CN111944721A - Lactobacillus rhamnosus with immunoregulation effect and application thereof - Google Patents
Lactobacillus rhamnosus with immunoregulation effect and application thereof Download PDFInfo
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- CN111944721A CN111944721A CN202010834655.4A CN202010834655A CN111944721A CN 111944721 A CN111944721 A CN 111944721A CN 202010834655 A CN202010834655 A CN 202010834655A CN 111944721 A CN111944721 A CN 111944721A
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- lactobacillus rhamnosus
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- 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.
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Abstract
Lactobacillus rhamnosus (L.rhamnosus)1.0320 strain with immunoregulation effect and application thereof are applied to fermentation process of yoghourt or pickled vegetables or used for preparing freeze-dried bacterial powder. The lactobacillus rhamnosus provided by the invention has high intestinal wall adhesion rate (more than 11.76%), low pH resistance (pH is lower than 3) and bile salt resistance (the colony number is 10 after the lactobacillus rhamnosus is incubated in 0.3% high-concentration bile salt for 4 hours7CFU/mL). Transformation value of Lactobacillus rhamnosus 1.0320 (Lactobacillus: cells: 1) on splenic lymphocytes and abdominal cavityThe macrophage secretes NO with strong regulating ability, and the immunoregulation ability is obviously higher than that of a reference strain Lactobacillus rhamnosus GG (p is less than 0.05). The immunoregulation strength and concentration of lactobacillus rhamnosus 1.0320 are positively correlated, and when the ratio of lactobacillus rhamnosus 1.0320 to cells is 100: when 1 hour, the conversion value of the spleen lymphocyte is 7.5 times of that of the positive control group, and the proliferation index of the spleen lymphocyte reaches more than 1.
Description
Technical Field
The invention belongs to the technical field of lactobacillus, and particularly relates to lactobacillus rhamnosus with an immunoregulation effect and an application thereof.
Background
Cyclophosphamide (CTX) is one of the most commonly used alkylating antineoplastic drugs, and like most chemotherapy drugs, it also has an inhibitory effect on the immune system of a patient while destroying tumor cells, which is a problem in the application of the Cyclophosphamide in tumor chemotherapy. Immunosuppression can lead to increased incidence of hypersensitivity diseases, autoimmune diseases, infectious diseases, and tumor formation. The prior commonly used immunopotentiator in clinic comprises chemical synthesis medicines of levamisole, isoprinosine and the like, but the immunopotentiator has great harm to liver and kidney functions, and can cause adverse reactions such as nausea, vomiting, abdominal pain and the like after being taken by part of people. Therefore, it is crucial to find safe and stable natural immunomodulators.
The lactobacillus is one of the well-known food safety probiotics, and has the effects of resisting infection, regulating immunity, resisting oxidation, regulating intestinal microecology and the like. Many clinical and animal experiments have confirmed that lactic acid bacteria can regulate innate and acquired immune responses in a host, and oral administration of probiotics has a positive effect on the composition of intestinal flora and colonization resistance to pathogenic bacteria, and can produce appropriate immune regulation responses. Lactobacilli are important probiotics that are present in the gut in a fixed manner and can elicit specific and non-specific immune responses in the host by binding to pattern recognition receptors expressed by immune cells and many other tissues in the gut, including the intestinal epithelium. It was found that the expression of anti-inflammatory cytokines (e.g., IL-10) and immunoglobulin M (IgM) was induced by Lactobacillus. Meanwhile, lactobacillus can enhance macrophage phagocytic activity and stimulate secretion of lysosomal enzyme, and improve host immunity by up-regulating macrophage activity and promoting proliferation and differentiation of T cells. The metabolite bacteriocin of lactobacillus can kill pathogenic bacteria in intestinal tract to improve intestinal immunity. Lactobacilli and their metabolites have also been proposed as delivery vehicles for antigens in place of inactivated vaccines and in the production of nutraceuticals. At present, the number of probiotics which can be added into food according with the regulations of national ministry of health is quite limited, and the actual immune efficacy of different strains in the same genus is obviously different, and no clear rule can be followed.
Disclosure of Invention
The invention aims to solve the problems that probiotics which can be added into food at present are limited and the like, provides a probiotic functional lactobacillus rhamnosus with strong immunoregulation effect and application thereof, and defines the regulation effect and the optimal dosage of lactobacillus rhamnosus 1.0320 on immunodeficiency, provides theoretical support for developing safe and efficient immunoregulation biological agents, and has important significance for enriching national strain libraries.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the lactobacillus rhamnosus strain is a strain 1.0320 of lactobacillus rhamnosus (L.rhamnosus) with the immunoregulation function, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.15557, the preservation address is No. 3 of No.1 Siro of Beijing Ing-oriented region, the institute of microbiology of China academy of sciences, and the preservation date is No. 08 of 2018, 04 months.
An application of the lactobacillus rhamnosus with the immunoregulation function is to apply the lactobacillus rhamnosus in the fermentation process of yoghourt or pickled vegetables.
An application of the lactobacillus rhamnosus with the immunoregulation function is that the lactobacillus rhamnosus is used for preparing freeze-dried bacterium powder.
Compared with the prior art, the invention has the beneficial effects that:
the lactobacillus rhamnosus of the invention has the capability of producing bacteriocin, high intestinal wall adhesion rate (more than 11.76 percent), low pH resistance (pH is lower than 3), and bile salt resistance (the colony count is 10 after the lactobacillus rhamnosus is incubated in 0.3 percent of high-concentration bile salt for 4 hours7CFU/mL)。
Compared with the immunoregulation effect of the rest 7 strains of lactobacillus, the lactobacillus rhamnosus provided by the invention has better regulation effects on spleen lymphocyte transformation, spleen lymphocyte proliferation, abdominal cavity macrophage phagocytosis and NO secretion of abdominal cavity macrophages, and the immunoregulation ability is obviously higher than that of a reference strain lactobacillus rhamnosus GG and has obvious difference. When the addition amount and the cell ratio are 100:1, the effect is optimal, the spleen lymphocyte transformation value is 7.5 times of that of a positive control group, the proliferation index of the spleen lymphocytes reaches more than 1, the phagocytic capacity of macrophages is not obviously different from that of the positive control group, and an unpredictable technical effect is obtained.
After the lactobacillus rhamnosus is cultured under optimized conditions (ultraviolet induction, pH, nitrogen source and NaCl addition), the diameter of an inhibition zone is increased from (26.24 +/-0.31) mm to (36.68 +/-0.51) mm, and is increased by 39.79%.
The lactobacillus rhamnosus provided by the invention is applied as a biological preparation with an immunoregulation function, and has the capabilities of recovering the body weight, the T lymphocyte proliferation capability, the abdominal cavity macrophage phagocytosis capability, the NK cell activity, the carbon particle clearance capability, the delayed hypersensitivity and the level of a cell inflammatory factor of an immunodeficient mouse, and the optimal dosage is as follows: (109CFU/mL)/0.02kg/d。
Drawings
FIG. 1 is a graph showing the effect of eight strains of Lactobacillus on the spleen lymphocyte transformation values of mice;
FIG. 2 is a graph showing the effect of eight strains of Lactobacillus on the mouse spleen lymphocyte Proliferation Index (PI);
FIG. 3 is a graph showing the effect of eight strains of Lactobacillus on the ability of macrophages in the abdominal cavity of a mouse to phagocytose neutral red;
FIG. 4 is a graph showing the effect of eight strains of Lactobacillus on the NO content secreted by macrophages in the abdominal cavity of a mouse;
FIG. 5 is a graph of the effect of Lactobacillus rhamnosus 1.0320 on mouse body weight;
FIG. 6 is a graph of the effect of Lactobacillus rhamnosus 1.0320 on mouse spleen T lymphocyte proliferation;
FIG. 7 is a graph of the effect of Lactobacillus rhamnosus 1.0320 on the phagocytic capacity of mouse macrophages;
FIG. 8 is a graph showing the effect of Lactobacillus rhamnosus 1.0320 on NK cell activity in mice;
FIG. 9 is a graph showing the effect of Lactobacillus rhamnosus 1.0320 on carbon clearance index in mice;
FIG. 10 is a graph showing the effect of Lactobacillus rhamnosus 1.0320 on delayed type hypersensitivity in mice;
FIG. 11 is a graph showing the effect of Lactobacillus rhamnosus 1.0320 on the IL-12/IL-10 ratio in mouse serum;
FIG. 12 is a graph of the effect of Lactobacillus rhamnosus 1.0320 on IgG in mouse serum;
FIG. 13 is a colony morphology of Lactobacillus rhamnosus 1.0320;
FIG. 14 is a cell morphology of Lactobacillus rhamnosus 1.0320.
Detailed Description
The technical solutions of the present invention are further described below with reference to the drawings and the embodiments, but the present invention is not limited thereto, and modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
The probiotic lactobacillus rhamnosus (L.rhamnosus)1.0320 strain with high bacteriocin yield is a gram-positive bacterium separated from a traditional marmilk wine sample. Firstly, a lactobacillus rhamnosus 1.0320 with strongest regulating effects on spleen lymphocyte transformation, spleen lymphocyte proliferation, abdominal cavity macrophage phagocytosis and NO content secretion of abdominal cavity macrophages is obtained from traditional mare milk wine and intestinal tracts of healthy human bodies through wide screening, and the optimal dose of the lactobacillus rhamnosus on the effect of recovering immunodeficiency is determined.
The first embodiment is as follows: the embodiment describes a strain of lactobacillus rhamnosus with an immunoregulation effect, namely a lactobacillus rhamnosus (L.rhamnosus)1.0320 strain, wherein the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.15557, the preservation address is No. 3 of Beijing university Hokko No.1 of sunward West Chen, and the preservation date is No. 08 of 2018 monthly 04.
The second embodiment is as follows: in the first embodiment, the lactobacillus rhamnosus with the immunoregulation function is separated from the traditional marmilk wine.
The third concrete implementation mode: in a specific embodiment, the lactobacillus rhamnosus with the immunoregulation function is shown in fig. 13 and 14, the bacterial colony of the lactobacillus rhamnosus is milk white, the edge is neat, the surface is smooth and convex, and the thallus is in a short rod shape.
The fourth concrete implementation mode: the application of the lactobacillus rhamnosus with the immunoregulation effect disclosed in any one of the first embodiment to the third embodiment is to apply the lactobacillus rhamnosus to the fermentation process of yoghourt or pickled vegetables.
The fifth concrete implementation mode: the application of lactobacillus rhamnosus with immunoregulation effect disclosed in any one of the first to third embodiments, wherein the lactobacillus rhamnosus is used for preparing freeze-dried bacterial powder.
Isolation and purification of Lactobacillus rhamnosus (L.rhamnosus) strain 1.0320: 1mL of a traditional marmilk wine sample and 1g of intestinal contents of a healthy human body are dissolved in 10mL of sterile PBS (0.01M, pH 7.4) respectively to prepare suspensions, the suspensions are inoculated into a sterilized MRS broth culture medium in an inoculation amount of 2%, the operations are repeated after the culture is carried out at a constant temperature of 37 ℃ for 18 hours, and the second generation is activated. Taking 100 mu L of fully activated second-generation bacterium liquid, carrying out three-zone streaking on a sterilized MRS-agar culture medium in a plate streaking mode, carrying out constant-temperature culture at 37 ℃ for 24h, and picking out a single colony. Inoculating the strain in a sterilized MRS broth culture medium for activation passage twice, and culturing at 37 ℃ for 12-16h at constant temperature to generate turbidity. Taking 100 mu L of bacterial liquid to perform three-zone lineation on a sterilized MRS-agar culture medium, culturing for 24h at the constant temperature of 37 ℃, selecting a single bacterial colony, observing the bacterial colony under a microscope, and observing the strain form and whether the bacterial colony is free from mixed bacteria pollution. After the strains are determined to be qualified strains, the strains are sent to Jilin Ku Mei Biotech limited in the form of bacterial liquid, and the species identification is carried out on the bacteria by using a 16S rDNA sequence sequencing method. 3 strains isolated from the marburg sample were identified and named Lactobacillus plantarum 69-2, Lactobacillus plantarum 1.0628, and Lactobacillus rhamnosus 1.0320; the 3 strains isolated from the intestinal tract of a healthy human body are named as lactobacillus plantarum 117-1, lactobacillus plantarum 23-1 and lactobacillus rhamnosus 118-1. Lactobacillus rhamnosus GG (ATCC53103), deposited in the American Type Culture Collection (ATCC) of the authorized depository, was purchased from the ATCC China office agency, Beijing Central plains, Inc. Lactobacillus plantarum P0002(CMCC-P0002) is purchased from China medical bacteria preservation and management center (CMCC), and researches prove that Lactobacillus rhamnosus GG and Lactobacillus plantarum P0002 have the function of regulating immunity and can be used as reference strains for screening Lactobacillus with the function of regulating immunity.
The spleen lymphocyte transformation, the spleen lymphocyte proliferation (equivalent cell culture solution as a negative control group; ConA with a final concentration of 5 mu g/mL as a positive control group), the phagocytic capacity of the abdominal cavity macrophages and the content of the abdominal cavity macrophages secreting NO are respectively carried out on 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 rhamnosus 1.0320 and lactobacillus rhamnosus 118-1 (equivalent cell culture solution as a negative control group; LPS with a final concentration of 30 mu g/mL as a positive control group), so that the conversion value of the spleen lymphocytes and the content of the abdominal cavity macrophages secreting NO of lactobacillus rhamnosus 1.0320 (lactobacillus: cells 1:1) are remarkably higher than those of the other 7 strains of lactobacillus (P < 0.05) (FIG. 1, B), Fig. 4). Wherein, the immunoregulatory ability of lactobacillus rhamnosus 1.0320 is significantly higher than that of the reference strain lactobacillus rhamnosus GG. The immunoregulation intensity of lactobacillus rhamnosus 1.0320 is positively correlated with the concentration, the spleen lymphocyte transformation value of lactobacillus rhamnosus 1.0320 (lactobacillus: cells are 100: 1) is 7.5 times of that of a positive control group (figure 1), the spleen lymphocyte Proliferation Index (PI) reaches more than 1 (figure 2), and the phagocytic capacity of abdominal cavity macrophages is not significantly different from that of the positive control group (p is more than 0.05) (figure 3). The result shows that the immunoregulation capability of the lactobacillus rhamnosus 1.0320 is obviously stronger than that of the rest 7 strains of lactobacillus.
Example 1:
optimization of Lactobacillus rhamnosus 1.0320 bacteriocin yield based on orthogonal analysis
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, K2HPO42.0 g、CH3COONa 5.0g、C6H5O7(NH4)32.0g、MgSO4 0.2g、MnSO40.05 g, and 1000mL of distilled water.
Influence of ultraviolet mutagenesis on bacteriocin yield: a plurality of 10mL of activated three-generation lactobacillus rhamnosus 1.0320 thallus suspension is put in a sterilized plate in a dark room, and the plate is respectively irradiated by 25W ultraviolet lamps for 0s, 20s, 40s, 60s, 80s, 100s and 120s at a distance of 30 cm. Escherichia coli is used as an indicator bacterium, and an oxford cup double-layer agar diffusion method is adopted for carrying out a bacteriostasis test.
Influence of initial pH of the medium on bacteriocin yield: inoculating activated second-generation Lactobacillus rhamnosus 1.0320 with 2% inoculum size in MRS culture medium with pH of 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, and 7.0, respectively, and culturing at 37 deg.C for 18 h. Escherichia coli is used as an indicator bacterium, and an oxford cup double-layer agar diffusion method is adopted for carrying out a bacteriostasis test.
③ Effect of the ratio of two important nitrogen sources on the yield of bacteriocins: the second generation of activated lactobacillus rhamnosus 1.0320 was inoculated in 2% inoculum size in MRS medium with yeast powder and tryptone ratio (basal medium ratio of 4: 10) adjusted to 2.5:11.5, 3:11, 3.5:10.5, 4:10, 4.5:9.5, 5:9, 5.5:8.5, respectively, and cultured at 37 ℃ for 18 h. Escherichia coli is used as an indicator bacterium, and an oxford cup double-layer agar diffusion method is adopted for carrying out a bacteriostasis test.
Influence of NaCl addition on bacteriocin yield: the second-generation activated lactobacillus rhamnosus 1.0320 is respectively inoculated in MRS culture media with NaCl addition of 0%, 0.5%, 1%, 1.5%, 2%, 2.5% and 3% in an inoculation amount of 2%, and cultured for 18h at 37 ℃. Escherichia coli is used as an indicator bacterium, and an oxford cup double-layer agar diffusion method is adopted for carrying out a bacteriostasis test.
The order of magnitude of range R is given by the factors in Table 21＞R3＞R4＞R2The main and secondary relationship of influence on the yield of the lactobacillus rhamnosus 1.0320 bacteriocin is that ultraviolet induction is greater than nitrogen source ratio, NaCl is greater than pH. From the K-values it can be determined that the best combination of lactobacillus rhamnosus 1.0320 bacteriocin production is: ultraviolet induction for 80s, adjustment of nitrogen source ratio (yeast powder: tryptone ═ 3.5:10.5), addition of NaCl of 1%, and initial pH of 6.4. The diameter of the zone of inhibition is increased from (26.24 +/-0.31) mm to (36.68 +/-0.51) mm.
TABLE 1 influence of single factors on the yield of bacteriocin of Lactobacillus rhamnosus 1.0320
TABLE 2 orthogonal test design and results table for yield optimization of Lactobacillus rhamnosus 1.0320 bacteriocin
Example 2:
immunoregulation effect of lactobacillus rhamnosus 1.0320 on immunodeficient model mice
Activating: 1mL of Lactobacillus rhamnosus 1.0320 preserved at-80 ℃ is taken out, streaked on an MRS-agar medium, and cultured at a constant temperature of 37 ℃ for 24 hours, and a single colony is picked. Inoculating the strain in a sterilized MRS broth culture medium for activation passage twice, and culturing at 37 ℃ for 12-16h at constant temperature to generate turbidity. Taking 100 mu L of bacterial liquid to perform three-zone lineation on a sterilized MRS-agar culture medium, culturing for 24h at the constant temperature of 37 ℃, selecting a single bacterial colony, observing under a microscope, and obtaining the qualified product without the pollution of mixed bacteria.
Preparing a sample: determining viable count of Lactobacillus rhamnosus 1.0320 bacteria liquid by plate counting method, centrifuging (4000r/min, 10min), washing for 3 times, and resuspending with sterile PBS (0.01M, pH 7.4) to 5 × 107CFU/mL、5×108CFU/mL、5×10910mL of each CFU/mL bacterial suspension was prepared in sterile PBS (0.01M, pH 7.4) in an amount of 15 mL.
Thirdly, molding: SPF-grade BALB/c female mice (weight 18 + -1 g; 6 weeks old) were randomly assigned: a blank control group (N), a model control group (M), a positive control group (P), a lactobacillus rhamnosus 1.0320 low dose group (1.0320-L), a lactobacillus rhamnosus 1.0320 medium dose group (1.0320-M) and a lactobacillus rhamnosus 1.0320 high dose group (1.0320-H). Except for a blank control group (replaced by equal volume of sterile PBS), cyclophosphamide is injected at a dose of 80mg/kg every day, and the weight of the mice is observed to continuously decrease for three consecutive days, which indicates that the cyclophosphamide causes the damage of the immune system of the mice, and the establishment of an immunosuppression model is successful.
Fourthly, intragastric administration: equal volumes of each group were given: sterile PBS, levamisole (2.5mg/mL), Lactobacillus rhamnosus 1.0320 (5X 10)7CFU/mL), Lactobacillus rhamnosus 1.0320 (5X 10)8CFU/mL) and Lactobacillus rhamnosus 1.0320 (5X 10)9CFU/mL) of the strain, wherein the weight, the proliferation capacity of T lymphocytes, the phagocytic capacity of abdominal macrophages, the activity of NK cells, the carbon clearance capacity, delayed hypersensitivity and the level of a cell inflammatory factor of the mouse are measured after continuous intervention for 30 days, as shown in the figure, the lactobacillus rhamnosus 1.0320 is found to be capable of remarkably relieving the weight reduction caused by immunodeficiency (figure 5), the improvement of the activity of immune cells is positively correlated with the viable count of lactobacillus rhamnosus 1.0320, and the high dose ((10) is used for (10)9CFU/mL)/0.02kg) of lactobacillus rhamnosus 1.0320 can promote phagocytic ability of macrophages (fig. 7) and carbon particle clearance ability (fig. 9) well, and has no significant difference (p > 0.05) from a positive control group; the effects of promoting T lymphocyte proliferation (fig. 6) and enhancing NK cell activity (fig. 8) were 1.06-fold and 1.54-fold, respectively, that of the positive control group. The delayed type hypersensitivity results are shown in figure 10, and simultaneously, lactobacillus rhamnosus 1.0320 can regulate the level of the cell inflammatory factor, play a role in relieving immunodeficiency by increasing the ratio of the IL-10 (figure 11) of the anti-inflammatory cytokine to the IL-12 of the proinflammatory cytokine and the level of immunoglobulin (figure 12), and determine the optimal dosage as (10)9CFU/mL)/0.02kg/d。
Example 3:
application of lactobacillus rhamnosus 1.0320 as freeze-dried bacterium powder with immune strengthening function
Activating: 1mL of Lactobacillus rhamnosus 1.0320 preserved at-80 ℃ is taken out, streaked on an MRS-agar medium, and cultured at a constant temperature of 37 ℃ for 24 hours, and a single colony is picked. Inoculating to sterilized MRS broth culture medium, activating and passaging twice, culturing at 37 deg.C for 12-16h, observing under microscope, and transferring to 40L fermentation tank for amplification culture after detection reaches standard.
Determining the viable count: adding 0.5mL of activated and amplified fermentation broth into 4.5mL of sterilized physiological saline, sequentially performing 10-fold gradient dilution, and selecting 10-8、10-9、10-10Diluting, spreading 200 μ L of diluent on sterile MRS-agar culture medium, culturing at 37 deg.C for 24 hr, counting, and determining viable count of fermentation broth to be 109CFU/mL。
Freeze-drying: centrifuging the fermentation liquor (4000r/min, 10min), collecting thalli cells, resuspending the thalli cells by using an equal-volume sterile PBS solution, wherein a freeze-drying protective agent is 5% sucrose skim milk powder (the concentration of the skim milk powder is 20%, and the concentration of sucrose is 5% (w/v)), and the rhamnose lactobacillus 1.0320 suspension and the sucrose skim milk powder freeze-drying protective agent are mixed according to the proportion of 1: 1(v/v), placing in a low-temperature refrigerator, and pre-cooling to-40 ℃. And simultaneously, the temperature of a freezing chamber of the freeze dryer is reduced to be below-40 ℃. After the sample is put in, a vacuum pump of a freeze dryer is opened to start freeze drying, the freeze drying time is about 24-72h, and the freeze-dried powder of lactobacillus rhamnosus 1.0320 is stored at-80 ℃, and the survival rate is about 49.6%.
The freeze-dried powder of Lactobacillus rhamnosus 1.0320 has immunity enhancing effect, and can be directly used as health product for storage at 0-4 deg.C, with viable count of 2 × 108CFU/g. Or used as an immune enhancing factor to be added into functional milk powder and prebiotics solid beverage. Has effects in improving proliferation ability of immune immunocyte, regulating phagocytic ability of macrophage, reducing inflammatory reaction, and regulating intestinal microecology.
Example 4:
application of lactobacillus rhamnosus 1.0320 and commercial leavening agent in food compounding
Activating: 1mL of Lactobacillus rhamnosus 1.0320 preserved at-80 ℃ is taken out, streaked on an MRS-agar medium, and cultured at a constant temperature of 37 ℃ for 24 hours, and a single colony is picked. Inoculating to sterilized MRS broth culture medium, activating and passaging twice, culturing at 37 deg.C for 12-16h, and observing under microscope without contamination.
Centrifugation: the fermentation broth was centrifuged (4000r/min, 10min), the cells were collected and resuspended in an equal volume of sterile 5% glucose solution.
③ fermenting the yoghourt: pretreating and standardizing raw milk, cooling to a fermentation temperature, and then inoculating a starter with an inoculation amount of 2%, wherein the composite starter is lactobacillus rhamnosus: streptococcus thermophilus: lactobacillus bulgaricus ═ 2: 4: and 4, the temperature of the fermentation tank is 42-45 ℃, the fermentation time is 2.5-3h, and when the pH value reaches 4.7, the temperature is reduced to 0-7 ℃ for stirring, so that the immune fortified yoghourt is obtained.
Fourthly, fermentation of pickled vegetables: fully washing fresh raw materials, adding 4% of salt and 2.5% of sucrose (w/w), inoculating a compound fermentation agent (lactobacillus rhamnosus 1.0320: lactobacillus plantarum 1:1) in an inoculation amount of 3%, fermenting at a constant temperature of 30 ℃ for 5 days to complete pickle, and preserving the pickle fermented by using lactobacillus rhamnosus 1.0320 as the fermentation agent for a longer time.
Claims (5)
1. A lactobacillus rhamnosus strain with immunoregulation function is characterized in that: lactobacillus rhamnosus (L.rhamnosus)1.0320 strain, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.15557, the preservation address is No. 3 of Xilu No.1 of Beijing Kogyo area, and the preservation date is No. 08 of 04.2018 at the institute of microbiology of China academy of sciences.
2. The lactobacillus rhamnosus strain with immunoregulation effect according to claim 1, characterized in that: the lactobacillus rhamnosus is isolated from traditional marmilk wine.
3. The lactobacillus rhamnosus strain with immunoregulation effect according to claim 1, characterized in that: the bacterial colony of the lactobacillus rhamnosus is milk white, the edge is neat, the surface is smooth and convex, and the thallus is in a short rod shape.
4. Use of lactobacillus rhamnosus with immunomodulatory effects according to any of claims 1 to 3, characterized in that: the lactobacillus rhamnosus is applied to the fermentation process of yoghourt or pickled vegetables.
5. Use of lactobacillus rhamnosus with immunomodulatory effects according to any of claims 1 to 3, characterized in that: the Lactobacillus rhamnosus is used for preparing freeze-dried powder.
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| CN112940985A (en) * | 2021-03-31 | 2021-06-11 | 盐城维康生物科技有限公司 | Lactobacillus rhamnosus preparation for enhancing human immunity and preparation method thereof |
| CN113621572A (en) * | 2021-08-03 | 2021-11-09 | 东北农业大学 | Application of lactobacillus rhamnosus in adhering colon cells |
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| CN114774315B (en) * | 2022-04-13 | 2023-12-19 | 微康益生菌(苏州)股份有限公司 | Application of lactobacillus rhamnosus strain LRa05 in preparation of immunity enhancing product and/or eczema relieving product |
| CN115851535A (en) * | 2022-12-10 | 2023-03-28 | 万益生物科技(山东)有限公司 | Lactobacillus rhamnosus WFP52 with function of regulating immunity and application thereof |
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