusted to 109 cfu/mL to obtain VSL# 3 bacterial solution.
DSS造模后每天记录小鼠体重、饮食和饮水情况，同时观察小鼠的粪便性状及粪便隐血情况，分别在第1天、第3天、第5天和第7天计算小鼠的疾病活动指数(缩写DAI)，DAI评分标准详见表1。实验结束后处死小鼠，所有小鼠取血、脱颈、取结肠、拍照、称重、量取结肠长度。结肠组织保存于-80℃冰箱和多聚甲醛中。其中，正常组的记录时间与DSS造模相同。After DSS modeling, the weight, diet and drinking water of mice were recorded every day, and the fecal characteristics and fecal occult blood of mice were observed. The disease activity index (abbreviated as DAI) of mice was calculated on the 1st day, 3rd day, 5th day and 7th day. The DAI scoring standard is shown in Table 1. After the experiment, the mice were killed, and blood was collected from all mice, the neck was dislocated, the colon was removed, photographed, weighed, and the colon length was measured. The colon tissue was stored in a -80℃ refrigerator and paraformaldehyde. Among them, the recording time of the normal group was the same as that of the DSS modeling.
表1 DAI指数评分表Table 1 DAI Index Score Table
表1中的“大便性状”，“正常”大便是指成形大便，“松散”大便是指不粘附于肛门的糊状、半成型大便，“稀便”是指可粘附于肛门的稀样水便。“大便隐血/弱眼血便”中，“正常”是指小鼠便血为阴性；“肉眼血便”是指肉眼可以直接观察到红色或褐色便血；“隐血阳性”是指不明显的肉眼血便，使用四甲基联苯胺进行检测为便血阳性。DAI指数等于体重、“大便性状”以及“大便隐血/弱眼血便”三个积分之和。In the "stool characteristics" in Table 1, "normal" stool refers to formed stool, "loose" stool refers to pasty, semi-formed stool that does not adhere to the anus, and "loose stool" refers to watery stool that can adhere to the anus. In "Occult blood in stool/weak blood in stool", "normal" means that the mouse stool is negative for blood; "blood in stool with naked eyes" means that red or brown blood in stool can be directly observed with the naked eye; "Occult blood positive" refers to blood in stool that is not obvious with naked eyes, and is positive for blood in stool when tested with tetramethylbenzidine. The DAI index is equal to the sum of the three scores of body weight, "stool characteristics" and "Occult blood in stool/weak blood in stool".
二、结果及分析2. Results and Analysis
1.体重变化1. Weight changes
统计小鼠分别在第1天、第3天、第5天和第7天的体重，各组小鼠的平均体重如表2和图1所示。The body weights of the mice were counted on the 1st day, the 3rd day, the 5th day and the 7th day, and the average body weights of the mice in each group are shown in Table 2 and Figure 1.
表2 各自小鼠平均体重Table 2 Average body weight of each mouse
表2中，“Control”是指正常组，“益生菌”即益生菌组合物治疗组，“VSL#3”即VSL#3治疗组。“*”是指模型组相对于正常组小鼠体重的差异显著水平P＜0.05，“**”是指模型组相对于正常组小鼠体重的差异显著水平P＜0.01，“▲”是指“益生菌”和“VSL#3”两组小鼠相对于模型组的差异显著水平P＜0.05。In Table 2, "Control" refers to the normal group, "Probiotics" refers to the probiotic composition treatment group, and "VSL# 3" refers to the VSL# 3 treatment group. "*" refers to the significant difference in weight between the model group and the normal group, P < 0.05, "**" refers to the significant difference in weight between the model group and the normal group, P < 0.01, and "▲" refers to the significant difference between the "Probiotics" and "VSL# 3" groups of mice and the model group, P < 0.05.
表2和图1的结果显示，正常组小鼠的体重呈缓慢升高的趋势，DSS诱导的小鼠，即模型组、“益生菌”组和“VSL#3”组，三组的体重均持续下降，模型组与对照组相比，在第3天体重下降开始显著(即*P＜0.05)，第7天，二者之间的差异显著程度更加明显(即**P＜0.01)。而益生菌组合物和VSL#3的干预可以减缓UC小鼠体重的下降，在第7天，“益生菌”和“VSL#3”这两组小鼠的体重下降的控制相对于模型组比较显著(即▲P＜0.05)。说明益生菌组合物和VSL#3可以控制UC引起的体重下降情况。并且，在第7天益生菌组合物组小鼠(即“益生菌”)的体重略高于VSL#3，说明益生菌组合物在控制UC小鼠体重降低的效果略优于VSL#3。The results in Table 2 and Figure 1 show that the weight of mice in the normal group showed a trend of slow increase, and the weight of mice induced by DSS, namely the model group, the "probiotics" group and the "VSL# 3" group, continued to decrease. Compared with the control group, the weight loss of the model group began to be significant on the 3rd day (i.e. *P < 0.05), and on the 7th day, the difference between the two was more obvious (i.e. **P < 0.01). The intervention of the probiotic composition and VSL# 3 can slow down the weight loss of UC mice. On the 7th day, the weight loss of the "probiotics" and "VSL# 3" groups of mice was significantly controlled compared with the model group (i.e. ▲P < 0.05). It shows that the probiotic composition and VSL# 3 can control the weight loss caused by UC. Moreover, on the 7th day, the weight of the mice in the probiotic composition group (i.e. "probiotics") was slightly higher than that of VSL# 3, indicating that the effect of the probiotic composition in controlling the weight loss of UC mice is slightly better than that of VSL# 3.
2.DAI的变化2. Changes in DAI
DSS诱导的溃疡性肠炎的小鼠由于体重下降、大便性状和便血情况的变化引起DAI指数的变化，小鼠DAI指数在第1天、第3天、第5天和第7天的统计值如表3和图2。表3中，各组小鼠的DAI取各组小鼠的平均值。The DAI index of mice with DSS-induced ulcerative enteritis changed due to weight loss, changes in stool characteristics and blood in stool. The statistical values of the DAI index of mice on the 1st, 3rd, 5th and 7th days are shown in Table 3 and Figure 2. In Table 3, the DAI of each group of mice was the average value of each group of mice.
表3 小鼠DAI值Table 3 DAI values of mice
表3中，“Control”是指正常组，“益生菌”即益生菌组合物治疗组，“VSL#3”即VSL#3治疗组。“*”是指模型组相对于正常组小鼠DAI指数的差异显著水平P＜0.05，“**”是指模型组相对于正常组小鼠DAI指数的差异显著水平P＜0.01，“▲”是指“益生菌”和“VSL#3”两组小鼠相对于模型组的差异显著水平P＜0.05。In Table 3, "Control" refers to the normal group, "Probiotics" refers to the probiotic composition treatment group, and "VSL# 3" refers to the VSL# 3 treatment group. "*" refers to the significant difference in the DAI index of the model group relative to the normal group of mice at the level of P < 0.05, "**" refers to the significant difference in the DAI index of the model group relative to the normal group of mice at the level of P < 0.01, and "▲" refers to the significant difference in the DAI index of the "Probiotics" and "VSL# 3" groups of mice relative to the model group at the level of P < 0.05.
表3和图2数据显示，正常组小鼠的DAI基本持平，而随着DSS诱导，模型组、益生菌组和VSL#3组小鼠的DAI逐渐升高，与对照组相比，第3天模型组小鼠DAI变得开始显著(即*P＜0.05)，第7天模型组小鼠DAI达到最高水平(即相对于control组**P＜0.01)。益生菌的干预可以控制DAI的升高，益生菌组小鼠在第5天和第7天的DAI值相对于模型组得到了显著的控制(即▲P＜0.05)，并且，在第7天益生菌干预组小鼠的DAI略低于VSL#3，说明本例的益生菌组合物在控制UC小鼠DAI升高的效果优于VSL#3。The data in Table 3 and Figure 2 show that the DAI of mice in the normal group remained basically the same, while with the induction of DSS, the DAI of mice in the model group, probiotic group and VSL# 3 group gradually increased. Compared with the control group, the DAI of mice in the model group began to become significant on the 3rd day (i.e. *P < 0.05), and the DAI of mice in the model group reached the highest level on the 7th day (i.e. **P < 0.01 relative to the control group). The intervention of probiotics can control the increase of DAI. The DAI values of mice in the probiotic group on the 5th and 7th days were significantly controlled relative to the model group (i.e. ▲P < 0.05), and the DAI of mice in the probiotic intervention group on the 7th day was slightly lower than that of VSL # 3, indicating that the probiotic composition of this example is better than VSL # 3 in controlling the increase of DAI in UC mice.
3.结肠长度的变化3. Changes in colon length
UC模型小鼠的结肠组织会发生改变，主要是因为溃疡和炎症的发生导致结肠组织缩短，治疗结束后，通过解剖测量的小鼠结肠长度如表4所示。The colon tissue of UC model mice changes, mainly because of the occurrence of ulcers and inflammation, which leads to shortening of the colon tissue. After the treatment, the length of the mouse colon measured by dissection is shown in Table 4.
表4 小鼠结肠长度Table 4 Colon length of mice
表4中，“Control”是指正常组，“益生菌”即益生菌组合物治疗组，“VSL#3”即VSL#3治疗组。“**”是指模型组相对于正常组小鼠结肠长度差异显著水平P＜0.01，“▲”是指“益生菌”和“VSL#3”两组小鼠相对于模型组小鼠结肠长度差异显著水平P＜0.05。In Table 4, "Control" refers to the normal group, "Probiotics" refers to the probiotic composition treatment group, and "VSL# 3" refers to the VSL# 3 treatment group. "**" refers to the significant difference in colon length between the model group and the normal group, P < 0.01, and "▲" refers to the significant difference in colon length between the "Probiotics" and "VSL# 3" groups of mice and the model group, P < 0.05.
表4的结果显示，在进行DSS诱导7天后的小鼠，模型组的结肠组织缩短情况比较严重，与control组相比较非常显著(**P＜0.01)。而益生菌组合物和VSL#3的干预可以显著控制小鼠结肠的缩短，相对于模型组得到了显著的控制(*P＜0.05)。通过表中数据可以发现益生菌组合物组小鼠的结肠长度比VSL#3组小鼠结肠长度长，可以说明益生菌组合物在控制UC小鼠结肠缩短的能力强于VSL#3。The results in Table 4 show that the shortening of the colon tissue in the model group was more serious after 7 days of DSS induction, which was significantly different from that in the control group (**P<0.01). The intervention of the probiotic composition and VSL# 3 can significantly control the shortening of the colon in mice, which was significantly controlled compared with the model group (*P<0.05). The data in the table show that the colon length of the mice in the probiotic composition group was longer than that of the mice in the VSL# 3 group, which indicates that the probiotic composition is more capable than VSL# 3 in controlling the shortening of the colon in UC mice.
表2至表4以及图1和图2的结果显示，本例的益生菌组合物对溃疡性肠炎具有治疗和预防作用，并且其治疗效果略优于现有的VSL#3产品。The results in Tables 2 to 4 and Figures 1 and 2 show that the probiotic composition of this example has therapeutic and preventive effects on ulcerative colitis, and its therapeutic effect is slightly better than that of the existing VSL# 3 product.
实施例二Embodiment 2
本例将实施例一证实具有溃疡性肠炎治疗和预防效果的组合物制成常见的食品，具体如下：In this example, the composition confirmed to have the therapeutic and preventive effects on ulcerative enteritis in Example 1 is made into a common food, specifically as follows:
将牛奶、维生素C和白糖等辅料，与培养的加氏乳杆菌TF08-1、嗜酸乳杆菌AM13-1和假小链状双歧杆菌TM12-14按照表5的配方混合，制备具有溃疡性肠炎治疗和预防功能的食品。The auxiliary materials such as milk, vitamin C and white sugar are mixed with cultured Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1 and Bifidobacterium pseudocatenulatus TM12-14 according to the formula of Table 5 to prepare a food with the function of treating and preventing ulcerative enteritis.
表5 含益生菌组合物的食品配方Table 5 Food formula containing probiotic composition
按照表5的配方比例混合牛奶、白糖，搅拌至完全混合，预热，20Mpa压力均质，90℃左右杀菌5-10分钟，冷却至40-43℃，混入保护剂(即维生素C)，并接种1-100×106cfu/g的加氏乳杆菌Lactobacillus gasseri TF08-1、嗜酸乳杆菌Lactobacillus acidophilusAM13-1和假小链状双歧杆菌Bifidobacterium pseudocatenulatum TM12-14三种混合益生菌的食品组合物。Milk and sugar were mixed according to the formula ratio in Table 5, stirred until completely mixed, preheated, homogenized at 20 MPa pressure, sterilized at about 90° C. for 5-10 minutes, cooled to 40-43° C., mixed with a protective agent (i.e., vitamin C), and inoculated with 1-100×106 cfu/g of a food composition of three mixed probiotics, namely, Lactobacillus gasseri TF08-1, Lactobacillus acidophilusAM13-1, and Bifidobacterium pseudocatenulatum TM12-14.
将本例的牛乳产品加入DSS造模的小鼠饲料中，按照实施例一的益生菌组合物治疗组进行饲喂和检测，所不同的是，本例仅仅在饲料中添加了本例的牛乳产品，不额外灌胃益生菌组合物菌液。检测结果显示，本例的牛乳产品同样能够控制UC小鼠体重降低、降低小鼠疾病活动指数DAI、改善肠道病变，具有溃疡性肠炎治疗和预防效果。The cow's milk product of this example was added to the feed of mice modeled with DSS, and the feeding and testing were carried out according to the probiotic composition treatment group of Example 1, except that in this example, only the cow's milk product of this example was added to the feed, and no additional probiotic composition liquid was gavaged. The test results showed that the cow's milk product of this example could also control the weight loss of UC mice, reduce the disease activity index DAI of mice, and improve intestinal lesions, and had the effect of treating and preventing ulcerative enteritis.
实施例三Embodiment 3
本例将实施例一证实具有溃疡性肠炎治疗和预防效果的益生菌组合物制成用于治疗溃疡性肠炎的药物，配方如表6所示：In this example, the probiotic composition confirmed to have the therapeutic and preventive effects on ulcerative enteritis in Example 1 was prepared into a medicine for treating ulcerative enteritis, and the formula is shown in Table 6:
表6 含益生菌组合物的药品配方Table 6 Pharmaceutical formulations containing probiotic compositions
按照表6的比例将乳糖、酵母粉、蛋白胨以纯净水混合均匀，预热到60-65℃，20Mpa压力均质，90℃左右杀菌20-30分钟，冷却至36-38℃，混入保护剂(即维生素C)，分别接入1-50×106cfu/mL加氏乳杆菌Lactobacillus gasseri TF08-1、嗜酸乳杆菌Lactobacillusacidophilus AM13-1和假小链状双歧杆菌Bifidobacterium pseudocatenulatum TM12-14活菌，36-38℃发酵至pH值为6.0，离心，冷冻干燥至水份含量小于3％，即制备组合益生菌冷冻干燥物。称取0.5克冷冻干燥物与等量的麦芽糊精混合后装入胶囊中，即制成含加氏乳杆菌Lactobacillus gasseri TF08-1、嗜酸乳杆菌Lactobacillus acidophilus AM13-1和假小链状双歧杆菌Bifidobacterium pseudocatenulatum TM12-14三株组合益生菌的胶囊剂药物组合物。Lactose, yeast powder, peptone and purified water were mixed evenly according to the proportions in Table 6, preheated to 60-65°C, homogenized at 20 MPa pressure, sterilized at about 90°C for 20-30 minutes, cooled to 36-38°C, mixed with a protective agent (i.e., vitamin C), and inoculated with 1-50×106 cfu/mL of Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1 and Bifidobacterium pseudocatenulatum TM12-14 live bacteria, respectively, fermented at 36-38°C to a pH value of 6.0, centrifuged, and freeze-dried to a moisture content of less than 3%, thereby preparing a combined probiotic freeze-dried product. 0.5 g of the freeze-dried product was weighed and mixed with an equal amount of maltodextrin and then filled into a capsule to prepare a capsule pharmaceutical composition containing three strains of combined probiotics including Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1 and Bifidobacterium pseudocatenulatum TM12-14.
采用本例的胶囊剂药物组合物替换实施例一益生菌组合物治疗组中的益生菌组合物菌液，按照实施例一相同的方式，灌胃本例的胶囊剂药物组合物，每天灌胃一粒胶囊，按照实施例一相同的方法进行检测。结果显示，本例的胶囊剂药物组合物同样能够控制UC小鼠体重降低、降低小鼠疾病活动指数DAI、改善肠道病变，具有溃疡性肠炎治疗和预防效果。The capsule pharmaceutical composition of this example was used to replace the probiotic composition bacterial solution in the probiotic composition treatment group of Example 1, and the capsule pharmaceutical composition of this example was gavaged in the same manner as Example 1, with one capsule gavaged per day, and the test was performed in the same manner as Example 1. The results showed that the capsule pharmaceutical composition of this example could also control the weight loss of UC mice, reduce the disease activity index DAI of mice, and improve intestinal lesions, and had the therapeutic and preventive effects of ulcerative enteritis.
实施例四Embodiment 4
一种用于治疗溃疡性肠炎(UC)的药物的制备方法A method for preparing a drug for treating ulcerative colitis (UC)
1、菌液准备：将1×109cfu/mL加氏乳杆菌Lactobacillus gasseri TF08-1、嗜酸乳杆菌Lactobacillus acidophilus AM13-1和假小链状双歧杆菌Bifidobacteriumpseudocatenulatum TM12-14分别进行厌氧培养，厌氧培养基采用PYG培养基，经过37℃厌氧发酵2-3天，然后进行等比例混合，终浓度约1×109cfu/mL。1. Preparation of bacterial solution: 1×109 cfu/mL Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1 and Bifidobacteriumpseudocatenulatum TM12-14 were cultured anaerobically respectively. The anaerobic culture medium was PYG medium. After anaerobically fermentation at 37°C for 2-3 days, the mixture was mixed in equal proportions to a final concentration of about 1×109 cfu/mL.
2、生长因子制备：将脱脂牛奶、酪蛋白进行混合、离心、超滤获得牛奶生长因子粗提物，其中含有维生素类物质、嘌呤类物质、嘧啶类物质等成分。2. Preparation of growth factors: skim milk and casein are mixed, centrifuged and ultrafiltered to obtain a crude extract of milk growth factors, which contains vitamins, purines, pyrimidines and other ingredients.
3、药物剂型制作：将5体积生长因子和1体积的保护剂(即维生素C)加入到100体积的发酵的菌液混合物中，充分搅拌混匀，然后加入淀粉辅料，如麦芽糊精，制备药物剂型。3. Preparation of drug dosage form: Add 5 volumes of growth factor and 1 volume of protective agent (i.e., vitamin C) to 100 volumes of fermented bacterial liquid mixture, stir well to mix, and then add starch auxiliary materials, such as maltodextrin, to prepare drug dosage form.
该药物剂型可以进一步制成浆料、散剂或颗粒剂等药品。The drug dosage form can be further prepared into medicines such as slurry, powder or granule.
将本例的药物剂型，直接添加到DSS造模的小鼠饲料中，按照实施例一的益生菌组合物治疗组进行饲喂和检测，所不同的是，本例仅仅在饲料中添加了本例的药物剂型，不额外灌胃益生菌组合物菌液。检测结果显示，本例的药物产品也能够控制UC小鼠体重降低、降低小鼠疾病活动指数DAI、改善肠道病变，具有溃疡性肠炎治疗和预防效果。The drug formulation of this example was directly added to the feed of DSS-modeled mice, and the feeding and testing were performed according to the probiotic composition treatment group of Example 1, except that in this example, only the drug formulation of this example was added to the feed, and no additional probiotic composition liquid was gavaged. The test results showed that the drug product of this example can also control the weight loss of UC mice, reduce the disease activity index DAI of mice, and improve intestinal lesions, and has the effect of treating and preventing ulcerative enteritis.
以上各实施例证明加氏乳杆菌、嗜酸乳杆菌和假小链状双歧杆菌三者联合使用能够治疗和预防溃疡性肠炎；并且，三种菌的组合物可以制成各种食品或药品使用；当然，可以理解，三种菌的组合物既然可以制成各种食品和药品，同样也可以制成各种保健品或食品添加剂。The above examples demonstrate that the combination of Lactobacillus gasseri, Lactobacillus acidophilus and Bifidobacterium pseudocatenulatus can treat and prevent ulcerative enteritis; and the combination of the three bacteria can be made into various foods or medicines for use; of course, it can be understood that since the combination of the three bacteria can be made into various foods and medicines, it can also be made into various health products or food additives.
另外，研究显示，三种菌的治疗效果很大程度上是基于微生态的改善，而这种微生态的改善不仅对溃疡性肠炎有治疗和预防效果，对其它与微生态相关疾病，例如普通肠炎、胃炎等，同样具有效果；因此，本申请的组合物可以用于预防或治疗炎症或炎症相关疾病，特别是各种肠炎、胃炎。In addition, studies have shown that the therapeutic effect of the three bacteria is largely based on the improvement of the microecology, and this improvement in the microecology not only has a therapeutic and preventive effect on ulcerative enteritis, but also has an effect on other microecological-related diseases, such as common enteritis, gastritis, etc.; therefore, the composition of the present application can be used to prevent or treat inflammation or inflammation-related diseases, especially various enteritis and gastritis.
以上内容是结合具体的实施方式对本申请所作的进一步详细说明，不能认定本申请的具体实施只局限于这些说明。对于本申请所属技术领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本申请的保护范围。The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application, which should be deemed to fall within the scope of protection of the present application.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2018/089314WO2019227414A1 (en) | 2018-05-31 | 2018-05-31 | Composition and uses thereof |
| Publication Number | Publication Date |
|---|---|
| CN112236154A CN112236154A (en) | 2021-01-15 |
| CN112236154Btrue CN112236154B (en) | 2024-10-18 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201880093354.1AActiveCN112236154B (en) | 2018-05-31 | 2018-05-31 | A composition and its application |
| Country | Link |
|---|---|
| CN (1) | CN112236154B (en) |
| WO (1) | WO2019227414A1 (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107927789A (en)* | 2017-12-01 | 2018-04-20 | 刘峰 | A kind of prebiotics food and preparation method thereof |
| GB202015687D0 (en)* | 2020-10-02 | 2020-11-18 | Microbiotica Ltd | Therapeutic composition |
| CN120624307A (en)* | 2025-08-13 | 2025-09-12 | 浙江大学 | A strain for preventing and/or treating ulcerative colitis, mixed probiotics and use thereof |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101119636A (en)* | 2005-03-16 | 2008-02-06 | 艾蒂尔药物有限公司 | Mixture of at least 6 species of lactic acid bacteria and/or bifidobacteria in the manufacture of sourdough |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ID29150A (en)* | 1999-01-15 | 2001-08-02 | Entpr Ireland Cs | USE OF LACTOBACILLUS SALIVARIUS |
| AU2002329043A1 (en)* | 2001-07-30 | 2003-02-24 | Claudio De Simone | Treatment of radiation-induced diarrhea with probiotics |
| AU2011202559B2 (en)* | 2003-12-17 | 2012-06-21 | N.V. Nutricia | Lactic Acid Producing Bacteria and Lung Function |
| ITMI20042189A1 (en)* | 2004-11-16 | 2005-02-16 | Anidral Srl | COMPOSITION BASED ON PROBIOTIC BACTERIA AND ITS USE IN THE PREVENTION OF E-OR IN THE TREATMENT OF PATHOLOGIES AND-OR RESPIRATORY INFECTIONS AND IN THE IMPROVEMENT OF INTESTINAL FUNCTIONALITY |
| CN100506973C (en)* | 2006-11-29 | 2009-07-01 | 康哲医药研究(深圳)有限公司 | Lactobacillus fermentum CMS-H002 and its application |
| CN101485687B (en)* | 2008-01-17 | 2012-06-27 | 杜灵广 | Medicament for treating gastrointestinal tract disease using biological viable bacteria |
| ES2389547B1 (en)* | 2010-12-07 | 2013-08-08 | Consejo Superior De Investigaciones Científicas (Csic) | BIFIDOBACTERIUM CECT 7765 AND ITS USE IN THE PREVENTION AND / OR TREATMENT OF OVERWEIGHT, OBESITY AND ASSOCIATED PATHOLOGIES. |
| ITRM20110477A1 (en)* | 2011-09-09 | 2013-03-10 | Giovanni Mogna | COMPOSITION INCLUDING N-ACETYLCISTEIN AND / OR LYSOZYME MICROINCAPSULATE GASTROPROTECT IN ASSOCIATION WITH PROBIOTIC BACTERES ABLE TO RESTORE THE BARRIER EFFECT OF THE STOMACH THAT IS LOST DURING THE PHARMACOLOGICAL TREATMENT OF |
| ES2526986B1 (en)* | 2013-07-18 | 2015-11-02 | Biopolis, S.L. | New strain of Bifidobacterium animalis subsp. lactis CECT 8145 and its use for the treatment and / or prevention of overweight and obesity and associated diseases |
| WO2017000249A1 (en)* | 2015-06-30 | 2017-01-05 | Perfect (China) Co., Ltd. | Bifidobacteria as probiotic foundation species of gut microbiota |
| KR20170032815A (en)* | 2015-09-15 | 2017-03-23 | 경희대학교 산학협력단 | Novel lactic acid bacteria and composition for preventing, improving or treating neurodegenerative diseases or cognitive dysfunction |
| MA45288A (en)* | 2016-06-08 | 2019-04-17 | Sofar Spa | New medical use of probiotics |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101119636A (en)* | 2005-03-16 | 2008-02-06 | 艾蒂尔药物有限公司 | Mixture of at least 6 species of lactic acid bacteria and/or bifidobacteria in the manufacture of sourdough |
| Publication number | Publication date |
|---|---|
| WO2019227414A1 (en) | 2019-12-05 |
| CN112236154A (en) | 2021-01-15 |
| Publication | Publication Date | Title |
|---|---|---|
| CN112218646B (en) | A composition and its application | |
| US11116806B2 (en) | Composite probiotic lactic acid bacteria powder and preparation method and use thereof | |
| CN108721337B (en) | A microbial agent for preventing intestinal toxicity of tumor chemotherapy | |
| PL188804B1 (en) | Enteral dietetic compositions and their application | |
| CN108076643A (en) | Lactobacillus rhamnosus bacteria for the treatment of e.g. bacterial vaginosis | |
| WO2022206300A1 (en) | Composition capable of facilitating defecation and use thereof | |
| CN116286551B (en) | Application of Bifidobacterium longum subsp. infantis in regulating fat metabolism in the body, shaping fat, reducing fat and improving obesity | |
| CN114933994B (en) | Composition with ulcerative colitis relieving function and application thereof | |
| CN116064313B (en) | Application of lactobacillus plantarum CCFM1281 in relieving exercise fatigue | |
| CN112236154B (en) | A composition and its application | |
| CN117917475B (en) | Lactobacillus plantarum P16 for regulating intestinal flora and its application, product and method | |
| CN116617273A (en) | Use of lactobacillus paracasei LC86 for the preparation of a product for the prevention, alleviation or treatment of muscle atrophy associated with aging | |
| CN117305171A (en) | A strain of Bifidobacterium breve and its application | |
| CN117866831A (en) | Lactobacillus rhamnosus and application thereof | |
| CN114657084A (en) | A kind of Bifidobacterium longum for relieving ulcerative colitis and its application | |
| CN116121154B (en) | Leuconostoc lactis and application thereof | |
| WO2005030230A1 (en) | Compositions and methods for treatment or prevention of psoriasis and related disorders | |
| WO2018112739A1 (en) | Bifidobacterium pseudocatenulatum, culture method therefor and application thereof | |
| CN114933992B (en) | Application of bifidobacterium longum and composite preparation thereof in relieving ulcerative colitis | |
| CN112236155B (en) | A composition and its application | |
| CN113041266B (en) | A strain of Lactobacillus casei that improves the pathological characteristics of psoriasis-like mice and its application | |
| CN112546074B (en) | A strain of Bifidobacterium breve capable of inhibiting the release of IL-23 and Th17 axis-related inflammatory factors and its application | |
| CN117264826A (en) | Bifidobacterium longum capable of regulating abundance of intestinal flora and intestinal health and application thereof | |
| RU2735717C1 (en) | Bifidobacterium bifidum strain used as a probiotic | |
| CN119410559B (en) | Bifidobacterium breve BBr37 with blood sugar reducing capability, and application, product and method thereof |
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| EE01 | Entry into force of recordation of patent licensing contract | Application publication date:20210115 Assignee:BGI PRECISION NUTRITION (SHENZHEN) TECHNOLOGY CO.,LTD. Assignor:BGI SHENZHEN Contract record no.:X2022440020021 Denomination of invention:A composition and its application License type:Common License Record date:20221107 | |
| EE01 | Entry into force of recordation of patent licensing contract | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |