CN118059188A - Bifidobacterium longum SF-B-60 metazoan bacterial agent and preparation method and application thereof - Google Patents
Bifidobacterium longum SF-B-60 metazoan bacterial agent and preparation method and application thereof Download PDFInfo
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- CN118059188A CN118059188A CN202410495445.5A CN202410495445A CN118059188A CN 118059188 A CN118059188 A CN 118059188A CN 202410495445 A CN202410495445 A CN 202410495445A CN 118059188 A CN118059188 A CN 118059188A
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- bifidobacterium longum
- metazoan
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- fermentation
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Abstract
The invention relates to the technical field of biological agents, in particular to a bifidobacterium longum SF-B-60 metazoan microbial agent, a preparation method and application thereof. The bifidobacterium longum SF-B-60 metazoan agent is prepared by fermenting a substrate to be fermented through bifidobacterium (Bifidobacterium longum) SF-B-60; the fermented substrate comprises the following components in parts by weight: 1 part of liquorice, 1 part of dendrobium, 1 part of medlar, 1 part of kudzuvine root, 1 part of orange peel and 25 parts of purified water; bifidobacterium longum SF-B-60 has been deposited in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at 11 and 15 days 2023, and has a deposit number of CGMCC No.29022, and a deposit address of North Star, west Hill No. 1, kogyo area of Beijing. The Bifidobacterium longum SF-B-60 metazoan agent has the functions of regulating immune cells and improving diarrhea.
Description
Technical Field
The invention relates to the technical field of biological agents, in particular to a bifidobacterium longum SF-B-60 metazoan microbial agent, a preparation method and application thereof.
Background
There are billions of beneficial bacteria in the human body, most of which are located in the digestive system. The digestive system is an important component of the systemic immune system and it contains the submucosal lymphoid tissues of the intestinal tract, including lymph nodes, lymphatic vessels, spleen, mesentery, etc. These tissues and organs play an important role in protecting against invasion of external pathogens, maintaining environmental stability in the intestinal tract, and the like. There is a complex interaction of beneficial bacteria in the digestive system with the immune system. In daily life, people can improve their immunity through diet and lifestyle, for example, intake of foods rich in probiotics (such as yogurt, fermented bean curd, etc.) or supplementation of probiotic preparations.
The metazoan is also called as inactivated probiotics, and is the metabolite component of the probiotics after the probiotics are processed and treated, and the metazoan comprises thalli and metabolites. Compared with probiotics, the metazoan has high safety, can withstand higher temperature, is convenient to store and transport, and has wide application range. The use of the metazoan can avoid the problems of narrow application industry, low bioavailability, unstable effect, easy transfer of drug resistance genes and the like of the live bacteria while obtaining the similar probiotics efficacy of the probiotics, and is widely applied to the industries of health-care food, food processing and the like.
Most of the existing metazoan products are prepared by mixing a plurality of strains with other components, such as metazoan milk tablets disclosed in CN112544721A and a preparation method thereof, and the metazoan products are complex in preparation method and unstable in product performance due to the fact that metazoan preparation of a plurality of probiotics is involved. In addition, few reports on the preparation of immunity-improving metaplasia products with the functions of regulating immune cells and improving diarrhea after fermentation of single strain bifidobacterium longum are made in the prior art.
Disclosure of Invention
Aiming at the technical problems that a plurality of strains are required to be mixed for a metazoan product in the prior art, the preparation method is complex, and the immunity enhancing function is to be enhanced, the invention provides a bifidobacterium longum SF-B-60 metazoan microbial agent, the preparation method and the application thereof, and the bifidobacterium longum SF-B-60 is fermented by a fermented substrate, so that the obtained metazoan microbial agent has good immunity enhancing effect.
In a first aspect, the invention provides a bifidobacterium longum SF-B-60 metazoan agent prepared by fermenting a substrate to be fermented by bifidobacterium longum (Bifidobacterium longum) SF-B-60; the fermented substrate comprises the following components in parts by weight: 1 part of liquorice, 1 part of dendrobium, 1 part of medlar, 1 part of radix puerariae, 1 part of orange peel and 25 parts of purified water; bifidobacterium longum SF-B-60 has been deposited in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at 11 and 15 days 2023, and has a deposit number of CGMCC No.29022, and a deposit address of North Star, west Hill No.1, kogyo area of Beijing.
Further, the bifidobacterium longum SF-B-60 metazoan agent also comprises isomaltooligosaccharide.
In a second aspect, the invention provides a preparation method of a bifidobacterium longum SF-B-60 metazoan agent, which comprises the following steps:
(1) Uniformly mixing the components of the liquid culture medium, inoculating 1% of bifidobacterium longum SF-B-60, and performing anaerobic culture to obtain an inoculant, wherein the culture temperature is 37 ℃, the culture time is 36h, and the liquid culture medium comprises the following components in parts by weight: 20g of glucose, 10g of brown sugar, 10g of skimmed milk powder, 6g of peptone, 4g of yeast extract powder, 2g of sodium acetate, 0.5g of dipotassium hydrogen phosphate, 0.5g of ammonium citrate, 0.05g of manganese sulfate, 0.1g of magnesium sulfate, 0.5g of L-cysteine hydrochloride and 1000g of water;
(2) Mixing the components of the substrate to be fermented, sieving with a 120-mesh sieve, sterilizing at 121 ℃ for 15min, inoculating an inoculant into the sterilized substrate to be fermented, performing anaerobic fermentation at 37 ℃ for 24h, performing anaerobic fermentation at 28 ℃ for 6h, and performing anaerobic fermentation at 37 ℃ for 24h to obtain a fermentation liquor;
(3) Breaking the wall of the fermentation liquor to obtain wall-broken fermentation liquor;
(4) Spray drying the wall-broken fermentation liquid to obtain wall-broken fermentation powder, mixing the wall-broken fermentation powder with isomaltooligosaccharide, and preparing into the bifidobacterium longum SF-B-60 metabacteria agent with the total colony count of 1.0X10 10 CFU/g.
Further, the mass ratio of the wall-broken fermentation powder to the isomaltooligosaccharide is 1:0.5-1.5.
Further, the mass ratio of the wall-broken fermentation powder to the isomaltooligosaccharide is 1:1.
In a third aspect, the invention provides an application of a bifidobacterium longum SF-B-60 metazoan agent in preparation of a product for regulating immune cell functions.
The product for regulating immune cell function is a medicine for regulating immune cell function.
Further, modulating immune cell function includes increasing proliferation capacity of spleen lymphocytes, increasing phagocytic capacity of mononuclear-macrophages, and increasing NK cell activity.
In a fourth aspect, the invention provides an application of a bifidobacterium longum SF-B-60 metazoan agent in preparing a product for improving diarrhea.
The diarrhea-improving product is a diarrhea-improving medicine.
The invention has the beneficial effects that:
The bifidobacterium longum SF-B-60 metazoan agent provided by the invention does not need to be compounded by a plurality of strains, has a simple preparation method, can regulate the immune cell function, can improve diarrhea, and can effectively enhance immunity. Experiments show that compared with the post-biotic bacteria agent prepared from commercial bifidobacterium longum and the post-biotic bacteria agent of bifidobacterium longum SF-B-60 obtained under different fermentation conditions, the post-biotic bacteria agent of bifidobacterium longum SF-B-60 provided by the invention can regulate immune cell functions, including improving the proliferation capacity of spleen lymphocytes, improving the phagocytic capacity of mononuclear-macrophages and NK cell activity, and can also relieve diarrhea caused by immunity reduction to a certain extent and strengthen physique.
The fermented substrate components of the bifidobacterium longum SF-B-60 metazoan agent have the following effects:
Licorice root: tonifying qi, strengthening middle-jiao, eliminating phlegm, relieving cough, detoxifying, and relieving pain;
Dendrobe: stomach benefiting, salivation promoting, yin nourishing and heat clearing;
medlar: nourishing liver and kidney, replenishing essence, improving eyesight, and enhancing immunity;
Radix Puerariae: clearing and activating the channels and collaterals, ascending yang and stopping diarrhea;
orange peel: promoting qi circulation, invigorating spleen, regulating stomach function, relieving vomit, eliminating dampness, and eliminating phlegm.
Detailed Description
In order to better understand the technical solutions of the present invention, the following description will clearly and completely describe the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
EXAMPLE 1 isolation of seed
(1) Bacterial source: yoghurt, 11 months 2022, was collected in the inner Mongolian municipality of China.
(2) Isolation of strains:
Sampling the yoghurt by using a sterilized sampling spoon, avoiding pollution in the sampling process, immediately preserving the yoghurt in a refrigerating box after sampling, numbering and recording, weighing 1g of the yoghurt sample, dissolving in 10mL of physiological saline, uniformly mixing, carrying out ten-time gradient dilution on the sample liquid, uniformly coating 100 mu L of each gradient on an improved MRS flat plate culture medium, then taking out the sample, placing the sample in an anaerobic tank, and carrying out anaerobic culture at 37 ℃ for overnight; after the cultivation is finished, different colonies with different forms and sizes grow on the flat plate, the colony features are selected to be round, convex, milky white or white, opaque and smooth and soft, mycelium is not formed, microscopic examination is carried out, after the microscopic examination is finished, the mycelium of bifidobacterium is selected as a microscopic examination result, further scribing and purifying treatment are carried out, and the flat plate anaerobic tank is activated for 1 day, so that the culture is obtained. And then strain identification is carried out.
Example 2 identification of species
(1) Authentication unit
Bioengineering (Shanghai) Co., ltd.
(2) Primer(s)
27F：5'-AGAGTTTGATCCTGGCTCAG-3'；
1492R：5'-GGTTACCTTGTTACGACTT-3'。
(3) Identification of sequences
The identification sequences are shown below:
AGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGGATCCATCAAGCTTGCTTGGTGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATACACCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGTTCCAGTTGATCGCATGGTCTTCTGGGAAAGCTTTCGCGGTATGGGATGGGGTCGCGTCCTATCAGCTTGACGGCGGGGTAACGGCCCACCGTGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTATCGGGGAGCAAGCGAGAGTGAGTTTACCCGTTG-AATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGTCGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGACGGTCGTAGAGATACGGCTTCCCTTCGGGGCGGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCCGTGTTGCCAGCGGATTATGCCGGGAACTCACGGGGGACCGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGACGCGGCGACGCGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGAATCAGCAACGTCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGCACCCGAAGCCGGTGGCCTAACCCCTTGTGGGATGGAGCCGTCTAAGGTGAGGCTCGTGATTGGGACTAAGTCGTA
(4) Identification result
The strain was identified as Bifidobacterium, presumably Bifidobacterium longum (Bifidobacterium longum), designated as Bifidobacterium longum SF-B-60, and was sent to China general microbiological culture Collection center for preservation with the following information:
The bifidobacterium longum (Bifidobacterium longum) SF-B-60 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on the 11 th month 15 of 2023, and has a preservation address of North Star Xiyu No. 1,3 in the Korean region of Beijing city and a preservation number of CGMCC No.29022.
Example 3
Bifidobacterium longum SF-B-60 metazoan agent
The fermented substrate comprises the following components in parts by weight: 1 part of liquorice, 1 part of dendrobium, 1 part of medlar, 1 part of radix puerariae, 1 part of orange peel and 25 parts of purified water;
The liquid culture medium comprises the following components in parts by weight: 20g of glucose, 10g of brown sugar, 10g of skimmed milk powder, 6g of peptone, 4g of yeast extract powder, 2g of sodium acetate, 0.5g of dipotassium hydrogen phosphate, 0.5g of ammonium citrate, 0.05g of manganese sulfate, 0.1g of magnesium sulfate, 0.5g of L-cysteine hydrochloride and 1000g of water;
The preparation method comprises the following steps:
(1) Uniformly mixing the components of the liquid culture medium, inoculating 1% of bifidobacterium longum SF-B-60, and performing anaerobic culture to obtain an inoculant, wherein the culture temperature is 37 ℃, and the culture time is 36 hours;
(2) Mixing the components of the substrate to be fermented, sieving with a 120-mesh sieve, sterilizing at 121 ℃ for 15min under high pressure, cooling to 37 ℃, inoculating an inoculant into the sterilized substrate to be fermented, performing anaerobic fermentation at 37 ℃ for 24h with an inoculum size of 1%, performing anaerobic fermentation at 28 ℃ for 6h, and performing anaerobic fermentation at 37 ℃ for 24h to obtain a fermentation liquor;
(3) Conveying the fermentation liquor to a high-pressure spraying wall breaking machine for wall breaking treatment to obtain wall breaking fermentation liquor;
(4) Spray drying the wall-broken fermentation liquid to obtain wall-broken fermentation powder, wherein the mass ratio of the wall-broken fermentation powder to the isomaltooligosaccharide is (1): 1 to prepare the bifidobacterium longum SF-B-60 metazoan bacterial agent with the total colony count of 1.0X10 10 CFU/g.
Example 4
Bifidobacterium longum SF-B-60 metazoan agent
The difference from example 3 is that the wall-broken fermentation powder and the isomaltooligosaccharide are mixed according to the mass ratio of 1:0.5, and preparing the bifidobacterium longum SF-B-60 metazoan bacterial agent with the total colony count of 1.0X10 10 CFU/g.
Example 5
Bifidobacterium longum SF-B-60 metazoan agent
The difference from example 3 is that the wall-broken fermentation powder and the isomaltooligosaccharide are mixed according to the mass ratio of 1:1.5, and preparing the bifidobacterium longum SF-B-60 metazoan bacterial agent with the total colony count of 1.0X10 10 CFU/g.
Comparative example 1
Bifidobacterium longum BL21 metazoan microbial inoculum
The difference from example 3 is that Bifidobacterium longum SF-B-60 is replaced with a commercially available Bifidobacterium longum BL21, bifidobacterium longum BL21 being purchased from Micro Kang Yisheng (Suzhou) Co.
Comparative example 2
Bifidobacterium longum SF-B-60 metazoan agent
The difference from example 3 is that, unlike the fermented substrate of bifidobacterium longum SF-B-60, the fermented substrate comprises the following components in parts by weight: 1 part of liquorice, 1 part of radix glehniae, 1 part of astragalus, 1 part of radix puerariae, 1 part of orange peel and 25 parts of purified water.
Wherein, the radix glehniae has the effects of clearing heat, nourishing yin, facilitating stomach and promoting fluid production; semen astragali Complanati has effects of invigorating kidney, supporting yang, nourishing liver, improving eyesight, and enhancing immunity.
Experimental example 1 mouse experiment
ConA-induced mouse spleen lymphocyte transformation assay (MTT method)
(1) Grouping and administration
SPF-class BALB/c male mice were first adaptively bred for one week and then randomly divided into 4 groups of 10, each of which was divided into a control group, a group of Bifidobacterium longum SF-B-60 metazoan agent 1 (Bifidobacterium longum SF-B-60 metazoan agent prepared in gavage example 3), a group of Bifidobacterium longum SF-B-60 metazoan agent 2 (Bifidobacterium longum SF-B-60 metazoan agent prepared in gavage comparative example 2) and a group of Bifidobacterium longum BL21 metazoan agent (Bifidobacterium longum BL21 metazoan agent prepared in gavage comparative example 1);
In the following four weeks, the corresponding metazoan bacteria are respectively irrigated to the stomach of the group 1 of the bifidobacterium longum SF-B-60 metazoan bacteria, the group 2 of the bifidobacterium longum SF-B-60 metazoan bacteria and the group 21 of the bifidobacterium longum BL21 metazoan bacteria, the amount of the irrigated stomach is 1 mg/day, and the contrast group is irrigated with normal saline with the same amount of the stomach.
(2) Experimental procedure
By the fifth week, splenocytes from mice were taken and assayed, each spleen cell suspension was added to a 24-well culture plate in two wells, 1mL per well, 75 μl of ConA solution was added to one well, and the other well was used as a control and placed in a 5% CO 2,37℃ CO2 incubator for 72h. 4h before the end of the culture, 0.7mL of supernatant was gently aspirated from each well, 0.7mL of RPMI1640 medium containing no calf serum was added, and 50. Mu.L/well of MTT (5 mg/mL) was added at the same time, and the culture was continued for 4h. After the cultivation is finished, 1mL of acidic isopropanol is added into each hole, and the mixture is blown and evenly mixed, so that the purple crystals are completely dissolved. Then, the cells were packed in 96-well plates, and 2 parallel wells were packed in each well, and the optical density value was measured at 570nm using an ELISA reader. The solution was also transferred directly into a 2mL cuvette and the OD was measured on a 721 spectrophotometer at a wavelength of 570 nm.
Subtracting the optical density value of the ConA-added hole from the optical density value of the ConA-added hole to represent the proliferation capacity of lymphocytes, and judging that the experimental result is positive if the optical density difference of the tested sample group is obviously higher than that of the control group.
(3) Experimental results
Experimental results of ConA-induced mouse spleen lymphocyte transformation assay (MTT assay) are shown in table 1:
TABLE 1 Effect of metabacteria on cellular immune function
* Indicating significant differences compared to the control group (P < 0.05); # The difference between the group 1 of the bifidobacterium longum SF-B-60 metazoan agent and the group 2 of the bifidobacterium longum SF-B-60 metazoan agent is obvious (P < 0.05).
As is clear from Table 1, the spleen lymphocyte transformation rate of the bifidobacterium longum SF-B-60 post-natant 1 group and the bifidobacterium longum SF-B-60 post-natant 2 group are significantly higher than those of the control group, which indicates that the bifidobacterium longum SF-B-60 post-natant of the example 3 and the comparative example 2 has the cell immunity function, the spleen lymphocyte transformation rate of the bifidobacterium longum SF-B-60 post-natant 1 group is significantly higher than that of the bifidobacterium SF-B-60 post-natant 2 group, it is shown that the bifidobacterium longum SF-B-60 post-natant 2 group is prepared by fermenting a substrate to be fermented by using the bifidobacterium longum SF-B-60, but the effect is better because the components of the substrate to be fermented are different, and the effect of the bifidobacterium longum SF-B-60 post-natant of the embodiment 3 is better in effect of regulating spleen lymphocytes and has the effect of improving immunity.
2. Carbon clearance test in mice
(1) Grouping and administration
Group (1) and administration in the ConA-induced mouse spleen lymphocyte transformation experiments.
(2) Experimental procedure
20. Mu.L of blood was taken from the inner canthus venous plexus 2min (t 2）、10min（t1) after each mouse was injected with ink, and immediately added to 2mL of 0.1% Na 2CO3 solution, and the optical density was measured at 600nm wavelength using 721 spectrophotometer (OD 1 is the optical density obtained by taking blood at 2min, OD 2 is the optical density obtained by taking blood at 10 min), and Na 2CO3 solution was used as a blank.
Mice were sacrificed, livers and spleens were taken, the viscera surface blood was blotted with filter paper, and each was weighed to indicate the ability of the mice to carbon clearance in terms of phagocytosis index. The phagocytosis index alpha is calculated as follows. The phagocytic index of the tested sample group is obviously higher than that of the control group, and the positive result of the test can be judged.
(3) Experimental results
The function of mononuclear-macrophages is phagocytic and foreign body digestion. The effect of the metazoan on mouse mononuclear-macrophage function was therefore known from the experimental results of the mouse carbon clearance experiments, and the results are shown in table 2:
TABLE 2 Effect of metazoan agents on mouse mononuclear-macrophage function
* Indicating significant differences compared to the control group (P < 0.05); # The difference between the group 1 of the bifidobacterium longum SF-B-60 metazoan agent and the group 2 of the bifidobacterium longum SF-B-60 metazoan agent is obvious (P < 0.05).
As can be seen from Table 2, the carbon clearance phagocytosis index of the group 1 of the bifidobacterium longum SF-B-60 post-natant and the group 2 of the bifidobacterium longum SF-B-60 post-natant are significantly higher than those of the control group, which indicates that the bifidobacterium longum SF-B-60 post-natant can improve the function of mononuclear macrophages, and the carbon clearance phagocytosis index of the group 1 of the bifidobacterium longum SF-B-60 post-natant is significantly higher than that of the group Yu Chang of the bifidobacterium SF-B-60 post-natant, which indicates that the bifidobacterium longum SF-B-60 post-natant of example 3 has better effect of improving the function of mononuclear macrophages and has stronger immune function.
NK cell Activity assay
(1) Grouping and administration
Group (1) and administration in the ConA-induced mouse spleen lymphocyte transformation experiments.
(2) Experimental procedure
Taking 100 mu L of target cells (24 Hyac-1 cells after passage are added into RPMI1640 complete culture solution, and the cell concentration is adjusted to be 4 multiplied by 10 5/ml) and effector cells (spleen suspension is prepared by taking spleen from a mouse) respectively (the effective target ratio is 50:1), and adding the target cells and the effector cells into a reaction well of a U-shaped 96-well culture plate; natural release Kong Jiaba cells of target cells and 100 μl of culture medium, maximum release Kong Jiaba cells of target cells and 100 μl of 1% NP 40; two duplicate wells are arranged on each of the above, the culture is carried out for 4 hours in a culture box with 5% CO 2 at 37 ℃, then a 96-well culture plate is centrifuged for 5 minutes at 1500r/min, 100 mu L of supernatant is sucked by each well and placed in a 96-well culture plate with a flat bottom, 100 mu L of LDH matrix liquid is added at the same time, the reaction is carried out for 3 minutes, 1mol/L of HCl is added in each well, and the optical density value (OD) is measured at 490nm of an enzyme-labeling instrument.
And calculating the NK cell activity according to the following formula, wherein the NK cell activity of the tested sample group is obviously higher than that of the control group, and the positive result of the test result can be judged.
(3) Experimental results
NK cells are heterogeneous multifunctional immune cells, natural killer cells unrelated to specific immune responses. NK cells do not need to be immunized or sensitized in advance, and the killing effect is earlier than that of other effector cells, so that the NK cells are the first defense line for resisting tumors of organisms. The NK cell activity improving compound has important significance for immunoregulation. The effect of the metazoan agent on NK cell activity was verified by the experimental result of NK cell activity assay (lactate dehydrogenase assay LDH assay). The results are shown in Table 3:
TABLE 3 Effect of metabacteria on NK cell Activity
* Indicating significant differences compared to the control group (P < 0.05); # The difference between the group 1 of the bifidobacterium longum SF-B-60 metazoan agent and the group 2 of the bifidobacterium longum SF-B-60 metazoan agent is obvious (P < 0.05).
As can be seen from Table 3, the NK cell activities of the bifidobacterium longum SF-B-60 post-natant 1 group and the bifidobacterium longum SF-B-60 post-natant 2 group are significantly higher than those of the control group, which indicates that the bifidobacterium longum SF-B-60 post-natant of example 3 and comparative example 2 can improve the NK cell activity and the immunity adjustment capability, and the NK cell activity of the bifidobacterium longum SF-B-60 post-natant 1 group is significantly higher than that of the bifidobacterium SF-B-60 post-natant 2 group, which indicates that the bifidobacterium longum SF-B-60 post-natant of example 3 has a stronger immunity enhancement function.
4. Relieving diarrhea
(1) Grouping and administration
SPF-grade BALB/c male mice of 6 weeks old, which are 18-20g in weight, are fed adaptively first in the first week and are randomly divided into 5 groups, each group of 10, specifically, a control group, a model group, a group of bifidobacterium longum SF-B-60 metazoan agents 1 (bifidobacterium longum SF-B-60 metazoan agent solution prepared in gavage example 3), a group of bifidobacterium SF-B-60 metazoan agents 2 (bifidobacterium longum SF-B-60 metazoan agent solution prepared in gavage comparative example 2) and a group of bifidobacterium longum BL21 metazoan agents (bifidobacterium longum BL21 metazoan agent solution prepared in gavage comparative example 1);
1g of the bifidobacterium longum SF-B-60 post-microbial agent of example 3, the bifidobacterium longum SF-B-60 post-microbial agent of comparative example 2 and the bifidobacterium longum BL21 post-microbial agent of comparative example 1 were taken and dissolved in 0.25ml of physiological saline, respectively, to obtain corresponding post-microbial agent solutions.
(2) Experimental procedure
In the next second week, the corresponding metazoan solutions of the metazoan agent 1 group of the bifidobacterium longum SF-B-60 metazoan agent 2 group of the bifidobacterium longum SF-B-60 metazoan agent and the metazoan agent group of the bifidobacterium longum BL21 are respectively irrigated with physiological saline with the same amount of the stomach of 0.2 ml/day, and the control group and the model group are irrigated with the physiological saline with the same amount of the stomach. In the third week, three groups, except the control group, were left to treat with streptomycin (5 g/liter) in drinking water for 3 days to disorder the intestinal flora of the mice. The water containing streptomycin was replaced with sterile water while feeding was stopped 18h before molding and from day 4 of the third week, the control group was filled with 3 times daily with normal saline, the model group was filled with 1 time normal saline and then 2 times with 1.2X10 11 CFU/mL ETEC O78: k80 suspension, each mouse was perfused with 0.2mL, four consecutive days with 2h intervals, the cage was replaced after intragastric ETEC, no padding was added, instead filter paper was laid in the cage, and diarrhea and mortality of the mice were observed every 2h for four days. The corresponding metazoan bacteria solution is first filled into the bifidobacterium longum SF-B-60 metazoan bacteria group and bifidobacterium longum BL21 metazoan bacteria group, and then the corresponding metazoan bacteria solution is filled into the stomach for 2 times per day, wherein the length of the metazoan bacteria solution is 1.2 multiplied by 10 11 CFU/mL, and the ETEC O78 is as follows: k80 suspension, 0.2mL per lavage of each mouse, the remainder of treatment was performed on mice of the same model group.
(3) Index detection
During the molding period, the mice were weighed at the same time each day. The day before mice were sacrificed, the mouse feces were collected, and the water content before and after lyophilization was measured, the feces water content (%) = (feces wet weight-feces dry weight)/feces wet weight×100%. After 24h of the last gastric lavage, blood is collected from the orbital venous plexus, the blood is kept still, 4000g is centrifuged for 10min, serum is centrifugally taken and stored in a refrigerator at-80 ℃, and the expression levels of inflammatory factors TNF-alpha, IL-6 and IL-10 in the serum are measured by an ELISA method. The level of AQP3, ST, TLR4 and IgA in the serum of the mice is detected by adopting an ELISA method.
(4) Experimental results
1) Weight change and fecal moisture content
Prolonged diarrhea can lead to weight loss in mice and disfigurement of feces. Thus, by recording the weight change and fecal moisture content of the mice, diarrhea symptom changes can be known. The results are shown in Table 4:
TABLE 4 effects of metazoan agents on weight changes and fecal moisture content in mice
* Indicating significant differences compared to the model group (P < 0.05); # Indicating that the difference between the group 1 of the bifidobacterium longum SF-B-60 metazoan agent and the group 2 of the bifidobacterium longum SF-B-60 metazoan agent is significant (P < 0.05); @ The difference between the group 1 of the bifidobacterium longum SF-B-60 metazoan agent and the group of the bifidobacterium longum BL21 metazoan agent is obvious (P < 0.05).
As can be seen from Table 4, the model group showed that the diarrhea model was successful because the feces were not molded due to diarrhea, the water content was too much and thin, and the weight was severely reduced, as compared with the control group. Compared with the model group, the water content of the mice in the three metazoan agent groups is obviously reduced, the weight reduction is slowed down, and the effects of improving diarrhea, reducing the water content of excrement and enhancing immunity of the three metazoan agents are shown, but the efficacy of the bifidobacterium longum SF-B-60 metazoan agent 1 group is obviously higher than that of the bifidobacterium longum SF-B-60 metazoan agent 2 group and the bifidobacterium longum BL21 metazoan agent group from the data.
2) The effect on serum inflammatory factors and the results are shown in table 5:
TABLE 5 Effect of metabacteria on serum inflammatory factors
* Indicating significant differences compared to the model group (P < 0.05); # Indicating that the difference between the group 1 of the bifidobacterium longum SF-B-60 metazoan agent and the group 2 of the bifidobacterium longum SF-B-60 metazoan agent is significant (P < 0.05); @ The difference between the group 1 of the bifidobacterium longum SF-B-60 metazoan agent and the group of the bifidobacterium longum BL21 metazoan agent is obvious (P < 0.05).
From Table 5, it is clear that the three metazoan agent groups have an effect of improving inflammatory factors at the levels of inflammatory factors IL-6, IL-10 and TNF- α, but the improvement effect of the bifidobacterium longum SF-B-60 metazoan agent group 1 is remarkably high in Yu Chang bifidobacterium SF-B-60 metazoan agent group 2 and bifidobacterium longum BL21 metazoan agent group.
3) Effects on diarrhea related proteins, results are shown in table 6:
TABLE 6 Effect of metazoan agents on diarrhea-associated proteins
* Indicating significant differences compared to model group drop (P < 0.05); # Indicating that the difference between the group 1 of the bifidobacterium longum SF-B-60 metazoan agent and the group 2 of the bifidobacterium longum SF-B-60 metazoan agent is significant (P < 0.05); @ The difference between the group 1 of the bifidobacterium longum SF-B-60 metazoan agent and the group of the bifidobacterium longum BL21 metazoan agent is obvious (P < 0.05).
As can be seen from table 6, in the index of diarrhea-related proteins, all of the three groups of post-natal microbial agents had an effect of adjusting diarrhea-related proteins, but the adjustment effect of group 1 of bifidobacterium longum SF-B-60 post-natal microbial agents was significantly higher than that of group 2 of bifidobacterium longum SF-B-60 post-natal microbial agents and group 2 of bifidobacterium longum BL21 post-natal microbial agents.
Experimental example 2 piglet experiment
(1) Experimental procedure
40 Healthy piglets, which were weaned 21 days old, were purchased from a farm in city of the Utility, and randomly divided into 4 groups, which were respectively a control group, a bifidobacterium longum SF-B-60 post-natant 1 group (fed with the bifidobacterium longum SF-B-60 post-natant prepared in example 3), a bifidobacterium longum SF-B-60 post-natant 2 group (fed with the bifidobacterium longum SF-B-60 post-natant prepared in comparative example 2), and a bifidobacterium BL21 post-natant group (fed with the bifidobacterium BL21 post-natant prepared in comparative example 1), 10 piglets were each group, and the test period was 14 days. Feeding feed to a control group; the corresponding metazoan agents are respectively added into the feed from the group 1 of the bifidobacterium longum SF-B-60 metazoan agents, the group 2 of the bifidobacterium longum SF-B-60 metazoan agents and the group 21 metazoan agents, so that the total colony count in the feed is 0.5 multiplied by 10 6 CFU/g. The weight of the piglets is recorded on days 0 and 14 of the test, and the whole-course feed intake and diarrhea of the piglets are recorded. Daily weight gain, daily feed intake, feed/meat ratio, diarrhea index (sum of diarrhea scores of piglets per group/(total number of piglets x total number of days)) and the like were calculated at the end of the experiment.
The diarrhea scoring criteria were as follows:
Dry manure, 1 minute; no diarrhea, normal dry feces, 2 minutes; slight diarrhea, slightly wet manure surface, semi-dry manure, 3 minutes; moderate diarrhea, soft manure, slightly thin, 4 minutes; severe diarrhea, watery feces, 5 minutes.
(2) Experimental results
As shown in Table 7, the weight of piglets in the group 1 of the post-production bacteria SF-B-60, the group 2 of the post-production bacteria SF-B-60 and the group 21 of the post-production bacteria BL21 of bifidobacterium were equivalent in 0 day, but the weight of piglets in the group 1 of the post-production bacteria SF-B-60 of bifidobacterium and the average daily gain were significantly increased as compared with the control group, and the weight of piglets in the group 1 of the post-production bacteria SF-B-60 of bifidobacterium was the fastest and significantly different from those in the group 2 of the post-production bacteria SF-B-60 of bifidobacterium and the group BL21 of bifidobacterium.
The meat-to-feed ratio of the group 1 of the bifidobacterium longum SF-B-60 metazoan agent is obviously lower than that of the other three groups; the diarrhea index is obviously lower than that of a control group and a bifidobacterium longum BL21 post-metazoan agent group; the serum of the three metazoan agent groups has the IgG content remarkably higher than that of the control group, and the IgG content of the bifidobacterium longum SF-B-60 metazoan agent 1 group is remarkably higher than that of the other three groups; the contents of TNF-alpha and IL-1 beta of the bifidobacterium longum SF-B-60 metazoan agent 2 group and the bifidobacterium longum BL21 metazoan agent group are obviously lower than those of a control group, and the contents of TNF-alpha and IL-1 beta of the bifidobacterium longum SF-B-60 metazoan agent 1 group are obviously lower than those of other three groups, which indicates that the bifidobacterium longum SF-B-60 metazoan agent 1 group can strengthen the constitution of piglets and improve the immunity.
TABLE 7 influence of metabacteria on piglet related indicators
Experimental example 3 volunteer experiment
The patients with diarrhea, who had abnormal indexes of immunoglobulin, neutrophil and leukocyte in the blood routine recruited to the hospital for treatment, were administered the bifidobacterium longum SF-B-60 post-natal agent of example 3 after having signed informed consent with the patients, and the experiment was performed for 30 days, and the patients were administered the bifidobacterium longum SF-B-60 post-natal agent 3g every day without changing the dietary structure and life habit of the patients themselves. During the test, the stool morphology of the test patients was recorded every 10 days, and after 30 days, the condition of the patients was counted.
According to statistics, the abnormal stool formation condition of diarrhea patients is improved, the normal dry stool form is presented, and the indexes of immunoglobulin, neutrophil and leucocyte in blood routine detection are also changed into normal.
Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.
Claims (8)
1. A bifidobacterium longum SF-B-60 metazoan agent is characterized in that the agent is prepared by fermenting a substrate to be fermented through bifidobacterium longum (Bifidobacterium longum) SF-B-60; the fermented substrate comprises the following components in parts by weight: 1 part of liquorice, 1 part of dendrobium, 1 part of medlar, 1 part of radix puerariae, 1 part of orange peel and 25 parts of purified water; bifidobacterium longum SF-B-60 has been deposited in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at 11 and 15 days 2023, and has a deposit number of CGMCC No.29022, and a deposit address of North Star, west Hill No. 1, kogyo area of Beijing.
2. The bifidobacterium longum SF-B-60 metazoan agent of claim 1, further comprising isomaltooligosaccharide.
3. A method for preparing the bifidobacterium longum SF-B-60 metazoan agent according to claim 1, comprising the steps of:
(1) Uniformly mixing the components of the liquid culture medium, inoculating 1% of bifidobacterium longum SF-B-60, and performing anaerobic culture to obtain an inoculant, wherein the culture temperature is 37 ℃, the culture time is 36h, and the liquid culture medium comprises the following components in parts by weight: 20g of glucose, 10g of brown sugar, 10g of skimmed milk powder, 6g of peptone, 4g of yeast extract powder, 2g of sodium acetate, 0.5g of dipotassium hydrogen phosphate, 0.5g of ammonium citrate, 0.05g of manganese sulfate, 0.1g of magnesium sulfate, 0.5g of L-cysteine hydrochloride and 1000g of water;
(2) Mixing the components of the substrate to be fermented, sieving with a 120-mesh sieve, sterilizing at 121 ℃ for 15min, inoculating an inoculant into the sterilized substrate to be fermented, performing anaerobic fermentation at 37 ℃ for 24h, performing anaerobic fermentation at 28 ℃ for 6h, and performing anaerobic fermentation at 37 ℃ for 24h to obtain a fermentation liquor;
(3) Breaking the wall of the fermentation liquor to obtain wall-broken fermentation liquor;
(4) Spray drying the wall-broken fermentation liquid to obtain wall-broken fermentation powder, mixing the wall-broken fermentation powder with isomaltooligosaccharide, and preparing into the bifidobacterium longum SF-B-60 metabacteria agent with the total colony count of 1.0X10 10 CFU/g.
4. The preparation method of the bifidobacterium longum SF-B-60 metabacteria agent as claimed in claim 3, wherein the mass ratio of the wall-broken fermentation powder to the isomaltooligosaccharide is 1:0.5-1.5.
5. The preparation method of the bifidobacterium longum SF-B-60 metabacteria agent as claimed in claim 4, wherein the mass ratio of the wall-broken fermentation powder to the isomaltooligosaccharide is 1:1.
6. Use of a bifidobacterium longum SF-B-60 metazoan agent according to claim 1 for the preparation of a product for modulating immune cell function.
7. The use of claim 6, wherein modulating immune cell function comprises increasing proliferation capacity of spleen lymphocytes, increasing phagocytic capacity of monocytes-macrophages, and increasing NK cell activity.
8. Use of a bifidobacterium longum SF-B-60 metazoan agent according to claim 1 for the preparation of a product for improving diarrhea.
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