CN118064332A - Lactococcus lactis subspecies JYLL-72 for promoting bone growth, application and preparation thereof - Google Patents
Lactococcus lactis subspecies JYLL-72 for promoting bone growth, application and preparation thereof Download PDFInfo
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of microorganisms, in particular to a lactococcus lactis subspecies JYLL-72 for promoting bone growth, and application and a preparation thereof. Lactococcus lactis subspecies (Lactococcus lactis subsp.lactis) JYLL-72 were deposited in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 3-11 days of 2024, and the deposited address is North Star Xiyu No. 1, 3 in the Korean region of Beijing, and the deposited number is CGMCC No.29993. According to the invention, the vitamin K2 content of the fermentation liquor is detected, and the lactococcus lactis subspecies JYLL-72 are screened out, so that the strain can participate in bone metabolism, promote skeletal development of young mice, and realize growth of the young mice in a short time.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a lactococcus lactis subspecies JYLL-72 for promoting bone growth, and application and a preparation thereof.
Background
At present, formula milk powder, formula health food and mineral supplements are several common bone growth promoting supplement options that function to promote bone growth primarily by providing the components of matter normally required by the human body. However, the sales price of formula milk powder and formula health food is usually expensive, and the cost is high after long-term administration; taking mineral supplements is prone to over-supplementation, causing damage to the body, thereby reducing the functioning of various aspects of the body. In recent years, the use of probiotics in contributing to long products has become a hotspot for research.
Current studies on bone growth promoting effects of probiotics have mainly focused on lactobacillus species such as lactobacillus acidophilus, lactobacillus fermentum, bifidobacterium lactis, bifidobacterium longum, lactobacillus johnsonii and the like. Chinese patent CN 116904378A discloses a strain of lactobacillus johnsonii (Lactobacillus johnsonii) JYLO-010 that promotes bone growth. Experiments prove that the strain can obviously improve the content of 25-hydroxy vitamin D in a mouse body, and further promote the apparent absorption rate of the mouse to calcium, so that the femoral bone density of the mouse is obviously improved; in addition, lactobacillus johnsonii JYLO-010 is effective in promoting proliferation, differentiation and maturation of osteoblasts and inhibiting osteoclast formation. Other, genus-classified probiotics have less research on bone growth promoting effects.
Meanwhile, with the continuous and deep research, vitamin K2 is found to have the effects of activating osteocalcin, increasing bone mineralization and the like, and has positive effects on bone metabolism.
Based on the above, the development of more different species and genus classification of probiotics with the effects of promoting bone growth and providing vitamin K2 has important significance.
Disclosure of Invention
Aiming at the technical problem of lack of lactococcus lactis with bone growth promoting effect at present, the invention provides a lactococcus lactis subspecies JYLL-72 for promoting bone growth, and application and preparation thereof.
In a first aspect, the present invention provides a lactococcus lactis subspecies (Lactococcus lactis subsp. Lactis) JYLL-72 for promoting bone growth, which has been deposited in China general microbiological culture Collection center, with a deposit address of Seagate No. 1, no. 3 of North Star, kogyo area, beijing, and a deposit number of CGMCC No.29993, at day 11 of 2024.
In a second aspect, the invention provides an application of the lactococcus lactis subspecies JYLL-72 in preparing fungus powder for promoting bone growth, and further the fungus powder or a preparation containing the fungus powder can be used as a medicine for promoting bone growth.
Furthermore, the fungus powder has the effect of producing vitamin K2 by fermentation.
Further, the bacterial powder has the effect of maintaining the levels of alanine aminotransferase, aspartic acid aminotransferase and alkaline phosphatase in serum.
Furthermore, the bacterial powder has the effects of increasing the expression level of OPG protein and reducing the expression level of RANKL and CTSK protein.
In a third aspect, the present invention provides a formulation for promoting bone growth comprising the bacterial powder of lactococcus lactis subspecies JYLL-72 as described above.
Further, glucose, isomaltooligosaccharide or fructooligosaccharide is also included.
Further, the number of cells of lactococcus lactis subspecies JYLL-72 in the preparation was 1.0X10. 10 10~2.0×1010 cfu/g.
Further, the preparation method of the bacterial powder comprises the following steps:
Performing stationary culture on activated lactococcus lactis subspecies JYLL-72 in an MRS liquid culture medium at a constant temperature of 37 ℃ for 12 hours to obtain seed liquid, inoculating the seed liquid into the MRS liquid culture medium, performing stationary culture at a constant temperature of 37 ℃ for 24 hours under anaerobic conditions to obtain bacterial liquid, and centrifuging, concentrating and freeze-drying the bacterial liquid to obtain bacterial powder.
The invention has the beneficial effects that:
According to the invention, the vitamin K2 content of the fermentation liquor is detected, and the lactococcus lactis subspecies JYLL-72 are screened out, so that the strain can participate in bone metabolism, promote skeletal development of young mice, and realize growth of the young mice in a short time. Lactococcus lactis subspecies JYLL-72 stimulate intestinal cells to secrete insulin-like growth factor-1 (IGF-1), a hormone known to act on bone growth, to promote osteoblast metabolism, including affecting bone formation and bone resorption, to mediate host growth and development. Lactococcus lactis subspecies JYLL-72 can induce an increase in IGF-1, thereby affecting bone growth and health.
The invention fully proves the effective action of lactococcus lactis subspecies JYLL-72 through growth and development index evaluation, fecal condition evaluation, alanine aminotransferase, aspartic acid aminotransferase and alkaline phosphatase level evaluation, IGF-1 concentration detection and bone immunohistochemical analysis of young mice.
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.
The preparation method of the MRS solid culture medium used in the specific embodiment of the invention comprises the following steps: 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 20g of glucose, 5g of sodium acetate, 2g of triammonium citrate, 2g of dipotassium hydrogen phosphate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 1mL of Tween 80 and 15g of agar powder are taken, dissolved by deionized water, fixed to 1L, pH is adjusted to 6.3, and sterilized by 115 ℃ high-pressure steam for 30 minutes, poured into a plate and cooled to form a solid.
The preparation method of the MRS liquid culture medium used in the specific embodiment of the invention comprises the following steps: taking 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 20g of glucose, 5g of sodium acetate, 2g of triammonium citrate, 2g of dipotassium hydrogen phosphate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate and 801 mL of tween, dissolving with deionized water, fixing the volume to 1L, adjusting the pH value to 6.3, sterilizing with 115 ℃ high-pressure steam for 30 minutes, pouring into a plate, and cooling to obtain the product.
Example 1 isolation, screening and identification of species
1. Isolation and purification of strains
(1) Sampling: taking cheese in Linzhi city of the Tibetan autonomous region, 7 months in 2022, and transporting the cheese back to a laboratory for preparing a sample by a cold chain;
(2) Preparing a sample: ① 1g of cheese is added into 10mL of sterile physiological saline, mixed evenly by shaking, and then diluted with the sterile physiological saline in a gradient way, wherein the concentration gradients are respectively 10 -1、10-2、10-3、10-4、10-5、10-6、10-7.
② 100 Mu L of each of 10 -5、10-6、10-7 gradient dilutions was applied to MRS solid medium, and the culture was allowed to stand at 37℃in an anaerobic incubator for 48 hours.
(3) Purification of the strain: according to colony characteristics of colony diameter of 0.5-1.5 mm, neat edge and milky yellow, single colony is selected to be subjected to three-region streaking on MRS solid culture medium, after streaking, the culture is carried out for 48 hours at 37 ℃ under anaerobic condition, the operation is repeated, six pure strains are obtained after total 3 streaking and purification, the pure strains are sequentially named as strain 1-strain 6, and the pure strains are added into an glycerol pipe to be preserved at-80 ℃.
2. Strain screening for fermented vitamin K2
Six pure strains are added into MRS liquid culture medium according to the inoculation amount of 2 percent by volume, the static culture is carried out for 24 hours at 37 ℃, the vitamin K2 content in the fermentation broth is measured, and the detection method is according to GB 5009.290-2023. The results are shown in Table 1 below.
TABLE 1 vitamin K2 content (unit: mg/L) in fermentation broths of different strains
From the data in Table 1, the highest vitamin K2 content in the fermentation broth of strain 2 resulted in the production of bone proteins from vitamin K2, which together with calcium produced bone mass and increased bone density.
3. Authentication and preservation
The strain 2 was sent to an identification unit of ataxia, a biotech company, and the primers used in the identification were as follows:
27F：5'-AGAGTTTGATCCTGGCTCAG-3'；
1492R：5'-CTACGGCTACCTTGTTACGA-3'。
the strain was identified as Lactococcus lactis subsp.
The identified strain is named JYLL-72, and is delivered to China general microbiological culture Collection center for storage, wherein the storage time is 2024, 3 months and 11 days, the storage address is North Star Xiyu No.1, 3 in the Korean region of Beijing, the storage number is CGMCC No.29993, and the strain is classified and named as lactococcus lactis subsp Lactococcus lactis subsp.
The gene sequence of the lactococcus lactis subspecies JYLL-72 is as follows:
TTTCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTTGAGCGCTGAAGGTTGGTACTTGTACCGACTGGATGAGCAGCGAACGGGTGAGTAACGCGTGGGGAATCTGCCTTTGAGCGGGGGACAACATTTGGAAACGAATGCTAATACCGCATAAAAACTTTAAACACAAGTTTTAAGTTTGAAAGATGCAATTGCATCACTCAAAGATGATCCCGCGTTGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGATGATACATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGGTAGAGAAGAACGTTGGTGAGAGTGGAAAGCTCATCAAGTGACGGTAACTACCCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGTGGTTTATTAAGTCTGGTGTAAAAGGCAGTGGCTCAACCATTGTATGCATTGGAAACTGGTAGACTTGAGTGCAGGAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGCCTGTAACTGACACTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGATGTAGGGAGCTATAAGTTCTCTGTATCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATACTCGTGCTATTCCTAGAGATAGGAAGTTCCTTCGGGACACGGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTGTTAGTTGCCATCATTAAGTTGGGCACTCTAACGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTCGCGAGACAGTGATGTTTAGCTAATCTCTTAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGGGAGTTGGGAGTACCCGAAGTAGGTTGCCTAACCGCAAGGAGGGCGCTTCCTAAGGTAAGACCGATGACTGGGGTGAAGTCGTAA.
EXAMPLE 2 preparation of lactococcus lactis subspecies JYLL-72 powder
Activating lactococcus lactis subspecies JYLL-72 preserved at-80 ℃ on an MRS solid culture medium, picking and transferring one single colony after activation into 100mL of MRS liquid culture medium, standing at a constant temperature of 37 ℃ for 12 hours to obtain seed liquid, inoculating the seed liquid into the MRS liquid culture medium according to an inoculum size of 1% (v/v), and standing at a constant temperature of 37 ℃ for 24 hours under anaerobic conditions to obtain bacterial liquid with a viable count of 1.0X10 10 cfu/mL.
And (3) centrifuging the bacterial liquid, concentrating, freezing and drying to obtain bacterial powder.
EXAMPLE 3 preparation of lactococcus lactis subspecies JYLL-72 powder
Activating lactococcus lactis subspecies JYLL-72 preserved at-80 ℃ on an MRS solid culture medium, picking and transferring one single colony after activation into 100mL of MRS liquid culture medium, standing at a constant temperature of 37 ℃ for 12 hours to obtain seed liquid, inoculating the seed liquid into the MRS liquid culture medium according to an inoculum size of 2% (v/v), and standing at a constant temperature of 37 ℃ for 24 hours under anaerobic conditions to obtain bacterial liquid with a viable count of 2.0X10 10 cfu/mL.
And (3) centrifuging the bacterial liquid, concentrating, freezing and drying to obtain bacterial powder.
EXAMPLE 4 preparation of lactococcus lactis subspecies JYLL-72 powder
Activating lactococcus lactis subspecies JYLL-72 preserved at-80 ℃ on an MRS solid culture medium, picking 1 single colony after activation, transferring into 100mL of MRS liquid culture medium, standing at a constant temperature of 37 ℃ for 12 hours to obtain seed liquid, inoculating the seed liquid into the MRS liquid culture medium according to an inoculum size of 1% (v/v), and standing at a constant temperature of 37 ℃ for 24 hours under anaerobic conditions to obtain bacterial liquid with a viable count of 1.5X10 10 cfu/mL.
And (3) centrifuging the bacterial liquid, concentrating, freezing and drying to obtain bacterial powder.
Example 5 animal experiments
SD pups were purchased from the university of Nanchang laboratory animal center. After weaning of the 3-week-old SD pups, 40 male SD pups were randomly divided into a control group, JYLL-72 low dose group, JYLL-72 medium dose group, and JYLL-72 high dose group, 10 each. These pups were kept in iron cages at room temperature of 25 ℃ and fed with sufficient water and food to maintain a 12 hour alternating shade.
The control group is filled with 0.2mL of sterilized normal saline every day, the JYLL-72 low-dose group is filled with 0.2mL of bacterial liquid every day (the viable count is 0.2X10 8 cfu/mL), the JYLL-72 medium-dose group is filled with 0.2mL of bacterial liquid every day (the viable count is 0.5X10 8 cfu/mL), the JYLL-72 high-dose group is filled with 0.2mL of bacterial liquid every day (the viable count is 1X 10 8 cfu/mL), and each treatment group bacterial liquid is prepared by mixing and diluting the lactococcus lactis subspecies JYLL-72 bacterial powder prepared in the example 2 with the sterilized normal saline.
After 28 days of gastric lavage, young mice are sacrificed by diethyl ether, blood samples are immediately collected via ocular veins, and serum is taken after centrifugation and stored in a refrigerator at-80 ℃ for later use. Taking the tibia of the young mouse, and placing the tibia into neutral tissue fixing solution for fixing and preserving.
1. Assessment of growth and development index
The length of the pups (from the tip of the nose to the anus) was measured every two weeks and the results are shown in table 2 below.
TABLE 2 different groups of different growth time young mice length (unit: cm)
Note that: * P <0.05 compared to the control group.
As can be seen from the data in Table 2, the length of each group of young mice is not obviously different in the initial first day, and the length of each group of young mice is different in 14 days, which shows that JYLL-72 fungus powder can obviously promote the growth of the length of the young mice and promote the whole growth and development.
2. Fecal condition scoring
The stool of the pups was collected on day 14 of the experiment and scored for stool status of the pups.
The normal feces should be brown in color, cylindrical in shape and moderate in hardness; no obvious peculiar smell; the particles should be uniform and have moderate size.
The faeces of the young mice were observed and scored according to the following scoring criteria.
Appearance: 0 is no abnormality in color, shape and texture; a score 1 indicates a slight abnormality in color, shape, or texture; a score 2 indicates a moderate anomaly in color, shape, or texture; a score 3 indicates a severe anomaly in color, shape, or texture;
wherein, the abnormal color of the excrement is mainly black or white, the abnormal shape of the excrement is mainly liquid, and the abnormal texture is mainly too dry or too thin.
Smell: 0 score indicates no obvious off-flavor; score 1 indicates slight off-flavors; 2 points represent moderate odor; score 3 indicates severe off-flavors.
Fecal particles: a 0 division indicates that the particles are very uniform (size uniformity ratio > 80%); 1 denotes that the particle size is substantially uniform (size uniformity ratio >50% and 80%); 2 parts represent that the particle size is not uniform (the uniform size accounts for more than 10 percent and less than or equal to 50 percent); the 3-division indicates that the particle size is completely nonuniform (the uniform size is less than or equal to 10%).
Adding the scores to obtain a total score, and judging whether the fecal condition is good or not according to the total score:
the total division is 0-3: indicating good fecal condition and healthy intestinal tract.
The total division is 4-6: indicating moderate abnormality in fecal condition and moderate problems in intestinal health.
The total division is 7-9: indicating severe abnormalities in fecal condition, and significant problems with intestinal health.
As shown in Table 3, the control group had a moderate problem in fecal condition, and the fecal condition was well strained with increasing JYLL-72 doses, indicating that JYLL-72 powder could significantly improve the fecal condition of young mice, thereby maintaining intestinal health and ensuring normal growth.
Table 3 fecal condition score for 14 days in different groups of pups
Note that: * P <0.05 compared to the control group.
3. Detection of serum indicators
Alanine aminotransferase (Alanine aminotransferase, ALT), aspartic acid aminotransferase (ASPARTATE AMINOTRANSFERASE, AST) and alkaline phosphatase (Alkaline phosphatase, ALP) levels in serum were detected using an automated biochemical analyzer (Chemray, 240). IGF-ELISA (enzyme linked immunosorbent assay, ELISA) kit (Shanghai, ind.) detects IGF-1 concentration in serum.
As can be seen from the data in table 4, the ALT, AST and ALP levels in serum of JYLL-72 and JYLL-72 high dose groups were significantly different from those of the control group, and the JYLL-72 powder was able to maintain alanine aminotransferase, aspartic acid aminotransferase and alkaline phosphatase levels in serum, thereby maintaining liver function, which is the main secretion organ of IGF-1, and thus increasing IGF-1 levels in serum.
TABLE 4 serum index levels of different groups of mice
Note that: * P <0.05 compared to control group; # indicates P <0.01 compared to the control group.
4. Bone immunohistochemical analysis
The tibial sections were analyzed by immunohistochemistry. The tibia was removed from the neutral tissue fixative and decalcified in 10% formic acid-formalin solution for one month. The decalcified tibia was cut longitudinally, embedded in paraffin, and cut into slices. Then adding the ready-to-use pepsin repairing liquid on the slices, and placing the wet box containing the slices into a baking oven at 37 ℃ for 20 minutes for antigen repairing. The sections were then placed in PBS solution (ph=7.4) and washed 3 times for 5 minutes each on a decolorizing shaker. The sections were then incubated in 3% hydrogen peroxide solution at room temperature for 25 minutes in the dark to block endogenous peroxidase. The decoloring process of the previous step is then repeated. Sections were incubated with 3% bovine serum albumin for 30 minutes at room temperature. Primary antibodies (OPG primary antibody was purchased from Servicebio, RANKL primary antibody was purchased from hawk, CTSK primary antibody was purchased from Abcam) were dropped onto the sections and placed in wet boxes for incubation overnight at 4 ℃. The incubated sections were washed 3 times with 5 minutes each in PBS solution on a destaining shaker. The sections were then incubated with horseradish peroxidase-labeled secondary antibody (purchased from Servicebio company) for 50 minutes at room temperature, followed by staining with DAB. All sections were counterstained with hematoxylin for 3 min. The sections were dehydrated, examined microscopically and images were collected, and the Average optical density values (AVERAGE INTEGRATED optical density, average IOD) in the sections were quantitatively assessed using Image-Pro Plus 6.0 software to analyze the levels of OPG, RANKL and CTSK protein expression in the tibia, the results are shown in table 5.
TABLE 5 expression levels of OPG, RANKL and CTSK proteins in the tibia of different groups of young mice
Note that: # indicates P <0.01 compared to the control group.
Both OPG and RANKL are osteoblast secreted proteins, and OPG can inhibit the physiological effects of RANKL and promote bone formation, and RANKL promotes the formation of osteoclasts. CTSK protein is highly expressed in osteoclasts, while RANKL is capable of stimulating expression of CTSK, further promoting osteoclast activity.
As can be seen from the data in table 5, all three test groups significantly increased the expression level of OPG protein, reduced the expression level of RANKL and CTSK protein, indicating that JYLL-72 bacterial powder can significantly affect the expression level of OPG, RANKL and CTSK protein in tibia, and significantly maintain the normal bone growth of young mice.
EXAMPLE 6 preparation of preparation for promoting bone growth
The lactococcus lactis subspecies JYLL-72 powder prepared in example 2 was mixed with glucose, and a preparation containing 1.0X10 10 cfu/g of cells was prepared, and glucose was purchased from shan east king sugar industry Co.
EXAMPLE 7 preparation of preparation for promoting bone growth
The lactococcus lactis subspecies JYLL-72 powder prepared in example 3 was mixed with isomaltooligosaccharide, and a preparation containing 2.0X10. 10 10 cfu/g of the cells was prepared, and the isomaltooligosaccharide was purchased from Botrytis cinerea Co., ltd.
EXAMPLE 8 preparation of preparation for promoting bone growth
The lactococcus lactis subspecies JYLL-72 powder prepared in example 4 was mixed with fructooligosaccharides, and a preparation containing 1.5X10 10 cfu/g of cells was prepared, and the fructooligosaccharides were purchased from Botrytis cinerea Co., ltd.
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 (9)
1. The lactococcus lactis subspecies JYLL-72 for promoting bone growth is characterized in that the lactococcus lactis subspecies (Lactococcus lactis subsp. Lactis) JYLL-72 are preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 3-11 days of 2024, and the preservation address is North Star Xiyu No. 1, 3 in the Korean region of Beijing, and the preservation number is CGMCC No.29993.
2. Use of the lactococcus lactis subspecies JYLL-72 according to claim 1 for the preparation of a powder for promoting bone growth.
3. The use according to claim 2, wherein the bacterial powder has the effect of producing vitamin K2 by fermentation.
4. The use according to claim 2, wherein the bacterial powder has the effect of maintaining the levels of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase in serum.
5. The use according to claim 2, wherein the bacterial powder has the effect of increasing the expression level of OPG protein and decreasing the expression level of RANKL and CTSK protein.
6. A formulation for promoting bone growth comprising the bacterial powder of lactococcus lactis subspecies JYLL-72 as defined in claim 1.
7. The preparation of claim 6, wherein the preparation method of the bacterial powder comprises the following steps:
Performing stationary culture on activated lactococcus lactis subspecies JYLL-72 in an MRS liquid culture medium at a constant temperature of 37 ℃ for 12 hours to obtain seed liquid, inoculating the seed liquid into the MRS liquid culture medium, performing stationary culture at a constant temperature of 37 ℃ for 24 hours under anaerobic conditions to obtain bacterial liquid, and centrifuging, concentrating and freeze-drying the bacterial liquid to obtain bacterial powder.
8. The formulation of claim 6, further comprising glucose, isomaltooligosaccharide, or fructooligosaccharide.
9. A formulation according to any one of claims 6 to 8, wherein the number of cells of lactococcus lactis subspecies JYLL-72 in the formulation is 1.0 x 10 10~2.0×1010 cfu/g.
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