Lactococcus lactis subsp. Lactis JYLL-28 with function of inhibiting formation of fatty liver, bacterial powder and application

Abstract

The invention relates to the technical field of microorganisms, in particular to lactococcus lactis subsp. Lactis JYLL-28 with a function of inhibiting fatty liver formation, bacterial powder and application, the lactococcus lactis subsp. Lactis JYLL-28 is preserved in the China General Microbiological Culture Collection Center on July 24, 2023, the preservation address is No. 3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.1089. The lactococcus lactis subsp. Lactis JYLL-28 is JYLL-28. The preservation number is CGMCC (China General Microbiological Culture Collection Center) The lactococcus lactis subsp. Lactis JYLL-28 is used for inhibiting weight increase of rats, regulating and controlling the blood fat level, reducing the liver inflammation level and the serum endotoxin level, inhibiting fatty liver formation and preventing liver fatty degeneration, and has no toxic or side effect and a lasting curative effect.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Lactococcus lactis subspecies JYLL-28 with the function of inhibiting the formation of fatty liver, bacterial powder and application thereof. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) includes simple fatty liver (SFL), non-alcoholic steatohepatitis (NASH) and related cirrhosis. It refers to a clinical pathological syndrome characterized by excessive fat deposition in hepatocytes, excluding alcohol and other clear liver-damaging factors. NAFLD is an acquired metabolic stress liver injury closely related to insulin resistance and genetic susceptibility. It has become an important cause of chronic liver disease in economically developed areas. In these areas, the prevalence of NAFLD in ordinary adults is about 10%-30%, of which 10%-20% is NASH, and the latter has a cirrhosis incidence rate of up to 25% within 10 years. Non-alcoholic fatty liver disease (NAFLD) refers to a clinical pathological syndrome characterized by excessive fat deposition in hepatocytes, excluding alcohol and other clear liver-damaging factors. It is an acquired metabolic stress liver injury closely related to insulin resistance and genetic susceptibility. In addition to directly causing decompensated cirrhosis, hepatocellular carcinoma, and recurrence of transplanted liver, nonalcoholic fatty liver disease can also affect the progression of other chronic liver diseases and participate in the onset of type 2 diabetes and atherosclerosis. Metabolic syndrome-related malignant tumors, arteriosclerotic cardiovascular and cerebrovascular diseases, and cirrhosis are important factors affecting the quality of life and life expectancy of patients with nonalcoholic fatty liver disease.

[0003] The treatment methods for non-alcoholic fatty liver disease include basic treatment, drug treatment and surgical treatment. Basic treatment refers to adjusting the patient's living habits. In terms of dietary habits, the total intake of calories, sugar and fat in food should be controlled and the intake of dietary fiber should be increased; in terms of exercise habits, moderate-intensity aerobic exercise should be performed regularly. Basic treatment is slow to take effect and is only suitable for patients with mild symptoms, with the risk of recurrence. Drug treatment mainly refers to the use of drugs for liver protection and anti-fibrosis treatment. If non-alcoholic fatty liver disease is combined with other diseases, targeted treatment measures should be taken for other diseases on the basis of liver protection and anti-fibrosis treatment. Long-term use of drugs can cause liver and kidney damage, and can also cause the body to develop drug resistance and drug resistance. The treatment effect decreases as the time of taking drugs increases. Surgical treatment is mainly for patients with terminal non-alcoholic fatty liver disease and patients with severe liver failure. This treatment method is mainly liver resection or liver transplantation, which causes great damage to the patient's body and brings great mental and economic pressure to the patient. In addition, follow-up treatment of surgical treatment often requires continued medication, and it is difficult to avoid side effects caused by drugs. Summary of the invention

[0004] In view of the technical problems of long medication cycle and obvious side effects in the drug treatment method of non-alcoholic fatty liver disease in the prior art, the present invention provides a strain of Lactococcus lactis subspecies JYLL-28 with the function of inhibiting the formation of fatty liver, bacterial powder and application thereof. Lactococcus lactis subspecies JYLL-28 is used to inhibit the weight gain of rats, regulate blood lipid levels, reduce liver inflammation levels and serum endotoxin levels, inhibit the formation of fatty liver, and prevent liver fatty degeneration, with no toxic side effects and long-lasting therapeutic effect.

[0005] In a first aspect, the present invention provides a strain of Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. lactis ) JYLL-28, Lactococcus lactis subspecies JYLL-28 was deposited in the General Microbiology Center of China Culture Collection Administration on July 24, 2023. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 27995.

[0006] In a second aspect, the present invention provides a bacterial powder containing the Lactococcus lactis subsp. lactis JYLL-28 as described above, and a method for preparing the bacterial powder comprises the following steps: Step 1: activating Lactococcus lactis subspecies JYLL-28 on MRS solid medium to obtain activated colonies; Step 2: Pick an activated bacterial colony and transfer it to MRS liquid medium for anaerobic culture to obtain seed liquid; Step 3: inoculating the seed solution into a new MRS liquid culture medium for anaerobically culturing to obtain a culture solution; Step 4: Centrifuge and freeze-dry the culture solution to obtain JYLL-28 bacterial powder.

[0007] Further, the MRS solid culture medium includes the following raw materials: 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of potassium hydrogen phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate, 15g of agar, and 1000mL of distilled water. The MRS solid culture medium is sterilized before use, the sterilization temperature is 121°C, the sterilization pressure is 0.1MPa, and the sterilization time is 20min.

[0008] Further, the MRS liquid culture medium includes the following raw materials: 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of potassium dihydrogen phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate, and 1000mL of distilled water. The MRS liquid culture medium is sterilized before use, the sterilization temperature is 121°C, the sterilization pressure is 0.1MPa, and the sterilization time is 20min.

[0009] Furthermore, in step 2, the amount of MRS liquid culture medium is 100 mL, the temperature of anaerobic culture is 37° C., and the time of anaerobic culture is 12 h.

[0010] Furthermore, in step three, the inoculation amount of the seed solution is 1% by volume, the temperature of the anaerobic culture is 37° C., and the time of the anaerobic culture is 24 h.

[0011] Furthermore, in step 3, the viable bacterial count of the culture solution is 1.0×10 10 cfu / mL.

[0012] In a third aspect, the present invention also provides a use of the bacterial powder as described above in the preparation of a medicine for treating non-alcoholic fatty liver disease.

[0013] Furthermore, non-alcoholic fatty liver disease is simple fatty liver.

[0014] The beneficial effects of the present invention are: The invention provides a strain of Lactococcus lactis subspecies JYLL-28 with the function of inhibiting the formation of fatty liver, bacterial powder and application thereof. Lactococcus lactis subspecies JYLL-28 is used to inhibit the weight gain of rats, reduce the lipid level in the liver, and effectively inhibit the formation of fatty liver; the levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein (LDL) in the serum of rats are reduced, and the level of high-density lipoprotein (HDL) is increased, thereby reducing the incidence of fatty liver combined with hyperlipidemia and preventing the progression of fatty liver; the levels of tumor necrosis factor TNF-α and serum endotoxin in the liver of rats are reduced, thereby preventing the progression of fatty liver to non-alcoholic steatohepatitis and fatty liver fibrosis. DETAILED DESCRIPTION

[0015] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0016] Example 1 Isolation and identification of strains 1. Screening and purification of strains (1-1) Strain source: Sterile MRS liquid culture medium placed in a pepper shed for one week and collected in Qingzhou City, Shandong Province, China on April 17, 2023.

[0017] (1-2) Sample preparation: S1: Add 1 mL of the culture solution in step (1-1) to 9 mL of 0.85 wt% sterile saline, shake and mix evenly to obtain a mixed solution for later use.

[0018] S2: Use sterile saline to dilute the mixture obtained in step S1 by 10 times in a gradient manner to 10% of the original concentration. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 times, and label them in sequence as sample 1, sample 2, sample 3, sample 4, sample 5, sample 6 and sample 7 for use.

[0019] (1-3) Preparation of MRS solid medium: Mix 10 g of peptone, 5 g of beef powder, 5 g of sodium acetate trihydrate, 2 g of potassium hydrogen phosphate heptahydrate, 1 mL of Tween 80, 0.05 g of manganese sulfate tetrahydrate, 2 g of triammonium citrate, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate, 15 g of agar and 1000 mL of distilled water, stir evenly at a natural pH value, and then sterilize at 121°C and 0.1 MPa for 20 min to obtain a sterilized MRS medium. Pour the sterilized MRS medium into a plate and let it cool to obtain an MRS solid medium for standby use.

[0020] (1-4) Cultivation: 100 µL of liquid was taken from each of samples 1 to 7, and spread on the MRS solid culture medium in steps (1-3), and cultured at 37°C under anaerobic conditions for 48 h.

[0021] (1-5) Selecting colonies: After the colonies are formed, single colonies are selected according to the following colony characteristics: diameter 1-2mm, round colonies, neat edges, slightly white color with a bulge in the middle.

[0022] (1-6) Separation and purification: The single colony selected in step (1-5) was inoculated onto MRS solid culture medium by streak method and cultured at 37°C under anaerobic conditions for 48 h. The streak purification was repeated three times for the single colony to obtain six pure strains. The six pure strains were marked as alternative strain 1, alternative strain 2, alternative strain 3, alternative strain 4, alternative strain 5 and alternative strain 6, respectively. Alternative strains 1 to alternative strain 6 were added to glycerol tubes and stored at -80°C.

[0023] 2. Screening of strains with cholesterol-lowering and triglyceride-lowering functions (2-1) Preparation of MRS liquid culture medium: Mix 10 g of peptone, 5 g of beef powder, 5 g of sodium acetate trihydrate, 2 g of potassium hydrogen phosphate heptahydrate, 1 mL of Tween 80, 0.05 g of manganese sulfate tetrahydrate, 2 g of triammonium citrate, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate and 1000 mL of distilled water, stir evenly at a natural pH value, and then sterilize at 121°C and 0.1 MPa for 20 min to obtain a sterilized MRS liquid culture medium, and let the sterilized MRS liquid culture medium cool for standby use.

[0024] (2-2) Preparation of MRS high cholesterol medium (MRS-Choesterol, MRS-CHO): First, weigh 1g of cholesterol, dissolve it in anhydrous ethanol and dilute to 100mL to prepare a cholesterol ethanol solution with a concentration of 10.0g / L. Then use a filter membrane with a pore size of 0.45μm to filter out bacteria in the cholesterol ethanol solution to obtain a cholesterol ethanol sterile solution. Then add the cholesterol ethanol sterile solution to the MRS liquid culture medium and mix well to prepare an MRS high cholesterol culture medium with a cholesterol concentration of 0.1g / L.

[0025] (2-3) Preparation of MRS-Triglyceride medium (MRS-TG): ①Preparation of triglyceride emulsion: First, prepare 150 mL of 2 wt% polyvinyl alcohol (PVA, average degree of polymerization is 1750) solution, and then use a filter membrane with a pore size of 0.45 μm to filter out bacteria in the polyvinyl alcohol aqueous solution to obtain a sterile polyvinyl alcohol aqueous solution. Take 50 mL of vegetable oil and mix it with the sterile polyvinyl alcohol aqueous solution to obtain a premix. Use an ultrasonic tissue homogenizer to perform high-speed homogenization on the premix three times in a row, and each high-speed homogenization treatment lasts for 5 minutes to fully dissolve the lipids in the polyvinyl alcohol aqueous solution to obtain a milky white triglyceride emulsion. The triglyceride emulsion is stored in a refrigerator at -20°C and ultrasonically treated before use.

[0026] ②Prepare MRS triglyceride culture medium: 40 mL of triglyceride emulsion was added into 1000 mL of MRS liquid culture medium and mixed evenly to obtain MRS triglyceride culture medium.

[0027] (2-4) Preparation of ammonium ferric sulfate solution First, 4.463 g of ammonium ferric sulfate was dissolved in 100 mL of 85 wt% phosphoric acid, and the mixture was evenly mixed to obtain an ammonium ferric sulfate-phosphoric acid solution. Then, 10 mL of the ammonium ferric sulfate-phosphoric acid solution was dissolved in concentrated sulfuric acid and the volume was fixed to 100 mL to obtain an ammonium ferric sulfate solution.

[0028] (2-5) Strain preparation: 2-5-1: The candidate strains 1 to 6 stored at -80°C were inoculated into MRS liquid culture medium respectively, placed in a constant temperature incubator and cultured aseptically at 37°C for 24 hours to obtain seed solutions of the candidate strains 1 to 6.

[0029] 2-5-2: Inoculate 1% of the seed liquid of alternative strains 1 to 6 into new MRS liquid culture medium, place it in a constant temperature incubator and culture it aseptically at 37°C for 24 hours.

[0030] 2-5-3: Repeat step 2-5-2 three times to fully restore the activity of alternative strains 1 to alternative strains 6 to obtain the culture fluid of activated alternative strains 1 to alternative strains 6.

[0031] 2-5-4: The culture solution of the activated candidate strains 1 to 6 was centrifuged at 8000 r / min for 10 min, the supernatant was discarded, and the precipitate obtained by centrifugation was washed three times with sterilized PBS buffer to obtain a bacterial suspension of the candidate strains 1 to 6. The concentration of the bacterial suspension of the candidate strains 1 to 6 was adjusted to 1×10 9 cfu / mL, and the activated bacterial suspensions of alternative strains 1 to 6 were obtained for use.

[0032] (2-6) Drawing cholesterol standard curve using ammonium ferrous sulfate method Use anhydrous ethanol to prepare a cholesterol standard solution with a concentration of 0.1g / L. Take 0μL, 200μL, 400μL, 600μL, 800μL, 1000μL, 1200μL, 1400μL and 1600μL of the standard solution and add them to sterilized dry centrifuge tubes. The capacity of the centrifuge tube is 10mL. Then add anhydrous ethanol to each centrifuge tube to control the total volume of the liquid in each centrifuge tube to 2mL. Then slowly add 2mL of the ammonium ferric sulfate solution prepared in step (2-4) to each centrifuge tube, and use a vortex mixer to shake and mix. After shaking and mixing, place each centrifuge tube at room temperature for 30 minutes to fully develop color. Finally, read the absorbance value A of the standard solution in each centrifuge tube at a wavelength of 560nm on a spectrophotometer. 560 The concentration of the standard solution in each centrifuge tube is used as the horizontal axis, A 560 Plot a standard curve for the ordinate.

[0033] (2-7) Determination of cholesterol degradation efficiency The activated bacterial suspensions of candidate strains 1 to 6 were inoculated into MRS-CHO medium at a volume fraction of 10%, and cultured at 37°C for 48 hours in a constant temperature shaker at a speed of 160 r / min to obtain fermentation broths of candidate strains 1 to 6. Then, the cholesterol degradation efficiencies of candidate strains 1 to 6 were detected respectively.

[0034] The cholesterol degradation efficiency was determined as follows: Thoroughly mix the fermentation broth in each MRS-CHO to obtain a suspension. Take 200 μL of the suspension and dilute it to 5000 μL with anhydrous ethanol to obtain a fermentation dilution. Place the fermentation dilution on a vortex mixer and mix thoroughly, then centrifuge at 8000 r / min for 10 min, take 2 mL of the supernatant of the fermentation dilution, and then slowly add 2 mL of the ammonium ferric sulfate solution prepared in step (2-4) to the supernatant of 2 mL of the fermentation dilution, and place it on a vortex mixer for shaking and mixing. Then place it at room temperature for 30 minutes to fully develop color, and finally read the A value of each sample to be tested at a wavelength of 560 nm on a spectrophotometer. 560 The blank sample is operated in the same way as above. 560 The value was tested seven times and the average value was taken. Combined with the cholesterol standard curve, the cholesterol concentration of the supernatant of the fermentation dilution of the candidate strain 1 to the candidate strain 6 was obtained. The cholesterol concentration of the supernatant of the fermentation dilution of the candidate strain 1 to the candidate strain 6 was substituted into the following formula to calculate the cholesterol degradation efficiency of the candidate strain 1 to the candidate strain 6. The calculation results are shown in Table 1.

[0035] Cholesterol degradation efficiency %=(initial cholesterol concentration of MRS-CHO-cholesterol concentration of supernatant of fermentation dilution of alternative strain) / initial cholesterol concentration of MRS-CHO×100%.

[0036] Table 1 Cholesterol degradation efficiency of candidate strains 1 to 6

[0037] As shown in Table 1, the cholesterol degradation efficiency of candidate strain 3 was the highest after being cultured at 37°C for 48 h.

[0038] (2-8) Determination of triglyceride degradation efficiency The activated bacterial suspensions of candidate strains 1 to 6 were inoculated into MRS-TG medium at a volume fraction of 10%, and cultured at 37°C for 48 hours in a constant temperature shaker at a speed of 160 r / min to obtain fermentation broths of candidate strains 1 to 6. Then, the triglyceride degradation efficiency of candidate strains 1 to 6 was detected respectively.

[0039] The method for determining the triglyceride degradation efficiency is carried out in accordance with the operating and culture instructions of the triglyceride determination kit. The triglyceride determination kit was purchased from Nanjing Jiancheng Biotechnology Co., Ltd. During the determination process, MRS-TG was used as a control sample, and the fermentation broth of the candidate strain 1-candidate strain 6 was used as the sample to be tested. Before the test, the fermentation broth in each MRS-TG was fully mixed, and then the fermentation broth was centrifuged at a speed of 8000r / min for 10min. After centrifugation, the supernatant was taken for determination. During the determination process, the blank tube was zeroed, and the absorbance value of each sample was read colorimetrically at a wavelength of 546nm. The absorbance value of each sample to be tested at a wavelength of 546nm was repeated seven times to take the average value. The triglyceride concentration of the supernatant of the candidate strain 1-candidate strain 6 was substituted into the following formula to calculate the triglyceride degradation efficiency of the candidate strain 1-candidate strain 6, and the calculation results are shown in Table 2.

[0040] Triglyceride degradation efficiency % = (absorbance value of control sample - average absorbance value of measured samples) / absorbance value of control sample × 100%.

[0041] Table 2 Triglyceride degradation efficiency of candidate strains 1 to 6

[0042] As shown in Table 2, the triglyceride degradation efficiency of candidate strain 3 was the highest after being cultured at 37°C for 48 h.

[0043] 3. Identification and preservation The candidate strain 3 separated and purified in steps (1-6) was sent for identification by Sangon Biotech (Shanghai) Co., Ltd. During the identification process, the primers used were as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'.

[0044] 1492R: 5'-GGTTACCTTGTTACGACTT-3'.

[0045] The obtained gene sequence is as follows:

[0046] The identification result is: the strain is Lactococcus lactis subsp. Lactococcus lactis subsp. lactis .

[0047] The identified strain was named Lactococcus lactis subsp. lactis JYLL-28 and sent to the General Microbiology Center of China Microbiological Culture Collection Administration for preservation. The preservation information is as follows: Classification name: Lactococcus lactis subsp. lactis Lactococcus lactis subsp. lactis , deposit date: July 24, 2023, deposit unit: General Microbiology Center of China Microbiological Culture Collection Administration, deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC No.27995.

[0048] Example 2 Preparation of Lactococcus lactis subsp. lactis JYLL-28 bacterial powder Step 1: Activate Lactococcus lactis subsp. lactis JYLL-28 stored at -80°C on MRS solid culture medium to obtain activated colonies.

[0049] Step 2: Pick an activated colony and transfer it to 100 mL of MRS liquid culture medium. Incubate it at 37°C for 12 hours to obtain seed solution.

[0050] Step 3: Inoculate the seed solution into a new MRS liquid medium at a 1% (v / v) inoculation volume, and incubate at 37°C for 24 h to obtain a viable bacterial count of 1.0×10 10 cfu / mL of culture medium.

[0051] Step 4: Centrifuge and freeze-dry the culture solution to obtain JYLL-28 bacterial powder.

[0052] Example 3 Animal Experiments with Lactococcus lactis subsp. lactis JYLL-28 1. Experimental Grouping: 60 6-week-old male Wistar rats weighing 180-190g purchased from the SPF Animal Experiment Center of Dalian Medical University were raised in a barrier environment with a temperature of 22-25°C and a humidity of 50%±10%. The rats were allowed to eat and drink freely in the barrier environment and were raised in alternating periods of 12h light and 12h darkness. All rats were adaptively raised for one week, and ordinary feed was used to feed the rats during the adaptive feeding period. After the adaptive feeding was completed, the rats were randomly divided into three groups, with 20 rats in each group. The three groups of rats were marked as blank control group (referred to as blank group), fatty liver model group (referred to as model group) and JYLL-28 group (referred to as experimental group), and the intervention experiment was carried out on each group of rats in the following way: Blank group: The mice were fed with ordinary feed and gavaged with 0.2 mL of sterile saline at 9:00 a.m. every day. The ordinary feed was purchased from Beijing Keao Xieli Company.

[0053] Model group: The mice were fed TP 4055 feed and gavaged with 0.2 mL of sterile saline at 9:00 a.m. every day. TP 4055 feed is the Tipoe model feed for non-alcoholic fatty liver disease modeling. TP 4055 feed was purchased from Nantong Trophy Feed Technology Co., Ltd.

[0054] Experimental group: The mice were fed TP 4055 feed and gavaged with 0.2 mL of JYLL-28 bacterial solution at 9:00 a.m. every day. TP 4055 feed was Tipoe model feed for non-alcoholic fatty liver disease modeling. TP 4055 feed was purchased from Nantong Trophy Feed Technology Co., Ltd. The JYLL-28 bacterial solution was prepared by mixing and diluting JYLL-28 bacterial powder with sterile saline. The number of viable bacteria in the JYLL-28 bacterial solution was 5.0×10 6 cfu / 0.2mL.

[0055] During the intervention experiment, rats in each group were free to eat and drink. The intervention experiment lasted for four weeks. After the intervention experiment, the rats were anesthetized by intraperitoneal injection of chloral hydrate and killed. Then blood was drawn from the heart and portal vein, and serum, liver and other tissue specimens were collected for subsequent testing and analysis.

[0056] II. General indicators of animal monitoring During the intervention experiment, the feed of each group of rats was weighed and replaced every day, and the vital signs, food intake, water intake, hair condition, mental state and activity, and accidental death of each group of rats were monitored. The monitoring results are shown in Table 3.

[0057] Table 3 Statistics of general indicators of rats

[0058] As shown in Table 3, compared with the rats in the model group, the rats in the experimental group had smooth and shiny hair, normal mental state and activity when fed with the same TP 4055 feed. It can be seen that Lactococcus lactis subsp. lactis JYLL-28 can significantly improve the hair, mental state and activity of rats.

[0059] 3. Changes in rat body weight, Lee's index and liver coefficient 1. Changes in rat body weight The rats in each group were weighed before and after the intervention experiment. The weight of the rats before the intervention experiment was recorded as the starting weight, and the weight of the rats after the intervention experiment was recorded as the final weight. The weight gain of the rats was calculated based on the starting weight and the final weight. The starting weight, final weight and weight gain of the rats in each group are shown in Table 4.

[0060] Table 4 Body weight changes of rats in each group

[0061] Note: The data in Table 4 are based on ±SD (n=20), and different letters indicate statistically significant differences among the groups (P<0.05).

[0062] As shown in Table 4, the initial weight of rats in each group was 180-190g, and there was no significant difference in the initial weight (P>0.05). After four weeks of intervention experiments on rats in each group, the final weight of the rats changed significantly, among which the rats in the model group had the largest weight gain, which was the most significant compared with the blank group and the experimental group (P<0.05). There was no significant difference in the weight gain of rats between the experimental group and the blank group (P>0.05). From the above, it can be concluded that Lactococcus lactis subsp. lactis JYLL-28 can significantly inhibit the weight gain of rats.

[0063] 2. Lee's index and liver coefficient of rats The body length of the rats was measured. The body length of the rats was the distance from the nose tip to the outer edge of the anus. The Lee's index of each group of rats was calculated according to the following formula: .

[0064] After the rats were killed, the whole liver was taken out to weigh the liver weight of the rats, and the liver coefficient of the rats was calculated according to the following formula. The Lee's index and liver coefficient of the rats in each group are shown in Table 5.

[0065] Liver coefficient = liver weight / body weight × 100%.

[0066] Table 5 Lee's index and liver coefficient of rats in each group

[0067] Note: The data in Table 5 are based on ±SD (n=20), and different letters indicate statistically significant differences among the groups (P<0.05).

[0068] As shown in Table 5, after four weeks of intervention experiments on rats in each group, the Lee's index of rats in the model group was the highest, and there was a significant difference between the blank group and the experimental group (P<0.05). The Lee's index of rats in the experimental group was close to that of the blank group, and there was no significant difference (P>0.05). It can be seen that Lactococcus lactis subspecies JYLL-28 can significantly improve the obesity of rats. The liver coefficient of rats in the model group was the highest, which was significantly different from the blank group and the experimental group (P<0.05). The liver coefficient of rats in the experimental group was the lowest, and there was also a significant difference compared with the liver coefficient of rats in the blank group (P<0.05). It can be seen that Lactococcus lactis subspecies JYLL-28 can significantly reduce the liver coefficient, thereby preventing the formation of fatty liver.

[0069] 4. Experimental rat serum test 1. Detection of blood lipid indexes in rats The coagulated heart blood samples were centrifuged at 3000 r / min at 4°C using a low-temperature centrifuge for 20 min. The upper serum was collected after centrifugation and packaged and frozen in a -20°C refrigerator. According to the Elisa kit operating instructions, the concentrations of total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) in the serum of each group of rats were detected. The test results are shown in Table 6.

[0070] Table 6 Blood lipid index test results of rats in each group

[0071] The test results in Table 6 are all based on ±SD (n=20), and different letters indicate statistically significant differences among the groups (P<0.05).

[0072] As shown in Table 6, after four weeks of continuous intervention, the TC, TG and LDL of the model group rats were significantly higher than those of the blank group and the experimental group (P<0.05), while the HDL was significantly lower (P<0.05). Compared with the model group, the TC, TG and LDL of the experimental group rats were lowered to varying degrees, while the HDL was higher; compared with the blank group and the model group, the TC and HDL of the experimental group rats were significantly different (P<0.05). This indicates that Lactococcus lactis subspecies JYLL-28 can reduce the concentrations of TC, TG and LDL in serum and increase the concentration of HDL, thereby preventing the formation of fatty liver.

[0073] 2. Detection of tumor necrosis factor in rat liver and serum endotoxin Liver TNF-α and serum endotoxin levels play an important role in the process of liver fatty degeneration and inflammation. The coagulated blood samples from the portal vein were centrifuged at 3000 r / min at 4°C using a low-temperature centrifuge for 20 minutes. The upper serum was collected after centrifugation, and the upper serum was divided and frozen in a refrigerator at -20°C. The concentrations of tumor necrosis factor (TNF-α) and serum endotoxin (Endotoxin) in the serum of rats in each group were detected according to the operating instructions of the Elisa tumor necrosis factor (TNF-α) kit and the operating instructions of the horseshoe crab kit. The test results are shown in Table 7.

[0074] Table 7 Liver TNF-α and serum endotoxin concentrations of rats in each group

[0075] Note: The test results in Table 7 are based on ±SD (n=20), and different letters indicate statistically significant differences among the groups (P<0.05).

[0076] As shown in Table 7, compared with the model group, the concentrations of TNF-α and Endotoxin in the experimental group were significantly reduced (P<0.05); compared with the blank group, there was no significant difference in the concentrations of TNF-α and Endotoxin in the experimental group (P>0.05), indicating that Lactococcus lactis subspecies JYLL-28 can significantly reduce the levels of TNF-α and endotoxin, thereby preventing the formation of fatty liver.

[0077] Example 4 Changes in rat indicators after cessation of intervention with Lactococcus lactis subsp. lactis JYLL-28 1. Experimental Grouping: Sixty 6-week-old male Wistar rats weighing 180-190g purchased from the SPF Animal Experiment Center of Dalian Medical University were raised in a barrier environment with a temperature of 22-25°C and a humidity of 50%±10%. The rats were free to eat and drink in the barrier environment and were raised in alternating 12h light and 12h night. All rats were adaptively raised for one week, and ordinary feed was used to feed the rats during the adaptive feeding period. After the adaptive feeding was completed, the rats were randomly divided into three groups, with 20 rats in each group. The three groups of rats were marked as the second blank control group (referred to as the second blank group), the second fatty liver model group (referred to as the second model group) and the second JYLL-28 group (referred to as the second experimental group), and the intervention experiment was carried out on each group of rats in the following way: The second blank group was fed with ordinary feed and gavaged with 0.2 mL of sterile saline at 9 a.m. every day. After four weeks, gavage was stopped and the mice were fed with free food and water for another four weeks. The ordinary feed was purchased from Beijing Keao Xieli Company.

[0078] The second model group: fed TP 4055 feed, 0.2 mL of sterile saline was gavaged at 9 am every day. After four weeks, gavage was stopped and the mice were fed with ordinary feed instead. They were allowed to eat and drink freely for another four weeks. TP 4055 feed is the Tipoe model feed for non-alcoholic fatty liver disease modeling. TP 4055 feed was purchased from Nantong Trofi Feed Technology Co., Ltd., and ordinary feed was purchased from Beijing Keao Xieli Company.

[0079] The second experimental group was fed TP 4055 feed and gavaged with 0.2 mL of JYLL-28 bacterial solution at 9:00 a.m. every day. TP 4055 feed was Tipoe model feed for non-alcoholic fatty liver disease modeling. TP 4055 feed was purchased from Nantong Trophy Feed Technology Co., Ltd. The JYLL-28 bacterial solution was prepared by mixing and diluting JYLL-28 bacterial powder with sterile saline. The number of viable bacteria in the JYLL-28 bacterial solution was 5.0×10 6 After four weeks, the intragastric administration of JYLL-28 bacterial solution was stopped, and the mice were fed with ordinary feed with free access to food and water for another four weeks. The ordinary feed was purchased from Beijing Keao Xieli Company.

[0080] During the intervention experiment, rats in each group were free to eat and drink. After the intervention experiment, the rats were anesthetized by intraperitoneal injection of chloral hydrate and then blood was drawn from the heart and portal vein, and serum, liver and other tissue specimens were collected for subsequent testing and analysis.

[0081] 2. Changes in rat body weight, Lee's index and liver coefficient 1. Changes in rat body weight The rats in each group were weighed before and after the intervention experiment. The weight of the rats before the intervention experiment was recorded as the starting weight, and the weight of the rats after the intervention experiment was recorded as the final weight. The weight gain of the rats was calculated based on the starting weight and the final weight. The starting weight, final weight and weight gain of the rats in each group are shown in Table 8.

[0082] Table 8 Body weight changes of rats in each group

[0083] Note: The data in Table 8 are based on ±SD (n=20), and different letters indicate statistically significant differences among the groups (P<0.05).

[0084] As shown in Table 8, there was no significant difference in the initial weight of rats in the second blank group, the second model group, and the second experimental group (P>0.05). After the intervention experiment, the final weight of rats in each group changed significantly, among which the weight gain of rats in the second experimental group was 224-235g, which was significantly different from that in the second blank group and the second model group (P<0.05). Four weeks after stopping the intragastric administration of JYLL-28 bacterial solution, the final weight of rats in the second experimental group was lower than that in the second blank group and the second model group, which shows that Lactococcus lactis subspecies JYLL-28 can reduce the weight growth rate of rats.

[0085] 2. Lee's index and liver index of rats The body length of the rats was measured. The body length of the rats was the distance from the nose tip to the outer edge of the anus. The Lee's index of each group of rats was calculated according to the following formula: .

[0086] After the rats were killed, the whole liver was taken out to weigh the liver weight of the rats, and the liver coefficient of the rats was calculated according to the following formula. The Lee's index and liver coefficient of the rats in each group are shown in Table 9.

[0087] Liver coefficient = liver weight / body weight × 100%.

[0088] Table 9 Lee's index and liver coefficient of rats in each group

[0089] Note: The data in Table 9 are based on ±SD (n=20), and different letters indicate statistically significant differences among the groups (P<0.05).

[0090] As shown in Table 9, the Lee's index of rats in the second model group was the highest, and there was a significant difference compared with the second blank group and the second experimental group (P<0.05). Although the Lee's index of rats in the second blank group and the second experimental group was not much different, the average Lee's index of rats in the second experimental group was lower than that in the second blank group, which showed that Lactococcus lactis subsp. lactis JYLL-28 could significantly reduce the obesity of rats.

[0091] The liver coefficient of the rats in the second experimental group was significantly different from that of the second blank group and the second model group (P<0.05). The liver coefficient of the rats in the second experimental group was the lowest, and the liver coefficient of the rats in the second model group was the highest. Four weeks after stopping the intragastric administration of JYLL-28 bacterial solution, the liver coefficient of the rats in the second experimental group was still lower than that of the second blank group and the second model group. This shows that Lactococcus lactis subspecies JYLL-28 can effectively reduce the liver coefficient, thereby reducing the incidence of fatty liver.

[0092] 3. Experimental rat serum test The blood lipid indexes, tumor necrosis factor (TNF-α) and serum endotoxin (Endotoxin) concentrations in the rat serum were detected respectively, and the detection method was the same as Example 3. The detection results of blood lipid indexes are shown in Table 10, and the detection results of tumor necrosis factor (TNF-α) and serum endotoxin (Endotoxin) concentrations are shown in Table 11.

[0093] 1. Results of blood lipid profile in rats Table 10 Test results of blood lipid indexes in the serum of rats in each group

[0094] Note: The test results in Table 10 are based on ±SD (n=20), and different letters indicate statistically significant differences among the groups (P<0.05).

[0095] As shown in Table 10, the concentrations of TC, TG and LDL in the second model group were significantly increased compared with the second blank group and the second experimental group (P<0.05), while HDL was significantly decreased (P<0.05). The second experimental group reduced the concentrations of TC, TG, and LDL to varying degrees, and increased the concentration of HDL, and there were significant differences compared with the second blank group and the second model group (P<0.05). Compared with the second model group, the blood lipid indexes of the rats in the second experimental group were still well improved after four weeks of stopping oral administration of JYLL-28 bacterial solution. It can be seen that Lactococcus lactis subspecies JYLL-28 can reduce the concentrations of TC, TG and LDL in serum, increase the concentration of HDL, and thus reduce the incidence of fatty liver.

[0096] 2. Results of rat liver tumor necrosis factor and serum endotoxin test Table 11 TNF-α in rat liver and endotoxin levels in serum

[0097] Note: The test results in Table 11 are based on ±SD (n=20), and different letters indicate statistically significant differences among the groups (P<0.05).

[0098] As shown in Table 11, there were significant differences in the levels of tumor necrosis factor TNF-α and serum endotoxin in the second experimental group compared with the second model group (P<0.05), and the levels of TNF-α and serum endotoxin in the second experimental group were significantly lower than those in the second model group; although there were no significant differences in the levels of tumor necrosis factor TNF-α and serum endotoxin in the second experimental group compared with the second blank group (P>0.05), the average values ​​were lower than those in the second blank group. It can be seen that Lactococcus lactis subspecies JYLL-28 significantly improved the levels of TNF-α and endotoxin, thereby reducing the incidence of fatty liver.

[0099] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily conceive of changes or substitutions within the technical scope disclosed by the present invention, and such changes or substitutions shall be within the scope of protection of the present invention.

Claims

1. A strain of Lactococcus lactis subspecies JYLL-28 having the function of inhibiting the formation of fatty liver, characterized in that: Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. lactis ) JYLL-28 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on July 24, 2023. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 27995.

2. A bacterial powder containing Lactococcus lactis subsp. lactis JYLL-28 according to claim 1, characterized in that: The preparation method of bacterial powder comprises the following steps: Step 1: activating Lactococcus lactis subspecies JYLL-28 on MRS solid medium to obtain activated colonies; Step 2: Pick an activated bacterial colony and transfer it to MRS liquid medium for anaerobic culture to obtain seed liquid; Step 3: inoculating the seed solution into a new MRS liquid culture medium for anaerobically culturing to obtain a culture solution; Step 4: Centrifuge and freeze-dry the culture solution to obtain JYLL-28 bacterial powder.

3. The bacterial powder of Lactococcus lactis subspecies lactis JYLL-28 according to claim 2, characterized in that MRS solid culture medium includes the following raw materials: 10 g of peptone, 5 g of beef powder, 5 g of sodium acetate trihydrate, 2 g of dipotassium hydrogen phosphate heptahydrate, 801 mL of Tween, 0.05 g of manganese sulfate tetrahydrate, 2 g of triammonium citrate, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate, 15 g of agar, and 1000 mL of distilled water.

4. The bacterial powder of Lactococcus lactis subspecies lactis JYLL-28 according to claim 2, characterized in that The MRS liquid culture medium includes the following raw materials: 10 g of peptone, 5 g of beef powder, 5 g of sodium acetate trihydrate, 2 g of dipotassium hydrogen phosphate heptahydrate, 801 mL of Tween, 0.05 g of manganese sulfate tetrahydrate, 2 g of triammonium citrate, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate, and 1000 mL of distilled water.

5. The bacterial powder of Lactococcus lactis subspecies lactis JYLL-28 according to claim 2, characterized in that: In step 2, the amount of MRS liquid culture medium used is 100 mL, the temperature of anaerobic culture is 37° C., and the time of anaerobic culture is 12 h.

6. The bacterial powder of Lactococcus lactis subsp. lactis JYLL-28 according to claim 2, characterized in that: In step 3, the inoculation amount of the seed solution is 1% by volume, the temperature of the anaerobic culture is 37° C., and the time of the anaerobic culture is 24 h.

7. The bacterial powder of Lactococcus lactis subsp. lactis JYLL-28 according to claim 2, characterized in that: In step 3, the viable bacterial count of the culture medium is 1.0×10 10 cfu / mL.

8. Use of the bacterial powder according to any one of claims 2 to 7 in the preparation of a medicament for treating non-alcoholic fatty liver disease.

9. The use according to claim 8, characterized in that Nonalcoholic fatty liver disease is simple fatty liver.

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