WO2022100125A1 - Novel application of lactobacillus paracasei et-22 in anti-aging and improving innate immunity - Google Patents
Novel application of lactobacillus paracasei et-22 in anti-aging and improving innate immunity Download PDFInfo
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Definitions
- the present invention relates to the technical field of microorganisms, in particular to a kind of Lactobacillus paracasei ET-22 (deposit number CGMCC No. 15077) which has anti-aging properties, can improve the innate immunity of organisms, and/or enhance the Novel use in compositions for efficacy against Staphylococcus aureus infection resistance.
- the immune system is the most vulnerable.
- the immune system is in constant contact with most organs and cells in the body, and its changes are bound to affect the tissue cells of these organs.
- the function of the immune system gradually declines, and both the innate immune response and the acquired immune response decline, which induces some diseases that seriously affect the tissues and organs, and exacerbates the aging of the body's various systems.
- Immunosenescence refers to the phenomenon of susceptibility to chronic and acute diseases due to the abnormal regulation of immune function.
- the decline of immune function is one of the important factors causing the aging of the body, so the strengthening of the immune function of the body can reduce the occurrence of pathological aging.
- Probiotics are a class of single or well-defined mixtures of microorganisms that produce beneficial effects on the host by altering the composition of the microbiota in a certain part of the host.
- Prebiotic effect refers to the beneficial physiological effect of a microbial preparation or fermented product on the host by regulating the immune function of the host mucosa and system and improving the balance of intestinal nutrition and flora.
- functional foods that can provide prebiotic effects, control aging and prolong life are more and more popular.
- Caenorhabditis elegans (C. elegans) is a common, free-living, small soil nematode belonging to the subclass Rhabditia, the order Rhabditidia, and the family Rhabditoidea. Feeding on bacteria, its life cycle is about 3d, with an average lifespan of 3 weeks. It is worm-like, with an adult length of about 1.0 to 1.5 mm and a body diameter of about 70.0 ⁇ m.
- Caenorhabditis elegans is bilaterally symmetrical, with a stratum corneum covering on the body surface, without segments, with 4 main epidermal cords and a fluid-filled prosthetic cavity. The biological characteristics of C.
- elegans such as simple structure, transparent body, easy observation, and short reproductive cycle, endow it with the premise of becoming a model organism.
- This experiment uses the model animal Caenorhabditis elegans to observe its lifespan to explore the anti-aging effect of probiotics in its body, and to provide a theoretical reference for the development and utilization of probiotic products for anti-aging and improving innate immunity.
- An object of the present invention is to provide a new use of Lactobacillus paracasei ET-22.
- the present invention provides a strain of Lactobacillus paracasei ET-22 with a deposit number of CGMCC No. 15077, which is preserved in the China Type Culture Collection and the General Microorganism Center of the China Committee for the Preservation of Microorganisms .
- Lactobacillus paracasei ET-22 strain is a biological material that has been published in CN110964653A and is publicly available.
- the present invention finds that the Lactobacillus paracasei ET-22 strain has the effects of anti-aging, improving the innate immunity of organisms, and/or improving the resistance of organisms to Staphylococcus aureus infection.
- the present invention provides Lactobacillus paracasei (Lactobacillus paracasei) in the preparation with anti-aging, can improve the innate immunity of organism and/or improve the composition of organism to Staphylococcus aureus infection resistance effect.
- the Lactobacillus paracasei is the Lactobacillus paracasei of the deposit number CGMCC No.15077.
- improving the resistance of an organism to Staphylococcus aureus infection described in the present invention includes: improving the ability of an individual to prevent Staphylococcus aureus infection, reducing the ability of an individual to infect an individual with Staphylococcus aureus, and/or Relief of symptoms of food poisoning, enteritis, pneumonia, skin infections, wound ulcers and/or meningitis in individuals caused by Staphylococcus aureus infection.
- the anti-aging includes delaying the phenomenon of aging and even death caused by Staphylococcus aureus infection.
- the Lactobacillus paracasei described in the present invention is used to prepare the composition in the form of a solid or liquid bacterial preparation.
- the composition of the present invention may comprise a food composition, a feed composition, a cosmetic composition or a pharmaceutical composition.
- the organism in the application of the composition of the present invention, is an animal or a human.
- the compositions of the present invention can be administered to animals or humans.
- the composition may also include ingredients conventional in the art.
- an appropriate amount of adjuvants may be included, and the adjuvants may be excipients, diluents, fillers, and/or absorption enhancers, and the like.
- the Lactobacillus paracasei of the present invention can be produced according to the food containing Lactobacillus paracasei in the prior art, and the composition can adopt different forms according to the needs of the recipient. For example, powders, lozenges, granules, microcapsules and/or liquid preparations and the like.
- the composition of the present invention is used to relieve symptoms such as food poisoning, enteritis, pneumonia, skin infection, wound ulceration and/or meningitis caused by Staphylococcus aureus infection;
- the application amount of the Lactobacillus paracasei is 1.0 ⁇ 10 3 CFU to 1.0 ⁇ 10 12 CFU/day, preferably 1.0 ⁇ 10 7 CFU to 1.0 ⁇ 10 11 CFU/day.
- the composition of the present invention is used for anti-aging (including prolonging lifespan); in specific application, the application amount of the Lactobacillus paracasei is 1.0 ⁇ 10 3 CFU ⁇ 1.0 ⁇ 10 12 CFU/day, preferably 1.0 ⁇ 10 7 CFU to 1.0 ⁇ 10 11 CFU/day.
- the composition of the present invention is used to improve innate immunity; in specific application, the application amount of the Lactobacillus paracasei is 1.0 ⁇ 10 3 CFU to 1.0 ⁇ 10 12 CFU/day, It is preferably 1.0 ⁇ 10 7 CFU to 1.0 ⁇ 10 11 CFU/day.
- the composition of the present invention is a food composition
- the food is a fermented milk product (such as fermented milk, flavored fermented milk, fermented milk beverage, etc.), cheese, milk-containing beverage, Solid drinks or milk powder.
- the composition of the present invention is a pharmaceutical composition
- the medicine may be an oral preparation or an external preparation, such as a smeared ointment and the like.
- the composition of the present invention is a cosmetic with anti-aging effects, can improve the innate immunity of an organism, and/or improve the resistance of an organism to Staphylococcus aureus infection.
- the present invention also provides a method for anti-aging, improving the innate immunity of an organism, and/or improving the resistance of an organism to Staphylococcus aureus infection, the method comprising administering an effective amount of paracheese milk to the organism Bacillus; wherein, the Lactobacillus paracasei is the Lactobacillus paracasei with the deposit number CGMCC No.15077.
- the improvement of the organism's resistance to Staphylococcus aureus infection includes: improving the individual's ability to prevent Staphylococcus aureus infection, reducing the ability of Staphylococcus aureus to infect an individual, and/ Or relieve food poisoning, enteritis, pneumonia, skin infection, wound ulceration and/or meningitis in individuals caused by Staphylococcus aureus infection.
- the application amount of the Lactobacillus paracasei is 1.0 ⁇ 10 3 CFU to 1.0 ⁇ 10 12 CFU/day.
- the application amount of the Lactobacillus paracasei is preferably 1.0 ⁇ 10 7 CFU to 1.0 ⁇ 10 11 CFU/day.
- the present invention provides a new use of Lactobacillus paracasei ET-22, which has the effects of anti-aging, improving the innate immunity of organisms, and/or improving the resistance of organisms to Staphylococcus aureus infection, and the like, It can be used to prepare foods, medicines, cosmetics and feeds with anti-aging and innate immune functions, and has wide application prospects.
- Figure 1 shows the pathogenicity of S. aureus on Caenorhabditis elegans and the effect of adding probiotic strains; the intervening substances (probiotics) added in each group in the figure are the same as those in the culture stage.
- Figure 2 shows the survival rate of C. elegans after S. aureus and probiotics intervened for 3 days; the intervening substances (probiotics) added in each group in the figure were the same as those in the culture stage.
- Figure 3 shows the bacteriostatic effect of probiotics on S. aureus.
- Example 1 Test of anti-aging effect of probiotics on Caenorhabditis elegans
- Lactobacillus paracasei ET-22 was purchased from Inner Mongolia Yili Industrial Group Co., Ltd.; Lactobacillus rhamnosus HN001 was purchased from Danisco; Bifidobacterium animalis subsp.lactis BB-12 was purchased from ChrHansen; Bifidobacterium lactis HN019 strain was purchased from DuPont Danisco; Lactobacillus fermentum LC40 was purchased from Biosearch life; wild-type Caenorhabditis elegans (C.elegans) was purchased from Biopolis SL Bio, Spain technology company; Staphylococcus aureus ATCC25923 was purchased from Biopolis SL biotechnology company in Spain.
- Lactobacillus in MRS medium Bifidobacterium lactis in MRS+Cys medium, cultured and activated at 37°C, cells were collected and eluted with normal saline, adjusted to 1 ⁇ 10 8 CFU in NGM medium for use.
- nematodes Age-matched nematodes were obtained and cultured in petri dishes containing nematode agar medium (nematode medium containing Escherichia coli OP50 as food), and different probiotics (1 ⁇ 10 8 CFU) were added for co-cultivation. After the nematodes became adults, they were transferred to petri dishes inoculated with Staphylococcus aureus ATCC25923 at 10 8 -10 9 CFU/mL to simulate infection with Staphylococcus aureus. The intervening substances (probiotics) added in the infection stage of each group were the same as those in the culture stage.
- Two controls were used, one without pathogenic bacteria (nematode petri dishes containing Escherichia coli OP50, ie OP50 group), and those with pathogenic bacteria Staphylococcus aureus infection without any intervention (containing only Staphylococcus aureus). the petri dish, that is, the S. aureus group).
- a group in which an intervening substance was added in the culture phase and no intervening substance in the infection phase, and a group in which the intervening substance was not added in the cultivation phase but added in the infection phase were set.
- Lifespan is the most direct reflection of changes in C. elegans before and after exposure.
- Lactobacillus paracasei group ET-22 could significantly prolong the lifespan of nematodes (p ⁇ 0.001).
- Lactobacillus paracasei ET-22 can significantly prolong the lifespan of C. elegans (p ⁇ 0.05).
- the experiments of the present invention show that the survival rate of C. elegans in the group in which the probiotic ET-22 intervention is added in the culture stage and no intervention is added in the infection stage is significantly higher than that in the culture stage and the infection stage without any intervention. group of things.
- Example 2 Analysis of the antibacterial properties of probiotics against Staphylococcus aureus
- the antibacterial activity of probiotics against S. aureus was analyzed by disc diffusion method. Pipette 1 mL of bacterial suspension with a concentration of 1 ⁇ 10 8 CFU/mL into 15 mL of RCA medium (50°C), mix it evenly, and pour it into a plate. Each petri dish was affixed with 3 paper sheets of the same drug at even and moderate intervals. After standing for 5 min, the plate was turned over and incubated at a constant temperature of 37 °C under microaerophilic conditions. After 48 h, the diameter of the inhibition zone was measured and recorded.
- Lactobacillus paracasei ET-22 (Deposit No. CGMCC No. 15077) has good potential in anti-aging and improving innate immunity, and can be used in food, such as fermented milk, cheese, milk-containing beverages, milk Powder or any other food containing the strain and its derivatives.
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Abstract
An application of Lactobacillus paracasei ET-22 in the preparation of a composition resisting aging, improving innate immunity of organisms, and/or enhancing the effective resistance of organisms to Staphylococcus aureus infections. Lactobacillus paracasei is a Lactobacillus paracasei with the preservation number CGMCC No. 15077.
Description
本发明涉及微生物技术领域，尤其是涉及一种副干酪乳杆菌(Lactobacillus paracasei)ET-22(保藏编号CGMCC No.15077)在制备具有抗衰老、可提高生物体先天免疫、和/或提升生物体对金黄色葡萄球菌感染抵御能力功效的组合物中的新用途。The present invention relates to the technical field of microorganisms, in particular to a kind of Lactobacillus paracasei ET-22 (deposit number CGMCC No. 15077) which has anti-aging properties, can improve the innate immunity of organisms, and/or enhance the Novel use in compositions for efficacy against Staphylococcus aureus infection resistance.
在体内各种各样的生理过程中，免疫系统是最容易受到攻击的。免疫系统与体内大多数器官和细胞保持持续接触，它的改变势必影响这些器官的组织细胞。随着年龄的增长，免疫系统的功能逐步减退，先天性免疫应答和获得性免疫应答都会随之下降，由此诱发出一些严重影响组织器官的疾病，加剧了机体各系统的衰老。免疫衰老(immunosenescence)就是指由于免疫功能调节失常而导致的易患慢性和急性疾病的现象。免疫功能的衰退是造成机体衰老的重要因素之一，因此机体免疫功能的加强能减少病理性衰老的发生。Of the various physiological processes in the body, the immune system is the most vulnerable. The immune system is in constant contact with most organs and cells in the body, and its changes are bound to affect the tissue cells of these organs. With the increase of age, the function of the immune system gradually declines, and both the innate immune response and the acquired immune response decline, which induces some diseases that seriously affect the tissues and organs, and exacerbates the aging of the body's various systems. Immunosenescence refers to the phenomenon of susceptibility to chronic and acute diseases due to the abnormal regulation of immune function. The decline of immune function is one of the important factors causing the aging of the body, so the strengthening of the immune function of the body can reduce the occurrence of pathological aging.
益生菌是一类通过改变宿主某一部位菌群的组成，从而产生有利于宿主健康作用的单一或组成明确的混合微生物。益生作用则是指某种微生物制剂或发酵制品通过调节宿主黏膜与系统免疫功能以及改善肠道营养与菌群平衡对宿主所产生的有益的生理作用。随着衰老人群的增加，能提供益生作用，控制老化和延长寿命的功能性食品越来越受到青睐。HIROMI KIMOTO-NIRA等采用快速老化小鼠模型(SAM)中的SAMP6，实验发现SAMP6小鼠服用热灭活的Lactococcus lactis subsp.cremoris H61后小鼠脾细胞产生更多的IL-12和IFN–γ，该研究认为Lactococcus lactis subsp.cremoris H61之所以能够抑制某些老化现象可能与其免疫调节作用相关。Probiotics are a class of single or well-defined mixtures of microorganisms that produce beneficial effects on the host by altering the composition of the microbiota in a certain part of the host. Prebiotic effect refers to the beneficial physiological effect of a microbial preparation or fermented product on the host by regulating the immune function of the host mucosa and system and improving the balance of intestinal nutrition and flora. With the increase of the aging population, functional foods that can provide prebiotic effects, control aging and prolong life are more and more popular. HIROMI KIMOTO-NIRA et al. used SAMP6 in a rapidly aging mouse model (SAM), and found that the spleen cells of SAMP6 mice produced more IL-12 and IFN-γ after taking heat-killed Lactococcus lactis subsp. cremoris H61 , the study believes that the reason why Lactococcus lactis subsp. cremoris H61 can inhibit some aging phenomena may be related to its immunomodulatory effect.
秀丽隐杆线虫(C.elegans)是一种常见的、自由生活的小型土壤线虫，属于小杆亚纲(Rhabditia)、小杆目(Rhabditidia)、小杆总科(Rhabditoidea)。以细菌为食，其生命周期大约为3d，平均寿命为3周。其呈蠕虫状、成体长约1.0～1.5mm，身体直径约70.0μm。秀丽隐杆线虫为两侧对称，体表有一层角质层覆盖物，无分节，有4条主要的表皮索状组织及1个充满体液的假体腔。秀丽隐杆线虫的结构简单、身体透明、易于观 察、繁殖周期短等生物学特征赋予了其成为模式生物的前提。通过研究线虫的衰老和寿命控制机制，并将其成果应用于其他生物体，对延长人的寿命及提高人的生活质量有很大意义。本实验借助模式动物秀丽线虫，通过对其寿命进行观察，探究益生菌在其体内的抗衰老作用，为抗衰老、提高先天免疫的益生菌产品开发利用提供理论参考。Caenorhabditis elegans (C. elegans) is a common, free-living, small soil nematode belonging to the subclass Rhabditia, the order Rhabditidia, and the family Rhabditoidea. Feeding on bacteria, its life cycle is about 3d, with an average lifespan of 3 weeks. It is worm-like, with an adult length of about 1.0 to 1.5 mm and a body diameter of about 70.0 μm. Caenorhabditis elegans is bilaterally symmetrical, with a stratum corneum covering on the body surface, without segments, with 4 main epidermal cords and a fluid-filled prosthetic cavity. The biological characteristics of C. elegans, such as simple structure, transparent body, easy observation, and short reproductive cycle, endow it with the premise of becoming a model organism. By studying the aging and lifespan control mechanism of nematodes and applying the results to other organisms, it is of great significance to prolong human lifespan and improve human life quality. This experiment uses the model animal Caenorhabditis elegans to observe its lifespan to explore the anti-aging effect of probiotics in its body, and to provide a theoretical reference for the development and utilization of probiotic products for anti-aging and improving innate immunity.
发明内容SUMMARY OF THE INVENTION
本发明的一个目的在于提供副干酪乳杆菌ET-22的新用途。An object of the present invention is to provide a new use of Lactobacillus paracasei ET-22.
根据本发明的具体实施方案，本发明提供副干酪乳杆菌(Lactobacillus paracasei)ET-22菌株，保藏编号CGMCC No.15077，保藏于中国典型培养物保藏中心以及中国微生物菌种保藏管理委员会普通微生物中心。副干酪乳杆菌(Lactobacillus paracasei)ET-22菌株是已在CN110964653A中公布的生物材料，是公众可以得到的。According to a specific embodiment of the present invention, the present invention provides a strain of Lactobacillus paracasei ET-22 with a deposit number of CGMCC No. 15077, which is preserved in the China Type Culture Collection and the General Microorganism Center of the China Committee for the Preservation of Microorganisms . Lactobacillus paracasei ET-22 strain is a biological material that has been published in CN110964653A and is publicly available.
本发明发现副干酪乳杆菌(Lactobacillus paracasei)ET-22菌株具有抗衰老、可提高生物体先天免疫、和/或提升生物体对金黄色葡萄球菌感染抵御能力的功效。The present invention finds that the Lactobacillus paracasei ET-22 strain has the effects of anti-aging, improving the innate immunity of organisms, and/or improving the resistance of organisms to Staphylococcus aureus infection.
从而，一方面，本发明提供了副干酪乳杆菌(Lactobacillus paracasei)在制备具有抗衰老、可提高生物体先天免疫、和/或提升生物体对金黄色葡萄球菌感染抵御能力功效的组合物中的应用，其中所述副干酪乳杆菌为保藏编号CGMCC No.15077的副干酪乳杆菌。Thus, on the one hand, the present invention provides Lactobacillus paracasei (Lactobacillus paracasei) in the preparation with anti-aging, can improve the innate immunity of organism and/or improve the composition of organism to Staphylococcus aureus infection resistance effect. Application, wherein the Lactobacillus paracasei is the Lactobacillus paracasei of the deposit number CGMCC No.15077.
根据本发明的具体实施方案，本发明中所述提升生物体对金黄色葡萄球菌感染抵御能力包括：提升个体预防金黄色葡萄球菌感染的能力，降低金黄色葡萄球菌感染个体的能力，和/或缓解金黄色葡萄球菌感染引起的个体食物中毒、肠炎、肺炎、皮肤感染、伤口溃烂和/或脑膜炎等症状。According to a specific embodiment of the present invention, improving the resistance of an organism to Staphylococcus aureus infection described in the present invention includes: improving the ability of an individual to prevent Staphylococcus aureus infection, reducing the ability of an individual to infect an individual with Staphylococcus aureus, and/or Relief of symptoms of food poisoning, enteritis, pneumonia, skin infections, wound ulcers and/or meningitis in individuals caused by Staphylococcus aureus infection.
根据本发明的具体实施方案，本发明的组合物的应用中，所述抗衰老包括延缓金黄色葡萄球菌感染导致的衰老甚至死亡现象。According to a specific embodiment of the present invention, in the application of the composition of the present invention, the anti-aging includes delaying the phenomenon of aging and even death caused by Staphylococcus aureus infection.
根据本发明的具体实施方案，本发明中所述副干酪乳杆菌以固态或液态菌制剂的形式用于制备所述组合物。According to a specific embodiment of the present invention, the Lactobacillus paracasei described in the present invention is used to prepare the composition in the form of a solid or liquid bacterial preparation.
根据本发明的具体实施方案，本发明的所述组合物可以包括食品组合物、饲料组合物、化妆品组合物或药品组合物。According to specific embodiments of the present invention, the composition of the present invention may comprise a food composition, a feed composition, a cosmetic composition or a pharmaceutical composition.
根据本发明的具体实施方案，本发明的组合物的应用中，所述生物体为动物或人。本发明的所述组合物可施用于动物或是人类。所述组合物还可包括所属领域中的常规 用料组分。例如，对于药物组合物，可包括适量的辅料，所述辅料可以为赋型剂、稀释剂、填充剂和/或吸收促进剂等。对于食品组合物，本发明的副干酪乳杆菌可以按照现有技术中含副干酪乳杆菌的食品进行生产，所述组合物可根据受施予者的需要，而采用不同形态。例如粉剂、锭剂、造粒、微胶囊和/或液体制剂等。According to a specific embodiment of the present invention, in the application of the composition of the present invention, the organism is an animal or a human. The compositions of the present invention can be administered to animals or humans. The composition may also include ingredients conventional in the art. For example, for pharmaceutical compositions, an appropriate amount of adjuvants may be included, and the adjuvants may be excipients, diluents, fillers, and/or absorption enhancers, and the like. As for the food composition, the Lactobacillus paracasei of the present invention can be produced according to the food containing Lactobacillus paracasei in the prior art, and the composition can adopt different forms according to the needs of the recipient. For example, powders, lozenges, granules, microcapsules and/or liquid preparations and the like.
根据本发明的具体实施方案，本发明的所述组合物是用于缓解由于金黄色葡萄球菌感染导致的食物中毒、肠炎、肺炎、皮肤感染、伤口溃烂和/或脑膜炎等症状；具体应用时，所述副干酪乳杆菌的应用量为1.0×10
3CFU～1.0×10
12CFU/天，优选为1.0×10
7CFU～1.0×10
11CFU/天。
According to a specific embodiment of the present invention, the composition of the present invention is used to relieve symptoms such as food poisoning, enteritis, pneumonia, skin infection, wound ulceration and/or meningitis caused by Staphylococcus aureus infection; The application amount of the Lactobacillus paracasei is 1.0×10 3 CFU to 1.0×10 12 CFU/day, preferably 1.0×10 7 CFU to 1.0×10 11 CFU/day.
根据本发明的具体实施方案，本发明的所述组合物是用于抗衰老(包括延长寿命)；具体应用时，所述副干酪乳杆菌的应用量为1.0×10
3CFU～1.0×10
12CFU/天，优选为1.0×10
7CFU～1.0×10
11CFU/天。
According to a specific embodiment of the present invention, the composition of the present invention is used for anti-aging (including prolonging lifespan); in specific application, the application amount of the Lactobacillus paracasei is 1.0×10 3 CFU～1.0×10 12 CFU/day, preferably 1.0×10 7 CFU to 1.0×10 11 CFU/day.
根据本发明的具体实施方案，本发明的所述组合物是用于提高先天免疫；具体应用时，所述副干酪乳杆菌的应用量为1.0×10
3CFU～1.0×10
12CFU/天，优选为1.0×10
7CFU～1.0×10
11CFU/天。
According to a specific embodiment of the present invention, the composition of the present invention is used to improve innate immunity; in specific application, the application amount of the Lactobacillus paracasei is 1.0×10 3 CFU to 1.0×10 12 CFU/day, It is preferably 1.0×10 7 CFU to 1.0×10 11 CFU/day.
在本发明的一具体实施方案中，本发明的所述组合物为食品组合物，所述食品为发酵乳制品(例如发酵乳、风味发酵乳、发酵乳饮料等)、乳酪、含乳饮料、固体饮料或乳粉。In a specific embodiment of the present invention, the composition of the present invention is a food composition, and the food is a fermented milk product (such as fermented milk, flavored fermented milk, fermented milk beverage, etc.), cheese, milk-containing beverage, Solid drinks or milk powder.
在本发明的另一具体实施方案中，本发明的所述组合物为药物组合物，所述药物可以是口服制剂，也可以是外用制剂，例如涂抹用药膏等。In another specific embodiment of the present invention, the composition of the present invention is a pharmaceutical composition, and the medicine may be an oral preparation or an external preparation, such as a smeared ointment and the like.
在本发明的另一具体实施方案中，本发明的所述组合物为具有抗衰老、可提高生物体先天免疫、和/或提升生物体对金黄色葡萄球菌感染抵御能力等功效的化妆品。In another specific embodiment of the present invention, the composition of the present invention is a cosmetic with anti-aging effects, can improve the innate immunity of an organism, and/or improve the resistance of an organism to Staphylococcus aureus infection.
另一方面，本发明还提供了一种抗衰老、提高生物体先天免疫、和/或提升生物体对金黄色葡萄球菌感染抵御能力的方法，所述方法包括给予生物体有效量的副干酪乳杆菌；其中，所述副干酪乳杆菌为保藏编号CGMCC No.15077的副干酪乳杆菌。In another aspect, the present invention also provides a method for anti-aging, improving the innate immunity of an organism, and/or improving the resistance of an organism to Staphylococcus aureus infection, the method comprising administering an effective amount of paracheese milk to the organism Bacillus; wherein, the Lactobacillus paracasei is the Lactobacillus paracasei with the deposit number CGMCC No.15077.
根据本发明的具体实施方案，本发明中，所述提升生物体对金黄色葡萄球菌感染抵御能力包括：提升个体预防金黄色葡萄球菌感染的能力，降低金黄色葡萄球菌感染个体的能力，和/或缓解金黄色葡萄球菌感染引起的个体食物中毒、肠炎、肺炎、皮肤感染、伤口溃烂和/或脑膜炎。According to a specific embodiment of the present invention, in the present invention, the improvement of the organism's resistance to Staphylococcus aureus infection includes: improving the individual's ability to prevent Staphylococcus aureus infection, reducing the ability of Staphylococcus aureus to infect an individual, and/ Or relieve food poisoning, enteritis, pneumonia, skin infection, wound ulceration and/or meningitis in individuals caused by Staphylococcus aureus infection.
根据本发明的具体实施方案，本发明中，所述副干酪乳杆菌的应用量为 1.0×10
3CFU～1.0×10
12CFU/天。
According to a specific embodiment of the present invention, in the present invention, the application amount of the Lactobacillus paracasei is 1.0×10 3 CFU to 1.0×10 12 CFU/day.
根据本发明的具体实施方案，本发明中，所述副干酪乳杆菌的应用量优选为1.0×10
7CFU～1.0×10
11CFU/天。
According to a specific embodiment of the present invention, in the present invention, the application amount of the Lactobacillus paracasei is preferably 1.0×10 7 CFU to 1.0×10 11 CFU/day.
综上所述，本发明提供了副干酪乳杆菌ET-22的新用途，该菌具有抗衰老、可提高生物体先天免疫、和/或提升生物体对金黄色葡萄球菌感染抵御能力等功效，可以用于制备具有抗衰老、提高先天免疫功效的食品、药物、化妆品及饲料等，具有广泛的应用前景。To sum up, the present invention provides a new use of Lactobacillus paracasei ET-22, which has the effects of anti-aging, improving the innate immunity of organisms, and/or improving the resistance of organisms to Staphylococcus aureus infection, and the like, It can be used to prepare foods, medicines, cosmetics and feeds with anti-aging and innate immune functions, and has wide application prospects.
图1为S.aureus对秀丽隐杆线虫的致病性和添加益生菌菌株的影响；图中各组别在感染阶段添加的干预物(益生菌)情况分别与培养阶段相同。Figure 1 shows the pathogenicity of S. aureus on Caenorhabditis elegans and the effect of adding probiotic strains; the intervening substances (probiotics) added in each group in the figure are the same as those in the culture stage.
图2为S.aureus和益生菌干预3天后秀丽隐杆线虫的存活率；图中各组别在感染阶段添加的干预物(益生菌)情况分别与培养阶段相同。Figure 2 shows the survival rate of C. elegans after S. aureus and probiotics intervened for 3 days; the intervening substances (probiotics) added in each group in the figure were the same as those in the culture stage.
图3为益生菌对S.aureus的抑菌效果。Figure 3 shows the bacteriostatic effect of probiotics on S. aureus.
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现结合具体实施例及对本发明的技术方案进行以下详细说明，应理解这些实例仅用于说明本发明而不用于限制本发明的范围。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the following detailed description is now given in conjunction with specific embodiments and technical solutions of the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope. In the examples, each original reagent material can be obtained commercially, and the experimental methods without specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions suggested by the instrument manufacturer.
实施例1：益生菌对秀丽隐线虫抗衰老作用测试Example 1: Test of anti-aging effect of probiotics on Caenorhabditis elegans
1.1材料与仪器1.1 Materials and Instruments
副干酪乳杆菌(Lactobacillus paracasei)ET-22来自内蒙古伊利实业集团股份有限公司；鼠李糖乳杆菌(Lactobacillus rhamnosus)HN001购自Danisco；乳双歧杆菌(Bifidobacterium animalis subsp.lactis)BB-12购自ChrHansen；乳双歧杆菌(Bifidobacterium lactis)HN019菌株购自杜邦丹尼斯克公司；发酵乳杆菌(Lactobacillus fermentum)LC40购自Biosearch life；野生型秀丽线虫(Caenorhabditis elegans，C.elegans)购自西班牙Biopolis SL生物技术公司；金黄色葡萄球菌(Staphylococcus aureus ATCC25923)购自西班牙Biopolis SL生物技术公司。Lactobacillus paracasei ET-22 was purchased from Inner Mongolia Yili Industrial Group Co., Ltd.; Lactobacillus rhamnosus HN001 was purchased from Danisco; Bifidobacterium animalis subsp.lactis BB-12 was purchased from ChrHansen; Bifidobacterium lactis HN019 strain was purchased from DuPont Danisco; Lactobacillus fermentum LC40 was purchased from Biosearch life; wild-type Caenorhabditis elegans (C.elegans) was purchased from Biopolis SL Bio, Spain technology company; Staphylococcus aureus ATCC25923 was purchased from Biopolis SL biotechnology company in Spain.
实验所用其他试剂均购于国药集团化学试剂有限公司。SPX-150B-Z型生化培养箱：上海博讯实业有限公司医疗设备厂；奥林巴斯显微镜(OlympusBX41)：南京艾朗仪器有限公司；LDZX-50KBS立式压力蒸汽灭菌锅：上海申安医疗器械厂；T6紫外/可见分光光度计：北京普析通用仪器有限公司；Multiskan FC型酶标仪：赛默飞世尔(上海)仪器有限公司。All other reagents used in the experiment were purchased from Sinopharm Chemical Reagent Co., Ltd. SPX-150B-Z biochemical incubator: Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory; Olympus Microscope (OlympusBX41): Nanjing Ailang Instrument Co., Ltd.; LDZX-50KBS Vertical Pressure Steam Sterilizer: Shanghai Shen'an Medical Instrument Factory; T6 UV/Vis Spectrophotometer: Beijing Puxi General Instrument Co., Ltd.; Multiskan FC Type Microplate Reader: Thermo Fisher (Shanghai) Instrument Co., Ltd.
1.2实验方法1.2 Experimental method
1.2.1益生菌活化1.2.1 Activation of probiotics
取乳杆菌于MRS培养基中，乳双歧杆菌于MRS+Cys培养基中，37℃培养活化，细胞收集并使用生理盐水洗脱，调整至1×10
8CFU于NGM培养基中备用。
Lactobacillus in MRS medium, Bifidobacterium lactis in MRS+Cys medium, cultured and activated at 37°C, cells were collected and eluted with normal saline, adjusted to 1×10 8 CFU in NGM medium for use.
1.2.2秀丽线虫寿命测定1.2.2 C. elegans lifespan assay
获取了年龄一致的线虫并将其培养在含线虫琼脂培养基(线虫培养基中含有大肠杆菌OP50为食物)的培养皿中，添加了不同益生菌(1×10
8CFU)进行共培养。在线虫成年后，它们被转移到接种了金黄色葡萄球菌ATCC25923的培养皿中，添加量为10
8～10
9CFU/mL，来模拟受到金黄色葡萄球菌感染的情况。各组别在感染阶段添加的干预物(益生菌)情况分别与培养阶段相同。使用了两个对照，分别是没有致病菌的条件(线虫培养皿中含有大肠杆菌OP50，即OP50组)，以及有致病菌金黄色葡萄球菌感染但没有任何干预物的(只含有金黄色葡萄球菌的培养皿，即S.aureus组)。另设置培养阶段添加干预物而感染阶段没有任何干预物的组别、以及培养阶段未添加干预物而感染阶段添加干预物的组别。
Age-matched nematodes were obtained and cultured in petri dishes containing nematode agar medium (nematode medium containing Escherichia coli OP50 as food), and different probiotics (1×10 8 CFU) were added for co-cultivation. After the nematodes became adults, they were transferred to petri dishes inoculated with Staphylococcus aureus ATCC25923 at 10 8 -10 9 CFU/mL to simulate infection with Staphylococcus aureus. The intervening substances (probiotics) added in the infection stage of each group were the same as those in the culture stage. Two controls were used, one without pathogenic bacteria (nematode petri dishes containing Escherichia coli OP50, ie OP50 group), and those with pathogenic bacteria Staphylococcus aureus infection without any intervention (containing only Staphylococcus aureus). the petri dish, that is, the S. aureus group). In addition, a group in which an intervening substance was added in the culture phase and no intervening substance in the infection phase, and a group in which the intervening substance was not added in the cultivation phase but added in the infection phase were set.
将线虫培养几天后，对它们的存活率进行每日计数。如果线虫对铂丝没有反应，则可被认为是死亡。每种条件均进行了两次独立测定。After the nematodes had been cultured for several days, their survival was counted daily. If the worm does not respond to the platinum wire, it can be considered dead. Two independent assays were performed for each condition.
对存活曲线进行了统计学的比较分析，用GraphPad Prism 4统计学软件包进行了log rank survival显著性分析。关于对线虫存活率的影响各组间在每一天体现的差异，先用Two-way ANOVA进行分析，再用Tukey’s post hoc test进行各组间的比较。用One-way ANOVA与Dunnett’s post hoc test分析各组存活率的显著性差异。Statistical comparative analysis of survival curves was performed, and log rank survival significance analysis was performed using the GraphPad Prism 4 statistical software package. Regarding the effect on the survival rate of nematodes, the differences between the groups in each day were first analyzed by Two-way ANOVA, and then Tukey's post hoc test was used to compare the groups. One-way ANOVA and Dunnett's post hoc test were used to analyze the significant differences in survival among the groups.
1.2.3实验结果1.2.3 Experimental results
寿命是秀丽线虫暴露前后变化最直接的反映。Lifespan is the most direct reflection of changes in C. elegans before and after exposure.
实验结果如图1所示，秀丽线虫经S.aureus感染后，在第3天开始，存活率显著下降，第5天秀丽线虫全部死亡。于之相比，10
8CFU副干酪乳杆菌ET-22处 理过的秀丽线虫组，在第3天，存活率为58.35±0.65％，从第5d存活率为13.3±0.7％，与S.aureus组存活率具有显著性差异(p<0.001)。发酵乳杆菌LC 40处理的秀丽线虫组，在第三天，存活率为25.85±0.85％，而在第5天线虫存活率为0％，与S.aureus组未有显著性差异。
The experimental results are shown in Figure 1. After the infection of C. elegans by S. aureus, the survival rate decreased significantly from the third day, and all the C. elegans died on the fifth day. In contrast, the C. elegans group treated with 10 8 CFU L. paracasei ET-22 had a survival rate of 58.35 ± 0.65% on day 3 and 13.3 ± 0.7% from day 5, and S. aureus Group survival rates were significantly different (p<0.001). The C. elegans group treated with Lactobacillus fermentum LC 40 had a survival rate of 25.85±0.85% on the third day, while the survival rate of the fifth antennae was 0%, which was not significantly different from the S. aureus group.
由图2可知，在第3天，与乳双歧杆菌BB-12，发酵乳杆菌LC 40处理的线虫组相比，副干酪乳杆菌组ET-22能够极显著延长线虫寿命(p<0.001)，与乳双歧杆菌HN019，鼠李糖乳杆菌HN001相比，副干酪乳杆菌ET-22能够显著延长秀丽线虫寿命(p<0.05)。As can be seen from Figure 2, on the 3rd day, compared with the nematode group treated with Bifidobacterium lactis BB-12 and Lactobacillus fermentum LC 40, the Lactobacillus paracasei group ET-22 could significantly prolong the lifespan of nematodes (p<0.001). , Compared with Bifidobacterium lactis HN019 and Lactobacillus rhamnosus HN001, Lactobacillus paracasei ET-22 can significantly prolong the lifespan of C. elegans (p<0.05).
此外，本发明的实验显示，培养阶段添加了益生菌ET-22干预物而感染阶段没有添加任何干预物的组别的秀丽隐杆线虫存活率，显著高于培养阶段及感染阶段均未添加干预物的组别。In addition, the experiments of the present invention show that the survival rate of C. elegans in the group in which the probiotic ET-22 intervention is added in the culture stage and no intervention is added in the infection stage is significantly higher than that in the culture stage and the infection stage without any intervention. group of things.
实施例2：益生菌对金黄色葡萄球菌的抑菌特性分析Example 2: Analysis of the antibacterial properties of probiotics against Staphylococcus aureus
采用纸片扩散法分析益生菌对S.aureus的抗菌活力。吸取1mL浓度为1×10
8CFU/mL的菌悬液于15mL RCA培养基(50℃)中，混和均匀后倾注到平皿中，待琼脂表面凝固后取药敏纸片贴于琼脂表面，每个培养皿贴3张间隔均匀适中的同种药物纸片，静置5min后翻转平皿,，微需氧条件下37℃恒温培养，48h后测量并记录抑菌圈的直径。
The antibacterial activity of probiotics against S. aureus was analyzed by disc diffusion method. Pipette 1 mL of bacterial suspension with a concentration of 1×10 8 CFU/mL into 15 mL of RCA medium (50°C), mix it evenly, and pour it into a plate. Each petri dish was affixed with 3 paper sheets of the same drug at even and moderate intervals. After standing for 5 min, the plate was turned over and incubated at a constant temperature of 37 °C under microaerophilic conditions. After 48 h, the diameter of the inhibition zone was measured and recorded.
为分析益生菌对秀丽线虫延长寿命的影响是否受抑菌能力的影响，进一步通过纸片扩散法对益生菌菌株的抑菌能力进行评价。结果表明，没有任何益生菌会在平板中产生透明圈，表明本发明涉及的益生菌菌株对S.aureus不会产生抑菌作用(图3)。益生菌并不是通过抑制S.aureus进而对秀丽线虫产生抗衰老、提高先天免疫的作用。In order to analyze whether the effect of probiotics on the lifespan extension of C. elegans is affected by the antibacterial ability, the antibacterial ability of the probiotic strains was further evaluated by the disc diffusion method. The results showed that none of the probiotics produced a transparent circle in the plate, indicating that the probiotic strains involved in the present invention did not have a bacteriostatic effect on S. aureus ( FIG. 3 ). Probiotics do not have anti-aging and innate immunity effects on C. elegans by inhibiting S. aureus.
以上结果表明副干酪乳杆菌(Lactobacillus paracasei)ET-22(保藏编号CGMCC No.15077)在抗衰老、提高先天免疫方面具有良好潜力，可以用于食品，如发酵乳、乳酪、含乳饮料、乳粉或其它含有该菌株及其衍生物的任何一种食品。The above results show that Lactobacillus paracasei ET-22 (Deposit No. CGMCC No. 15077) has good potential in anti-aging and improving innate immunity, and can be used in food, such as fermented milk, cheese, milk-containing beverages, milk Powder or any other food containing the strain and its derivatives.
Claims (13)
- 一种副干酪乳杆菌(Lactobacillus paracasei)在制备具有抗衰老、可提高生物体先天免疫、和/或提升生物体对金黄色葡萄球菌感染抵御能力功效的组合物中的应用，其中所述副干酪乳杆菌为保藏编号CGMCC No.15077的副干酪乳杆菌。A kind of Lactobacillus paracasei (Lactobacillus paracasei) in the preparation with anti-aging, can improve the innate immunity of organisms, and/or enhance the application of organisms to Staphylococcus aureus infection resistance effect in the composition, wherein said paracasei Lactobacillus is Lactobacillus paracasei with deposit number CGMCC No.15077.
- 根据权利要求1所述的应用，其中，所述提升生物体对金黄色葡萄球菌感染抵御能力包括：提升个体预防金黄色葡萄球菌感染的能力，降低金黄色葡萄球菌感染个体的能力，和/或缓解金黄色葡萄球菌感染引起的个体食物中毒、肠炎、肺炎、皮肤感染、伤口溃烂和/或脑膜炎。The use according to claim 1, wherein the enhancing the organism's resistance to Staphylococcus aureus infection comprises: enhancing the individual's ability to prevent Staphylococcus aureus infection, reducing the ability of the individual to infect the individual with Staphylococcus aureus, and/or Relief of food poisoning, enteritis, pneumonia, skin infection, wound ulceration and/or meningitis in individuals due to Staphylococcus aureus infection.
- 根据权利要求1所述的应用，其中，所述副干酪乳杆菌以固态或液态菌制剂的形式用于制备所述组合物；优选地，所述组合物包括食品组合物、饲料组合物、化妆品组合物或药品组合物。The application according to claim 1, wherein the Lactobacillus paracasei is used to prepare the composition in the form of a solid or liquid bacterial preparation; preferably, the composition includes a food composition, a feed composition, a cosmetic composition or pharmaceutical composition.
- 根据权利要求3所述的应用，其中，所述药品组合物还包括药学上可接受的辅料，所述辅料包括赋型剂、稀释剂、填充剂和/或吸收促进剂。The use according to claim 3, wherein the pharmaceutical composition further comprises pharmaceutically acceptable adjuvants, the adjuvants comprising excipients, diluents, fillers and/or absorption enhancers.
- 根据权利要求3所述的应用，其中，所述食品组合物包括粉剂、锭剂、造粒、微胶囊、液体制剂和/或其他剂型。The use according to claim 3, wherein the food composition comprises powders, lozenges, granules, microcapsules, liquid preparations and/or other dosage forms.
- 根据权利要求1所述的应用，其中，所述组合物是用于缓解由于金黄色葡萄球菌感染导致的食物中毒、肠炎、肺炎、皮肤感染、伤口溃烂和/或脑膜炎；The use according to claim 1, wherein the composition is for relieving food poisoning, enteritis, pneumonia, skin infection, wound ulceration and/or meningitis due to Staphylococcus aureus infection;优选地，其中，所述副干酪乳杆菌的应用量为1.0×10 3CFU～1.0×10 12CFU/天，更优选为1.0×10 7CFU～1.0×10 11CFU/天。 Preferably, the application amount of the Lactobacillus paracasei is 1.0×10 3 CFU to 1.0×10 12 CFU/day, more preferably 1.0×10 7 CFU to 1.0×10 11 CFU/day.
- 根据权利要求1所述的应用，其中，所述组合物是用于抗衰老：The use according to claim 1, wherein the composition is for anti-aging:优选地，其中，所述副干酪乳杆菌的应用量为1.0×10 3CFU～1.0×10 12CFU/天，更优选为1.0×10 7CFU～1.0×10 11CFU/天。 Preferably, the application amount of the Lactobacillus paracasei is 1.0×10 3 CFU to 1.0×10 12 CFU/day, more preferably 1.0×10 7 CFU to 1.0×10 11 CFU/day.
- 根据权利要求1所述的应用，其中，所述组合物是用于提高先天免疫；The use according to claim 1, wherein the composition is for enhancing innate immunity;优选地，其中，所述副干酪乳杆菌的应用量为1.0×10 3CFU～1.0×10 12CFU/天，更优选为1.0×10 7CFU～1.0×10 11CFU/天。 Preferably, the application amount of the Lactobacillus paracasei is 1.0×10 3 CFU to 1.0×10 12 CFU/day, more preferably 1.0×10 7 CFU to 1.0×10 11 CFU/day.
- 根据权利要求6-8中任一项所述的应用，其中，所述组合物为食品组合物。The use according to any one of claims 6-8, wherein the composition is a food composition.
- 根据权利要求9所述的应用，其中，所述食品为发酵乳制品、乳酪、含乳饮料、固体饮料或乳粉。The application according to claim 9, wherein the food is fermented dairy product, cheese, milk-containing beverage, solid beverage or milk powder.
- 一种抗衰老、提高生物体先天免疫、和/或提升生物体对金黄色葡萄球菌感染 抵御能力的方法，所述方法包括给予生物体有效量的副干酪乳杆菌；其中，所述副干酪乳杆菌为保藏编号CGMCC No.15077的副干酪乳杆菌。A method for anti-aging, improving the innate immunity of an organism, and/or improving the resistance of an organism to Staphylococcus aureus infection, the method comprising giving an organism an effective amount of Lactobacillus paracasei; wherein, the paracasei milk The bacillus is Lactobacillus paracasei of deposit number CGMCC No.15077.
- 根据权利要求11所述的方法，其中，所述提升生物体对金黄色葡萄球菌感染抵御能力包括：提升个体预防金黄色葡萄球菌感染的能力，降低金黄色葡萄球菌感染个体的能力，和/或缓解金黄色葡萄球菌感染引起的个体食物中毒、肠炎、肺炎、皮肤感染、伤口溃烂和/或脑膜炎。The method of claim 11, wherein the enhancing the organism's ability to resist S. aureus infection comprises: enhancing the individual's ability to prevent S. aureus infection, reducing the ability of the individual to infect the individual with S. aureus, and/or Relief of food poisoning, enteritis, pneumonia, skin infection, wound ulceration and/or meningitis in individuals due to Staphylococcus aureus infection.
- 根据权利要求11或12所述的方法，其中，所述副干酪乳杆菌的应用量为1.0×10 3CFU～1.0×10 12CFU/天；优选为1.0×10 7CFU～1.0×10 11CFU/天。 The method according to claim 11 or 12, wherein the application amount of the Lactobacillus paracasei is 1.0×10 3 CFU～1.0×10 12 CFU/day; preferably 1.0×10 7 CFU～1.0×10 11 CFU /sky.
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