CN115449497A - Mucous membrane lactobacillus capable of regulating liver lipid metabolism and application thereof - Google Patents
Mucous membrane lactobacillus capable of regulating liver lipid metabolism and application thereof Download PDFInfo
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- CN115449497A CN115449497A CN202211310474.7A CN202211310474A CN115449497A CN 115449497 A CN115449497 A CN 115449497A CN 202211310474 A CN202211310474 A CN 202211310474A CN 115449497 A CN115449497 A CN 115449497A
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- lactobacillus
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- liver
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a mucous membrane mucus lactobacillus for regulating liver lipid metabolism and application thereof, belonging to the technical field of microorganisms. The invention screens out a mucous membrane mucus lactobacillus CCFM1277 with the preservation number of GDMCC No:62777. the lactobacillus mucosus CCFM1277 provided by the invention has the effect of relieving non-alcoholic fatty liver, and is specifically embodied in that: effectively reduce the lipid deposition in the liver of non-alcoholic fatty liver disease individuals caused by high-fat diet, reduce the size of fat cells, and up-regulate the expression of mRNA of enzymes related to lipolysis in the liver, including lipoprotein lipase LPL, fatty triglyceride lipase ATGL, hormone sensitive lipase HSL, serine hydrolase MGL and the like, and a key enzyme CPT-1 for fatty acid beta-oxidation.
Description
Technical Field
The invention relates to a mucous membrane mucus lactobacillus for regulating liver lipid metabolism and application thereof, belonging to the technical field of microorganisms.
Background
As dietary structure shifts to high fat and high protein, the incidence of Non-alcoholic Fatty Liver Disease (NAFLD) induced thereby increases year by year and progresses in a low age state, and NAFLD has become one of the most common chronic Liver diseases worldwide. As the prevalence of NAFLD has increased significantly in recent years, it now accounts for one fourth of the total population. In recent years, the incidence of NAFLD has also increased year by year in our country, becoming the second largest chronic liver disease, second only to viral hepatitis. NAFLD prevalence is higher in obese people. Research shows that the death rate of the patient population suffering from NAFLD is obviously higher than that of the normal population. NAFLD is a major causative factor of various chronic liver diseases, such as liver cirrhosis and liver cancer. Because the pathogenesis of NAFLD is complex, no ideal therapeutic medicine and scheme exist at present.
Gut microbes include a variety of microbes (primarily bacteria) that aid in digestion, energy extraction and antagonism of pathogenic colonization, and also stimulate the immune system of the gastrointestinal tract by competitive uptake of nutrients and space, thereby reducing pathogens. Gut microbial dysbiosis refers to the destruction of the normal gut flora, which may be caused by a range of environmental, immunological or host factors as well as bile flow, altered gastric pH or intestinal motility disorders. There is a large body of evidence that gut microbial dysregulation, linked to the pathogenesis of human liver disease, plays an important role in NAFLD and its associated metabolic disorders. Intervention in nonalcoholic fatty liver using probiotics has therefore become a recent hotspot for research, with a large body of evidence that probiotics may alleviate NAFLD by modulating hepatic lipolysis, reducing hepatic lipid deposition.
Chinese patent application CN 113265361A discloses a composite probiotic preparation for relieving non-alcoholic fatty liver, a preparation method and application thereof, but the relieving effect of single bacteria on non-alcoholic fatty liver is not researched. The research focuses on the relieving effect of single probiotics on NAFLD, and a mucous membrane lactobacillus strain with good relieving effect is obtained.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, obtains a mucous membrane lactobacillus CCFM1277 capable of preventing nonalcoholic fatty liver through in-vivo research of animal experiments, and has the functions of reducing lipid deposition in liver, reducing the size of fat cells, up-regulating the expression of enzymes related to lipolysis in liver, such as lipoprotein lipase LPL, fatty triglyceride lipase ATGL, hormone sensitive lipase HSL, serine hydrolase MGL and the like, up-regulating fatty acid beta-oxidation key enzyme CPT-1 and the like.
In order to achieve the above purpose, the invention provides a strain of lactobacillus mucosus (limolactibacillus mucosae) CCFM1277, which has been deposited at the Guangdong province collection of microorganisms and strains in 26 months 9 and 2022, with the deposit number being GDMCC No:62777, the preservation address is Guangzhou city's Renlie Dayu No. 100, no. 59, building 5.
In one embodiment, the lactobacillus mucosae CCFM1277 has the following biological properties:
the characteristics of the thallus are as follows: is creamy yellow;
colony characteristics: bacterial colonies on the MRS solid plate are creamy yellow and convex, have irregular edges and are gram-positive bacteria;
growth characteristics: the culture was carried out in MRS medium for about 16h to the end of log under aerobic conditions at a constant temperature of 37 ℃.
The invention also provides application of the lactobacillus mucosus CCFM1277 in preparing a medicament for preventing and/or treating non-alcoholic fatty liver.
In one embodiment, the viable count of lactobacillus mucosus CCFM1277 in the medicament is not less than 1 × 10 10 CFU/g。
In one embodiment, the medicament contains the lactobacillus mucosus CCFM1277, a pharmaceutical carrier and/or a pharmaceutical excipient.
The invention also provides application of the lactobacillus mucosae CCFM1277 in preparing health-care products which are helpful for controlling fat in vivo and/or maintaining the healthy level of blood fat.
The invention also provides a product containing the lactobacillus mucosae CCFM1277.
In one embodiment, the product is a starter culture containing lactobacillus mucosus CCFM1277.
In one embodiment, the fermentation agent is obtained by culturing the lactobacillus mucosae CCFM1277 in a culture medium for a period of time, collecting bacterial cells in a cell culture solution, and directly using the bacterial cells as the fermentation agent or treating the bacterial cells to obtain the fermentation agent.
In one embodiment, the processing includes, but is not limited to: washing, adding a protective agent, drying and the like.
In one embodiment, the preparation method of the leavening agent is as follows: inoculating lactobacillus mucosus CCFM1277 into a culture medium according to the inoculation amount accounting for 1-5% of the total mass of the culture medium, and culturing at 37 ℃ for 18h to obtain a culture solution; centrifuging the culture solution to obtain thalli; the cells were resuspended in physiological saline to obtain a starter culture.
In one embodiment, the medium is MRS medium.
In one embodiment, the viable count of the lactobacillus mucosus CCFM1277 in the product is not less than 1 × 10 10 CFU/g。
In one embodiment, the product comprises a food, nutraceutical, or pharmaceutical product.
In one embodiment, the medicament comprises the lactobacillus mucosus CCFM1277, a pharmaceutical carrier and/or a pharmaceutical excipient.
In one embodiment, the food product comprises a health food product comprising said lactobacillus mucosae CCFM1277.
In one embodiment, the food product comprises a dairy product, a bean product, a meat product or a fruit and vegetable product produced by using the fermentation agent of the lactobacillus mucosus CCFM1277.
In addition, the invention also provides application of the lactobacillus mucosus CCFM1277 in food additives, wherein the application comprises but is not limited to being used as a food leavening agent.
Has the advantages that: the invention proves that the mucous membrane lactobacillus strain CCFM1277 has the functions of reducing lipid deposition in the liver, reducing the size of fat cells, up-regulating the expression of enzymes related to lipolysis in the liver, such as lipoprotein lipase LPL, fatty triglyceride lipase ATGL, hormone sensitive lipase HSL, serine hydrolase MGL and the like, and up-regulating the fatty acid beta-oxidation key enzyme CPT-1 and the like. Shows better treatment or prevention effect on the non-alcoholic fatty liver, so that the probiotic food, the health care product and the medicine for preventing and treating the non-alcoholic fatty liver can be prepared, and have very wide application prospect.
Biological material preservation
The lactobacillus mucosus CCFM1277 is classified and named as Limosilactibacillus mucosae, is preserved in Guangdong province microorganism strain preservation center in 26 th 9 th 2022, and has the preservation address of No. 59 floor 5 of Michelia Tokyo 100 Mr. Guangzhou, the preservation number is GDMCC No:62777.
drawings
FIG. 1 shows oil red staining of liver tissue from each group, indicating that Lactobacillus mucosae CCFM1277 reduces fat deposition in the liver; in the figure: the upper letters of the histogram being different represent significant differences (P < 0.05).
FIG. 2 shows HE staining of perirenal adipose tissue in each group, indicating that Lactobacillus mucosae CCFM1277 decreases adipocyte size; in the figure: the upper letters of the histogram being different represent significant differences (P < 0.05).
FIG. 3 shows the effect of Lactobacillus mucosus CCFM1277 on the mRNA relative expression level of lipoprotein lipase LPL in mice of each group, which indicates that Lactobacillus mucosus CCFM1277 can significantly improve the mRNA relative expression of mouse LPL; in the figure: in the figure: the upper letters of the histogram being different represent significant differences (P < 0.05).
FIG. 4 shows the relative expression level of mRNA of ATGL, which is a fatty triglyceride lipase in each group, indicating that Lactobacillus mucosus CCFM1277 can increase the relative expression level of mRNA of ATGL in mice; in the figure: in the figure: the upper letters of the histogram being different represent significant differences (P < 0.05).
FIG. 5 shows the effect of Lactobacillus mucosae CCFM1277 on changes in mouse hormone-sensitive lipase HSL, indicating that Lactobacillus mucosae CCFM1277 can significantly increase the relative expression of mRNA of mouse HSL; in the figure: in the figure: the upper letters of the histogram being different represent significant differences (P < 0.05).
FIG. 6 shows the effect of Lactobacillus mucosae CCFM1277 on mouse serine hydrolase MGL, indicating that Lactobacillus mucosae CCFM1277 can significantly increase the relative expression of mRNA of mouse MGL; in the figure: in the figure: the upper letters of the histogram being different represent significant differences (P < 0.05).
FIG. 7 shows the effect of Lactobacillus mucosae CCFM1277 on mouse carnitine palmitate transferase CPT-1, indicating that Lactobacillus mucosae CCFM1277 can significantly increase the relative expression of mRNA of mouse CPT-1; in the figure: in the figure: the upper letters of the histogram being different represent significant differences (P < 0.05).
Detailed Description
The invention will be better understood from the following examples.
In the present invention, "%" or percentages used to indicate concentrations or ratios are weight percentages unless otherwise specified.
The present invention relates to the following media:
MRS liquid culture medium: 10g of tryptone, 10g of beef extract, 5g of yeast powder, 20g of glucose, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate monohydrate, 80 mL of Tween, and water till 1000mL.
MRS solid medium was obtained by adding 1.5% agar to the total weight of liquid medium on the above basis.
Example 1: collection, separation and identification of lactobacillus mucosus CCFM1277
Taking 1g of a stool sample collected from 5-year-old boys in Guangdong Lianzhou, performing gradient dilution, coating the stool sample on an MRS solid culture medium, placing the stool sample in an aerobic environment, culturing for 72 hours at 37 ℃, observing and recording colony morphology, selecting and recording colony morphology, removing colony streaking and purifying, then culturing for 48 hours at 37 ℃ in an MRS liquid culture medium, performing gram staining on the obtained colony, recording strain morphology, removing gram-negative strains and gram-positive cocci in the colony, selecting and obtaining gram-positive bacilli, removing catalase-positive strains after catalase analysis, reserving catalase-negative strains, detecting by using fructose-6-phosphokinase to remove negative strains, and identifying the obtained bacterial strains as mucomyxolactobacillus mucosae by 16S rDNA sequencing, wherein the bacterial strains are named as CCFM1277. Subculturing the obtained mucous membrane lactobacillus, collecting thallus, centrifuging at 3000rpm in a centrifuge tube for 10min, washing, repeating for 3 times, and adding the thallus into matrix protectant for cryopreservation. Another strain of the lactobacillus mucosus screened in the same batch is named as FGSYC17L3.
16S rDNA amplification conditions: 5min at 95 ℃;35 cycles (95 ℃ 30s,55 ℃ 30s,72 ℃ 2 min); 72 ℃ for 10min
An amplification primer: 27F: (5'-AGAGTTTGATCCTGGCTCAG-3'), 1492R: (5'-TACGGCTACCTTGTTACGACT T-3') purification and sequence alignment of the amplified products were carried out according to the method described in the literature (Turroni F et al. Expanding the Diversity of the bipolar amplification in the Human endogenous transform [ J ]. Appl Environ Microb.2009;75 (6): 1534-45).
Example 2: preparation of mucous membrane lactobacillus strain CCFM1277 bacterial suspension
(1) Activation culture:
adopting MRS liquid culture medium, and carrying out static culture in a common incubator at 37 ℃.
The culture target: selecting a single colony of the frozen and preserved thallus, placing the single colony in a liquid MRS liquid culture medium, and standing and culturing for about 24 hours in a common incubator at 37 ℃ to activate the mucous membrane lactobacillus CCFM1277.
(2) First-stage culture:
adopting MRS liquid culture medium, and static culturing in a common incubator at 37 ℃.
Culturing a target: activated lactobacillus mucosus CCFM1277 is transferred to MRS liquid culture medium by 1 percent of inoculation amount based on the volume of the culture medium, and two generations are carried out.
(3) Secondary culture:
transferring the lactobacillus mucosus CCFM1277 subjected to primary culture into a 1LMRS liquid culture medium according to the inoculation amount of 1 percent of the volume of the culture medium, performing static culture in a common incubator at 37 ℃ for about 24 hours, and collecting the thallus. Washed twice with PBS pH7.4, then resuspended in 30% glycerol solution with viable count of 2.2X 10 10 CFU/mL, when used in subsequent experiments, glycerol was discarded by centrifugation, washed once with physiological saline and resuspended in physiological saline.
Example 3: determination of gastrointestinal fluid tolerance of lactobacillus mucosus CCFM1277
Assay method to simulate survival in gastrointestinal fluids: the artificial simulated gastrointestinal fluid needs to be prepared fresh. Pepsin (Shanghai test, 64008860) was dissolved in PBS (pH3.0) to a final concentration of 3g/L, and was filtered through a 0.22 μm filter to prepare a simulated gastric fluid. Trypsin (shanghai bio-engineering ltd., a 003319-0001) was dissolved in PBS having a ph of 8.0 to a final concentration of 1g/L, and filtered through a 0.22 μm filter to prepare a simulated intestinal fluid. Centrifuging cultured mucous membrane lactobacillus at 4 deg.C at 6000rpm for 10min, collecting bacterial mud, re-suspending with 0.85% normal saline, and regulating bacterial liquid density to 1 × 10 in simulated gastric juice (pH 3.0) 9 CFU/mL. After mixing uniformly, placing the mixture at 37 ℃ for culturing for 2h, and counting the number of viable bacteria. Adding 1mL of the bacterial liquid treated by simulated gastric juice into 9mL of simulated intestinal juice (pH8.0), uniformly mixing, culturing at 37 ℃, and detecting the number of viable bacteria after 4 hours. The percentage of the ratio of the number of the treated living bacteria to the number of the initial living bacteria is the survival rate.
The results showed that the survival rate of CCFM1277 in simulated gastric fluid was 87.66% and in simulated intestinal fluid was 78.81%
Example 4: mucous membrane lactobacillus strain CCFM1277 for improving liver fat accumulation of non-alcoholic fatty liver mice
1. Laboratory animal
Male SPF grade C57BL/6J mice (4 weeks old, weight 20-23 g), from Wentonlifwa laboratory animals technology, inc., zhejiang, china. Mice were housed in polypropylene cages containing food and water at controlled temperature (22 ℃), relative humidity (50 ± 10%), free drinking water, control mice fed on standard diet, model building and CCFM1277 mice fed on 60% high fat diet.
2. Experimental methods
(1) Establishment of non-alcoholic fatty liver mouse model
The control mice are fed with standard feed, the model-making and CCFM1277 mice are fed with 60% high-fat feed, drinking water is freely added, the water lasts for 12 weeks, the weight is weighed every week, and after 12 weeks, the weight of the model-making group is 25% higher than that of the control group, namely the model making is considered to be successful.
(2) Experimental groups and dosing
32C 57BL/6J mice were randomly divided into 4 groups, i.e., a control group, a modeling group, a Lactobacillus mucosus CCFM 1277-treated group, and a Lactobacillus mucosus FGSYC17L 3-treated group, each of which contained 8 mice. The administration mode of the mucous membrane lactobacillus treatment group is intragastric administration, and the intragastric administration dosage is 4 multiplied by 10 10 CFU/100. Mu.L/day. The mucous membrane mucus lactobacillus treatment model is perfused with the mucous membrane mucus lactobacillus every day, and normal group and model group are perfused with normal saline as control until the experiment is finished.
During the mouse molding, the body weight of the mouse was weighed weekly.
After 12 weeks of molding, the weight of the molding group is 25.81 percent higher than that of the control group, the molding is considered to be successful, after the mice are sacrificed, part of liver tissues are dissected, the blood is washed clean by normal saline, 4 percent paraformaldehyde is fixed for 24 hours, and the steps of dehydration, paraffin embedding and freezing section are carried out. Then, the ultrathin sections (4 μm) were washed with isopropanol and stained with oil red O staining solution. The stained sections were coverslipped with neutral gum as adhesive. A pathological section scanner is used to record the micrographs.
The fat accumulation in the liver of the mouse with the non-alcoholic fatty liver disease is increased, the number of fat drops is increased in an oil red slice, the area of the fat drops is increased, and the severity of the non-alcoholic fatty liver disease can be reflected. The slice fat ratio was analyzed using the software Image Pro Plus, as shown in fig. 1, the fat ratio was significantly increased from 2.74% to 22.61% (p < 0.05) in the modeling group compared to the blank group, whereas the fat area of the lactobacillus mucosus CCFM1277 group was 10.84%, which significantly alleviated the fat accumulation caused by high fat diet (p < 0.05), whereas FGSYC17L3 did not have this effect.
Example 5: mucous membrane lactobacillus strain CCFM1277 for improving perirenal fat accumulation of nonalcoholic fatty liver mice
The molding and grouping processing are performed in the same manner as in example 4.
Dissecting partial perirenal adipose tissue, washing blood with normal saline, fixing with 4% paraformaldehyde for 24 hr, dehydrating, embedding in paraffin, and slicing. Then, ultrathin sections (4 μm) were taken and stained with Hematoxylin and Eosin (HE). The stained sections were coverslipped with neutral gum as adhesive. A pathological section scanner is used to record the photomicrograph.
The fat cells of the non-alcoholic fatty liver mouse have larger volume and loose arrangement. The size of the adipocytes in the section was analyzed using software FIJI, as shown in FIG. 2, the area of the adipocytes in the molded group was 1020.09 μm compared to the blank group 2 Increased significantly to 2928.23 μm 2 (p<0.05 And the lactobacillus mucosae CCFM1277 group is 2380.25 μm 2 This situation (p) can be significantly alleviated<0.05 FGSYC17L3 has no such effect.
Example 6: lactobacillus mucosus CCFM1277 for regulating relative expression of non-alcoholic fatty liver disease mouse lipoprotein lipase mRNA
The molding and grouping processing are performed in the same manner as in example 4.
After 24h of the last gavage, a part of liver tissue was taken and stored in a refrigerator at-80 ℃ for subsequent experiments. During the experiment, liver tissues are taken out, total RNA of adipose tissues is extracted by using a Trizol method, and after the total RNA is reversely transcribed into cDNA, the relative expression quantity of lipoprotein lipase (LPL) mRNA of mouse liver tissues is detected by using a quantitative PCR (qPCR) method.
The lipoprotein lipase can catalyze the hydrolysis of triglyceride in lipoprotein and participate in the metabolism of chylomicron and very low density lipoprotein, and the expression of LPL in liver can promote the lipolysis in liver and avoid the deposition of liver lipid. As shown in FIG. 3, the relative expression level of mRNA of LPL in the model mice is reduced to 0.64, while the expression level of Lactobacillus mucosus CCFM1277 is significantly increased to 2.78 (p < 0.05) compared with the control group.
Example 7: lactobacillus mucosus CCFM1277 for regulating relative expression of fatty triglyceride lipase mRNA of nonalcoholic fatty liver mice
The molding and grouping processing are performed in the same manner as in example 4.
After 24h of the last gavage, a part of liver tissue was taken and stored in a refrigerator at-80 ℃ for subsequent experiments. During the experiment, liver tissues are taken out, total RNA of adipose tissues is extracted by using a Trizol method, and after the total RNA is reversely transcribed into cDNA, the relative expression quantity of the mRNA of the fatty triglyceride lipase (ATGL) of the mouse liver tissues is detected by using a quantitative PCR (qPCR) method.
The triglyceride lipase can specifically hydrolyze the first ester bond of triglyceride, promote the decomposition of triglyceride in liver, and reduce liver lipid deposition. As shown in FIG. 4, the relative expression level of mRNA of ATGL in the model mice is up to 0.33 compared with that of the control group, while Lactobacillus mucosae CCFM1277 has a significant effect of increasing the transcription level of ATGL of the mice to 1.18 (p < 0.05), while FGSYC17L3 has no effect.
Example 8: lactobacillus mucosus CCFM1277 for regulating relative expression of non-alcoholic fatty liver disease mouse hormone sensitive lipase mRNA
The molding and grouping processing are performed in the same manner as in example 4.
After 24h of the last gavage, a part of liver tissue was taken and stored in a refrigerator at-80 ℃ for subsequent experiments. During the experiment, liver tissues are taken out, total RNA of adipose tissues is extracted by using a Trizol method, and after the total RNA is reversely transcribed into cDNA, the relative expression quantity of the mRNA of the mouse liver tissue Hormone Sensitive Lipase (HSL) is detected by using a quantitative PCR (qPCR) method.
The hormone sensitive lipase can directly act on fat to hydrolyze triglyceride into diglyceride, promote decomposition of triglyceride in liver, and reduce liver lipid deposition. As shown in FIG. 5, the relative expression amount of mRNA of HSL in the model group mice is reduced to 0.43, and the expression amount of mRNA of Lactobacillus mucosus CCFM1277 in the model group mice is increased to 3.87, so that the HSL transcription level of the mice is remarkably increased (p < 0.05).
Example 9: lactobacillus mucosus CCFM1277 for regulating relative expression of serine hydrolase mRNA of mouse with nonalcoholic fatty liver
The molding and grouping processing are performed in the same manner as in example 4.
After 24h of the last gavage, a part of the liver tissue was taken and stored in a refrigerator at-80 ℃ for subsequent experiments. During the experiment, liver tissues are taken out, total RNA of adipose tissues is extracted by using a Trizol method, and after the total RNA is reversely transcribed into cDNA, the relative expression quantity of serine hydrolase (MGL) mRNA of mouse liver tissues is detected by using a quantitative PCR (qPCR) method.
The serine hydrolase can decompose triacylglycerol into free fatty acid and glycerol, promote decomposition of triglyceride in liver, and reduce liver lipid deposition. As shown in fig. 6, the relative mRNA expression level of MGL in the model mice was significantly reduced to 0.28 (p < 0.05) and the lactobacillus mucosus CCFM1277 group was significantly increased to 2.07, compared to the control group, indicating that lactobacillus mucosus CCFM1277 could well increase the HSL transcription level, whereas FGSYC17L3 did not have this effect.
Example 10: lactobacillus mucosus CCFM1277 for regulating relative expression of non-alcoholic fatty liver disease mouse carnitine palmitate transferase mRNA
The molding and grouping processing are performed in the same manner as in example 2.
After 24h of the last gavage, a part of liver tissue was taken and stored in a refrigerator at-80 ℃ for subsequent experiments. In the experiment, liver tissues are taken out, total RNA of adipose tissues is extracted by using a Trizol method, and after the total RNA is reversely transcribed into cDNA, the carnitine palmitate transferase (CPT-1) of mouse liver tissues is detected by using a quantitative PCR (qPCR) method
Relative mRNA expression level.
Carnitine palmitate transferase can transfer long-chain acyl coenzyme A outside mitochondria into mitochondria matrix for beta oxidation, is a rate-limiting enzyme for fatty acid oxidation, and maintains the balance of blood sugar and energy supply by catalyzing fatty acid to enter mitochondria for oxidation. As shown in FIG. 7, the mRNA relative expression level of CPT-1 in the mice of the model group is reduced to 0.75, while the Lactobacillus mucosae CCFM1277 has a significant effect on increasing the CPT-1 transcription level of the mice, the CPT-1 transcription level of the Lactobacillus mucosae CCFM1277 group is significantly increased to 3.08 (p < 0.05), and FGSYC17L3 has no effect.
The results show that the lactobacillus mucosae CCFM1277 can effectively reduce lipid deposition in the liver, reduce the size of fat cells and up-regulate enzymes related to lipolysis in the liver: the effects of expression of mRNA (messenger ribonucleic acid) such as lipoprotein lipase LPL, fatty triglyceride lipase ATGL, hormone sensitive lipase HSL, serine hydrolase MGL, critical enzyme CPT-1 for increasing the fatty acid beta-oxidation and the like can be used for preparing medicaments or health care products for preventing and treating nonalcoholic fatty liver, or producing weight-losing food, for example, the weight-losing food can be used as food additives of probiotic beverages, sour soybean milk, fermented jelly, fermented tea beverages or dairy products (such as yoghourt, cheese products, lactic acid bacteria and milk powder).
Example 11: preparation of tablet containing lactobacillus mucosus CCFM1277 microbial inoculum
The specific manufacturing process comprises the following basic steps: activation of bacterial strain → enlarged culture → collection of bacterial strain → preparation of bacterial suspension → freeze drying → total mixing → tabletting
1. Activating strains: the lactobacillus mucosus CCFM1277 is statically cultured in MRS liquid culture medium in a common incubator at 37 ℃ in an inoculation amount of 1 percent of the volume of the culture medium, and is continuously activated for two generations.
2. And (3) amplification culture: inoculating the activated lactobacillus mucosus CCFM1277 with an inoculum size of 1% of the volume of the culture medium into 1L of MRS liquid culture medium for amplification culture, and performing static culture in a common incubator at 37 ℃ for 24h.
3. Collecting thalli and preparing bacterial suspension: after the completion of the scale-up culture, the cells were collected by centrifugation at a low temperature of 4 ℃ and washed twice with PBS (pH 7.4), and then made into 1X 10 cells by using a 13% by weight skim milk aqueous solution 10 CFU/mL of bacterial suspension.
4. And (3) freeze drying: and preparing the bacterial suspension into bacterial powder according to a conventional freeze drying process.
5. Total mixing: adding stearic acid 2% of the total weight of the fungus powder as a lubricant and CMC-Na 3% of the total weight of the fungus powder as a binding agent, and uniformly mixing.
6. Tabletting: tabletting was performed by a tabletting machine according to conventional tabletting process.
Example 12: preparation of powder containing lactobacillus mucosus CCFM1277 microbial inoculum
The specific manufacturing process comprises the following basic steps: the steps of strain activation → enlarged culture → collection of thallus → preparation of bacterial suspension → freeze drying of strain activation, enlarged culture, collection of thallus and preparation of bacterial suspension are as described above.
And (3) freeze drying: the bacterial suspension is prepared into freeze-dried thallus powder according to the conventional freeze-drying process.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A lactobacillus mucosae (Lactobacillus mucosae) CCFM1277 is deposited in 26.9.2022 in Guangdong province of microorganism culture collection with the deposit number of GDMCC No:62777.
2. a product containing Lactobacillus mucosae CCFM1277 according to claim 1.
3. The product according to claim 2, characterized in that it is a starter culture containing lactobacillus mucosus CCFM1277.
4. The product of claim 3, wherein the starter culture is prepared by a method comprising: culturing the lactobacillus mucosus CCFM1277 in a culture medium for a period of time, collecting bacterial cells in a cell culture solution, and directly using the bacterial cells as a leavening agent or treating the bacterial cells to obtain the leavening agent.
5. The product of claim 3, wherein the product is a food product, a nutraceutical product, or a pharmaceutical product.
6. A product according to any one of claims 2 to 5, wherein the viable count of Lactobacillus mucosus CCFM1277 in the product is not less than 1 x 10 10 CFU/g。
7. The product according to claim 5, wherein the drug comprises the Lactobacillus mucosus CCFM1277, a pharmaceutical carrier and/or a pharmaceutical excipient.
8. Use of lactobacillus mucosae CCFM1277 according to claim 1 for the preparation of a medicament for the prevention and/or treatment of non-alcoholic fatty liver disease.
9. Use of lactobacillus mucosae CCFM1277 according to claim 1 for the preparation of a health product useful for controlling body fat and/or for maintaining healthy levels of blood lipids.
10. Use of lactobacillus mucosus CCFM1277 according to claim 1 in a food additive.
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