CN113208114B - Application of streptococcus thermophilus MN002 in lipid metabolism regulation and dietary supplement - Google Patents
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
- A23C9/156—Flavoured milk preparations ; Addition of fruits, vegetables, sugars, sugar alcohols or sweeteners
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
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- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The invention discloses application of streptococcus thermophilus MN002 in lipid metabolism regulation and a dietary supplement, and discloses application of streptococcus thermophilus MN002 in preparation of a product with a lipid metabolism regulation function, wherein the product is used for inhibiting expression of a fat-related transcription factor and inhibiting differentiation of preadipocytes into mature adipocytes, so that accumulation of the adipocytes is reduced, and secretion of the fat-related cytokines is reduced; the invention also provides a dietary supplement containing inactivated streptococcus thermophilus MN002 and having the function of regulating lipid metabolism. The invention has the effect of achieving the function of regulating lipid metabolism.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to application of a strain in regulation of lipid metabolism, and also relates to a dietary supplement added with the strain.
Background
Obesity is a metabolic disease resulting from an imbalance in energy intake and consumption, and is one of the major causes of cardiovascular disease, type 2 diabetes, and the increased incidence of certain tumors in developed countries. Increasing research results indicate that adipose tissue is not only a passive energy storage organ, but also an endocrine organ capable of secreting a variety of hormonal substances; the differentiation and regulation of fat cells are closely related to various human diseases such as obesity, diabetes, fatty liver, hyperlipidemia and breast cancer.
The essence of cell differentiation is the shift in gene expression pattern. The accumulation of cell fat and morphological changes accompanying adipogenesis are caused by the induced expression of specific genes in cells during differentiation. In this sense, changes in the expression and activity of transcription factors determine the differentiation process. Studies of cultured preadipocyte and fibroblast cell lines have yielded much data on transcription factors that promote adipogenesis and adipocyte differentiation. There are three major transcription factors identified to date that have a direct effect on adipogenesis and adipocyte differentiation, namely PPAR γ, C/EBPs and ADD1/SREBP1. Tumor necrosis factor alpha (TNF-alpha) can reduce gene expression of adipocytes by inducing fat depletion, e.g., weakening normal biological function of fat, and TNF-alpha can inhibit differentiation of cultured 3T3-L1 preadipocytes by activating MAPK signaling pathway.
Differentiation of adipocytes is affected by factors such as hormones, growth factors, extracellular matrix, etc., and most products aiming at regulation of fat metabolism are currently on the market. Currently, there are two main types of clinical means for weight control:
one class is weight-losing medicine, leptin, UCP, peroxidase proliferation activated receptor gamma (PPAR-gamma receptor) synthesized by adipose tissue and the like all have important relation with the occurrence of obesity, and the medicine acting on the substances can directly improve the fat distribution accumulation condition. The drugs for weight control mainly include adrenergic drugs, 5-hydroxytryptamine (5-HT) receptor agonists, monoamine reuptake inhibitors, lipase inhibitors, etc., and the mechanisms of action are different, but adverse reactions are more, such as anxiety, insomnia, sexual irritation, nausea, vomiting, blood sugar reduction, lymph node abnormality, etc.
The other class is probiotic products, which are live bacterial preparation products, and the live bacterial preparation products have the following defects to influence the efficacy of the products, including: (1) When the intestinal canal is immature, inflammation or immune function is incomplete, the live bacteria break through the intestinal wall and invade the organism, thereby initiating inflammation; (2) Is easily affected by antibiotics and also easily causes drug-resistant gene transfer; (3) The long-term use of live bacterial preparations increases the risk of phage infection and outbreak; (4) Poor heat resistance, easy pollution, product instability and other processing characteristics; and (5) the storage conditions of products such as low temperature and light shielding are harsh.
In the probiotic products disclosed in the prior art, the species of bacteria capable of achieving the weight-reducing effect are usually lactobacillus, bifidobacterium and the like, and live bacteria are also needed.
Disclosure of Invention
Therefore, the technical problem to be solved by the present invention is to provide a new use of Streptococcus thermophilus (Streptococcus thermophilus) MN002, that is, a use in weight reducing efficacy, wherein the MN002 can effectively inhibit expression of fat-related transcription factors, inhibit pre-adipocytes from differentiating into mature adipocytes, further reduce accumulation of adipocytes, and reduce secretion of fat-related cytokines; the invention also provides a dietary supplement containing the MN002 strain and capable of effectively controlling weight.
The application of streptococcus thermophilus MN002 in preparing a product with a lipid metabolism regulation function is used for inhibiting the expression of fat-related transcription factors and inhibiting preadipocytes from differentiating into mature adipocytes, so that the accumulation of the adipocytes is reduced, and the secretion of the fat-related cytokines is reduced. The Streptococcus thermophilus MN002 (Streptococcus thermophilus) is preserved in China general microbiological culture Collection center at 5.7.2010, the preservation number is CGMCC No.3817, and the preservation address is China academy of sciences microorganism research institute No.3 of West Lu No. 1 North Chen West district of Chaozhou Tokyo, beijing. The Streptococcus thermophilus MN002 is also called Streptococcus thermophilus MN-ZLW-002.
The streptococcus thermophilus MN002 is inactivated streptococcus thermophilus MN002.
The product is a dietary supplement, and the content of the streptococcus thermophilus MN002 in the dietary supplement is more than or equal to 100 hundred million/g; preferably, the content of the inactivated streptococcus thermophilus MN002 in the dietary supplement is more than or equal to 140 hundred million/g.
The product is microbial agent, food, health product or medicine.
The dietary supplement with the function of regulating lipid metabolism comprises prebiotics or/and food raw materials and microbial inoculum; the microbial inoculum is inactivated streptococcus thermophilus MN002.
The addition amount of the streptococcus thermophilus MN002 is more than or equal to 100 hundred million/g. Preferably, the addition amount of the streptococcus thermophilus MN002 is more than or equal to 140 hundred million/g.
The content of the prebiotics is 45-75% by mass.
The prebiotics comprise inulin and at least one of isomaltooligosaccharide, fructo-oligosaccharide, stachyose and lactitol.
The food raw material comprises at least one of skimmed milk powder, resistant dextrin and fruit and vegetable powder.
The dietary supplement of the present invention also includes the adjunct maltodextrin or/and citric acid.
The invention comprises the following components in parts by weight:
further, the invention comprises the following components in parts by weight:
a method for preparing dietary supplement with lipid metabolism regulating effect comprises mixing raw materials, and packaging.
Preferably, the mixing step is:
(1) Weighing skim milk powder and fruit and vegetable powder, mixing uniformly and marking as material A;
(2) Weighing maltodextrin, resistant dextrin and inactivated MN002 bacterial powder, uniformly mixing and recording as a material B;
(3) Weighing isomaltooligosaccharide, fructo-oligosaccharide, stachyose, lactitol, inulin and citric acid;
(4) Mixing material A and material B with the weighed isomaltooligosaccharide, fructo-oligosaccharide, stachyose, lactitol, inulin and citric acid uniformly.
The technical scheme of the invention has the following advantages:
1. the invention provides a new application of streptococcus thermophilus MN002, which can inhibit expression of fat-related transcription factors, inhibit preadipocytes from differentiating into mature adipocytes, reduce accumulation of the adipocytes and secretion of fat-related cytokines, and further achieve a lipid metabolism regulation function.
2. The invention adopts the inactivated strain as an effective component for the first time, and specifically adopts the inactivated streptococcus thermophilus MN002 to develop a product with the function of regulating lipid metabolism. Compared with the live bacteria, the inactivated strain has better heat resistance and pH stability, is easy to process and is not limited by food forms; meanwhile, the method has the advantages of stable bacteria content, easily controlled quality and no pollution to the production line; the inactivated strains are not required to be refrigerated and are not influenced by external conditions such as production, transportation and the like; the medicine is not influenced by antibiotics, has no risk of drug-resistant gene transfer, and has very obvious effect;
in addition, the inactivated streptococcus thermophilus MN002 is used as a microbial inoculum in the product, so that the effect of inactivating strains can be brought, macrophages can be activated to secrete related cell factors so as to regulate fat metabolism, the expression of fat related transcription factors (such as PPAR-gamma and C/EBP-alpha) is inhibited, pre-adipocyte differentiation into mature adipocyte is inhibited, the accumulation of the adipocyte is reduced, the secretion of ADP and other fat related cell factors is reduced, and the effect is very remarkable.
3. In the invention, inactivated streptococcus thermophilus MN002 is adopted to develop a dietary supplement with the function of regulating lipid metabolism; the main functional component of the dietary supplement is inactivated streptococcus thermophilus MN002, and isomaltooligosaccharide, fructooligosaccharide, stachyose, lactitol and inulin are used as prebiotics; the macrophage is further activated through the synergistic interaction among the main functional components, so that the macrophage secretes related cytokines to better regulate fat metabolism, and the effects of better inhibiting the expression of fat related transcription factors (such as PPAR-gamma and C/EBP-alpha) and inhibiting the differentiation of preadipocytes into mature adipocytes are achieved, thereby reducing the accumulation of the adipocytes and reducing the secretion of fat related cytokines such as ADP and the like.
4. According to the invention, the maltodextrin, the resistant dextrin, the skim milk powder, the citric acid, the fruit and vegetable powder and other components are selected as the food raw materials and the addition auxiliary agents, so that the mouthfeel can be further improved and the quality guarantee period can be prolonged on the premise of meeting the synergistic interaction between main functional components.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a morphological diagram of co-cultured cells of example 1 of the present invention, on a 20 μm scale, after staining with oil Red O;
FIG. 2 is a morphological diagram of co-cultured cells of example 2 of the present invention, on a 20 μm scale, after staining with oil Red O;
FIG. 3 is a morphological diagram of co-cultured cells of example 3 of the present invention, on a 20 μm scale, after staining with oil Red O;
FIG. 4 is a morphological diagram of the co-cultured cells of example 4 of the present invention, on a 20 μm scale, after staining with oil Red O;
FIG. 5 is a morphological picture of co-incubated cells of example 5 of the invention, stained with oil Red O, on a scale of 20 μm;
FIG. 6 is a morphological diagram of co-cultured cells of example 6 of the present invention, stained with oil Red O, on a scale of 20 μm;
FIG. 7 is a morphological diagram of the co-cultured cells of comparative example 1 of the present invention, on a 20 μm scale, after staining with oil Red O;
FIG. 8 is a morphological picture of cells co-cultured with the blank supernatant group of the present invention after staining with oil red O, on a scale of 20 μm;
FIG. 9 is a graphical representation of the appearance of co-incubated cells of the control group of the present invention after staining with oil Red O, on a scale of 50 μm;
FIG. 10 is a morphological diagram of cells co-cultured with the PGN group of the present invention, stained with oil Red O, on a scale of 20 μm;
FIG. 11 is a morphological picture of co-cultured cells of example 7 of the present invention, on a 20 μm scale, after staining with oil Red O;
FIG. 12 is a morphological diagram of the co-cultured cells of example 2 of the present invention after staining with oil red O, on a scale of 20 μm.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
The preparation method of the inactivated streptococcus thermophilus MN002 bacterial powder in the following embodiment comprises the following steps:
(1) Activating and culturing the preserved strain Mn002 with MRS liquid culture medium, standing and culturing at 37 deg.C for 16-20 hr until the viable count reaches 10 9 cfu/mL or more;
(2) And (3) centrifugally collecting wet thalli, adding a protective agent, and performing vacuum drying at 70-80 ℃ to obtain bacterial powder with inactivated streptococcus thermophilus MN002.
Example 1
A dietary supplement with lipid metabolism regulating effect comprises skimmed milk powder, maltodextrin, resistant dextrin, isomaltooligosaccharide, fructo-oligosaccharide, stachyose, lactitol, citric acid, inulin, semen Sojae Atricolor powder, and inactivated Streptococcus thermophilus MN002. The specific formulation is shown in table 1.
The preparation method of the inactivated streptococcus thermophilus MN002 comprises the following steps:
(1) Activating and culturing the preserved strain Mn002 with MRS liquid culture medium, standing and culturing at 37 deg.C for 18h to logarithmic phase, wherein viable count reaches 10 9 cfu/mL or more;
the formula of the MRS liquid culture medium is as follows:
the volume is fixed to 1L and the pH value is adjusted to 6.2.
(2) Centrifugally collecting wet thalli, adding a protective agent, and performing vacuum drying at 75 ℃ to obtain bacterial powder with inactivated streptococcus thermophilus MN 002; the amount of inactivated streptococcus thermophilus MN002 in the bacterial powder used in this example was about 2500 hundred million/g.
TABLE 1
| Example 1 | Example 2 | Example 3 | Example 4 | Example 5 | Example 6 | Example 7 | Comparative example 1 | |
| MN002 bacterial powder (g) | 30 | 16 | 25 | 9 | 16 | 16 | 15 | 0 |
| Skimmed milk powder (g) | 12 | 20 | 30 | 8 | 0 | 20 | 12 | 20 |
| Maltodextrin (g) | 8 | 15 | 5 | 20 | 58 | 123 | 8 | 31 |
| Resistant dextrin (g) | 35 | 15 | 20 | 10 | 15 | 0 | 35 | 15 |
| Isomaltooligosaccharide (g) | 20 | 18 | 25 | 10 | 18 | 0 | 20 | 18 |
| Fructo-oligosaccharide (g) | 100 | 25 | 15 | 20 | 25 | 0 | 100 | 25 |
| Stachyose (g) | 30 | 10 | 25 | 16 | 10 | 0 | 30 | 10 |
| Lactitol (g) | 30 | 25 | 18 | 10 | 25 | 0 | 30 | 25 |
| Inulin (g) | 30 | 15 | 20 | 10 | 15 | 0 | 30 | 15 |
| Citric acid (g) | 30 | 15 | 10 | 20 | 0 | 15 | 30 | 15 |
| Black bean powder (g) | 2 | 8 | 10 | 5 | 0 | 8 | 2 | 8 |
| Bacterial count (hundred million/g) | 252 | 220 | 308 | 163 | 220 | 220 | 120 | 0 |
According to the mixture ratio in the table 1, the preparation method comprises the following steps:
(1) Weighing the skim milk powder and the black bean powder according to the formula amount, and uniformly mixing to obtain a material A;
(2) Weighing maltodextrin, resistant dextrin and inactivated MN002 bacterial powder, uniformly mixing and recording as a material B;
(3) Weighing isomaltooligosaccharide, fructo-oligosaccharide, stachyose, lactitol, inulin and citric acid;
(4) Mixing material A and material B with weighed isomaltooligosaccharide, fructo-oligosaccharide, stachyose, lactitol, inulin and citric acid, and packaging.
In this example, the dietary supplement is packaged in 2g portions, and 3 dietary supplements are recommended daily.
Example 2
The difference in this embodiment is only that the composition and the ratio in example 6 are adopted in this embodiment, and the difference is only that MN002 in this embodiment is a viable bacteria preparation, and the preparation method of the MN002 viable bacteria preparation is as follows:
culturing the activated streptococcus thermophilus MN002 for 8 hours at 40 ℃ in two stages; inoculating the culture into an enrichment culture medium, wherein the inoculation amount is 2.5%, and stirring and fermenting for 18 hours under the conditions that the temperature is 40 ℃ and the pH value is 7.1 to obtain streptococcus thermophilus fermentation liquor; centrifuging the fermentation liquor of the streptococcus thermophilus MN002 for 18 minutes at 4 ℃, and taking the lower-layer precipitate for later use; mixing the precipitate with lyophilized protectant, and vacuum freeze drying at-50 deg.C for 12 hr to obtain Streptococcus thermophilus MN002 powder with viable count of 2.5 × 10 11 cfu/g。
Test examples
1. Design of experiments
The dietary supplements prepared in the embodiments are adopted, meanwhile, a Peptidoglycan (PGN) positive control and a blank control are set, the peptidoglycan positive control and the blank control are co-cultured with a mouse mononuclear macrophage line RAW264.7, co-culture supernatant is taken to co-culture with mouse preadipocytes 3T3-L1, fat differentiation is induced simultaneously, after induction is finished, secretion conditions of cytokines (ADP and TNF-alpha) and expression levels of mRNA (PPAR-gamma and C/EBP-alpha) are measured, cell morphology is observed by oil red 0 staining, and influence of inactivated streptococcus thermophilus MN002 dietary supplement mediated macrophages on preadipocyte differentiation and adipocyte function is analyzed.
2. Experimental method
2.1 RAW264.7 cell culture
Taking out the frozen RAW264.7 cells from the liquid nitrogen tank, thawing in 37 deg.C water bath for 1-2min, sucking 1ml of frozen stock solution after the frozen stock solution is melted, adding 5ml of complete culture medium, placing in 15ml centrifuge tube, blowing, beating, mixing, and transferring to 25mm 2 Culturing in a flask at 37 deg.C and 5% CO 2 Culturing in an incubator.
Cell morphology and growth were observed daily, with medium changes every 1-2 days. Passage was performed when the cell monolayer was 80% confluent, or transferred to 75mm 2 Culture bottles: discarding old culture solution, washing with PBS for 3 times, adding 4 deg.C PBS 3ml to stimulate cells for 3-4min, and removing by suction when cells become round and brightPBS, carefully blowing off the bottom cells with complete medium, passaging in new flasks at a ratio of 1.
2.2 RAW264.7 cells were co-cultured with test samples
The dietary supplements of examples 1 to 7 and comparative examples 1 and 2 were prepared into 10% by mass of suspensions with sterilized physiological saline, and diluted 10-fold with RPMI-1640 medium to prepare working solutions.
Each well of a 24-well plate was filled with 0.9mL of 5X 10 5 Cell suspension of RAW264.7 cells at 37 ℃ 5% 2 Cells were attached to the wall by culturing in an incubator for 2 h. 0.1mL of the working solution of example 1 to example 7, the working solution of comparative example 1, the working solution of example 2, and RPMI-1640 medium with a PGN concentration of 20. Mu.g/mL were added to each well, 3 parallel wells were set for each group, 37 ℃ and 5% CO 2 And collecting supernatant after 24h of co-culture in an incubator.
2.3 Co-culture of test supernatants with 3T3-L1 cells
3T3-L1 cells at 2.5X 10 4 The density of/mL was inoculated into 12-well plates, the culture continued for 2d after monolayer fusion to 100%, old medium was aspirated, PBS washed 2 times and DM1 (high-sugar DMEM complete medium containing 10 μ g/mL insulin, 0.5 mmol/L3-isobutyl-1-methylxanthine, 0.25 μmol/L dexamethasone) was added 2 mL/well, 2.2 supernatants were added, respectively, example 1-example 7 set to inactivate MN002 dietary supplement (supernatant of inactivated dietary supplement intervention of example 1-example 7 was added at 10% by volume), comparative 1 set (supernatant of dietary supplement intervention of comparative 1 was added at 10% by volume), example 2 set (supernatant of dietary supplement intervention of example 2 was added at 10% by volume), PGN set (supernatant of PGN intervention was added at 10% by volume), blank supernatant set (blank supernatant of control set at 10% by volume), control set (high-sugar complete medium was added at 10% by volume), DMEM was added in parallel for each set, 3 DMEM. After 3 days of culture, the medium was aspirated, washed 2 times with PBS, DM2 (high-glucose DMEM complete medium containing 10. Mu.g/mL of insulin, 2 mL/well) was added, and the culture was continued for 3 days, and the supernatant and cells were collected.
2.4 cytokine detection and oil Red O staining
The supernatant and cells were used for index detection: the content of Adiponectin (ADP) and TNF-alpha in the supernatant was determined using ADP and TNF-alpha ELISA kits (R & D, USA), respectively. Total RNA extraction kit (Kyofu biotechnology Co., ltd.) and cDNA synthesis kit (BIO-RAD Co., U.S.A.) TRIzol method are adopted to extract plate bottom cell RNA, after reverse transcription to cDNA, qPCR kit (BIO-RAD Co., U.S.A.) is utilized to determine the relative expression quantity of PPAR-gamma and C/EBP-alpha (beta-actin is used as an internal reference). The cells were stained with oil red O by a conventional method, and the cell morphology was observed under a mirror and photographed.
3. Results of the experiment
The contents of Adiponectin (ADP) and TNF-alpha in the supernatant, the relative expression amounts of PPAR-gamma and C/EBP-alpha are shown in tables 2 and 3, and the morphology of cells stained with oil red O is shown in FIGS. 1 to 12. Table 2 shows the expression level (fold of internal control) of mRNA of the adipose-related transcription factor after the intervention of the supernatant, and Table 3 shows the secretion level (pg/ml) of the adipose-related cytokine after the intervention of the supernatant.
TABLE 2
Note: in the above-mentioned table 2, the following, a comparison with blank supernatant group, p<0.05； b Comparison with PGN group, P<0.05； c Comparison with control group, P<0.05。
TABLE 3
Note: in the above-mentioned table 3, the following, a comparison with blank supernatant group, P<0.05； b Comparison with PGN group, P<0.05； c Compared with the control group,P<0.05; n.d indicates no detection.
As can be seen from tables 2 and 3 above: the relative expression amount of genes of PPAR-gamma and C/EBP-alpha and the secretion amount of an adipose-related cytokine ADP of an inactivated streptococcus thermophilus MN002 dietary supplement group are both obviously lower than those of a blank supernatant group and a control group, no statistical difference (P < 0.05) exists between the inactivated streptococcus thermophilus MN002 dietary supplement group and the control group, and the secretion amount of TNF-alpha is higher than those of the blank supernatant group and the control group.
After staining the cells with oil red O, it was observed under the microscope that the inactivated streptococcus thermophilus MN002 dietary supplement group of examples 1-7 and the live streptococcus thermophilus MN002 dietary supplement group of example 2 had lower fat differentiation rate and smaller fat particles than the control group, and similar morphology to the PGN group, as shown in fig. 1-12.
Preadipocytes play a very important role in the development of obesity, and if proliferation or hypertrophy of preadipocytes can be effectively inhibited, the development of obesity can be effectively controlled. In the experiment, the supernatant obtained after co-culture of the inactivated streptococcus thermophilus MN002 dietary supplement and the RAW264.7 obviously inhibits fat differentiation of fibroblast 3T3-L1, and fat accumulation in cells is obviously reduced compared with a control group, which indicates that the inactivated streptococcus thermophilus MN002 dietary supplement effectively inhibits fat differentiation and maturation.
The process of adipogenesis is accompanied by an increase in the expression of a series of adipogenesis-associated transcription factors and adipocyte-specific genes, particularly PPAR-gamma, which is a "master switch" that regulates adipocyte differentiation, with increased expression during the early stages of adipocyte differentiation. C/EBP-alpha plays an important role in the later stage of adipocyte differentiation, and can promote the high expression of PPAR-gamma and maintain the phenotype of differentiated cells. In the experiment, the gene expression of PPAR-gamma and C/EBP-alpha of the inactivated streptococcus thermophilus MN002 dietary supplement group is obviously inhibited, which indicates that the initiation and the maintenance of the differentiation of preadipocytes are inhibited.
ADP plays an important role in the process of regulating fat metabolism and insulin resistance, and is a possible therapeutic target for cardiovascular diseases, and the level of ADP secretion by cells in the inactivated streptococcus thermophilus MN002 dietary supplement group is lower than that of a control group in the experiment, which indicates that the fat accumulation capacity of fat cells is inhibited, and the secretion function of the fat cells is also obviously inhibited; on the contrary, the TNF-alpha can regulate fat metabolism by inhibiting the intake of fatty acid, inhibiting the generation of fat and promoting the decomposition of fat, and the inactivated Streptococcus thermophilus MN002 dietary supplement and the TNF-alpha in the RAW264.7 coculture supernatant can directly inhibit the differentiation of preadipocytes, can also promote the production of the TNF-alpha by the adipocytes and inhibit the differentiation of the adipocytes into mature adipocytes.
The test results show that the inactivated streptococcus thermophilus MN002 dietary supplement can regulate fat metabolism by activating macrophages to secrete relevant cytokines, inhibit the expression of fat-related transcription factors (PPAR-gamma and C/EBP-alpha), inhibit preadipocytes from differentiating into mature adipocytes, reduce the lipopexia of cells and reduce the secretion of fat-related cytokines such as ADP and the like. The secretion of TNF-alpha of the cell is increased, and the differentiation and formation of fat can be further inhibited. From this, we can conclude that the group of inactivated streptococcus thermophilus MN002 dietary supplements can suppress fat differentiation by modulating immune metabolism and affect the metabolic function of adipocytes.
Experimental results show that the inactivated streptococcus thermophilus MN002 dietary supplement can regulate fat metabolism by activating macrophages and enabling the macrophages to secrete relevant cytokines, inhibit the expression of fat-related transcription factors (PPAR-gamma and C/EBP-alpha), and inhibit preadipocytes from differentiating into mature adipocytes, so that the lipopexia of cells is reduced, and the secretion of fat-related cytokines such as ADP is reduced. The secretion of TNF-alpha of the cell is increased, and the differentiation and formation of fat can be further inhibited.
Meanwhile, the invention also verifies the effect of only the microbial inoculum, and the verification proves that: in the case of having the same bacteria content as in example 6 and example 2, the effect of the addition of the food materials and the auxiliary agents on the effect of the present application was not significant. That is, the effect of directly using the same amount of the inactivation microbial agent was substantially the same as the effect of adding the food material and the auxiliary agent in example 6, and the effect of directly using the same amount of the viable microbial agent was substantially the same as the effect of adding the food material and the auxiliary agent in example 2.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (16)
1. The streptococcus thermophilus MN002 has the preservation number of CGMCC No.3817 and can be used for preparing products with the function of regulating lipid metabolism.
2. Use according to claim 1, wherein the Streptococcus thermophilus MN002 is inactivated Streptococcus thermophilus MN002.
3. Use according to claim 2, characterized in that the product is a dietary supplement in which the content of Streptococcus thermophilus MN002 is > 100 million/g.
4. Use according to claim 3, characterized in that the content of inactivated Streptococcus thermophilus MN002 in the dietary supplement is greater than or equal to 140 million/g.
5. Use according to claim 1 or 2, wherein the product is a microbial agent, a food product, a nutraceutical product or a pharmaceutical product.
6. The dietary supplement with the function of regulating lipid metabolism comprises prebiotics or/and food raw materials, and a microbial inoculum; the method is characterized in that the microbial inoculum is inactivated streptococcus thermophilus MN002.
7. A dietary supplement with lipid metabolism regulation function according to claim 6, wherein the Streptococcus thermophilus MN002 is added in an amount of 100 hundred million/g or more.
8. A dietary supplement with lipid metabolism regulation function according to claim 7, wherein the Streptococcus thermophilus MN002 is added in an amount of 140 hundred million/g or more.
9. A dietary supplement having a lipid metabolism-regulating function according to any one of claims 6 to 8, wherein the prebiotics comprise at least inulin and are present in an amount of 45 to 75% by weight.
10. A dietary supplement having lipid metabolism-regulating function according to claim 9, wherein said prebiotic further comprises at least one of isomaltooligosaccharide, fructooligosaccharide, stachyose, lactitol.
11. A dietary supplement having a lipid metabolism-regulating function according to any one of claims 6 to 8, wherein the food material comprises at least one of skim milk powder, resistant dextrin, fruit and vegetable powder.
12. A dietary supplement having a function of regulating lipid metabolism according to any of claims 6 to 8, further comprising an auxiliary agent, wherein the auxiliary agent is maltodextrin or/and citric acid.
13. A dietary supplement having lipid metabolism-regulating function according to any one of claims 6 to 8, comprising in parts by weight:
0-40 parts of skim milk powder;
0-45 parts of maltodextrin;
0-50 parts of resistant dextrin;
0-40 parts of isomaltooligosaccharide;
0-45 parts of fructo-oligosaccharide;
0-50 parts of stachyose;
0-40 parts of lactitol;
0-30 parts of inulin;
0-40 parts of citric acid;
0-40 parts of fruit and vegetable powder;
the inactivated streptococcus thermophilus MN002 is more than or equal to 100 hundred million/g.
14. A dietary supplement having lipid metabolism-regulating function according to claim 13, comprising, in parts by weight:
5-30 parts of skim milk powder;
5-20 parts of maltodextrin;
5-20 parts of resistant dextrin;
10-25 parts of isomaltooligosaccharide;
10-25 parts of fructo-oligosaccharide;
10-25 parts of stachyose;
10-25 parts of lactitol;
10-20 parts of inulin;
10-20 parts of citric acid;
5-10 parts of fruit and vegetable powder;
inactivated streptococcus thermophilus MN002 is more than or equal to 140 hundred million/g.
15. A method for preparing a dietary supplement having a function of regulating lipid metabolism, comprising the steps of uniformly mixing the raw materials of the dietary supplement having a function of regulating lipid metabolism according to any one of claims 6 to 14, and packaging.
16. A method of preparing a dietary supplement having lipid metabolism-regulating function according to claim 15, wherein the mixing step comprises:
(1) Weighing the skimmed milk powder and the fruit and vegetable powder, and uniformly mixing to obtain a material A;
(2) Weighing maltodextrin, resistant dextrin and inactivated MN002 bacterial powder, uniformly mixing and recording as a material B;
(3) Weighing isomaltooligosaccharide, fructo-oligosaccharide, stachyose, lactitol, inulin and citric acid;
(4) Mixing material A and material B with the weighed isomaltooligosaccharide, fructo-oligosaccharide, stachyose, lactitol, inulin and citric acid uniformly.
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