Bifidobacterium longum subsp. Longum i772 metagen product with fat reducing function, composition and application

Abstract

The invention discloses a bifidobacterium longum subsp. Longum i772 metagen product with a fat reducing function, a composition and application, and belongs to the field of microbial engineering. Wherein the metabiotic product comprises an inactivated bifidobacterium longum subsp. Longum i772, an inactivated bifidobacterium longum subsp. The composition is prepared from the following raw materials in parts by weight: 0.0002 to 10 parts of bifidobacterium longum subsp. Longum i772 bacterial powder, 0.015 to 0.06 part of L-carnitine, 0.5 to 3 parts of long-chain inulin and 1 to 5 parts of isomaltooligosacharide. The bifidobacterium longum subsp. Longum i772 provided by the invention can achieve the effects of promoting digestion and metabolism and losing weight by increasing the abundance of colabacillus and ruminococcus in intestinal tracts. The method is suitable for preparing milk powder or other foods and health-care products with a fat-reducing function.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial engineering, and relates to a new application of Bifidobacterium longum subspecies longum i772, and specifically relates to a postbiotic product, a composition and an application of Bifidobacterium longum subspecies longum i772 with fat-reducing function. Background Art

[0002] Weight problems are usually measured by the body mass index (BMI). According to the BMI standard for the Chinese population, a BMI between 18.5-24 is defined as normal, between 24-28 is defined as overweight, and 28 and above is defined as obese. Currently, more than 30% of the population in China is overweight, of which more than 10% are obese. Studies have shown that obesity is closely related to a variety of diseases, such as diabetes, hypertension, and hyperuricemia. General recommendations for weight control focus on diet control and increased exercise, but most young white-collar workers do not have time for special exercise under greater work and life pressures, and it is difficult to stick to diet control.

[0003] The disorder of hormone level, fat and sugar metabolism causes a large amount of fat accumulation, which is a major cause of obesity. Simple exercise and diet control are not effective. It is necessary to start from the regulation mechanism of hormone production and fat and sugar metabolism to regulate this type of obese people.

[0004] Bifidobacterium longum subspecies longum is a type of Gram-positive, strictly anaerobic, non-spore-forming microorganisms, and its same strains often have certain effects in improving motor skills, relieving constipation, improving cognitive abilities, etc., and have good application prospects. At present, there are already applications of Bifidobacterium longum in weight loss, such as the Chinese invention patent with publication number CN113234640B, which discloses a strain of Bifidobacterium longum with weight loss and lipid-lowering function and its application. However, there is no research on the inactivated bacteria of Bifidobacterium longum subspecies longum in weight loss, and there is no in-depth study of the mechanism of the weight loss effect of the strain. Summary of the invention

[0005] The purpose of the present invention is to provide a postbiotic product, composition and application of Bifidobacterium longum subspecies longum i772 with fat-reducing function, so as to solve the problem that there is a lack of weight-loss mechanism in the current inactivated bacteria of Bifidobacterium longum subspecies longum.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A postbiotic product of Bifidobacterium longum subspecies longum i772 with fat-reducing function, comprising inactivated Bifidobacterium longum subspecies longum i772.

[0008] Among them, Bifidobacterium longum subsp. longum i772 (Latin name: Bifidobacterium longum subsp. longum), whose strain has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee on May 20, 2020, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 19856. The strain has been first disclosed in the Chinese invention patent with publication number CN112458015B and the name "Bifidobacterium longum subsp. longum i772, its separation and purification method and application".

[0009] As a limitation, the bacterial content of Bifidobacterium longum subspecies longum i772 is 2×10 6 -5×10 10 CFU / g or CFU / mL.

[0010] As another limitation, the fat-reducing function is achieved by increasing the abundance of Ruminococcus in the intestine.

[0011] The present invention also provides the use of the above-mentioned postbiotic product in the preparation of a product with fat-reducing function.

[0012] The present invention also provides a Bifidobacterium longum subspecies longum i772 composition with fat-reducing function. The raw materials for making the effective ingredients thereof include, by weight: 0.0002-10 parts of Bifidobacterium longum subspecies longum i772 bacterial powder, 0.015-0.06 parts of L-carnitine, 0.5-3 parts of long-chain inulin and 1-5 parts of isomaltooligosaccharide.

[0013] Among them, long-chain inulin refers to inulin with a degree of polymerization DP≥23.

[0014] As a limitation, the Bifidobacterium longum subspecies longum i772 bacterial powder includes live bacteria or inactivated bacteria.

[0015] As a further limitation, the live bacteria amount or inactivated bacteria content of the Bifidobacterium longum subspecies longum i772 powder is 1×10 11 -5×10 11 CFU / g or CFU / mL.

[0016] As another limitation, the fat-reducing function of the live bacteria is achieved by increasing the abundance of Phascolarctobacterium in the intestine.

[0017] The present invention also provides a product having the effects of promoting fat metabolism and preventing fat accumulation, and the product comprises the above-mentioned composition.

[0018] Due to the adoption of the above technical solution, the present invention has the following technical advances compared with the prior art:

[0019] ① The postbiotic product of Bifidobacterium longum subspecies longum i772 with fat-reducing function provided by the present invention achieves weight-reducing and lipid-lowering effects by increasing the abundance of Ruminococcus in the intestine. The inactivated bacteria can be applied to more types of products, and the inactivated bacteria agent has good stability. The product properties are more stable during the shelf life, and there will be no problems such as fermentation to produce post-acid, and there is no need to consider the maintenance of the number of live bacteria;

[0020] ② The Bifidobacterium longum subspecies longum i772 composition with fat-reducing function provided by the present invention can achieve weight-reducing and lipid-lowering effects, wherein the active Bifidobacterium longum subspecies longum i772 achieves weight-reducing and lipid-lowering effects by increasing the abundance of Phascolarctobacterium in the intestine, and the added components such as L-carnitine, long-chain inulin and oligosaccharides have a synergistic effect with the bacterial agent in weight-reducing and lipid-lowering. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph showing the weight measurement results of rats in Example 7;

[0022] Figure 2 This is a graph showing the measurement results of blood lipid indexes in rats in Example 7;

[0023] Figure 3 This is a graph showing the measurement results of the proportion of white fat around the kidney and epididymis of rats in Example 7;

[0024] Figure 4 This is the petal diagram of ASV analysis in Example 7;

[0025] Figure 5 This is a graph showing the results of the intestinal community richness analysis in Example 7;

[0026] Figure 6 This is a graph showing the results of intestinal community diversity analysis in Example 7;

[0027] Figure 7 This is a graph showing the results of the intestinal community uniformity analysis in Example 7;

[0028] Figure 8 It is a stacked bar chart of the percentage of dominant species in each group at the phylum level in Example 7;

[0029] Fig. 9 This is the clustering heat map of dominant species in each group at the genus level in Example 7;

[0030] Fig.10 This is a bubble chart of the Biomarker analysis of each group at the genus level in Example 7. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below by specific examples. It should be understood that the described examples are only used to explain the present invention, and are not intended to limit the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art.

[0033] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0034] In the following embodiments:

[0035] Bifidobacterium longum subsp. longum i772, this strain was deposited in the China General Microbiological Culture Collection Center on May 20, 2020, with the collection number CGMCC NO.19856, and the Latin name is Bifidobacterium longum subsp.longum.

[0036] Example 1

[0037] This embodiment provides a postbiotic product of Bifidobacterium longum subspecies longum i772 with fat-reducing function, comprising an inactivated bacterial agent of Bifidobacterium longum subspecies longum i772, the bacterial agent having a bacterial content of 2×10 6 CFU / g, the amount of live bacteria is less than 3000 CFU / g.

[0038] Example 2

[0039] This embodiment provides a postbiotic product of Bifidobacterium longum subspecies longum i772 with fat-reducing function, comprising an inactivated bacterial agent of Bifidobacterium longum subspecies longum i772, the bacterial agent having a bacterial content of 5×10 10 CFU / g, the amount of live bacteria is less than 3000 CFU / g.

[0040] Example 3

[0041] This embodiment provides a postbiotic product of Bifidobacterium longum subspecies longum i772 with fat-reducing function, comprising an inactivated bacterial agent of Bifidobacterium longum subspecies longum i772, the bacterial agent having a bacterial content of 3×10 8 CFU / g, the amount of live bacteria is less than 3000 CFU / g.

[0042] Example 4

[0043] This embodiment provides a Bifidobacterium longum subspecies longum i772 composition with fat-reducing function, which is prepared from 0.2 g of live bacterial powder of Bifidobacterium longum subspecies longum i772, 15 kg of L-carnitine, 0.5 kg of long-chain inulin and 1 kg of isomaltooligosaccharide.

[0044] Among them, the viable bacterial count of Bifidobacterium longum subspecies longum i772 was 1×10 11 CFU / g.

[0045] Example 5

[0046] This embodiment provides a Bifidobacterium longum subspecies longum i772 composition with fat-reducing function, which is prepared from 10 kg of inactivated bacterial powder of Bifidobacterium longum subspecies longum i772, 60 kg of L-carnitine, 3 kg of long-chain inulin and 5 kg of isomaltooligosaccharide.

[0047] Among them, the content of inactivated bacteria of Bifidobacterium longum subspecies longum i772 was 3×10 11 CFU / g.

[0048] Example 6

[0049] This embodiment provides a milk powder with the effect of promoting fat metabolism and preventing fat accumulation. The milk powder is added with 0.5wt% of a Bifidobacterium longum subspecies longum i772 composition, and the composition is made of 5kg of live bacterial powder of Bifidobacterium longum subspecies longum i772, 20kg of L-carnitine, 2kg of long-chain inulin and 3kg of isomaltooligosaccharide.

[0050] Among them, the viable bacteria of Bifidobacterium longum subspecies longum i772 were 5×10 11 CFU / g.

[0051] Example 7

[0052] This embodiment provides the use of inactivated bacteria of Bifidobacterium longum subspecies longum i772 and a live bacteria composition having fat-reducing function, which are used for weight loss and lipid reduction, and provides a control experiment, which specifically includes the following steps:

[0053] S1. Experimental Preparation

[0054] 1) Experimental animals

[0055] Sprague Dawley rats (SD rats), male, 10-12 weeks old;

[0056] The experimental animals were provided by Sbefor (Suzhou) Biological Co., Ltd. (China), certificate: SCXK(Su)2022-0006.

[0057] 2) Instruments, consumables and reagents

[0058] Disposable 5 ml syringe (Shanghai Kangdeli, China), electronic analytical balance (Precisa12A, Precisa, Switzerland), pyrogen-free ultrapure water system (Millipo3150, Millipore, USA), three-section rat gavage needle (Zhongke Life Science, China), electric constant temperature water bath (HW-SY11-KP2, Beijing Changfeng Instrument Co., Ltd., China), centrifuge tube (1.5 ml, Wuxi Nice Life Science Co., Ltd., China), automatic blood biochemical analyzer (URIT-8210vet, Urit, China);

[0059] Simvastatin (S407772-1ml, Shanghai Aladdin Biochemical Technology Co., Ltd., China), customized high-fat feed (Jiangsu Synergy Pharmaceutical Bioengineering Co., Ltd., China).

[0060] 3) Animal grouping and obesity modeling

[0061] The rats were kept in an SPF environment with an ambient temperature of 23±2℃, a humidity of 55±5%, and a 12h light and 12h dark cycle. After 7 days of adaptive feeding, the rats were divided into 8 groups: normal control group, model control group, positive control group, inactivated group, experimental group 1, experimental group 2, experimental group 3, and experimental group 4, with 12-15 rats in each group.

[0062] Except for the normal control group, all rats had their feed replaced with a customized high-fat feed (15.0% sucrose, 15.0% lard, appropriate amount of casein, calcium hydrogen phosphate, and stone powder were added to the maintenance feed), and normal drinking water was maintained. After 2 weeks of feeding, the rats in each group were weighed, and the obese insensitive individuals with no significant weight gain in the remaining groups except the normal control group were eliminated to ensure that the remaining rats could be allocated to 10 rats in each group;

[0063] After the obesity-insensitive individuals were eliminated after modeling verification, all rats except the normal control group continued to be fed with a high-fat diet for 6 consecutive weeks to construct a rat obesity model.

[0064] 4) Sample administration by intragastric gavage

[0065] After the rat obesity model was established, the rats in each group were gavaged daily. The rats in the normal control group and the model control group were gavaged with an equal volume of normal saline daily; the rats in the positive control group were gavaged with 40 mg / kg simvastatin (solvent was normal saline) daily, once a day for 28 consecutive days, and were fed with a high-fat diet during the gavage period.

[0066] The rats in the inactivated group were given inactivated Bifidobacterium longum subspecies longum i772 by gavage every day, and the dose was equivalent to 5x10 9 CFU, once a day for 28 consecutive days, and high-fat diet was maintained during gavage;

[0067] The rats in experimental group 1 were given active Bifidobacterium longum subspecies longum i772 by gavage every day, and the dose converted to human daily intake is 5x10 9 CFU, once a day for 28 consecutive days, and high-fat diet was maintained during gavage;

[0068] The rats in the experimental group 2 were given an active Bifidobacterium longum subspecies longum i772 composition by oral gavage every day. The specific ingredients were L-carnitine + long-chain inulin + oligosaccharide + active Bifidobacterium longum subspecies longum i772. The dosage was converted to 18 mg + 0.9 g + 1.5 g + 10 mg per day for humans. The amount of live bacteria of Bifidobacterium longum subspecies longum i772 was 1×10 11 CFU / g, once a day for 28 consecutive days, and high-fat diet was maintained during gavage;

[0069] The rats in the experimental group 3 were given a composition by oral gavage every day. The specific ingredients were L-carnitine + long-chain inulin + isomaltooligosaccharide. The dosage was converted to 36mg + 1.8g + 3g per day for humans, once a day for 28 consecutive days. The rats were fed a high-fat diet during the gavage period.

[0070] The rats in the experimental group 4 were given a composition of inactivated Bifidobacterium longum subspecies longum i772 by oral gavage every day. The specific ingredients were L-carnitine + long-chain inulin + oligosaccharide + inactivated Bifidobacterium longum subspecies longum i772. The dosage was equivalent to 36mg + 1.8g + 3g + 10mg per day for human body. The content of inactivated Bifidobacterium longum subspecies longum i772 was 1×10 11 CFU / g, once a day for 28 consecutive days, and high-fat diet was maintained during gavage.

[0071] Reference for equivalent dose conversion between rats and humans: Anroop B. Nair, Shery Jacob. A simple practice guide for dose conversion between animals and human. Journal of Basic and Clinical Pharmacy, Vol. 7, Issue 2, March-May 2016.

[0072] S2. Experiment on the efficacy of reducing fat and losing weight

[0073] During the whole experiment, the weight changes of rats in each group were recorded once a week. Figure 1 As shown;

[0074] Depend on Figure 1It can be seen that compared with the model control group, the body weight of rats in the inactivated group, experimental group 1, experimental group 2, experimental group 3 and experimental group 4 decreased significantly during the oral gavage intervention period.

[0075] 24 hours after the last intragastric administration, blood was collected from each group of rats, and the serum was separated by centrifugation and then the total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in the serum of each group of rats were detected by automatic biochemical analyzer. Figure 2 As shown;

[0076] Depend on Figure 2 It can be seen that after oral gavage intervention, compared with the model control group, the triglyceride and total cholesterol levels of rats in the inactivated group, experimental group 1, experimental group 2 and experimental group 4 were significantly decreased; there was no significant difference in the total cholesterol content of rats in experimental group 3, and the triglyceride content decreased significantly.

[0077] After blood sampling, rats in each group were anesthetized, their kidneys and epididymis were removed, adipose tissue was separated, and the fat / body ratio was calculated as the white fat ratio of these two tissues. Figure 3 As shown;

[0078] Depend on Figure 3 It can be seen that compared with the model control group, the proportion of white fat around the epididymis of rats in the inactivation group, experimental group 1, experimental group 2 and experimental group 4 decreased significantly; the proportion of white fat around the kidneys of rats in experimental group 1, experimental group 2 and experimental group 4 decreased significantly; there was no significant change in experimental group 3.

[0079] S3. Gut microbiota biochemistry experiment

[0080] Except for experimental group 3 and experimental group 4, the colon contents of rats in each group were taken for intestinal flora analysis by 16s amplicon. After double-end splicing, low-quality sequences and chimera sequences were filtered out for the original data, high-quality data statistics were performed, among which the ratio of Q20 (data with data quality ≥ Q20 in valid data, base sequencing accuracy of 99%) to Q30 (data with data quality ≥ Q30 in valid data, base sequencing accuracy of 99.9%) was mainly investigated. The results are shown in Table 1:

[0081] Table 1 Valid data statistics

[0082]

[0083]

[0084]

[0085] Note: In the table, Valid% indicates the ratio of valid data (Valid) to raw data (Raw); Q20% indicates the proportion of valid data with data quality ≥ Q20; Q30% indicates the proportion of valid data with data quality ≥ Q30; GC% indicates the GC content of valid data

[0086] As can be seen from Table 1, the Q20 ratio of each group of samples is above 90%, and the Q30 ratio is above 80%, indicating that the accuracy of this sequencing is sufficient.

[0087] Since the composition of mammalian intestinal flora is more complex, conventional OTU clustering methods have the limitation of underestimating microbial diversity. Therefore, this experiment no longer clusters based on sequence similarity, but clusters non-redundant sequences ASVs (or characteristic sequences) generated after deduplication and noise reduction to improve data accuracy and species resolution. The ASV analysis between multiple groups of samples is displayed in the form of a petal diagram, as shown in the figure below: Figure 4 As shown, the number of overlapping parts represents the number of ASVs shared by two or more groups, and the number of non-overlapping parts represents the number of ASVs unique to each group;

[0088] Depend on Figure 4 It can be seen that the unique sequences of the intestinal flora in the inactivated group and experimental group 2 decreased, and it is speculated that this may be due to the massive proliferation of specific probiotics in the intestinal samples of the rats in these experimental groups.

[0089] Based on the results of ASV analysis, a preliminary analysis of species distribution was conducted on each group of samples. The Observed_Species value mainly reflects the richness of the community and is used to estimate the number of species in the community. The Simpson value mainly reflects the diversity of the community. The larger the value, the more species there are in the sample and the more uniform the number of each species. The Pielou-e value mainly reflects the uniformity of the community. The results are shown in Figure 2. Figure 5-7 As shown;

[0090] Depend on Figure 5-7 It can be seen that the richness and diversity of intestinal flora in the inactivated products, active bacteria and live bacteria compositions of Bifidobacterium longum subspecies i772 decreased, and the uniformity of the community decreased, which once again verified that the intestinal samples of rats in the three experimental groups contained a large amount of specific probiotics (Bifidobacterium longum).

[0091] According to the ASV annotation results and the ASV abundance table of each sample, the species abundance table at the kingdom, phylum, class, order, family, genus, and species level was obtained, and the species composition of different samples (groups) was analyzed based on the species abundance table at different levels. According to the species annotation abundance table at each level, the top 30 species with the highest abundance were selected and classified. The results are as follows: Figure 8 , Fig. 9 As shown;

[0092] Depend on Figure 8 It can be seen that at the phylum level, the abundance of Bacteroidetes (Bacteroidetes) increased, while the abundance of Firmicutes (Firmicutes) decreased in experimental group 2 compared with the model control group;

[0093] Depend on Fig. 9 It can be seen that at the genus level, the relative abundance of the Bifidobacterium genus to which Bifidobacterium longum belongs was the lowest in the model control group, while the relative abundance in the inactivated group, experimental group 1, and experimental group 2 increased compared with the model control group, indicating that the results of this experiment are reliable, and the relative abundance of the Bacteroides genus in the three groups also increased compared with the model control group, among which the increase in experimental group 2 was the most obvious.

[0094] In order to compare between groups and emphasize statistical significance and biological relevance, and to find biomarkers with statistical differences between groups, this experiment conducted an indicator species analysis. The results are shown in Figure 2. Fig.10 As shown;

[0095] The size of the dots in the figure represents the sqrtIVt value, which can be used to compare the correlation between microorganisms and treatment groups. The larger the value of a species, the more likely it is to be used as a biomarker for the treatment group. The color represents the p-value, and the more significant the p-value, the more reliable the result. Fig.10 It can be seen that in addition to the common Enterobacter (Enterorhabdus), the biomarker of the model control group was also found to have a high correlation with Christensennellaceae, an intestinal pathogen belonging to the Firmicutes phylum, which is associated with fat deposition, inflammatory bowel disease and metabolic disorders; while the biomarker in experimental groups 1 and 2 was mainly Phascolarctobacterium; the biomarker in the inactivated group was Ruminococcus;

[0096] Pseudomonas aeruginosa has been found to be present at higher levels in the intestines of people who are more likely to lose weight, and its increased abundance can promote fat metabolism; Ruminococcus aeruginosa can promote digestion, and its abundance has been found to be reduced in people with ulcerative colitis;

[0097] The analysis results show that in a mammalian model, Bifidobacterium longum subspecies longum i772 can promote digestion metabolism and weight loss by increasing the abundance of Phascolarctobacterium and Ruminococcus in the intestine.

Claims

1. A postbiotic product of Bifidobacterium longum subspecies longum i772 with fat-reducing function, characterized in that: The postbiotic preparation comprises inactivated Bifidobacterium longum subsp. longum i772.

2. The postbiotic product according to claim 1, characterized in that The bacterial content of the Bifidobacterium longum subspecies longum i772 is 2×10 6 -5×10 10 CFU / g or CFU / mL.

3. The postbiotic product according to claim 1 or 2, characterized in that The fat-reducing function is achieved by increasing the Ruminococcus The abundance achieved.

4. Use of the postbiotic product according to claim 3 in the preparation of products with fat-reducing function.

5. A composition of Bifidobacterium longum subspecies longum i772 with fat-reducing function, characterized in that: The raw materials for making the effective component include, by weight: 0.0002-10 parts of powder of Bifidobacterium longum subspecies longum i772, 0.015-0.06 parts of L-carnitine, 0.5-3 parts of long-chain inulin and 1-5 parts of isomaltooligosaccharide.

6. The composition according to claim 5, characterized in that The Bifidobacterium longum subspecies longum i772 bacterial powder includes live bacteria or inactivated bacteria.

7. The composition according to claim 6, characterized in that The live bacteria amount or inactivated bacteria content of the Bifidobacterium longum subspecies longum i772 bacterial powder is 1×10 11 -5×10 11 CFU / g or CFU / mL.

8. The composition according to any one of claims 5 to 7, characterized in that The fat-reducing function of the live bacteria is to increase the Phascolarctobacterium The abundance achieved.

9. A product that promotes fat metabolism and prevents fat accumulation, characterized in that: The product comprises the composition of any one of claims 5-8.

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