Lactobacillus paracasei K56 metaplasm and application thereof in aspect of controlling in-vivo fat

Abstract

The invention relates to a lactobacillus paracasei K56 metagen and application of the lactobacillus paracasei K56 metagen in the aspect of controlling in-vivo fat, and particularly provides one or more applications of the lactobacillus paracasei K56 metagen selected from the group consisting of the following beneficial intestinal bacteria: the lactobacillus paracasei K56 metagen is used for promoting the proliferation of the beneficial intestinal bacteria, the beneficial intestinal bacteria are selected from one or more of norkfRuminococcus, Lactobacillus and Coriobacterium UCG-002, preferably one or more of norkfRuminococcus and Coriobacterium UCG-002, and the lactobacillus paracasei K56 metagen is used for promoting the proliferation of the beneficial intestinal bacteria, preferably one or more of the norkfRuminococcus, Lactobacillus and Coriobacterium UCG-002. The composition is used for regulating obesity (such as obesity caused by high-fat diet); for modulating (e.g., caused by high fat diet) lipid metabolism disorders and / or liver lipid accumulation; for modulating (e.g., caused by high fat diet) colon immune disorders; wherein the lactobacillus paracasei K56 is preserved in the German Colletion of Microorganisms and Cell Cultures, the preservation number of the lactobacillus paracasei K56 is DSM 27447, and the lactobacillus paracasei K56 is preserved in the German Microorganism Preservation Center (MCC), the preservation number of the lactobacillus paracasei K56 is DSM 27447, and the preservation number of the lactobacillus paracasei K56 is CGMCC NO. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number of the strain is CGMCC 15139, and the strain is also preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the strain is CGMCC 15139.

Description

Technical Field

[0001] The present invention relates to the fields of biomedicine and food, and relates to functional research of a postbiotic, in particular to a Lactobacillus paracasei K56 postbiotic and its use in controlling body fat. Background Art

[0002] The relationship between obesity and intestinal inflammation caused by a high-fat diet (HF) is currently a hot topic of research. Studies have reported that a high-fat diet interacts with the intestinal flora, which can aggravate intestinal inflammation. The high-fat diet and lifestyle of obese patients disrupt their intestinal flora, increasing intestinal-derived lipopolysaccharide (LPS) in the blood. LPS, acting as an inflammatory trigger, binds to the TLR4 receptor and activates the NF-κB signaling pathway, promoting the synthesis and release of inflammatory factors such as TNF-α, IL-6, and IL-1β, and exacerbating the body's inflammatory state.

[0003] A high-fat diet can disrupt the balance of intestinal flora, affecting metabolic function and energy homeostasis, leading to intestinal inflammation and obesity. The beneficial effects of probiotics in improving intestinal flora balance have been confirmed in numerous studies, and probiotics may provide new preventive and therapeutic approaches for obesity. Some studies have shown that bacteria such as Lactobacillus and Bifidobacterium, either alone or in combination, can prevent or control obesity. Over 85% of research reports indicate that feeding Lactobacillus and Bifidobacterium can reduce animal weight gain, fat accumulation, and white adipose tissue.

[0004] The most effective way to prevent or improve obesity is to adjust your diet and increase physical activity. Dietary supplements play an important supporting role in maintaining a healthy diet. Numerous studies have confirmed the beneficial effects of probiotics in improving gut microbiome balance and obesity. Obesity and gut microbiota are closely linked: obesity can disrupt gut microbiota, while disrupted gut microbiota can exacerbate obesity. Therefore, research into the preventive and ameliorative effects of probiotics on obesity and the intestinal inflammation it causes is of great significance to human health.

[0005] Postbiotics are defined as “biologically active components of microorganisms or their metabolites.” Postbiotics are more stable than live bacteria and maintain their effectiveness during food processing and storage.

[0006] Currently reported postbiotics include functional bacterial metabolites such as enzymes (glutathione peroxidase (GPX), superoxide dismutase (SOD), peptides, exopolysaccharides (EPS), organic acids, short-chain fatty acids, and bacterial components (peptidoglycan, teichoic acid, cell surface proteins, etc.). As one of the main postbiotic components, probiotic surface proteins have attracted widespread attention due to their ability to regulate health by directly mediating bacterial adhesion and colonization of intestinal epithelial cells. Summary of the Invention

[0007] The Lacticaseibacillus paracasei K56 strain was deposited at the German Collection of Microorganisms and Cell Cultures on June 27, 2013, with the accession number DSM 27447. Furthermore, L. paracasei K56 was also deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 29, 2017, with the accession number CGMCC 15139.

[0008] After detailed research, the inventors of the present application provide the application of Lactobacillus paracasei K56 postbiotics in regulating high-fat diet obesity and colon immune disorders.

[0009] In particular, the inventors of the present application found that the surface protein of Lactobacillus paracasei K56 can increase the abundance of beneficial intestinal bacteria norank_f_Ruminococcaceae, Lactobacillus and / or Coriobacteriaceae_UCG-002, especially can increase the abundance of beneficial intestinal bacteria norank_f_Ruminococcaceae and / or Coriobacteriaceae_UCG-002, and has high application value.

[0010] To this end, in a first aspect of the present invention, the present invention provides a use of a Lactobacillus paracasei K56 postbiotic in preparing a composition, wherein the composition has one or more uses selected from the following (1)-(8):

[0011] (1) Used to promote the proliferation of beneficial intestinal bacteria, wherein the beneficial intestinal bacteria are selected from one or more of norank_f_Ruminococcaceae, Lactobacillus, and Coriobacteriaceae_UCG-002, preferably one or more of norank_f_Ruminococcaceae and Coriobacteriaceae_UCG-002;

[0012] (2) It helps regulate intestinal flora, wherein regulating intestinal flora is to promote the proliferation of beneficial intestinal bacteria, wherein the beneficial intestinal bacteria are selected from one or more of norank_f_Ruminococcaceae, Lactobacillus, and Coriobacteriaceae_UCG-002, preferably one or more of norank_f_Ruminococcaceae and Coriobacteriaceae_UCG-002;

[0013] (3) Helps control body fat;

[0014] (4) Helps maintain healthy blood lipid (cholesterol / triglyceride) levels;

[0015] (5) Helps maintain healthy blood sugar levels;

[0016] (6) Used to regulate obesity (such as that caused by a high-fat diet);

[0017] (7) Used to regulate dyslipidemia and / or hepatic lipid accumulation (e.g., caused by a high-fat diet);

[0018] (8) Used to regulate colon immune disorders (such as those caused by high-fat diet);

[0019] The Lactobacillus paracasei K56 is deposited in the German Collection of Microorganisms and Cell Cultures with a deposit number of DSM 27447; it is also deposited in the China General Microbiological Culture Collection Center (CGMCC) with a deposit number of CGMCC 15139.

[0020] In some embodiments, the composition is a pharmaceutical composition, a food (regular food or health food), a food additive, a dietary supplement, or a feed.

[0021] In some embodiments, the composition is an oral dosage form. Preferably, the oral dosage form is selected from the group consisting of solutions, suspensions, emulsions, powders, lozenges, pills, syrups, lozenges, tablets, chewing gums, and capsules.

[0022] In some embodiments, the Lactobacillus paracasei K56 postbiotic is heat-killed Lactobacillus paracasei K56 bacteria or Lactobacillus paracasei K56 surface protein.

[0023] In some embodiments, the heat-killed bacteria of Lactobacillus paracasei K56 are obtained by subjecting Lactobacillus paracasei K56 to high-temperature steam sterilization.

[0024] In some embodiments, the temperature of the high temperature steam sterilization is 100-150°C, preferably 120-125°C, and most preferably 121°C.

[0025] In some embodiments, the high temperature steam sterilization time is 10-20 minutes, preferably 15 minutes.

[0026] In some embodiments, the Lactobacillus paracasei K56 surface protein comprises an LPXTG motif protein and a moonlighting protein.

[0027] In some embodiments, the LPXTG motif protein comprises KONBH9 (GenBank: CCK24055.1), KONA48 (GenBank: CCK23225.1), or KONAB9 (GenBank: CCK23405.1).

[0028] In some embodiments, the moonlighting proteins comprise A0A0K1KXQ2 (Gene ID: 57089615), K0N7K7 (GenBank: CCK23322.1), KOMU67 (GenBank: CCK22051.1), A0A0K1KYZ6 (Gene ID: 57090004), and K0N700 (GenBank: CCK23323.1).

[0029] In some embodiments, the surface protein of Lactobacillus paracasei K56 is as shown in Table A.

[0030] In some embodiments, the Lactobacillus paracasei K56 surface protein is prepared by the following method:

[0031] (1) Lactobacillus paracasei K56 was mixed with an acidic lithium chloride aqueous solution as an extraction reagent and incubated;

[0032] (2) centrifuging the mixture obtained in step (1) and taking the supernatant, wherein the supernatant is a surface protein extract of Lactobacillus paracasei K56.

[0033] In some embodiments, the method for preparing the surface protein of Lactobacillus paracasei K56 further has one or more technical features selected from the following (i)-(v):

[0034] (i) the ratio of the mass of the Lactobacillus paracasei K56 to the volume of the extraction reagent acidic lithium chloride aqueous solution is 1 g:3 mL;

[0035] (ii) the concentration of lithium chloride in the acidic lithium chloride aqueous solution is 1-10 mol / L, preferably 4-6 mol / L, more preferably 5 mol / L;

[0036] (iii) the pH of the acidic lithium chloride aqueous solution is 1.0-3.0, preferably 2.0;

[0037] (iv) the incubation time is at least 10 min, preferably 10-40 min, more preferably 30-40 min, and most preferably 30 min;

[0038] (v) The incubation is carried out in an ice water bath.

[0039] In a second aspect of the present invention, the present invention provides a postbiotic of Lactobacillus paracasei K56, which is selected from heat-killed bacteria of Lactobacillus paracasei K56 and surface protein of Lactobacillus paracasei K56;

[0040] Preferably, the heat-killed bacteria of Lactobacillus paracasei K56 are obtained by high-temperature steam sterilization as defined in the first aspect;

[0041] Preferably, the Lactobacillus paracasei K56 surface protein is the K56 surface protein defined in the first aspect, or the Lactobacillus paracasei K56 surface protein is obtained by the preparation method of the Lactobacillus paracasei K56 surface protein defined in the first aspect.

[0042] In a third aspect of the present invention, the present invention provides a composition comprising the Lactobacillus paracasei K56 postbiotic of the second aspect;

[0043] Preferably, the composition is a pharmaceutical composition, a food (regular food or health food), a food additive, a dietary supplement or a feed;

[0044] Preferably, the composition is in an oral dosage form, preferably, the oral dosage form is selected from the group consisting of solution, suspension, emulsion, powder, lozenge, pill, syrup, buccal lozenge, tablet, chewing gum, and capsule.

[0045] Beneficial effects

[0046] Live Lactobacillus paracasei K56 and its postbiotics can regulate blood lipid metabolism disorders and liver lipid accumulation, colon immune disorders, and intestinal flora imbalance caused by a high-fat diet. Among them, the K56 surface protein plays an important role in mediating its regulatory effects on high-fat diet-induced obesity and colon immune disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The weight gain trend of mice within 6 weeks of drug administration;

[0048] Figure 2 The effects of Lactobacillus paracasei K56 and postbiotics on lipid metabolism in mouse serum (n=10);

[0049] Figure 3 Effects of Lactobacillus paracasei K56 and postbiotics on fasting blood glucose (A); glucose tolerance test (B); area under the OGTT curve (C); and insulin level (D) in mice.

[0050] Figure 4 Effects of Lactobacillus paracasei K56 and postbiotics on leptin (A) and adiponectin (B) in mice;

[0051] Figure 5 Effects of Lactobacillus paracasei K56 and postbiotics on AST activity (A), ALT activity (B), and LPS content (C) in mouse serum.

[0052] Figure 6 Effects of Lactobacillus paracasei K56 and postbiotics on the changes of liver weight (A), liver coefficient (B), epididymal fat weight (C), and epididymal fat coefficient (D) in mice

[0053] Figure 7 Effects of Lactobacillus paracasei K56 and postbiotics on SOD activity (A), MDA content (B), and CAT activity (C) in mouse liver.

[0054] Figure 8 Effects of Lactobacillus paracasei K56 and postbiotics on HE staining of mouse epididymal fat (A) and colon HE staining (B);

[0055] Figure 9 The effects of Lactobacillus paracasei K56 and postbiotics on the free fatty acid content in mouse liver;

[0056] Figure 10 The effects of Lactobacillus paracasei K56 and postbiotics on the mRNA expression of adipose tissue-related factors FAS (A); PPAR-γ (B); CEBP-α (C) in mice;

[0057] Figure 11Effects of Lactobacillus paracasei K56 and postbiotics on inflammatory factors IL-1β(A); IL-6(B); IL-10(C); TNF-α(D) in mouse colon tissue;

[0058] Figure 12 Effects of Lactobacillus paracasei K56 and postbiotics on tight junction proteins Claudin-1 (A), ZO-1 (B), and Occludin (C) in mouse colon tissue;

[0059] Figure 13 Effects of Lactobacillus paracasei K56 and postbiotics on TLR4 protein expression (A), p50 protein expression (B), and p-p65 / p65 protein expression (C) in mouse colon.

[0060] Figure 14 The effect of Lactobacillus paracasei K56 and postbiotics on the protein expression of p-JNK / JNK in mouse colon;

[0061] Figure 15 The effects of Lactobacillus paracasei K56 and postbiotics on the OTU species of the intestinal flora of mice;

[0062] Figure 16 Alpha diversity analysis of intestinal flora species in the Lactobacillus paracasei K56 and postbiotic-treated mice;

[0063] Figure 17 This is the species dilution curve of the intestinal flora of mice in the Lactobacillus paracasei K56 and postbiotic treatment groups;

[0064] Figure 18 Beta diversity analysis of intestinal flora in mice treated with Lactobacillus paracasei K56 and postbiotics;

[0065] Figure 19 This is the composition analysis of the intestinal flora at the phylum level in the mice treated with Lactobacillus paracasei K56 and postbiotics;

[0066] Figure 20 This is the composition analysis of the intestinal flora of mice in the Lactobacillus paracasei K56 and postbiotic treatment groups at the genus level;

[0067] Figure 21 The changes of beneficial bacteria at the genus level in the intestinal microorganisms of mice in the Lactobacillus paracasei K56 and postbiotic treatment groups;

[0068] Figure 22 The changes of harmful bacteria at the genus level in the intestinal microorganisms of mice in the Lactobacillus paracasei K56 and postbiotic treatment groups. DETAILED DESCRIPTION

[0069] Below in conjunction with embodiment and accompanying drawing, embodiment of the present invention is described in detail, but those skilled in the art will understand that following embodiment and accompanying drawing are only used for illustrating the present invention, and are not limiting the scope of the present invention. According to the following detailed description of accompanying drawing and preferred embodiment, various purposes and advantages of the present invention will become apparent to those skilled in the art. Unless otherwise specified, each raw material and reagent all can be purchased commercially.

[0070] The Lactobacillus paracasei K56 used in the present invention was provided by Inner Mongolia Yili Industrial Group Co., Ltd., and the high-fat mice were provided by Nanjing Bioray Pharmaceutical Research Institute Co., Ltd.; various mouse ELISA kits were provided by Shanghai Zhuocai Biotechnology Co., Ltd.; and various enzyme activity assay kits were provided by Beijing Solebold Technology Co., Ltd.

[0071] The microscope, automatic biological tissue dehydrator, and slicer were provided by Leica Microsystems (Shanghai) Trading Co., Ltd.; the biological tissue freezing embedding machine, embedding machine freezing table, biological tissue spreader, and biological tissue baking machine were provided by Jinhua Yidi Medical Equipment Co., Ltd.

[0072] The present invention provides a postbiotic of Lactobacillus paracasei K56 that can regulate high-fat diet obesity and colon immune disorders. The research contents include: (1) establishing a high-fat diet mouse obesity model, evaluating the regulatory effect of Lactobacillus paracasei K56 postbiotic on mouse obesity, and its regulatory effect on liver lipid accumulation and colon immune disorders caused by obesity; (2) observing the effect of Lactobacillus paracasei K56 postbiotic on regulating intestinal flora, and jointly analyzing the regulation of Lactobacillus paracasei K56 on metabolic disorders caused by high-fat diet based on the "intestinal flora-intestine-liver axis"; (3) clarifying the differential effect of Lactobacillus paracasei K56 postbiotic in regulating obese colon immune disorders caused by high-fat diet.

[0073] Specifically, the technical solution adopted by the present invention is:

[0074] A Lactobacillus paracasei K56 postbiotic capable of regulating high-fat diet obesity and colon immune disorders is prepared, wherein the Lactobacillus paracasei K56 postbiotic comprises heat-killed Lactobacillus paracasei bacteria and Lactobacillus paracasei surface protein.

[0075] Provided is the regulatory effect of the above-mentioned Lactobacillus paracasei K56 postbiotic on obesity in mice.

[0076] Provide the role of the above-mentioned Lactobacillus paracasei K56 postbiotics in regulating blood lipid metabolism disorders and liver lipid accumulation caused by a high-fat diet.

[0077] Provided is the effect of the above-mentioned Lactobacillus paracasei K56 postbiotics on regulating colon immune disorders caused by a high-fat diet.

[0078] Provided is the use of the above-mentioned Lactobacillus paracasei K56 postbiotic in regulating intestinal flora imbalance caused by a high-fat diet.

[0079] Provided are the differential effects of the above-mentioned Lactobacillus paracasei K56 postbiotics in regulating obese colon immune disorders caused by a high-fat diet.

[0080] The present invention will be further explained below with reference to specific embodiments.

[0081] Example 1: Preparation of Lactobacillus paracasei K56 postbiotics

[0082] (1) Live Lactobacillus paracasei

[0083] The activated Lactobacillus paracasei was inoculated into MRS broth at a ratio of 1% (v / v) and cultured at 37°C for 16 hours. The cells were centrifuged at 3520 × g and 4°C for 10 minutes, the supernatant was discarded, and the bacterial pellet was collected. The cells were washed with sterilized 0.9% saline solution, and the concentration of the bacterial solution was adjusted to 10% with 0.9% sterile saline solution after three times. 8 CFU / mL is reserved.

[0084] (2) Heat-killed Lactobacillus paracasei

[0085] The activated Lactobacillus paracasei was inoculated into MRS broth at a ratio of 1% (v / v) and cultured at 37°C for 16 h. The bacterial precipitate was collected by centrifugation (3520×g, 10 min, 4°C), washed twice with sterile PBS, and resuspended in 0.9% sterile saline to adjust the bacterial solution concentration to 10 8 The suspension was sterilized by high-pressure steam at 121° C. for 15 min, and the sample prepared in this manner was used as the experimental sample in the present invention.

[0086] (3) Surface protein extraction

[0087] The activated Lactobacillus paracasei was inoculated into MRS broth at a 1% (v / v) ratio and cultured at 37°C for 16 hours. The mixture was centrifuged at 3520×g for 10 minutes at 4°C, the supernatant discarded, and the bacterial precipitate collected. A 5 mol / L, pH 2.0 acidic lithium chloride solution was added at a ratio of 9 mL per 3.0 g of bacterial cells. The cells and the acidic lithium chloride solution were incubated in an ice-water bath for 30 minutes, centrifuged at 10,000×g for 20 minutes, and the supernatant collected. The supernatant contained the surface protein.

[0088] Surface protein is secreted into the extracellular or is attached to cell surface after intracellular synthesis, performs various functions, as maintaining cell morphology, adhesion and regulating immunity, and surface protein mainly comprises S-layer protein, LPXTG motif protein and part-time protein etc.The present inventor, by the protein spectrum identification (Table A) to Lactobacillus paracasei K56 surface protein, finds that Lactobacillus paracasei K56 does not contain S-layer protein, mainly comprises LPXTG motif protein K0NBH9 (170kDa), KONA48 (30kDa), K0NAB9 (46kDa), and part-time protein A0A0K1KXQ2 (37kDa), K0N7K7 (58kDa), KOMU67 (47kDa), A0A0K1KYZ6 (44kDa), K0N700 (10kDa) etc.

[0089] Table A Surface protein spectrum identification results of Lactobacillus paracasei K56

[0090]

[0091] (4) Lactobacillus paracasei K56 surface protein-removing bacteria

[0092] The activated Lactobacillus paracasei was inoculated into MRS broth at a ratio of 1% (v / v) and cultured at 37°C for 16h. The cells were centrifuged at 3520 × g for 10min at 4°C, and the supernatant was discarded to collect the bacterial precipitate. 9mL of 5mol / L, pH 2.0 acidic lithium chloride solution was added to every 3.0g of cells. The cells and the acidic lithium chloride solution were incubated in an ice-water bath for 30min, centrifuged at 10000 × g for 20min, and the bacterial precipitate was collected. The cells were washed with sterilized 0.9% saline solution, and the concentration of the bacterial solution was adjusted to 10% with 0.9% sterile saline after three repetitions. 8 CFU / mL is reserved.

[0093] Example 2 Establishment of a high-fat diet model and the effect of Lactobacillus paracasei K56 and its postbiotics on mouse body weight Regulatory effect

[0094] C57BL / 6 male mice, 6 weeks old, weighing 15-20g. The temperature of the animal laboratory was 22±2℃, the humidity was 40-60%, and the light-dark cycle was 12h (lighting time 8:00A.M~8:00P.M). The mice were free to drink water and eat SPF bar feed, and were pre-fed for one week to adapt to the environment. All mice were divided into two groups, a normal group (15 mice) and a model group (65 mice). The normal group was fed with normal feed, and the model group was fed with high-fat feed. The mice were kept in separate cages, 5 mice per cage, and the weight of the mice was measured once a week. After 9 weeks of feeding, 10 normal mice (blank group (Con)) and 60 successfully modeled high-fat mice were taken.

[0095] Sixty high-fat diet mice were randomly divided into six groups (n=10): a model group (Mod, or HFD), a live Lactobacillus paracasei K56 group (LAL), a Lactobacillus paracasei K56 surface protein group (SLA), a Lactobacillus paracasei K56 surface protein-deficient group (LA), a killed Lactobacillus paracasei K56 group (DLA), and a drug-positive control group (ORT). The blank group continued to be fed a normal diet, while the remaining groups continued to be fed a high-fat diet. The specific gavage schedule for each group is shown in Table 1. Daily before treatment, the mice's growth status was recorded, and observations were made for mortality, mental state, hair gloss, and activity. Body weight was measured once a week at a fixed time before gavage.

[0096] Table 1 Grouping of mice and daily gavage

[0097]

[0098]

[0099] 1. Determination of mouse weight and body mass index

[0100] Mice were weighed weekly, and weight gain was recorded. Body length (the distance from the nose tip to the anus) was measured after sacrifice. Liver and epididymal fat were removed and weighed after dissection. Lee's index and organ index were calculated using the following formula:

[0101]

[0102]

[0103] The mice were weighed at a fixed time every week from the beginning of the intervention, and the weight gain of the mice was recorded. The intervention lasted for 16 weeks in total. A high-fat diet-induced mouse obesity model was established by feeding the mice with a high-fat diet for 9 weeks. Live, inactivated, and surface-protein-depleted Lactobacillus paracasei K56 bacteria and their surface proteins were administered orally to the mice on the high-fat diet. A high-fat diet for 9 weeks caused obesity in the mice. Compared with the mice in the CON group with a normal diet, the weight of the mice in the HFD group increased significantly (P < 0.05), with a weight gain of 15%-20%. The obese mouse model was successfully established. Afterwards, after 6 cycles of gavage intervention, if Figure 1As shown, the CON group gained 3.178g, and the HFD group gained 3.029g. Weight loss was more pronounced in the sixth week of drug intervention, likely due to gavage damaging the esophagus and suppressing appetite. The weight gains in the HFD+live K56 group, HFD+deproteinized K56 group, HFD+deproteinized K56 group, HFD+inactivated K56 group, and drug-positive group were 0.714g, 1.578g, 0.733g, 1.416g, and 0.878g, respectively. This suggests that live K56, surface protein, and inactivated Lactobacillus paracasei significantly inhibited weight gain in mice induced by a high-fat diet, with live K56 and surface protein being more effective, reducing the weight of mice induced by a high-fat diet. Deproteinized K56 also inhibited weight gain in mice to a certain extent.

[0104] In addition, after 6 weeks of oral gavage intervention, other items or indicators were also tested simultaneously. Please refer to the subsequent examples for details.

[0105] Example 3 Regulatory Effects of Lactobacillus paracasei K56 and Its Postbiotics on Obesity in Mice

[0106] 1. Mouse insulin sensitivity assay

[0107] Glucose tolerance test (OGTT): After fasting for 12 hours, baseline blood glucose levels were measured (0 minutes), followed by oral administration of 2 g / kg glucose. Blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 minutes.

[0108] Fasting blood glucose measurement in mice: Blood glucose was measured before killing mice, which had fasted for 12 hours. The mice were gently pricked in the tail with a lancet, a drop of blood was manually squeezed out, and then quickly dripped onto a blood glucose test strip. The result was then read using a rapid blood glucose meter.

[0109] Determination of fasting serum insulin in mice: Collect blood from mice to prepare serum. For insulin determination, aspirate 10 μL of serum and use it to determine the serum insulin level using the ELISA method, following the kit instructions.

[0110] Fasting blood sugar is an important indicator for determining the degree of obesity in mice. Insulin resistance can block the entry of glucose in the blood into cells, causing an increase in blood sugar levels. Figure 3 A shows that the fasting blood glucose of mice in the CON group was the lowest. There was a significant difference in the fasting blood glucose between the HFD group and the LAL group, SLA group and ORT group (P<0.05). Although there was no significant difference in the fasting blood glucose between the LA group and the DLA group and the HFD group, there was a certain downward trend. This indicates that Lactobacillus paracasei K56 and its postbiotics have a certain protective effect on the increase in fasting blood glucose in mice induced by a high-fat diet. Figure 3As shown in Figure 15, blood glucose levels in all groups increased 15 minutes after glucose injection, reaching a peak at 30 minutes and then showing a downward trend. Fasting blood glucose levels in mice in the CON group remained low throughout the experimental period, rising slightly and returning to normal levels after 2 hours. Fasting blood glucose levels in mice in the HFD group increased significantly, but decreased more slowly than in the other groups, remaining at a high level after 2 hours. Compared with the HFD group, blood glucose levels in all treatment groups were significantly lower within 2 hours than in the HFD group. Blood glucose trends in the LAL, SLA, and ORT groups were significantly more stable and closer to normal levels, indicating that live Lactobacillus paracasei bacteria and surface proteins have better tolerance to glucose injection. At 2 hours, blood glucose levels in the LAL group were not significantly different from those in the CON group, and were superior to those in the ORT group.

[0111] The statistics of the area under the curve (AUC) can illustrate the glucose tolerance of mice in different groups from another perspective. Figure 3 As shown in Figure C, the AUC values ​​of mice in the HFD group were significantly higher than those in the CON group, indicating that the high-fat diet disrupted blood glucose homeostasis in mice and led to abnormal glucose metabolism in mice fed a high-fat diet. Compared with the HFD group, the AUC values ​​of the LAL, SLA, LA, and ORT groups were significantly lower (P<0.05), demonstrating better glucose regulation. This indicates that live Lactobacillus paracasei K56, surface protein, Lactobacillus paracasei K56 without surface protein, and orlistat can all improve glucose tolerance in diet-induced obese mice, with live Lactobacillus paracasei K56 having the greatest effect.

[0112] Similar to blood sugar levels, insulin levels also increase in obesity. Insulin levels are also an important indicator for determining insulin sensitivity in mice. Figure 3 As shown in D, the insulin level of mice in the CON group was the lowest. Compared with the CON group, the insulin level of the HFD group was significantly increased. Compared with the HFD group, the serum insulin levels of mice in the LAL group, SLA group, and ORT group were significantly decreased, indicating that the live bacteria and surface protein of Lactobacillus paracasei K56 have a protective effect on hyperinsulinemia in mice induced by a high-fat diet.

[0113] 2. Determination of serum leptin, adiponectin, ALT, AST and blood lipids in mice

[0114] The serum of mice in each intervention group was collected, and the levels of blood biochemical indicators (TG, TC, HDL-C, LDL-C, leptin, adiponectin, ALT, AST) of mice in different groups were determined using the kit.

[0115] The four blood lipids include TC, TG, LDL-C and HDL-C, which are closely related to atherosclerosis, diabetes, coronary heart disease, obesity, etc. Figure 2 As shown, compared with the CON group, serum TG, TC, and LDL-C levels in the HFD group were significantly increased, while HDL-C levels were significantly decreased, indicating that the model mice developed abnormal lipid metabolism after continuous high-fat diet feeding. Compared with the HFD group, the LAL, SLA, LA, and ORT groups all significantly reduced serum TC, TG, and LDL-C levels and increased serum HDL-C levels (P < 0.05). The DLA group significantly reduced serum TC and TG levels and increased serum HDL-C levels (P < 0.05). Live Lactobacillus paracasei K56 bacteria, surface proteins, and orlistat had a more pronounced effect. These results indicate that Lactobacillus paracasei K56 and its postbiotics improve lipid metabolism disorders in mice induced by a high-fat diet by reducing serum TC, TG, and LDL-C levels and increasing HDL-C levels.

[0116] Leptin is a hormone that regulates lipid storage and enhances energy consumption. It has the functions of regulating blood sugar, lowering blood sugar levels, and maintaining blood sugar homeostasis. Figure 4 As shown in A, a high-fat diet significantly increased the serum leptin level. Compared with the HFD group, the serum leptin levels of mice in the LAL, SLA, and ORT groups were significantly decreased (P<0.05), indicating that live Lactobacillus paracasei K56 bacteria and surface protein can more effectively improve the leptin resistance caused by obesity in mice, which is of great significance for preventing obesity in mice.

[0117] Adiponectin is an insulin-sensitizing hormone that can improve insulin resistance and atherosclerosis in mice. Figure 4 As shown in B, compared with the CON group, the adiponectin level of mice in the HFD group was significantly decreased. After intervention in each treatment group, the adiponectin levels in the LAL group, SLA group, LA group, and ORT group were significantly increased compared with the HFD group (P<0.05). There was no significant difference in the adiponectin levels between the LAL group and the ORT group and the CON group. The results showed that live Lactobacillus paracasei K56, surface protein, Lactobacillus paracasei K56 without surface protein, and orlistat had a certain improvement effect on the decrease in adiponectin level induced by high-fat diet, and live Lactobacillus paracasei K56 could regulate adiponectin to normal levels.

[0118] ALT and AST are considered to be important indicators of liver damage caused by hepatic steatosis and inflammatory response. Figure 5As shown in A and 5B, the ASL and ALT activities in the serum of mice in the HFD group were significantly increased compared with those in the CON group (P<0.05). Compared with the HFD group, the ASL and ALT activities in the serum of mice in the LAL group, SLA group, and ORT group were significantly decreased (P<0.05). Although there were no significant differences in the ASL and ALT activities in the serum of mice in the LA group and DLA group compared with those in the HFD group, the overall activities were reduced, indicating that live Lactobacillus paracasei K56, surface protein, and orlistat had inhibitory effects on the ASL and ALT activities, and Lactobacillus paracasei K56 surface protein-depleted bacteria and dead Lactobacillus paracasei K56 also had certain effects.

[0119] The increase of LPS, the active component of endotoxin, will increase the synthesis and release of pro-inflammatory factors in the intestine, causing intestinal inflammation and intestinal mucosal barrier damage. Figure 5 As shown in Figure C, after long-term high-fat diet feeding, the serum LPS content of mice in the HFD group was significantly increased compared with the CON group (P<0.05). However, intervention with live Lactobacillus paracasei K56 significantly reduced the serum LPS content of mice with obese liver injury (P<0.05), indicating that live Lactobacillus paracasei K56 can effectively inhibit the increase in serum LPS content caused by a high-fat diet.

[0120] 3. Determination of lipid accumulation in mouse liver

[0121] HE staining: Embed the tissue in paraffin and cut into 4 μm sections. Bake at 60℃ for 2 hours. Dewax the paraffin sections and place them in the following order:

[0122]

[0123] Wash with tap water for 10 minutes. Hematoxylin staining: Stain sections in hematoxylin solution for 3-5 minutes, rinse with tap water for 2 minutes, differentiate in 1% hydrochloric acid ethanol solution for 10-15 seconds, and rinse with tap water for 20 minutes to return to blue. Eosin staining: Stain sections in eosin solution for 50 seconds, then rinse with tap water for 2 minutes. Dehydration and mounting: Place sections in 80% ethanol for 30 seconds, 95% ethanol for 30 seconds, anhydrous ethanol I for 1 minute, anhydrous ethanol II for 2 minutes, xylene I for 2 minutes, and xylene II for 5 minutes to clear, then mount sections with neutral gum. Microscopic examination and image acquisition and analysis. Interpretation: Cell nuclei appear blue, and cytoplasm appears red.

[0124] Determination of Free Fatty Acids in Mouse Liver: Fresh mouse livers of similar size are homogenized and the supernatant is used for FFA determination. The determination principle is that FFAs bind to copper ions to produce a yellow-green color, the depth of which is proportional to the concentration of the fatty acid. The FFA level in the sample is calculated by comparing the absorbance of the sample with that of a standard and a blank.

[0125] Determination of oxidative stress markers in mouse liver: Weigh an appropriate amount of liver and 9 times the weight of the tissue block in normal saline. Place the mixture in a manual homogenizer and grind in an ice bath. Once homogenized, centrifuge the liver homogenate at 2500×g for 10 minutes at 4°C, and remove the supernatant. An appropriate amount of the supernatant is used to assay liver oxidative markers (SOD, CAT, and MDA levels) using assay kits.

[0126] The liver is the main organ for fat deposition in obese mice. The mouse liver coefficient is one of the important indicators for evaluating the health and functional status of the mouse liver. The mouse abdominal epididymal fat coefficient is also an important indicator reflecting lipid metabolism. Figure 6 As shown, compared with the CON group, the liver weight, liver coefficient, epididymal fat weight, and epididymal fat coefficient of mice in the HFD group were significantly increased (P < 0.05), indicating that a high-fat diet can lead to abnormal liver status and excessive fat accumulation in mice. Compared with the HFD group, the liver weight, liver coefficient, epididymal fat weight, and epididymal fat coefficient of the LAL and SLA groups were significantly decreased (P < 0.05). The liver weight of the LAL group was significantly decreased, while the liver coefficient, epididymal fat weight, and epididymal fat coefficient did not differ significantly. These results indicate that live Lactobacillus paracasei K56 bacteria and surface proteins significantly inhibited high-fat diet-induced fat accumulation in mice. However, dead Lactobacillus paracasei K56 bacteria and those without surface proteins had a weaker ability to improve obesity in mice on a high-fat diet.

[0127] SOD in normal tissues is an important antioxidant metalloenzyme that removes ROS. A high-fat diet can induce an increase in the level of oxidative stress in the liver of mice and reduce the SOD content in the liver. Figure 7 As shown in Figure A, the SOD activity in the livers of mice in the HFD group was significantly lower than that in the CON group (P < 0.05), indicating successful modeling. This may be due to the rapid increase in oxidative stress in the liver, which leads to the depletion of the SOD enzyme in the liver that plays an antioxidant role. After oral administration of Lactobacillus paracasei K56 to each treatment group, the SOD activity in the liver of mice increased slightly, but there was no significant difference and it did not return to the level of the CON group.

[0128] Excessive accumulation of MDA, the final product of lipid peroxidation metabolism, can damage the structure of biological membranes. Figure 7 As shown in Figure B, after 16 weeks of high-fat diet feeding, the MDA content in the livers of mice in the HFD group was nearly three times higher than that in the CON group, a highly significant difference (P<0.05), indicating significant lipid peroxidation. Although there were no significant differences in MDA content in liver cells among the treatment groups, it decreased in the livers of mice in the LAL, SLA, DLA, and ORT groups, indicating a decrease in intracellular lipid peroxidation.

[0129] CAT can work together with GPx to remove the toxic substance hydrogen peroxide produced by cell metabolism and reduce its damage to cells. Figure 7 As shown in Figure C, compared with the CON group, the CAT activity in the liver of the HFD group was significantly decreased, and compared with the HFD group, the CAT activity in the liver of each treatment group was significantly increased (P<0.05).

[0130] These results indicate that after 16 weeks of high-fat diet feeding, the activities of SOD and CAT in the livers of mice were significantly lower than those in the CON group, while the MDA content was significantly higher. This may be due to the long-term accumulation of reactive oxygen species (ROS) in the body caused by the high-fat diet, which metabolizes and produces a large number of reactive oxygen species (ROS). This leads to a significant depletion of antioxidant enzymes such as SOD and CAT, and a reduction in the body's antioxidant defenses. Furthermore, the free radicals react with unsaturated fatty acids on cell membranes, accelerating lipid peroxidation and increasing MDA content, leading to severe damage to cell membranes. Compared with the HFD group, the activity of the antioxidant enzyme SOD in the livers of mice treated with Lactobacillus paracasei K56 was enhanced, CAT activity was significantly increased, and MDA content was reduced, thereby enhancing the body's antioxidant capacity and alleviating liver damage caused by the high-fat diet.

[0131] The results of histological analysis of epididymal fat ( Figure 8 A) shows that adipocytes in the CON group were round and regularly arranged; adipocytes in the HFD group were significantly larger than those in the other groups, while the LAL group significantly reduced adipocyte size. These results indicate that Lactobacillus paracasei K56 significantly inhibited high-fat diet-induced fat accumulation and adipocyte expansion in mice, further suppressing high-fat diet-induced obesity in mice.

[0132] Colon tissue sections stained with HE can directly reflect the pathological condition of mouse colon tissue. Figure 8 As shown in Figure B, the intestinal mucosa of the CON group mice showed neatly arranged colonic mucosa, without any damage or atrophy. The HFD group mice showed severe colonic damage, with reduced residual goblet cells and loose structure, severe mucosal epithelial damage, and disrupted crypt structure, indicating that a high-fat diet can cause abnormalities in the shape and function of goblet cells in the intestinal mucosa. The LAL group significantly improved the irregular arrangement, atrophy, and defects of the intestinal mucosa, increased the number of goblet cells, and reduced crypt cell loss. Therefore, in the context of high-fat diet-induced colonic inflammation in obese mice, Lactobacillus paracasei K56 intervention was able to repair morphological damage to the colon of obese mice.

[0133] The level of FFA in the liver is an important indicator for evaluating the degree of lipid accumulation in the mouse liver. FFA in the liver can damage liver cells by increasing the oxidative stress of liver cells. Figure 9It can be seen that compared with the CON group, the FFA level in the HFD group was significantly increased (P<0.05), and compared with the HFD group, the FFA levels in the LAL group and the ORT group were significantly decreased (P<0.05). This shows that continuous high-fat diet feeding can significantly increase the level of FFA in the liver, and live Lactobacillus paracasei K56 has a significant inhibitory effect on the increase of FFA level in the mouse liver.

[0134] 4. Effect of mRNA expression of related factors in mouse adipose tissue

[0135] The expression of FAS, PPAR-γ and CEBP-α mRNA in adipose tissue (epididymis) was detected by RT-PCR, and the RNA concentration was measured after RNAm was extracted.

[0136] FAS is a rate-limiting enzyme that plays an important role in fatty acid regeneration and has a rich enzyme system function. Obesity is caused by excessive expression of FAS, which leads to fat deposition in the body. Orlistat can inhibit the expression of FAS gene and thus inhibit fat formation. Figure 10 As shown in Figure A, compared with the CON group, the relative expression of the FAS gene in adipose tissue of mice in the HFD group was significantly upregulated (P<0.05). Compared with the HFD group, the relative expression of the FAS gene in the LAL, SLA, LA, and ORT groups was significantly downregulated (P<0.05), but still somewhat lower than that in the CON group. The DLA group also had a certain effect on the downregulation of the relative expression of the FAS gene. These results suggest that the intervention of live Lactobacillus paracasei K56, surface protein, Lactobacillus paracasei K56 without surface protein, and orlistat may downregulate the expression of lipase genes, thereby inhibiting fat synthesis.

[0137] Activation of peroxisome proliferator-activated receptors (PPARs) can regulate glucose and lipid metabolism, adipocyte differentiation, insulin resistance and inflammatory response. Figure 10 As shown in B, compared with the CON group, the expression of PPAR-γ gene in the epididymal adipose tissue of mice in the HFD group was significantly increased; compared with the HFD group, the expression of PPAR-γ gene in the epididymal adipose tissue of mice in the LAL group, SLA group and ORT group was decreased, and there were significant differences, indicating that the intervention of live Lactobacillus paracasei K56 bacteria and surface protein regulated the expression of PPAR-γ gene.

[0138] CEBP-α plays an important role in the differentiation of adipocytes. Increased expression of PPARγ in mice is also beneficial to the expression of CEBP-α. Figure 10As shown in C, compared with the CON group, the relative expression of CEBP-α gene in epididymal adipose tissue of mice in the HFD group was significantly increased (P<0.05). Compared with the HFD group, the relative expression of CEBP-α gene in the LAL group, SLA group, LA group, and ORT group was significantly decreased (P<0.05).

[0139] In summary, live Lactobacillus paracasei K56, surface protein, Lactobacillus paracasei K56 without surface protein, and orlistat can all inhibit the increased expression of FAS, CEBP-α, and PPARγ genes in epididymal adipose tissue of mice induced by a high-fat diet, and the live Lactobacillus paracasei K56 and surface protein have a stronger inhibitory effect, thereby reducing the formation of adipocytes, promoting fat oxidation, and reducing the fat content in mice.

[0140] Example 4: Lactobacillus paracasei K56 postbiotics regulate colon immune disorders induced by a high-fat diet

[0141] 1. Determination of inflammatory factor levels in mouse colon

[0142] After killing the mice, the colon tissue (from the cecum to the anus) was removed and washed with saline. The length of the colon was measured and recorded. The colon contents were then scraped with forceps and stored in a -80°C refrigerator. The proximal colon tissue was weighed and added with 19 volumes of saline (weight-to-volume ratio, e.g., 10 mg tissue 190 μL saline). The tissue was then homogenized in a tissue homogenizer for 40 seconds. The supernatant was then centrifuged at 4°C and 3500 × g for 15 minutes. The supernatant was then used to determine the levels of inflammatory factors (IL-6, IL-1β, IL-10, and TNF-α).

[0143] Increased LPS levels are associated with low-grade chronic inflammation in local tissues; TNF-α, IL-1β, and IL-6 are pro-inflammatory cytokines secreted by monocytes, macrophages, and intestinal epithelial cells. IL-10 is a classic anti-inflammatory cytokine in the intestine.

[0144] The proximal colon of mice was homogenized and the supernatant was used for ELISA analysis. Figure 11As shown, the HFD group fed a high-fat diet showed significant inflammation of the colonic tissue. Specifically, the levels of pro-inflammatory TNF-α, IL-1β, and IL-6 increased significantly, while the level of anti-inflammatory IL-10 decreased significantly (P < 0.05). Feeding Lactobacillus paracasei K56 had a certain inhibitory effect on high-fat diet-induced colonic inflammation. Compared with the HFD group, the LAL, SLA, and ORT groups significantly reduced the level of TNF-α, the LAL, LA, DLA, and ORT groups significantly reduced the levels of IL-6 and TNF-α, and the LAL, LA, and ORT groups significantly increased the level of IL-10 (P < 0.05). These results indicate that live Lactobacillus paracasei K56 significantly reduced the level of pro-inflammatory factors in the mouse colon, alleviated the elevated levels of inflammatory factors, and thus alleviated inflammation.

[0145] 2. Immunohistochemistry to detect the expression of tight junction proteins (Claudin, ZO-1, Occludin)

[0146] Dewax paraffin sections and dehydrate. Antigen retrieval: Prepare sodium citrate antigen retrieval solution: distilled water (1:49). Mix well and microwave on high for approximately 3 minutes until boiling. Reduce heat to low for 10 minutes. Remove and allow to cool naturally to room temperature (do not allow the temperature to drop suddenly). Remove sections from the retrieval solution and wash twice with distilled water, then three times with 1× PBS (5 minutes each). Blocking: Drain the edges of the tissue sections, circle the tissue with an immunohistochemical marker, add 3% hydrogen peroxide, and incubate in a humidified chamber at room temperature in the dark for 10 minutes. Blocking: After incubation, rinse three times with 1× PBS (5 minutes each). Drain the sections and dilute goat serum blocking solution with 1× PBST (1:10). Add blocking solution and block in a humidified chamber at room temperature for 30-60 minutes. Primary Antibody Incubation: Pour off the blocking solution and drip an appropriate amount of the corresponding primary antibody working solution (diluted with blocking solution according to the corresponding ratio) onto the tissue within the circle. Incubate in a humidified chamber at 4°C overnight. Secondary Antibody Incubation: The next day, remove the humidified chamber from the refrigerator, return it to room temperature, and rinse three times with 1× PBS solution for 5 minutes each. Drain the liquid and drip an appropriate amount of enzyme-labeled anti-mouse / rabbit secondary antibody solution (1:1000) and incubate at room temperature for 1 hour. DAB Counterstaining of Nuclei: After slightly drying the sections, drip DAB staining solution into the circle and incubate at room temperature in the dark for 2-10 minutes. Mounting: Add antifade mounting solution to the slide and gradually cover it with a coverslip from one side to avoid bubbles. Observe the slides under a microscope.

[0147] The distribution of tight junction proteins Claudin-1, ZO-1 and Occludin immunohistochemical staining was observed. After immunohistochemical staining, the expression of Claudin-1, ZO-1 and Occludin showed a brown-yellow, interwoven, continuous network structure, and the brown-yellow was continuously distributed along the membrane. Figure 12 As shown, intestinal epithelial cells in the CON group were neatly arranged and continuously distributed, with evenly distributed tight junction proteins and a high number of positive cells. In the HFD group, claudin-1, ZO-1, and occludin proteins were mostly discontinuous, diffuse, and weakly positive, and tight junction protein expression was significantly reduced. Compared with the HFD group, tight junction protein expression increased in all intervention groups, with the SLA and ORT groups almost returning to normal levels, close to those in the CON group. This suggests that the surface protein of Lactobacillus paracasei K56 can more effectively ameliorate the reduction of tight junction proteins induced by a high-fat diet, strengthen the intestinal barrier, protect the junctions between colonic epithelial cells in high-fat mice, significantly increase intestinal permeability, and promote intestinal integrity.

[0148] 3. Western blot detection of the expression of NF-κB / JNK inflammatory pathway-related proteins (TLR4, p65, p50, JNK) in colon tissue

[0149] SDS-PAGE electrophoresis: Clean the glass plates. After the plates have dried, pair one concave glass plate with another flat glass plate, with the flat glass facing outward and the concave glass facing inward. The space between the plates will be the gap for gel pouring. Place the plates in the gel dispenser and check that the bottoms are aligned to prevent gel leakage. Prepare a 10% separating gel according to your experimental needs. Add TEMED and immediately shake well before pouring the gel. Fill the separating gel to the appropriate level. Before pouring, test the gel with a comb; the comb teeth should be approximately 5-8 mm above the gel surface. Then, slowly and evenly add anhydrous ethanol to the gap until it is completely filled, taking care not to disperse the gel. Allow the separating gel to solidify in approximately 30 minutes. Pour off the anhydrous ethanol and blot with absorbent paper. Prepare a 5% stacking gel according to the manufacturer's instructions. Add TEMED and immediately shake well before pouring the gel. Fill the remaining space with stacking gel and insert the comb into the gel, making sure there are no bubbles under the comb. Once the stacking gel has solidified, remove the gel dispenser and carefully remove the comb. You are ready to begin electrophoresis. Place the gel caster in the electrophoresis tank, add sufficient electrophoresis buffer, and then add the protein marker and sample in order. Run at 80V for 20 minutes, then at 120V for 1 hour.

[0150] Transfer: Place a transfer clamp, two sponge pads, and two thick filter papers in a tray with transfer solution. Open the gel plate, cut the gel according to the size of the target protein, and place it in the tray. Cut the PVDF (0.45um) according to the size of the gel and activate the PVDF membrane in methanol. Open the clamp, white on the left and black on the right, add a sponge and a layer of thick filter paper on each side. Carefully place the gel on the black filter paper, cover the PVDF membrane on the gel, and avoid bubbles. Cover the membrane with a piece of filter paper and finally cover it with another sponge pad. Transfer conditions (wet transfer): Place the transfer tank in ice water for transfer, and transfer at a constant current of 200mA for 70 minutes.

[0151] Immunoreaction: Place the transferred membrane in an incubation tank filled with TBST, rinse quickly once, then add an appropriate amount of blocking solution, place on a decolorizing shaker, and block for 1 hour at room temperature. Dilute the primary antibody according to the antibody instructions. After preparation, pour out the blocking solution in the incubation tank, add the prepared primary antibody (1:1000 dilution of blocking solution), and incubate on a shaker at 4°C overnight (slow shaking). Rinse the membrane quickly three times with TBST, then add TBST and place on a decolorizing shaker for rapid elution, each time for 10 minutes, for three washes. Dilute the secondary antibody in blocking solution at a ratio of 1:5000, then add it to the incubation tank, place on a shaker for slow shaking, and incubate at room temperature for 1 hour. Pour out the secondary antibody, then add TBST again, and place on a decolorizing shaker for rapid elution, each time for 10 minutes, for three washes.

[0152] Exposure: In a darkroom, mix developer A and developer B in a ratio of 1:1. Develop one strip at a time, developing each strip in turn. Adjust the exposure conditions according to the different luminous intensities.

[0153] Membrane washing: Wash the membrane according to the instructions of the membrane regeneration solution (strong), and then perform the following steps such as sealing according to the above steps.

[0154] Reagent recipe: Electrophoresis buffer: 3.03g Tris + 14.4g glycine + 1g SDS, dilute to 1L with dH2O. Transfer buffer: 3.03g Tris + 14.4g glycine + 200mL methanol, dilute to 1L with dH2O. Blocking buffer: 5g skim milk powder + 100mL 0.1% TBST, mix thoroughly.

[0155] The present invention observed the changes in TLR4 / NF-κB in the intestine of mice fed with a high-fat diet, and detected the expression levels of TLR4, p50 and p-p65 / p65 proteins. The results showed that the LPS content in the serum of mice fed a high-fat diet increased significantly, and the level of inflammatory factors in the colon tissue also increased significantly. However, after the intervention of Lactobacillus paracasei K56 and its postbiotics, the levels of IL-6, IL-1β and TNF-α decreased to varying degrees, and the level of IL-10 increased. Therefore, we further measured the expression of related proteins in the TLR4 / NF-κB pathway to explore whether the alleviating effect of Lactobacillus paracasei K56 and its postbiotics on colon immune disorders caused by a high-fat diet involves this pathway. Figure 13As shown in the results, compared with the CON group, the protein expression level of TLR4 in the cell membrane and the expression of p50 and p-p65 / p65 proteins in the cell nucleus were significantly upregulated after high-fat diet feeding (P<0.05). These changes were inhibited after intervention with Lactobacillus paracasei K56 and its postbiotics, but the effects of the DLA and LA groups appeared to be weaker than those of the LAL, SLA, and ORT groups. These results were consistent with the trend of changes in related biochemical indicators in mouse serum, liver, and colon. These results indicate that a high-fat diet can induce an inflammatory response in the colon, and Lactobacillus paracasei K56 and its postbiotics can improve the occurrence of inflammation in the colon of obese mice fed a high-fat diet by inhibiting the expression of related proteins in the TLR4 / NF-κB signaling pathway.

[0156] The c-Jun N-terminal kinase (JNK) pathway is a cell stress pathway that can be used as a potential therapeutic target for obesity. After LPS acts on TLR4, it can also mediate extracellular signals into cells to regulate inflammatory responses by activating the MAPK signaling pathway. Figure 14 As shown in the data, compared with the CON group, the protein expression level of p-JNK / JNK in the colon of mice in the HFD group was significantly increased, indicating that a high-fat diet may induce an inflammatory response in the colon by activating the JNK signaling pathway. Compared with the HFD group, the protein expression levels of p-JNK / JNK in the LAL group, DLA group, LA group, SLA group and ORT group were significantly decreased (P<0.05), indicating that live Lactobacillus paracasei K56, Lactobacillus paracasei K56 with surface protein removed, dead Lactobacillus paracasei K56, surface protein and orlistat may regulate the occurrence of inflammation in the body by inhibiting p-JNK / JNK.

[0157] Example 5: Lactobacillus paracasei K56 postbiotics regulate intestinal flora imbalance caused by high-fat diet

[0158] Total bacterial DNA was extracted from mouse intestinal contents according to the QIAamp DNA Stool Mini Kit instructions. DNA was quantified using Nanodrop, and the quality of the extracted DNA was verified by 1.2% agarose gel electrophoresis. Primers were designed to amplify conserved regions of bacterial 16S rDNA using polymerase chain reaction (PCR), and sample-specific barcode sequences were added.

[0159] PCR conditions were: 95°C, 3 min; 24 cycles of 95°C, 5 s, 57°C, 90 s, 72°C, 10 s; and 72°C, 5 min. A 20 μL reaction system included: 2.5 μL TransStart buffer, 2 μL dNTPs, 1 μL forward primer, 1 μL reverse primer, 0.5 μL Pfu polymerase (all-gold), and 20 ng template DNA. The PCR amplification products were quantified using the Quant-iTPicoGreen dsDNA Assay Kit using a Microplate reader (BioTek, FLx800). Based on the fluorescence quantification results and the sequencing requirements for each sample, the samples were mixed in the appropriate proportions.

[0160] Sequencing libraries were then prepared using the TruSeq Nano DNA LT Library Prep Kit (Illumina). The positive and negative reads obtained from the double-end sequencing were first spliced ​​in pairs. Sequences containing N in the spliced ​​results were filtered, and sequences greater than 200 bp were retained. After quality filtering and purification of chimeric sequences, the resulting sequences were clustered into operational taxonomic units (OTUs) at 97% sequence homology using the QIIME2 dada2 analysis pipeline. These OTUs were aligned with the Greengenes database using PyNAST software and taxonomic information was annotated. α-diversity and β-diversity were analyzed using QIIME2 software (2019.4) and R software (V3.4.1). Principal coordinates analysis (PCoA) was performed on each group of samples using the Jaccard method, and distance box plots of the samples were drawn. Using the Galaxy online analysis platform, the LEfSe analysis method simultaneously performed differential analysis on all classification levels, drew LDA value distribution bar charts and species taxonomy branch diagrams of significantly different species, and searched for robust differential species between groups.

[0161] 1. Mouse intestinal flora species OTU

[0162] The Venn diagram illustrates the effects of Lactobacillus paracasei K56 and its postbiotics on the OTUs contained in high-fat diet-induced obese mice, indicating the similarity of the intestinal flora contained in different samples. OTU clustering was performed on non-repeated sequences (excluding single sequences) at 97% similarity, and chimeras were removed during the clustering process to obtain representative sequences of OTUs. Figure 15As shown, a Venn diagram illustrates the relationship between the microbial composition diversity of the control, model, orlistat, LAL, SLA, DLA, and LA groups. A total of 1152 OTUs were identified, including 483 unique OTUs in the control group, 103 of which were unique; 96 unique OTUs in the model group; 105 unique OTUs in the orlistat group; 171 unique OTUs in the LAL group; 152 unique OTUs in the LA group; 79 unique OTUs in the SLA group; and 66 unique OTUs in the DLA group. A total of 380 OTUs were shared among the seven groups, accounting for 33% of the total OTUs. Lactobacillus paracasei K56 and its postbiotic intervention influenced the OTU composition of the mouse gut microbiota to a certain extent, with the LAL group having the most unique OTUs and the DLA group having the least unique OTUs.

[0163] 2. Alpha diversity analysis of mouse intestinal flora species

[0164] Alpha diversity can reflect the abundance and diversity of microbial communities, including a series of statistical analysis indices to estimate the species abundance and diversity of environmental communities. It is mainly related to two factors. One is the number of species in the sample, that is, richness, which is measured by the Chao1 index. The higher the Chao1 index value, the richer the species in the community; the second is the relative density of each species in the sample, that is, the uniformity of individual distribution in the community, which is represented by the Shannon index and the Simpson index. The larger the Shannon index value, the higher the community diversity; the larger the Simpson index value, the lower the community diversity. The Goodscoverage indicator reflects the sequencing depth and coverage of the sample. The changes in the Alpha diversity of the intestinal flora after treatment in different groups are as follows. Figure 16 As shown. Compared with the Control group, the Chao1 index in the LAL group was significantly increased (P < 0.05), indicating that the LAL group had a higher species richness. Compared with the Control and Model groups, the Orlistat group had a lower Simpson index, while the Shannon index in the LA and Orlistat groups was significantly increased (P < 0.05), indicating higher community diversity. The Goodscoverage values ​​of all samples were greater than 0.995, indicating that the sequencing coverage of the samples was high.

[0165] 3. Species dilution curve of mouse intestinal flora

[0166] The rarefaction curve mainly uses the microbial alpha diversity index of each sample at different sequencing depths to construct a curve to reflect the microbial diversity of each sample at different sequencing quantities. If the diversity index is sobs (representing the number of species actually observed), when the curve tends to be flat, it means that the amount of sequencing data is reasonable, and more data will only produce a small number of new species. Figure 17 This shows that the sequencing depth of this experiment covers most species diversity.

[0167] The Shannon curve is constructed based on the sequence of each sample and the microbial diversity index at different sequencing depths. A flat curve indicates that the sequencing data volume is sufficient to reflect the vast majority of microbial diversity in the sample. The figure below shows that this experiment meets the requirements for further analysis.

[0168] The Rank-Abundance curve can be used to explain two aspects of diversity: species richness and community evenness. As shown in the figure below, the width of the curve reflects species richness horizontally; the wider the curve on the horizontal axis, the higher the species richness. The shape (flatness) of the curve reflects the evenness of the community within the sample; the flatter the curve, the more evenly distributed the species.

[0169] 4. Beta diversity analysis of mouse intestinal flora

[0170] Beta diversity refers to the differences in species composition between different environmental communities. Sample hierarchical cluster analysis, principal component analysis (PCA), principal coordinates analysis (PCoA), and non-metric multidimensional scaling (NMDS) are commonly used to reflect beta diversity between samples. To better reflect the diversity of microbial colony results between groups, PCoA analysis is used. When samples are close together, it indicates a similar species structure, so samples with high similarity are more likely to cluster together. The PCoA analysis method is similar to PCA analysis, but PCA is based on Euclidean distance, while PCoA is not restricted by distance algorithms.

[0171] Jaccard analysis was used to analyze the beta diversity of the intestinal flora of each group of mice. The profiles of the intestinal flora of the seven groups of samples were different, indicating that there were differences in the flora structure of the seven groups. Figure 18 A reflects the effects of ordinary feed and high-fat feed on the changes in intestinal flora structure. The sample composition between the Model group and the Control group was significantly different. Figure 18 B reflects the differences in sample composition between each treatment group and the Control group. The differences in sample composition between the LAL group, LA group, SLA group, Orlistat group and the Control group were significant, and there was some overlap in sample composition between the DLA group and the Control group. Figure 18 C reflects the differences in sample composition between the treatment groups and the Model group. The sample composition between the LAL group, LA group, SLA group and the Model group is similar, which may be because the basic diets of the treatment group and the Model group are all high-fat feed. The microbial aggregation of the SLA group is tighter than that of the Model group, and the parallelism is better. Figure 18 As shown in D, high-fat diet intervention caused significant changes in the structure of the microbial community. The microbial community also changed after intervention with Lactobacillus paracasei K56 and its postbiotics. The microorganisms in the LAL group, DLA group, and SLA group clustered more closely.

[0172] 5. Composition analysis of mouse intestinal flora at the phylum level

[0173] The intestinal flora of mice in the control group, model group, Lactobacillus paracasei K56 and its metabiotic group, Lactobacillus paracasei K56 surface protein-depleted group, and orlistat group were compared at the phylum and genus levels to analyze the differences in the flora. Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria were the dominant phyla in the cecal microbial community. The results of the phylum-level analysis are as follows: Figure 19 As shown, the intestinal microbiota of experimental mice was mainly composed of Firmicutes, Bacteroidetes, Actionobacteria, Desulfobacterota, Verrucomicrobiota, Patescibacteria, and Proteobacteria at the phylum level. Firmicutes and Bacteroidetes dominated the intestinal microbiota of mice, accounting for over 70%. Compared with the control group, the relative abundance of Firmicutes and Bacteroidetes in the model group was not significantly different, while the relative abundance of Actionobacteria was significantly increased (P < 0.05). Compared with the model group, the relative abundance of Firmicutes was significantly decreased in the DLA and orlistat groups (P < 0.05). The relative abundance of Bacteroidetes and Actionobacteria did not differ significantly among the treatment groups.

[0174] The Firmicutes / Bacteroidetes ratio is positively correlated with host fat storage, body weight, visceral fat, and subcutaneous fat. Probiotics and prebiotics can reduce the Firmicutes / Bacteroidetes (F / B) ratio. Compared with the control group, the F / B ratio was significantly decreased in the LAL, DLA, LA, SLA, and ORT groups (P < 0.05), indicating that Lactobacillus paracasei K56 and its postbiotics can prevent and improve obesity by reducing the Firmicutes-to-Bacteroidetes ratio.

[0175] 6. Composition analysis of mouse intestinal flora at the genus level

[0176] The changes in intestinal flora were further analyzed at the genus level. Figure 20-22 It can be seen that the intestinal flora of the seven groups of mice at the genus level mainly include beneficial bacteria such as norank_f_Muribaculaceae, Dubosiella, Lachnospiraceae_NK4A136 (Lachnospiraceae_NK4A136_group), norank_f_Ruminococcaceae, Lactobacillus, Enterobacter, Coriobacteriaceae_UCG-002, Akkermansia, and Bifidobacterium; and harmful bacteria such as Ileibacterium, Desulfovibrio, Clostridia UCG-014_norank, norank_f__Erysipelotrichaceae, and norank_f__Oscillospiraceae.

[0177] norank_f_Muribaculaceae can inhibit intestinal barrier dysfunction, lipid metabolism disorders, suppress harmful bacteria and oxidative stress, and improve intestinal mucosal inflammation. The abundance of Dubosiella is negatively correlated with body weight; Lachnospiraceae_NK4A136_group is negatively correlated with a variety of metabolic diseases and chronic inflammation; Ruminococcaceae is positively correlated with the proportion of carbohydrates in high-fat feeds, especially plant polysaccharides. Lachnospiraceae_NK4A136_group and Ruminococcaceae are bacteria rich in the production of short-chain fatty acids (SCFAs) in healthy intestines. SCFAs are beneficial to maintaining the anaerobic environment in the intestine. Lactobacillus can prevent pathogens from invading and colonizing the intestine, inhibit the production of pathogens and endotoxins, and can also produce lactic acid, acetic acid and propionic acid to promote intestinal health; Enterorhabdus is negatively correlated with the concentration of pro-inflammatory cytokines and plays a positive role in intestinal homeostasis. Coriobacteriaceae_UCG-002 can improve the steatosis of non-alcoholic fatty liver disease, and may have the function of improving steatosis by affecting the sphingolipid metabolic pathway, thereby reducing the steatosis of liver cells. Studies have shown that Coriobacteriaceae can alleviate insulin resistance and improve liver steatosis. Akkermansia is a microorganism that inhibits the intestinal mucus layer. Because Akkermansia can increase the amount of mucin and reduce colonic inflammation, Akkermania is believed to have a positive effect on maintaining intestinal barrier homeostasis. The abundance of beneficial bacteria such as norank_f_Muribaculaceae, Lachnospiraceae_NK4A136_group, and Akkermansia did not change significantly at the genus level. Compared with the Control group, the Model group significantly reduced the relative abundance of beneficial bacteria such as Bifidobacterium (P < 0.05). Compared with the Model group, the SLA group significantly increased the relative abundance of norank_f_Ruminococcaceae, Lactobacillus, and Coriobacteriaceae_UCG-002; the LA group significantly increased the relative abundance of Lactobacillus (P<0.05).

[0178] norank_f__Erysipelotrichaceae and Ileibacterium both belong to the Erysipelotrichaceae family and are closely associated with metabolic disorders. Studies have shown that the abundance of Ileibacterium can increase tenfold in models of obesity and high-fat diets. Ileibacterium is likely associated with metabolic diseases, and its specific role requires further investigation. Desulfovibrio is a bacterium associated with intestinal inflammation and is associated with LPS production in mice with high-fat diet-induced obesity. Reducing the relative abundance of Desulfovibrio may help improve intestinal integrity and reduce intestinal inflammation. ClostridiaUCG-014_norank, Clostridium, is highly abundant in the intestine of early-stage liver cancer. Increased abundance of some pro-inflammatory bacteria, such as ClostridiaUCG-014, can induce inflammatory responses and upregulate pro-inflammatory lipid metabolites. norank_f__Oscillospiraceae is a pathogenic intestinal bacterium associated with inflammation that may participate in mucin degradation, triggering intestinal barrier damage and systemic inflammatory responses. The relative abundance of norank_f__Oscillospiraceae did not change significantly in any of the groups. Compared with the Model group, the relative abundance of Ileibacterium was significantly decreased in the DLA and ORT groups, while the relative abundance of Desulfovibrio increased in the DLA group, the relative abundance of norank_o__Clostridia_UCG-014 increased in the LA group, and the relative abundance of norank_f__Erysipelotrichaceae increased significantly in the ORT group (P < 0.05).

[0179] In summary, our results suggest that a high-fat diet can, to a certain extent, cause intestinal microbial disturbances in mice. Lactobacillus paracasei K56 and its postbiotic intervention can modulate some of these microbiota, with differences observed between different groups. In particular, Lactobacillus paracasei K56 surface proteins significantly increased the relative abundance of the beneficial bacteria norank_f_Ruminococcaceae, Lactobacillus, and Coriobacteriaceae_UCG-002. In particular, the Lactobacillus paracasei K56 surface proteins significantly increased the relative abundance of the beneficial bacteria norank_f_Ruminococcaceae and Coriobacteriaceae_UCG-002 compared to the live LAL group, achieving unexpected results.

[0180] It should be understood that the invention described herein is not limited to specific methodology, experimental protocols or reagents, as these may vary. The discussion and examples provided herein are presented only to describe specific embodiments and are not intended to limit the scope of the invention, which is limited only by the claims.

Claims

1. Use of a Lactobacillus paracasei K56 postbiotic in preparing a composition, the composition having one or more uses selected from the following (1)-(8): (1) Used to promote the proliferation of beneficial intestinal bacteria, wherein the beneficial intestinal bacteria are selected from one or more of norank_f_Ruminococcaceae, Lactobacillus, and Coriobacteriaceae_UCG-002, preferably one or more of norank_f_Ruminococcaceae and Coriobacteriaceae_UCG-002; (2) It helps regulate intestinal flora, wherein regulating intestinal flora is to promote the proliferation of beneficial intestinal bacteria, wherein the beneficial intestinal bacteria are selected from one or more of norank_f_Ruminococcaceae, Lactobacillus, and Coriobacteriaceae_UCG-002, preferably one or more of norank_f_Ruminococcaceae and Coriobacteriaceae_UCG-002; (3) Helps control body fat; (4) Helps maintain healthy blood lipid (cholesterol / triglyceride) levels; (5) Helps maintain healthy blood sugar levels; (6) Used to regulate obesity (such as that caused by a high-fat diet); (7) Used to regulate dyslipidemia and / or hepatic lipid accumulation (e.g., caused by a high-fat diet); (8) Used to regulate colon immune disorders (such as those caused by high-fat diet); in, The Lactobacillus paracasei K56 is deposited in the German Collection of Microorganisms and Cell Cultures with a deposit number of DSM 27447; it is also deposited in the China General Microbiological Culture Collection Center (CGMCC) of the China National Committee for the Administration of Microbiological Culture Collection with a deposit number of CGMCC 15139.

2. The use according to claim 1, wherein The composition is a pharmaceutical composition, a food (ordinary food or health food), a food additive, a dietary supplement or a feed.

3. The use according to any one of claims 1 to 2, wherein The composition is in an oral dosage form. Preferably, the oral dosage form is selected from the group consisting of solutions, suspensions, emulsions, powders, lozenges, pills, syrups, buccal lozenges, tablets, chewing gums, and capsules.

4. The use according to any one of claims 1 to 3, wherein The Lactobacillus paracasei K56 postbiotic is heat-inactivated bacteria of Lactobacillus paracasei K56 or surface protein of Lactobacillus paracasei K56.

5. The use according to claim 4, wherein The heat-killed bacteria of Lactobacillus paracasei K56 are obtained by subjecting Lactobacillus paracasei K56 to high-temperature steam sterilization.

6. The use according to claim 5, wherein The temperature of the high temperature steam sterilization is 100-150°C, preferably 120-125°C, and most preferably 121°C.

7. The use according to any one of claims 5 to 6, wherein The high-temperature steam sterilization time is 10-20 minutes, preferably 15 minutes.

8. The use according to claim 4, wherein The surface protein of Lactobacillus paracasei K56 comprises an LPXTG motif protein and a moonlighting protein; Preferably, the LPXTG motif protein comprises K0NBH9 (GenBank: CCK24055.1), KONA48 (GenBank: CCK23225.1), and K0NAB9 (GenBank: CCK23405.1); Preferably, the part-time protein comprises A0A0K1KXQ2 (GeneID: 57089615), K0N7K7 (GenBank: CCK23322.1), KOMU67 (GenBank: CCK22051.1), A0A0K1KYZ6 (Gene ID: 57090004), and K0N700 (GenBank: CCK23323.1); Preferably, the surface protein of Lactobacillus paracasei K56 is as shown in Table A.

9. The use according to claim 4, wherein The Lactobacillus paracasei K56 surface protein is obtained by the following method: (1) Lactobacillus paracasei K56 was mixed with an acidic lithium chloride aqueous solution as an extraction reagent and incubated; (2) centrifuging the mixture obtained in step (1) and taking the supernatant, wherein the supernatant is a surface protein extract of Lactobacillus paracasei K56.

10. The use according to claim 9, wherein The method further has one or more technical features selected from the following (i)-(v): (i) the ratio of the mass of the Lactobacillus paracasei K56 to the volume of the extraction reagent acidic lithium chloride aqueous solution is 1 g:3 mL; (ii) the concentration of lithium chloride in the acidic lithium chloride aqueous solution is 1-10 mol / L, preferably 4-6 mol / L, more preferably 5 mol / L; (iii) the pH of the acidic lithium chloride aqueous solution is 1.0-3.0, preferably 2.0; (iv) the incubation time is at least 10 min, preferably 10-40 min, more preferably 30-40 min, and most preferably 30 min; (v) The incubation is carried out in an ice water bath.

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