WO2022036822A1 - 一株具有免疫调节作用的鼠李糖乳杆菌及其应用 - Google Patents

一株具有免疫调节作用的鼠李糖乳杆菌及其应用 Download PDF

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WO2022036822A1
WO2022036822A1 PCT/CN2020/120519 CN2020120519W WO2022036822A1 WO 2022036822 A1 WO2022036822 A1 WO 2022036822A1 CN 2020120519 W CN2020120519 W CN 2020120519W WO 2022036822 A1 WO2022036822 A1 WO 2022036822A1
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lactobacillus rhamnosus
rhamnosus
lacticaseibacillus
lactobacillus
strain
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侯俊财
姜瞻梅
王惋
高达
徐聪
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东北农业大学
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    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
    • A23L19/20—Products from fruits or vegetables; Preparation or treatment thereof by pickling, e.g. sauerkraut or pickles
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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    • C12R2001/225—Lactobacillus

Definitions

  • the invention belongs to the technical field of lactobacillus, in particular to a strain of lactobacillus rhamnosus with immunomodulatory effect and application thereof.
  • Cyclophosphamide is one of the most commonly used alkylating antitumor drugs. Like most chemotherapy drugs, it can destroy tumor cells and inhibit the patient's immune system. Problems in the application of chemotherapy. Immunosuppression can lead to an increased incidence of hypersensitivity disease, autoimmune disease, infectious disease, and tumor formation.
  • the commonly used immunopotentiating drugs in clinical practice include chemically synthesized drugs such as levamisole and isoprinosine, but they are extremely harmful to liver and kidney functions. Some people may cause nausea, vomiting, abdominal pain and other adverse reactions after taking them. Therefore, it is crucial to find safe and stable natural immune modulators.
  • Lactic acid bacteria is one of the recognized food-safety probiotics, which has the effects of anti-infection, immune regulation, anti-oxidation, and regulation of intestinal microecology. Many clinical and animal experiments have confirmed that lactic acid bacteria can modulate innate and adaptive immune responses in the host, and oral probiotics have a positive effect on the composition of intestinal flora and colonization resistance to pathogenic bacteria, and can generate appropriate immunity Modulate the response. Lactobacillus is an important probiotic that is fixed in the gut. Lactobacillus can elicit specific and non-specific immune responses in the host by binding to pattern recognition receptors expressed by immune cells in the gut and many other tissues, including the intestinal epithelium. .
  • Lactobacillus can induce the expression of anti-inflammatory cytokines (such as IL-10) and immunoglobulin M (IgM).
  • Lactobacillus can enhance the phagocytic activity of macrophages and stimulate the secretion of lysosomal enzymes, and improve host immunity by up-regulating macrophage activity and promoting the proliferation and differentiation of T cells.
  • Bacteriocin a metabolite of Lactobacillus, can improve intestinal immunity by killing pathogenic bacteria in the intestine.
  • Lactobacillus and its metabolites have also been proposed as delivery vehicles for antigens instead of inactivated vaccines and in the production of nutraceuticals.
  • the number of probiotics that can be added to food in accordance with the regulations of the Ministry of Health is still very limited, and the actual immune efficacy of different strains of the same genus is also significantly different, and there is no clear rule to follow.
  • the purpose of the present invention is to solve the problems such as the limited probiotics that can be added to food at present, provide a probiotic Lactobacillus rhamnosus with strong immune regulation effect and its application, and clarify that Lactobacillus rhamnosus 1.0320 is immune to Defective regulation and optimal dosage provide theoretical support for the development of safe and efficient immunomodulatory biological agents, and are of great significance for enriching the national strain library.
  • a strain of Lactobacillus rhamnosus with immunomodulatory effect L. rhamnosus 1.0320 strain, is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration Committee, with the preservation number of CGMCC No.15557, The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit date is April 8, 2018.
  • Lactobacillus rhamnosus is applied in the fermentation process of yogurt or pickles.
  • Lactobacillus rhamnosus An application of the above-mentioned Lactobacillus rhamnosus with immunomodulatory effect, wherein the Lactobacillus rhamnosus is used to prepare freeze-dried bacteria powder.
  • Lactobacillus rhamnosus of the present invention to produce bacteriocin is high intestinal wall adhesion rate (over 11.76%), low pH resistance (pH lower than 3), bile salt resistance (at a high concentration of 0.3% bile salts) After incubating for 4 h, the number of colonies was 10 7 CFU/mL).
  • the Lactobacillus rhamnosus of the present invention has better regulation on spleen lymphocyte transformation, spleen lymphocyte proliferation, phagocytic ability of peritoneal macrophages, and secretion of NO by peritoneal macrophages
  • the immunomodulatory ability was significantly higher than that of the reference strain Lactobacillus rhamnosus GG and the difference was significant.
  • the ratio of added amount to cells is 100:1
  • the effect is the best
  • the transformation value of spleen lymphocytes is 7.5 times that of the positive control group
  • the proliferation index of spleen lymphocytes is above 1
  • the phagocytic ability of macrophages is not significantly different from that of the positive control group. , and obtained unpredictable technical effects.
  • the diameter of the inhibition zone increases from (26.24 ⁇ 0.31) mm to (36.68 ⁇ 0.51) mm, an increase of 39.79% .
  • the Lactobacillus rhamnosus of the present invention is applied as a biological preparation with immunoregulatory function, and has the functions of restoring the body weight of immunodeficient mice, T lymphocyte proliferation ability, phagocytic ability of peritoneal macrophages, NK cell activity, carbon particle clearance ability, delayed type
  • the ability of hypersensitivity reaction, cellular inflammatory factor level, and the optimal dose is: (10 9 CFU/mL)/0.02kg/d.
  • Fig. 1 is a graph showing the influence of eight strains of Lactobacillus on the transformation value of mouse spleen lymphocytes
  • Figure 2 is a graph showing the influence of eight strains of Lactobacillus on the proliferation index (PI) of mouse spleen lymphocytes;
  • Figure 3 is a graph showing the effect of eight strains of Lactobacillus on the phagocytosis of neutral red by mouse peritoneal macrophages;
  • Figure 4 is a graph showing the effect of eight strains of Lactobacillus on the content of NO secreted by mouse peritoneal macrophages;
  • Figure 5 is a graph showing the effect of Lactobacillus rhamnosus 1.0320 on the body weight of mice;
  • Figure 6 is a graph showing the effect of Lactobacillus rhamnosus 1.0320 on the proliferation of mouse spleen T lymphocytes
  • Figure 7 is a graph showing the effect of Lactobacillus rhamnosus 1.0320 on the phagocytic ability of mouse macrophages
  • Figure 8 is a graph showing the effect of Lactobacillus rhamnosus 1.0320 on the activity of mouse NK cells
  • Figure 9 is a graph showing the effect of Lactobacillus rhamnosus 1.0320 on the clearance index of carbon particles in mice;
  • Figure 10 is a graph showing the effect of Lactobacillus rhamnosus 1.0320 on delayed-type hypersensitivity in mice;
  • Figure 11 is a graph showing the effect of Lactobacillus rhamnosus 1.0320 on the IL-12/IL-10 ratio in mouse serum;
  • Figure 12 is a graph showing the effect of Lactobacillus rhamnosus 1.0320 on IgG in mouse serum;
  • Figure 13 is a colony morphology diagram of Lactobacillus rhamnosus 1.0320;
  • Fig. 14 is a morphological diagram of Lactobacillus rhamnosus 1.0320.
  • the present invention has the probiotic function L. rhamnosus 1.0320 strain with high-yield bacteriocin, which is a gram-positive bacterium isolated from traditional kumiss samples.
  • L. rhamnosus 1.0320 strain with high-yield bacteriocin which is a gram-positive bacterium isolated from traditional kumiss samples.
  • bacteriocin a gram-positive bacterium isolated from traditional kumiss samples.
  • Embodiment 1 This embodiment describes a strain of Lactobacillus rhamnosus with immunomodulatory effect, L. rhamnosus 1.0320 strain, which is preserved in the General Committee for the Preservation and Administration of Microorganisms of China Microbiology Center, the deposit number is CGMCC No. 15557, the deposit address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit date is April 08, 2018.
  • Embodiment 2 A strain of Lactobacillus rhamnosus with immunomodulatory effect described in Embodiment 1, the Lactobacillus rhamnosus is isolated from traditional kumiss.
  • Embodiment 3 The Lactobacillus rhamnosus strain described in Embodiment 1 has an immunomodulatory effect, as shown in Figures 13 and 14, the colony of the Lactobacillus rhamnosus is milky white, with neat edges and a smooth surface , bulge, the cells are short rod-shaped.
  • Embodiment 4 An application of the Lactobacillus rhamnosus with immunomodulatory effect according to any one of Embodiments 1 to 3, the Lactobacillus rhamnosus is applied in the fermentation process of yogurt or pickles.
  • Embodiment 5 An application of the Lactobacillus rhamnosus with immunomodulatory effect according to any one of Embodiments 1 to 3, where the Lactobacillus rhamnosus is used to prepare freeze-dried bacteria powder.
  • L. rhamnosus strain 1.0320 Dissolve 1 mL of traditional kumiss and 1 g of healthy human intestinal contents with 10 mL of sterile PBS (0.01M, pH 7.4) to prepare a suspension. 2% of the inoculum was inoculated into the sterilized MRS broth medium, and after culturing at a constant temperature of 37°C for 18 hours, the above operation was repeated to activate the second generation. Take 100 ⁇ L of the fully activated second-generation bacterial solution and carry out three-district streaking on the sterilized MRS-agar medium by plate streaking, incubate at 37°C for 24 hours, and pick a single colony.
  • Lactobacillus plantarum 69-2 Three strains isolated from kumiss samples were identified and named Lactobacillus plantarum 69-2, Lactobacillus plantarum 1.0628 and Lactobacillus rhamnosus 1.0320; three strains isolated from healthy human gut were named Lactobacillus plantarum 117-1 , Lactobacillus plantarum 23-1 and Lactobacillus rhamnosus 118-1.
  • Lactobacillus rhamnosus GG ATCC53103
  • ATCC authorized depository American Type Culture Collection
  • ATCC-P0002 was purchased from the China Medical Bacteria Collection Center (CMCC). Studies have confirmed that Lactobacillus rhamnosus GG and Lactobacillus plantarum P0002 have the function of regulating immunity, and can be used to screen Lactobacillus with immune regulating function. reference strain.
  • Lactobacillus plantarum P0002 (CMCC-P0002), Lactobacillus plantarum 69-2, Lactobacillus plantarum 1.0628, Lactobacillus plantarum 117-1, Lactobacillus plantarum 23-1, Lactobacillus rhamnosus GG (ATCC53103), Lactobacillus saccharomyces 1.0320 and Lactobacillus rhamnosus 118-1 were used for transformation of spleen lymphocytes, proliferation of spleen lymphocytes (the same amount of cell culture medium was used as a negative control group; ConA with a final concentration of 5 ⁇ g/mL was used as a positive control group), peritoneal macrophages.
  • the phagocytic capacity of phagocytes and the content of NO secreted by peritoneal macrophages were measured (the same amount of cell culture medium was used as a negative control group; LPS with a final concentration of 30 ⁇ g/mL was used as a positive control group), and the results showed that Lactobacillus rhamnosus 1.0320 (Lactobacillus The transformation value of spleen lymphocytes and the content of NO secreted by peritoneal macrophages with a cell ratio of 1:1) were significantly higher than those of the other 7 strains of Lactobacillus (p ⁇ 0.05) (Fig. 1, Fig. 4).
  • the immunomodulatory ability of Lactobacillus rhamnosus 1.0320 was significantly higher than that of the reference strain Lactobacillus rhamnosus GG.
  • the immunomodulatory strength of Lactobacillus rhamnosus 1.0320 was positively correlated with the concentration, and the spleen lymphocyte transformation value of Lactobacillus rhamnosus 1.0320 (Lactobacillus:cell ratio of 100:1) was 7.5 times higher than that of the positive control group (Fig. 1).
  • the lymphocyte proliferation index (PI) reached above 1 (Fig. 2), and the phagocytic ability of peritoneal macrophages was not significantly different from that of the positive control group (p>0.05) (Fig. 3).
  • the results showed that the immunomodulatory ability of Lactobacillus rhamnosus 1.0320 was significantly stronger than the other 7 strains of lactic acid bacteria.
  • MRS basal medium preparation (initial pH 6.2): beef powder 5.0g, tryptone 10.0g, glucose 20.0g, yeast powder 4.0g, Tween 801.0mL, K 2 HPO 4 2.0g, CH 3 COONa 5.0g, C 6 H 5 O 7 (NH 4 ) 3 2.0 g, MgSO 4 0.2 g, MnSO 4 0.05 g, and distilled water 1000 mL.
  • 1Effect of ultraviolet mutagenesis on the production of bacteriocin Take a number of 10 mL of activated three-generation Lactobacillus rhamnosus 1.0320 cell suspension in a dark room in a sterilized plate, and irradiate it with a 25W ultraviolet lamp for 0s, 20s, and 40s at a distance of 30cm. , 60s, 80s, 100s, 120s.
  • Escherichia coli as the indicator bacteria, the antibacterial test was carried out by Oxford cup double-layer agar diffusion method.
  • the activated second-generation Lactobacillus rhamnosus 1.0320 was inoculated with a 2% inoculum in the ratio of yeast powder to tryptone (basal medium ratio was 4:10) In MRS medium adjusted to 2.5:11.5, 3:11, 3.5:10.5, 4:10, 4.5:9.5, 5:9, 5.5:8.5, cultured at 37°C for 18h.
  • MRS medium adjusted to 2.5:11.5, 3:11, 3.5:10.5, 4:10, 4.5:9.5, 5:9, 5.5:8.5, cultured at 37°C for 18h.
  • Escherichia coli as the indicator bacteria, the antibacterial test was carried out by Oxford cup double-layer agar diffusion method.
  • the effect of adding NaCl on the production of bacteriocin The activated second-generation Lactobacillus rhamnosus 1.0320 was inoculated with 2% inoculum in 0%, 0.5%, 1%, 1.5%, 2%, and 2.5% of NaCl. , 3% MRS medium, cultured at 37°C for 18h. Using Escherichia coli as the indicator bacteria, the antibacterial test was carried out by Oxford cup double-layer agar diffusion method.
  • 1Activation Take 1 mL of Lactobacillus rhamnosus 1.0320 stored at -80°C, streak it on MRS-agar medium, cultivate at 37°C for 24 hours, and pick a single colony. It was inoculated into sterilized MRS broth medium for activation and passage twice, and after 12-16 hours of constant temperature incubation at 37°C, turbidity appeared. Take 100 ⁇ L of bacterial liquid to carry out three-district streak on sterilized MRS-agar medium, cultivate at 37 °C for 24 h, pick a single colony for observation under a microscope, and there is no bacterial contamination, which is qualified.
  • Example preparation Determine the viable count of Lactobacillus rhamnosus 1.0320 bacterial solution by plate counting method, wash 3 times after centrifugation (4000r/min, 10min), resuspend with sterile PBS (0.01M, pH 7.4) to prepare Each of 10 mL of bacterial solutions with concentrations of 5 ⁇ 10 7 CFU/mL, 5 ⁇ 10 8 CFU/mL, and 5 ⁇ 10 9 CFU/mL was prepared, and 15 mL of sterile PBS (0.01M, pH 7.4) was prepared.
  • cyclophosphamide was given daily at an injection dose of 80 mg/kg for three consecutive days, and it was observed that the weight of the mice continued to decrease, indicating that cyclophosphamide caused the immune system of the mice. damage, the immunosuppression model was successfully established.
  • mice body weight T lymphocyte proliferation ability, peritoneal macrophages Phagocytosis, NK cell activity, carbon particle clearance, delayed-type hypersensitivity, and cellular inflammatory factor levels were determined.
  • Lactobacillus rhamnosus 1.0320 could significantly alleviate the weight loss caused by immunodeficiency (Fig. 5), the improvement of immune cell activity was positively correlated with the viable count of Lactobacillus rhamnosus 1.0320, and the high dose ((10 9 CFU/mL)/0.02kg) of Lactobacillus rhamnosus 1.0320 could well Promote the phagocytic ability of macrophages (Fig. 7) and the ability to clear carbon particles (Fig. 9), and there is no significant difference with the positive control group (p>0.05); promote the proliferation of T lymphocytes (Fig. 6) and enhance NK cells The effects of activity (Fig. 5).
  • Lactobacillus rhamnosus 1.0320 can also regulate the level of cellular inflammatory factors by increasing the levels of anti-inflammatory cytokines IL-10 and pro-inflammatory cytokines IL-12.
  • Lactobacillus rhamnosus 1.0320 as freeze-dried bacterial powder with immune-enhancing function
  • 1Activation Take 1 mL of Lactobacillus rhamnosus 1.0320 stored at -80°C, streak it on MRS-agar medium, cultivate at 37°C for 24 hours, and pick a single colony. Inoculated in sterilized MRS broth medium for activation and passage twice, and after culturing at 37°C for 12-16 hours, observed under a microscope, no bacterial contamination, qualified, and transferred to a 40L fermenter for amplification after reaching the standard. nourish.
  • 2Determination of the number of viable bacteria take 0.5mL of the activated and amplified fermentation broth and add it to 4.5mL of sterilized normal saline, do 10-fold gradient dilution in turn, select 10-8 , 10-9 , 10-10 dilution times, take 200 ⁇ L of the diluted solution was spread on sterile MRS-agar medium, incubated at 37°C for 24 hours, and counted on a plate to determine the viable bacterial count of the fermentation broth to be 10 9 CFU/mL.
  • Lyophilization the fermentation broth was centrifuged (4000r/min, 10min), the bacterial cells were collected, and resuspended with an equal volume of sterile PBS solution, and the freeze-drying protective agent was 5% sucrose skim milk powder (the concentration of skim milk powder was 20 %, the sucrose concentration is 5% (w/v)), the Lactobacillus rhamnosus 1.0320 suspension and the sucrose skim milk powder freeze-drying protective agent are mixed uniformly at 1:1 (v/v), and placed in a low-temperature refrigerator for pre-cooling to -40 degrees Celsius. At the same time, the temperature of the freezer of the freeze dryer was lowered to below -40°C.
  • the freeze-drying time is about 24-72 hours.
  • the Lactobacillus rhamnosus 1.0320 freeze-dried bacterial powder is stored at -80°C, and the survival rate is about 49.6%.
  • Lactobacillus rhamnosus 1.0320 freeze-dried bacterial powder has an immune strengthening function and can be directly stored as a health care product at 0-4° C.
  • the number of viable bacteria is 2 ⁇ 10 8 CFU/g.
  • 1Activation Take 1 mL of Lactobacillus rhamnosus 1.0320 stored at -80°C, streak it on MRS-agar medium, cultivate at 37°C for 24 hours, and pick a single colony. Inoculated into sterilized MRS broth medium for activation and passage twice, and after culturing at 37°C for 12-16 hours, observed under a microscope, there was no contamination by impurities, and it was qualified.
  • Centrifugation Centrifuge the fermentation broth (4000 r/min, 10 min), collect the bacterial cells, and resuspend with an equal volume of sterile 5% glucose solution.
  • Fermentation of yogurt the raw milk is pretreated and standardized, cooled to the fermentation temperature, and the starter is inoculated with 2% of the inoculum.
  • the temperature of the fermentation tank is 42-45°C, and the fermentation time is 2.5-3h. When the pH reaches 4.7, the temperature is lowered to 0-7°C for stirring to obtain immune-enhanced yogurt.

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Abstract

一株具有免疫调节作用的鼠李糖乳杆菌（L.rhamnosus）1.0320株及其应用，其应用于酸奶或泡菜的发酵过程中或用于制备冻干菌粉。该鼠李糖乳杆菌菌株1.0320的肠壁粘附率高（达11.76％以上），耐低pH（pH低于3），耐胆盐（在0.3％的高浓度胆盐中孵育4h后，菌落数为10 7CFU/mL），对脾淋巴细胞转化值和腹腔巨噬细胞分泌NO具有很强的调节能力（乳杆菌：细胞为1:1），且免疫调节能力显著高于参考菌株鼠李糖乳杆菌GG（p＜0.05），其免疫调节强度与浓度呈正相关，当其与细胞比例为100：1时，脾淋巴细胞转化值是阳性对照组的7.5倍，脾淋巴细胞增殖指数达到1以上。

Description

一株具有免疫调节作用的鼠李糖乳杆菌及其应用 技术领域
本发明属于乳杆菌技术领域，具体涉及一株具有免疫调节作用的鼠李糖乳杆菌及其应用。
背景技术
环磷酰胺(Cyclophosphamide，CTX)是最常用的烷化剂类抗肿瘤药物之一，与大部分化疗药物相同，在破坏肿瘤细胞的同时还对患者的免疫系统发生抑制作用，这是其在肿瘤化疗中应用的难题。免疫抑制可导致超敏性疾病、自身免疫疾病、感染性疾病和肿瘤形成的发生率增加。目前临床常用的免疫增强药有化学合成药物左旋咪唑、异丙肌苷等，但是其对肝肾功能伤害极大，部分人群服用后可引起恶心、呕吐、腹痛等不良反应。因此，寻找安全、稳定的天然免疫调节剂至关重要。
乳酸菌是公认的食品安全级益生菌之一，具有抗感染、免疫调节、抗氧化、调节肠道微生态等效果。许多临床和动物实验证实了乳酸菌可以调节宿主体内固有免疫和获得性免疫反应，口服益生菌对肠道菌群的组成和对致病菌的定殖抗性具有积极作用，并且能够产生适当的免疫调节反应。乳杆菌是肠道内固定存在的重要益生菌，乳杆菌可通过与肠道内免疫细胞和包括肠上皮在内的许多其他组织表达的模式识别受体结合，在宿主中引发特异性和非特异性免疫反应。研究发现，乳杆菌可诱导抑炎细胞因子(如：IL-10)以及免疫球蛋白M(IgM)表达。同时，乳杆菌可增强巨噬细胞吞噬活性并刺激溶酶体酶的分泌，并通过上调巨噬细胞活性和促进T细胞增殖分化来提高宿主的免疫力。乳杆菌的代谢产物细菌素能够通过杀伤肠道中的致病菌，以提高肠道免疫力。乳杆菌和其代谢产物也已被提议用作抗原的递送载体代替灭活疫苗以及生产营养保健品。目前，符合国家卫生部规定可添加到食品中的益生菌数目还十分有限，同属但不同菌株之间的实际免疫功效也存在显著差异，并且无明确的规律可循。
发明内容
本发明的目的是为了解决目前可添加到食品中的益生菌有限等问题，提供一株具有强免疫调节作用的益生功能鼠李糖乳杆菌及其应用，并明确鼠李糖乳杆菌1.0320对免疫缺陷的调节作用及最佳剂量，为开发安全高效的免疫调节生物制剂提供理论支撑，为丰富国家菌种库具有重要意义。
为实现上述目的，本发明采取的技术方案如下：
一株具有免疫调节作用的鼠李糖乳杆菌，鼠李糖乳杆菌(L.rhamnosus)1.0320株，该菌种保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.15557，保藏地址是北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，保藏日期为2018年04月08号。
一种上述具有免疫调节作用的鼠李糖乳杆菌的应用，将鼠李糖乳杆菌应用于酸奶或泡菜的发酵过程中。
一种上述具有免疫调节作用的鼠李糖乳杆菌的应用，鼠李糖乳杆菌用于制备冻干菌粉。
本发明相对于现有技术的有益效果为：
本发明的鼠李糖乳杆菌产细菌素的能力为，肠壁粘附率高(达11.76％以上)，耐低pH(pH低于3)，耐胆盐(在0.3％的高浓度胆盐中孵育4h后，菌落数为10 7CFU/mL)。
本发明的鼠李糖乳杆菌与其余7株乳杆菌的免疫调节作用相比，对于脾淋巴细胞转化、脾淋巴细胞增殖、腹腔巨噬细胞吞噬能力、腹腔巨噬细胞分泌NO具有较好的调节作用，免疫调节能力显著高于参考菌株鼠李糖乳杆菌GG且差异显著。当添加量与细胞比例为100:1时效果最佳，脾淋巴细胞转化值是阳性对照组的7.5倍，脾淋巴细胞增殖指数达到1以上，且巨噬细胞吞噬能力与阳性对照组差异不显著，获得了难以预料的技术效果。
本发明的鼠李糖乳杆菌在优化培养条件(紫外诱导、pH、氮源、NaCl添加量)后抑菌圈直径由(26.24±0.31)mm增加到(36.68±0.51)mm，增加了39.79％。
本发明的鼠李糖乳杆菌作为具有免疫调节功能的生物制剂应用，具有恢复免疫缺陷小鼠体重、T淋巴细胞增殖能力、腹腔巨噬细胞吞噬能力、NK细胞活性、碳粒廓清能力、迟发型超敏反应、细胞炎性因子水平的能力，且最佳剂量为：(10 9CFU/mL)/0.02kg/d。
附图说明
图1为八株乳杆菌对小鼠脾淋巴细胞转化值的影响图；
图2为八株乳杆菌对小鼠脾淋巴细胞增殖指数(PI)的影响图；
图3为八株乳杆菌对小鼠腹腔巨噬细胞吞噬中性红能力的影响图；
图4为八株乳杆菌对小鼠腹腔巨噬细胞分泌NO含量的影响图；
图5为鼠李糖乳杆菌1.0320对小鼠体重的影响图；
图6为鼠李糖乳杆菌1.0320对小鼠脾脏T淋巴细胞增殖的影响图；
图7为鼠李糖乳杆菌1.0320对小鼠巨噬细胞吞噬能力的影响图；
图8为鼠李糖乳杆菌1.0320对小鼠NK细胞活性的影响图；
图9为鼠李糖乳杆菌1.0320对小鼠碳粒廓清指数的影响图；
图10为鼠李糖乳杆菌1.0320对小鼠迟发型超敏反应的影响图；
图11为鼠李糖乳杆菌1.0320对小鼠血清中IL-12/IL-10比值的影响图；
图12为鼠李糖乳杆菌1.0320对小鼠血清中IgG的影响图；
图13为鼠李糖乳杆菌1.0320的菌落形态图；
图14为鼠李糖乳杆菌1.0320的菌体形态图。
具体实施方式
下面结合附图和实施例对本发明的技术方案作进一步的说明，但并不局限于此，凡是对本发明技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，均应涵盖在本发明的保护范围中。
本发明具有高产细菌素的益生功能鼠李糖乳杆菌(L.rhamnosus)1.0320株，是从传统马奶酒样品中分离出来的一株革兰氏阳性菌。首先经广泛筛选，从传统马奶酒和健康人体肠道中获得一株具有对脾淋巴细胞转化、脾淋巴细胞增殖、腹腔巨噬细胞吞噬能力、腹腔巨噬细胞分泌NO含量调节作用最强的鼠李糖乳杆菌1.0320，并确定所述的鼠李糖乳杆菌对恢复免疫缺陷作用的最佳剂量。
具体实施方式一：本实施方式记载的是一株具有免疫调节作用的鼠李糖乳杆菌，鼠李糖乳杆菌(L.rhamnosus)1.0320株，该菌种保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.15557，保藏地址是北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，保藏日期为2018年04月08号。
具体实施方式二：具体实施方式一所述的一株具有免疫调节作用的鼠李糖乳杆菌，所述鼠李糖乳杆菌分离于传统马奶酒中。
具体实施方式三：具体实施方式一所述的一株具有免疫调节作用的鼠李糖乳杆菌，如图13和14所示，所述鼠李糖乳杆菌的菌落呈乳白色、边缘整齐、表面平滑、凸起，菌体呈短杆状。
具体实施方式四：一种具体实施方式一至三任一项所述的具有免疫调节作用的鼠李糖乳杆菌的应用，将鼠李糖乳杆菌应用于酸奶或泡菜的发酵过程中。
具体实施方式五：一种具体实施方式一至三任一项所述的具有免疫调节作用的鼠李糖乳杆菌的应用，鼠李糖乳杆菌用于制备冻干菌粉。
鼠李糖乳杆菌(L.rhamnosus)1.0320株的分离纯化：将1mL传统马奶酒样本和1g健康人体肠道内容物分别用10mL无菌PBS(0.01M，pH 7.4)溶解配制成悬液，以2％的接种量接种至灭菌的MRS肉汤培养基中，37℃恒温培养18h后重复上述操作，活化二 代。取充分活化的二代菌液100μL以平板划线的方式于灭菌的MRS-琼脂培养基上进行三区划线，于37℃恒温培养24h，挑取单菌落。接种于灭菌的MRS肉汤培养基中活化传代两次，37℃恒温培养12-16h后，出现浑浊。取100μL菌液在灭菌的MRS-琼脂培养基上进行三区划线，于37℃恒温培养24h，挑取单菌落在显微镜下观察，观察菌株形态以及是否无杂菌污染。确定为合格菌株后以菌液形式送样至吉林库美生物科技有限公司利用16S rDNA序列测序的方法对细菌进行种属鉴定。鉴定出分离于马奶酒样本的3株菌株命名为植物乳杆菌69-2，植物乳杆菌1.0628和鼠李糖乳杆菌1.0320；分离于健康人体肠道的3株菌株命名为植物乳杆菌117-1，植物乳杆菌23-1和鼠李糖乳杆菌118-1。鼠李糖乳杆菌GG(ATCC53103)，保藏于授权保藏单位美国典型培养物保藏中心(ATCC)，购买于ATCC中国大陆官方代理北京中原公司。植物乳杆菌P0002(CMCC-P0002)购买于中国医学细菌保藏管理中心(CMCC)，研究证实鼠李糖乳杆菌GG和植物乳杆菌P0002具有调节免疫的功能，可用作筛选具有免疫调节功能乳杆菌的参考菌株。
分别对植物乳杆菌P0002(CMCC-P0002)、植物乳杆菌69-2、植物乳杆菌1.0628、植物乳杆菌117-1、植物乳杆菌23-1、鼠李糖乳杆菌GG(ATCC53103)、鼠李糖乳杆菌1.0320、鼠李糖乳杆菌118-1进行脾淋巴细胞转化、脾淋巴细胞增殖(等量细胞培养液作为阴性对照组；终浓度为5μg/mL的ConA作为阳性对照组)、腹腔巨噬细胞吞噬能力、腹腔巨噬细胞分泌NO含量的测定(等量细胞培养液作为阴性对照组；终浓度为30μg/mL的LPS作为阳性对照组)，结果鼠李糖乳杆菌1.0320(乳杆菌：细胞为1:1)的脾淋巴细胞转化值和腹腔巨噬细胞分泌NO含量显著高于其余7株乳杆菌(p＜0.05)(图1、图4)。其中，鼠李糖乳杆菌1.0320的免疫调节能力显著高于参考菌株鼠李糖乳杆菌GG。鼠李糖乳杆菌1.0320的免疫调节强度与浓度呈正相关，鼠李糖乳杆菌1.0320(乳杆菌：细胞为100：1)的脾淋巴细胞转化值是阳性对照组的7.5倍(图1)，脾淋巴细胞增殖指数(PI)达到1以上(图2)，且腹腔巨噬细胞吞噬能力与阳性对照组差异不显著(p＞0.05)(图3)。结果表明，鼠李糖乳杆菌1.0320的免疫调节能力显著强于其余7株乳酸菌。
实施例1：
基于正交分析对鼠李糖乳杆菌1.0320细菌素产量优化
MRS基础培养基配制(起始pH 6.2)：牛肉粉5.0g、胰蛋白胨10.0g、葡萄糖20.0g、酵母粉4.0g、吐温801.0mL、K 2HPO 42.0g、CH 3COONa 5.0g、C 6H 5O 7(NH 4) 32.0g、MgSO 40.2g、MnSO 40.05g、蒸馏水1000mL。
①紫外诱变对细菌素产量的影响：暗室中取若干10mL活化三代的鼠李糖乳杆菌 1.0320菌体悬浮液于灭菌的平皿中，距离30cm处分别用25W紫外灯照射0s、20s、40s、60s、80s、100s、120s。以大肠杆菌作为指示菌，采用牛津杯双层琼脂扩散法进行抑菌试验。
②培养基初始pH对细菌素产量的影响：将活化二代的鼠李糖乳杆菌1.0320以2％接种量分别接种于pH为5.8、6.0、6.2、6.4、6.6、6.8、7.0的MRS培养基中，37℃培养18h。以大肠杆菌作为指示菌，采用牛津杯双层琼脂扩散法进行抑菌试验。
③两种重要氮源的比例对细菌素产量的影响：将活化二代的鼠李糖乳杆菌1.0320以2％接种量分别接种于酵母粉与胰蛋白胨比例(基础培养基比例为4：10)调整为2.5:11.5、3:11、3.5:10.5、4:10、4.5:9.5、5:9、5.5:8.5的MRS培养基中，37℃培养18h。以大肠杆菌作为指示菌，采用牛津杯双层琼脂扩散法进行抑菌试验。
④添加NaCl对细菌素产量的影响：将活化二代的鼠李糖乳杆菌1.0320以2％接种量分别接种于NaCl添加量为0％、0.5％、1％、1.5％、2％、2.5％、3％的MRS培养基中，37℃培养18h。以大肠杆菌作为指示菌，采用牛津杯双层琼脂扩散法进行抑菌试验。
由表2中各因素的极差值大小顺序R 1＞R 3＞R 4＞R 2可知对鼠李糖乳杆菌1.0320细菌素产量影响的主次关系为紫外诱导＞氮源比例＞NaCl＞pH。从K值可以确定，鼠李糖乳杆菌1.0320细菌素生产的最佳组合为：紫外诱导80s、调整氮源比例(酵母粉：胰蛋白胨＝3.5:10.5)、NaCl添加量1％、初始pH为6.4。抑菌圈直径由(26.24±0.31)mm增加到(36.68±0.51)mm。
表1单因素对鼠李糖乳杆菌1.0320细菌素产量的影响表
Figure PCTCN2020120519-appb-000001
Figure PCTCN2020120519-appb-000002
表2鼠李糖乳杆菌1.0320细菌素产量优化的正交试验设计方案及结果表
Figure PCTCN2020120519-appb-000003
实施例2：
鼠李糖乳杆菌1.0320对免疫缺陷模型小鼠的免疫调节作用
①活化：将-80℃保藏的鼠李糖乳杆菌1.0320取出1mL，并划线于MRS-琼脂培养基，于37℃恒温培养24h，挑取单菌落。接种于灭菌的MRS肉汤培养基中活化传代两次，37℃恒温培养12-16h后，出现浑浊。取100μL菌液在灭菌的MRS-琼脂培养基上进行三区划 线，于37℃恒温培养24h，挑取单菌落在显微镜下观察，无杂菌污染，为合格。
②样品制备：采用平板计数法确定鼠李糖乳杆菌1.0320菌液的活菌数，离心(4000r/min，10min)后洗涤3次，用无菌PBS(0.01M，pH 7.4)重悬配置成浓度为5×10 7CFU/mL、5×10 8CFU/mL、5×10 9CFU/mL的菌液各10mL，并准备15mL的无菌PBS(0.01M，pH 7.4)。
③造模：对SPF级BALB/c雌性小鼠(体重18±1g；6周龄)随机分为：空白对照组(N)、模型对照组(M)、阳性对照组(P)、鼠李糖乳杆菌1.0320低剂量组(1.0320-L)、鼠李糖乳杆菌1.0320中剂量组(1.0320-M)和鼠李糖乳杆菌1.0320高剂量组(1.0320-H)。除空白对照组(等体积无菌PBS代替)外，每日给予注射剂量为80mg/kg的环磷酰胺，连续三天，观察到小鼠体重持续下降，说明环磷酰胺造成了小鼠免疫系统的损伤，免疫抑制模型建立成功。
④灌胃：各组分别给予等体积的：无菌PBS、无菌PBS、左旋咪唑(2.5mg/mL)、鼠李糖乳杆菌1.0320(5×10 7CFU/mL)、鼠李糖乳杆菌1.0320(5×10 8CFU/mL)和鼠李糖乳杆菌1.0320(5×10 9CFU/mL)各0.2mL，连续干预30天后，对小鼠体重、T淋巴细胞增殖能力、腹腔巨噬细胞吞噬能力、NK细胞活性、碳粒廓清能力、迟发型超敏反应、细胞炎性因子水平进行测定，如图所示，结果发现所述鼠李糖乳杆菌1.0320能够显著缓解免疫缺陷造成的体重下降(图5)，免疫细胞活性的提高与鼠李糖乳杆菌1.0320的活菌数呈正相关，其中高剂量((10 9CFU/mL)/0.02kg)的鼠李糖乳杆菌1.0320能够很好地促进巨噬细胞的吞噬能力(图7)、碳粒廓清能力(图9)，与阳性对照组差异不显著(p＞0.05)；对T淋巴细胞增殖的促进作用(图6)和增强NK细胞活性(图8)的影响分别是阳性对照组的1.06倍和1.54倍。迟发型超敏反应结果如图10所示，同时，鼠李糖乳杆菌1.0320还能够调节细胞炎性因子水平，通过升高抑炎性细胞因子IL-10与促炎性细胞因子IL-12的比值(图11)和免疫球蛋白的水平(图12)，以发挥缓解免疫缺陷的功能，并确定最佳剂量为(10 9CFU/mL)/0.02kg/d。
实施例3：
鼠李糖乳杆菌1.0320作为具有免疫强化功能的冻干菌粉的应用
①活化：将-80℃保藏的鼠李糖乳杆菌1.0320取出1mL，并划线于MRS-琼脂培养基，于37℃恒温培养24h，挑取单菌落。接种于灭菌的MRS肉汤培养基中活化传代两次，37℃恒温培养12-16h后，在显微镜下观察，无杂菌污染，为合格，检测达标后转至40L发酵罐中进行扩增培养。
②活菌数确定：取活化、扩增的发酵液0.5mL加入至4.5mL灭菌的生理盐水中，依次做10倍梯度稀释，选取10 -8、10 -9、10 -10稀释倍数，取200μL稀释液在无菌MRS-琼脂培养基上涂布，37℃恒温培养24h后，平皿计数，确定发酵液活菌数为10 9CFU/mL。
③冻干：将发酵液离心(4000r/min，10min)，收集菌体细胞，用等体积的无菌PBS溶液重悬，冷冻干燥保护剂为5％蔗糖脱脂乳粉(脱脂乳粉浓度为20％，蔗糖浓度为5％(w/v))，将鼠李糖乳杆菌1.0320悬液与蔗糖脱脂乳粉冻干保护剂按1：1(v/v)混合均匀，置于低温冰箱预冷至-40摄氏度。同时将冻干机的冷冻仓温度下降至-40℃以下。放入样品后，打开冻干机真空泵开始冻干，冻干时间约为24-72h，将鼠李糖乳杆菌1.0320冻干菌粉于-80℃保存，存活率约为49.6％。
上述的鼠李糖乳杆菌1.0320冻干菌粉具有免疫强化功能可以直接作为保健品于0-4℃储存，活菌数为2×10 8CFU/g。或作为免疫强化因子添加至功能性乳粉、益生元固体饮料中。具有提高免疫免疫细胞的增值能力，调节巨噬细胞的吞噬能力，降低机体炎性反映的发生，调节肠道微生态。
实施例4：
鼠李糖乳杆菌1.0320与商业发酵剂复合在食品中的应用
①活化：将-80℃保藏的鼠李糖乳杆菌1.0320取出1mL，并划线于MRS-琼脂培养基，于37℃恒温培养24h，挑取单菌落。接种于灭菌的MRS肉汤培养基中活化传代两次，37℃恒温培养12-16h后，在显微镜下观察，无杂菌污染，为合格。
②离心：将发酵液离心(4000r/min，10min)，收集菌体细胞，用等体积的无菌5％葡萄糖溶液重悬。
③酸奶发酵：将原料乳进行预处理和标准化，冷却至发酵温度后以2％的接种量接种发酵剂，复合发酵剂为鼠李糖乳杆菌：嗜热链球菌：保加利亚乳杆菌＝2：4：4，发酵罐温度为42-45℃，发酵时间为2.5-3h，当pH达4.7时，将温度降低至0-7℃进行搅拌，即得到免疫强化酸奶。
④泡菜发酵：将新鲜原料充分洗涤后，加入4％的食盐和2.5％的蔗糖(w/w)，以3％的接种量接种复合发酵剂(鼠李糖乳杆菌1.0320：植物乳杆菌＝1:1)，于30℃恒温发酵5d，泡菜完成，用鼠李糖乳杆菌1.0320作为发酵剂发酵的泡菜保存时间更长。

Claims (5)

  1. 一株具有免疫调节作用的鼠李糖乳杆菌，其特征在于：鼠李糖乳杆菌(L.rhamnosus)1.0320株，该菌种保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.15557，保藏地址是北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，保藏日期为2018年04月08号。
  2. 根据权利要求1所述的一株具有免疫调节作用的鼠李糖乳杆菌，其特征在于：所述鼠李糖乳杆菌分离于传统马奶酒中。
  3. 根据权利要求1所述的一株具有免疫调节作用的鼠李糖乳杆菌，其特征在于：所述鼠李糖乳杆菌的菌落呈乳白色、边缘整齐、表面平滑、凸起，菌体呈短杆状。
  4. 一种权利要求1～3任一项所述的具有免疫调节作用的鼠李糖乳杆菌的应用，其特征在于：将鼠李糖乳杆菌应用于酸奶或泡菜的发酵过程中。
  5. 一种权利要求1～3任一项所述的具有免疫调节作用的鼠李糖乳杆菌的应用，其特征在于：鼠李糖乳杆菌用于制备冻干菌粉。
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