Application of bifidobacterium animalis subsp. lactis combined microbial agent in preparation of weight loss preparations

Abstract

The present disclosure belongs to the technical field of probiotics, and specifically to application of bifidobacterium animalis subsp. lactis combined microbial agent in preparation of weight loss preparations. Body fat rates, BMI, blood lipid levels, and the content of inflammatory factors may be effectively reduced by using bifidobacterium animalis subsp. Lactis TY—SOl+U9 in weight loss preparations, and the proliferation of beneficial bacteria (Bifidobacterium and / or Blautia) may be effectively promoted to effectively improve obesity. (+ Fig. l)

Description

P2019 / NL APPLICATION OF BIFIDOBACTE'RIUM ANIMALIS SUBSP. LACTIS COMBINED MICROBIAL AGENT IN PREPARATION OF WEIGHT LOSS PREPARATIONS Technical Field The present disclosure belongs to the technical field of pro biotics, and specifically to application of bifidobacterium ani malis subsp. lactis combined microbial agent in preparation of weight loss preparations. Background Obesity may lead to a variety of diseases, such as cardiovas cular disease. In addition, obesity also leads to the release of inflammatory factors such as TNFd and C reactive protein (CRP) from adipocytes, which triggers lowgrade inflammation in a body to inhibit the oxidative energy supply of the adipocytes, so as to further promote the development of obesity, thereby posing a haz ard to human health. At present, obesity is managed clinically in three main meth odslifestyle interventions, weight loss drug interventions, and weight loss surgical interventions. However, these methods are difficult to adhere to or have certain side effects, such that it is necessary to develop novel drugs as direct or auxiliary means for existing antiobesity efforts. Because of the advantages of safety and no side effects, probiotics can improve obesity symp toms in various manners, such as inhibiting the proliferation of harmful intestinal bacteria, relieving inflammation, or the like. Based on this, developing a probiotic that may be used in weight loss preparations is of great significance. Summary In view of the above problems, the present disclosure is in tended to provide an application of a probiotic in preparation of weight loss preparations. The probiotic is bifidobacterium animal is subsp. Lactis TYSOl+U9 (the accession number CGMCC No. 21255 and CGMCC No. 11560), which may improve obesity by reducing body fat rates, Body Mass Indexes (BMI), blood lipid levels, and the content of inflammatory factors, and promoting the proliferation of beneficial bacteria. Culture preservation information: Bifidobacterium animalis subsp. Lactis TYSOl, is deposited with the China General Microbiological Culture Collection Center, the accession number is CGMCC No. 21255 (Address: Institute of Mi crobiology Chinese Academy of Sciences, No.1 West Beichen Road, Chaoyang District, Beijing 100101, China; Date: November 27, 2020), Classification named: Bifidobacterium animalis subsp.lactis. Bifidobacterium animalis subsp. Lactis U9, is deposited with the China General Microbiological Culture Collection Center, the accession number is CGMCC No. 11560 (Address: Institute of Micro biology Chinese Academy of Sciences, No.1 West Beichen Road, Chao yang District, Beijing 100101, China; Date: November 04, 2015), Classification named: Bifidobacterium animalis subsp.lactis. The strain has been disclosed in the patent document CN116270758A. A control isolated strain used in the present disclosure is: Bifidobacterium animalis subsp. lactis A6, is deposited with the China General Microbiological Culture Collection Center, the accession number is CGMCC No. 9273 (Address: Institute of Microbi ology Chinese Academy of Sciences, NO.1 West Beichen Road, Chao yang District, Beijing 100101, China; Date: June 05, 2014). The strain has been disclosed in the patent document CN113583923B. In some specific embodiments, the abovedescribed applica tions may include one or more combinations of the following appli cations: (1) an application of bifidobacterium animalis subsp. Lactis TYS01+U9 in preparation of preparations for reducing body fat rates; (2) an application of bifidobacterium animalis subsp. Lactis TYS01+U9 in preparation of preparations for reducing Body Mass Indexes (BMI); (3) an application of bifidobacterium animalis subsp. Lactis TYS01+U9 in preparation of preparations for reducing blood lipid levels; and (4) an application of bifidobacterium animalis subsp. Lactis TYS01+U9 in preparation of preparations for reducing the content of inflammatory factors, wherein the inflammatory factors comprise TNFd and CRP. It is to be noted that, the body fat rates, the BMIs, the blood lipid, and the inflammatory factors are terms known in the art and do not have a specific meaning. For example, the body fat rate is the proportion of fat weight in the human body in the to tal weight of the human body; the BMI is a standard that is inter nationally used to measure the fatness degree and health of the human body, and is calculated by using the following formula: BMI = body weight + heightz; the blood lipid is a general term for neutral fats (triglycerides) and lipids (phospholipids, glycoli pids, sterols, steroids) in plasma; and the inflammatory factors refer to various cytokines involved in inflammatory response, mainly including tumor necrosis factora (TNFd), Interleukin10 (IL10), Interleukin6 (IL6), transforming growth factor0 (TGF 0) or Inflammatory factors (CRP) etc. The body fat rates, the BMI, the blood lipid levels, and the content of inflammatory factors may be effectively reduced by using the bifidobacterium animalis subsp. Lactis TYSOl in the weight loss preparations. It is to be noted that, the bifidobacterium animalis subsp. Lactis TYS01+U9 may reduce a lowdensity lipoprotein cholesterol level and / or a highdensity lipoprotein cholesterol level and / or a total cholesterol level and / or a triglyceride level by decreasing fat areas, so as to reduce the blood lipid levels, and reduce the content of inflammatory factors by reducing the content of TNFd and / or the content of CRP. In some specific embodiments, the application is an applica tion for promoting proliferation of bifidobacterium and / or blautia in intestinal flora. It is to be noted that, the bifidobacterium animalis subsp. Lactis TYS01+U9 may control body weight by promoting the prolif eration of beneficial bacteria related to obesity. In some specific embodiments, in the above application, the weight loss preparations include drug medicament or a food prepa ration. It is to be noted that, the bifidobacterium animalis subsp. Lactis TYS01+U9 may be prepared into food or drugs. In ad dition, a dosage form of the food preparation may be a solid dos age form, such as a probiotic tablet, probiotic powder, a probi otic capsule, and a probiotic pill, or may be a liquid dosage form, such as a probiotic beverage, a probiotic stirred yogurt, etc., or may be a cream dosage form, such as a set yogurt, etc. It is also It is to be noted that, excipients need to be added when the bifidobacterium animalis subsp. Lactis TYS01+U9 is prepared into preparations. The added excipients are selected according to the dosage to be selected, and are well known in the art. The present disclosure has the following beneficial effects: Body fat rates, BMI, blood lipid levels, and the content of inflammatory factors may be effectively reduced by using bifidobacterium animalis subsp. Lactis TYS01+U9 in weight loss preparations, and the proliferation of beneficial bacteria (Bifidobacterium and / or Blautia) may be effectively promoted to effectively improve obesity. Brief Description of the Drawings Fig. 1 shows the impact of TYS01+U9 on the content of in flammatory factors (Serum tumor necrosis factor d (TNFd)) in a simple obesity population. Fig. 2 shows the impact of TYS01+U9 on the content of in flammatory factors (Serum Creactive protein (CRP)) in a simple obesity population. Fig. 3 shows the impact of TYS01+U9 on community composi tions at a phylum level of intestinal flora in two groups. Fig. 4 shows the impact of TYS01+U9 on community composi tions at a genus level of intestinal flora in two groups. Fig. 5 shows the impact of TYS01+U9 on the abundance of bifidobacterium in intestinal flora in two groups. Fig. 6 shows the impact of TYS01+U9 on the abundance of blautia in intestinal flora in two groups. Detailed Description of the Embodiments The given embodiments are intended to better illustrate the invention, but the invention is not limited to the given embodi ments. Therefore, technicians skilled in the field make non essential improvements and adjustments to the implementation plan according to the above invention, which still falls within the scope of protection of the invention. The endpoints and any values of the range disclosed herein are not limited to that exact range or value and shall be under stood to contain values close to these ranges or values. For nu merical ranges, between endpoint values, between endpoint values and individual point values, and individual point values may be combined to obtain one or more new numerical ranges which shall be considered as disclosed herein. The present invention will be described in detail below by embodiments. It should be understood that the following embodi ments are used only for exemplary further detailed explanation and description of the contents of the invention, and not for limiting the invention. In the following embodiments, a probiotic TYS01 is derived from Tianyou strain library. It is deposited with the China Gen eral Microbiological Culture Collection Center, the accession num ber is CGMCC No. 21255 (Address: Institute of Microbiology Chinese Academy of Sciences, NO.1 West Beichen Road, Chaoyang District, Beijing 100101, China; Date: November 27, 2020), Classification named: Bifidobacterium animalis subsp.lactis. Bifidobacterium animalis subsp. Lactis U9, is deposited with the China General Microbiological Culture Collection Center, the accession number is CGMCC No. 11560 (Address: Institute of Micro biology Chinese Academy of Sciences, NO.1 West Beichen Road, Chao yang District, Beijing 100101, China; Date: November 04, 2015), Classification named: Bifidobacterium animalis subsp.lactis. Bifidobacterium animalis subsp. Lactis A6, is deposited with the China General Microbiological Culture Collection Center, the accession number is CGMCC No. 9273 (Address: Institute of Microbi ology Chinese Academy of Sciences, NO.1 West Beichen Road, Chao yang District, Beijing 100101, China; Date: June 05, 2014). In the following embodiments, subjects are selected based on the following criteria: aged between 18 and 45 years old; adult BMI Z 28 kg / m2, or total fat percentage of >25% for male and >30% for female, with no abnormalities in blood lipids; consent to drink test products during the study; and agree to sign an in formed consent form. Exclusion criteria: patients having serious diseases of the heart, liver, kidneys and hematopoietic system, and mental disease; subjects having diseases that affect body weight, such as hypothyroidism and Cushing's syndrome; subjects who have taken things related to test functions for a short period of time, which affects determination of results; subjects who fail to take test samples as prescribed, causing the efficacy to be un able to be determined, or causing the efficacy and safety judgment to be affected due to incomplete information; and subjects who have participated in another clinical trial within the past month. In the following embodiments, information such as BMIs, body fat rates, visceral fat areas, muscle rates, and the like is rec orded by using a calibrated body composition meter (Inbody770); waistlines are measured at two fingers below the umbilicus; and hiplines are measured at the widest part of the hip. In the following embodiments, a diversity sequencing opera tion of intestinal flora is completed by Shanghai Majorbio Tech nology Co., Ltd; and an Illumina.PE300 sequencing platform is used for sequencing to perform high throughput sequencing on 16SrDNA. A specific flow for high throughput sequencing includes: col lecting faeces of subjects and preserving same at 80°C; and per forming high throughput sequencing on 16SrDNA, including DNA ex traction, PCR amplification and product purification, PCR product quantification and homogenization, construction of PE libraries, and sequencing. Details are shown as follows. DNA extraction: after genomic DNA extraction is completed, and the extracted genomic DNA is detected through 1% agarose gel electrophoresis. PCR amplification and product purification: the concentration and purity of DNA are measured by using NanoDrop2000; and PCR am plification is performed on a V3V4 region of a 16S rRNA gene by using 338F (5-ACTCCTACGGGAGGCAGCAG-3) and 806R (5'- GGACTACHVGGGTWTCTAAT3). After PCR products of the same sample are mixed, 2% agarose gel is used to recycle the PCR products, and a AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) is used to purify the recycled products; then 2% agarose gel electrophoresis detection is performed, and a QuantusnnFluo rometer (Promega, USA) is used to detect and quantify the recycled products. Construction of PE libraries includes: using NEXTflex TMRapidDNASquit (BiooScientific, USA) to construct a library: (1) linking connectors; (2) using magnetic beads for screening, so as to remove connector selfconnecting fragments; (3) performing enrichment of library templates by means of PCR amplification; and (4) using the magnetic beads to recycle the PCR products, so as to obtain a final library. Sequencing includes: 1) one end of a DNA fragment being com plementary to a primer base and immobilized on a chip; 2) preform ing PCR synthesis by using the DNA fragment as a template and a base sequence immobilized on the chip as a primer, so as to syn thesize, on the chip, a target DNA fragment to be detected; 3) performing denaturation and annealing, and the other end of the DNA fragment on the chip being randomly complementary to another primer nearby and also immobilized, so as to form a "bridge; 4) performing PCR amplification to generate a DNA cluster; 5) linear izing a DNA amplicon into a single strand; 6) adding modified DNA polymerase and dNTP having 4 fluorescent labels, only synthesizing one basic group in each cycle; 7) scanning a surface of a reaction plate by using laser, and reading a nucleotide species polymerized by the first round of reactions for each template sequence; 8) performing chemical cleavage on a fluorescence groupand a "termi nation group, restoring the viscosity of 3end, and continuously polymerizing second nucleotide; and 9) counting fluorescent signal results collected in each round, and obtaining a sequence of the template DNA fragment. In the following embodiments, bioinformatics and data statis tical analysis are performed based on an ISanger cloud platform (https: / / www.isanger.com / ). A bioinformatic analysis method in cludes: PE.reads obtained through Miseg sequencing first being spliced according to an overlap relationship, at the same time, performing quality control and filtration on sequence quality, performing OTU cluster analysis and taxonomic analysis of species after sample distinguishing, and based on taxonomic information, performing statistic analysis of community structures at each classification level. On the basis of the above analysis, a series of indepth statistical and visual analyses such as multivariate analysis and significance test for differences are performed on community compositions and phylogenetic information of a plurality of samples. Embodiment 1: Population experiments with probiotic combina tions on obesity symptoms 200 subjects were recruited according to the above subject criteria and were randomly grouped into two groups; there were 50 subjects in a placebo group, and each subject drank one bag of maltodextrin beverage every day during a feeding trial; there were 50 subjects in an TYS01+A6 group, 50 subjects in an TYS01+U9 group, and 50 subjects in an A6+U9 group, each subject drank one bag of probiotic solid beverage every day (a viable count being about 5><1010 CFU / bag, the bacterial counts of TYS01, A6, and U9 were proportioned respectively in 1:1 according to different com binations, the rest was supplemented with maltodextrin, and malto dextrin was used in the placebo group). Intervention was performed for a total of 12 weeks, body parameters related to obesity index es were respectively measured once before intervention and after intervention, blood and faeces samples were collected once, and a total of 190 subjects completed the trial (10 subjects withdrew from the group due to inability to perform postintervention sam pling due to the epidemic situation). The subjects were all simple obesity patients, and there were no significant differences in de mographic or baseline indicators; and during the trial, there was no significant difference in food and beverage situations of the subjects, and details were shown in Table 1 below. Table 1 Population baseline data m (n=50) group(n=49) (n=45) (n=46) Pvawe m Gender 1:4 1:6 9:16 3:10 1.00 (maema) BIVII (kg / mz) 26.5812.86 28.6315.18 27951322 29211502 0.21 Body fat rate (%) 36911438 38491491 37701459 38.115.17 0.37 d the probiotic groups; BMIs and body fat rates using continuous variables normally or approximately normally distributed were de scribed through mean 1 standard deviation; and age was described using a skewed distribution with median and interquartile spacing. Embodiment 2: Adjustment effect of bifidobacterium animalis subsp. Lactis TYSOl+U9 on parameters related to physical obesity in simple obesity population As shown in Table 2, after the subjects were subjected to 12 weeks of intervention, there was no significant change in BMI and body fat rate in the placebo group, the TYS01+A6 group, and the A6+U9 group compared with that before intervention, and the de crease was greater than that in the placebo group, while the TY S01+U9 group showed a significant decrease after intervention com pared with that before intervention (p<0.05); and there was no significant change in visceral fat area in the placebo group and the A6+U9 group after intervention, and the visceral fat area was significantly decreased in the TYS01+A6 group and the TYS01+U9 group compared with that before intervention (p<0.05) . It indicat ed that the TYS01+A6 group and the TYS01+U9 group had the poten tial to reduce body fat of obesity populations, and the TYS01+U9 group had a better effect. Table 2 Parameters related to physical obesity in obesity population before and after intervention + Intervention Before intervention After intervention subjects value Placebo 50 26.581286 26.5012.69 0.53 BlVll(kg / m2) TYSOl+A6 49 28.6315.18 28.2815.25 0.12 TYSOl+U9 45 27.9513.22 27.7813.38 0.04 m 0.71 m Bodyfat rate TY-SOl+A6 49 38.49i4.91 37.30i5.48 0.08 (%)W W W Visceral fat Placebo 50 0.74 152.60) 146.45) area (cm2) _ TY-S01+A6 49 143.00 (114.80, 128.50 (111.30, 0.03 199.00) 197.40) TY-SOl+U9 45 134.50(115.40,151.6) 120.5(106.7,139.3) Ê 123.60(110.20,149.0 _ A6+U9 46 5) 119.30(107.05,141.70) 0.06 m tion within the group; BMI and body fat rate were continuous vari ables that are normally or approximately normally distributed and were described through mean 1 standard deviation; and visceral fat area belonged to skewed distribution data and was described with median and interquartile spacing. Embodiment 3: Adjustment effect of bifidobacterium animalis subsp. Lactis TYSOl+U9 on lipid metabolism in simple obesity pop ulation Fasting venous blood of the subjects was collected to detect total cholesterol (TC), total triglyceride (TG), lowdensity lipo protein cholesterol (LDL_C), and highdensity lipoprotein choles terol (HDL_C), results were shown in Table 3, and there was no significant difference in the triglyceride level in each group be fore and after intervention; after 12 weeks of intervention, there were no significant changes in TC and LDL_C in the placebo group, the TYS01+A6 group, and the A6+U9 group before and after inter vention, and TC and LDL_C in the TYS01+U9 group were significant ly decreased (p<0.05); and HDL_C in the placebo group, the TY S01+A6 group, and the TYS01+U9 group after intervention was sig nificantly reduced compared with that before intervention (p<0.05). It indicated that TYS01+U9 had a better effect on re ducing blood lipid. Table 3 Population lipid index information before and after intervention + Intervention Before intervention After intervention P value subjects Triglyceride TY-SOl+A6 49 O.85(O.69,1.07) O.98(O.75,1.45) 0.12 (mmol / L)m 0.64 m 0.85 Cholesterolm (mmol / L) Ty-501+A5 49 4.261072 4.1010.80 W m m Low-densitym lipoprotein TY-SOl+A6 49 2.6910.62 2.5310.60 W cholesterol TY-SOl+U9 45 3.4110.84 3.1910.76 W (mmol / L) A6+U9 46 3.0510.62 2.9010.55 Ë m lipoprotein TY-501+A6 49 1.2811.27 1.1610.11 0.005 Cholesterol TYS01+U9 45 1.3210.28 1.2510.26 0.003 (mmol / L) A6+U9 46 1.221030 1.181032 0.20 m tion within the group; continuous variables normally or approxi mately normally distributed were described through mean 1 standard deviation, and a skewed distribution was described with median and interquartile spacing. Embodiment 4: Adjustment effect of bifidobacterium animalis subsp. Lactis TYSOl+U9 on inflammatory indicators In an obese state, adipose tissue secreted inflammatory fac tors, causing chronic inflammation throughout the body. In the ad ipose tissue, TNFd was an inflammatory factor that was mainly ex pressed by adipocytes and stromal vascular cells, and was able to inhibit the expression of genes involved in fatty acid oxidation and increase the expression of genes involved in cholesterol and fatty acid resynthesis. CRP was also an inflammatory marker, and was a risk indicator most related to obesityrelated cardiovascu lar disease. In this embodiment of the present disclosure, serum of the subjects was extracted and isolated by using a high speed centri fuge (at a rotary speed being 3500 rpm for 15 min), and the con tent of the tumor necrosis factor d (TNFd) and the C reactive protein (CRP) in serum samples was detected by using an ELISA kit. The content of the TNFd was shown in Fig. 1. There was no signif icant difference in the TNFd in the placebo group and the U9+A6 group before and after intervention, and the TNFd in the TY S01+U9 group was significantly reduced. The content of the CRP was shown in Fig. 2 (the placebo group was on the left, the TYS01+U9 group was in the middle, and the U9+A6 group was on the right), and the content of the CRP was significantly reduced in all the three groups. The above results showed that the TYS01+U9 group had the best effect on relieving inflammation. Embodiment 5: Impact of bifidobacterium animalis subsp. Lac- tis TYSOl+U9 on intestinal flora (1) Compositions of intestinal flora In order to learn community structure compositions of inter vention in two groups at each phylum level and genus level and learn the relative abundance of microorganisms in each group, sub stance compositions of intestinal flora of the subjects at phylum and genus levels (abundance>1%) were analyzed. The analysis re sults was shown in Fig. 3~4. (V0_A referred to preintervention in the placebo group, V2_A referred to postintervention in the pla cebo group, V0_B referred to preintervention in the U9+A6 group, and V2_B referred to postintervention in the U9+A6 group. V0_D referred to preintervention in the TYS01+U9 group, and V2_D re ferred to postintervention in the TYS01+U9 group), dominant bac teria in the intestinal flora in obesity population were Firmicu tes, Bacteroidota, Actinobacteriota and Proteobacteria, dominant bacterium in the intestinal flora in obesity population were Bac teroides, Faecalibacterium, Blautia, etc. (2) Abundance of beneficial bacteria After experimental intervention, abundances of Bifidobacte rium and Blautia in the TYS01+U9 group were significantly in creased (p<0.05), and there were no significant differences in the abundances of the Bifidobacterium and Blautia in the placebo group and the U9+A6 group before and after intervention (Fig. 5~6). It might be seen that, TYS01+U9 was able to promote the prolifera tion of intestinal beneficial bacteria. To sum up, intervention with the composition of the probiotic TYS01+U9 was able to reduce body fat and inflammation levels in the simple obesity population, and thus the probiotic had the po tential to be applied in fat reduction products. The above is only a preferred embodiment of the invention, and it should be noted that for persons of ordinary skill in the art of the invention without departing from the principle, the im proofs and touches shall also be regarded as the protection scope of the invention. CONCLUSIONS 1. Combined microbial agent of Bifidobacterium animalis subsp. Lactis, consisting of two Bifidobacterium animalis subsp. Lactis strains TYSOl and U9, in which the microbial agent is prepared by mixing the two strains in a ratio for what the number of viable cells is 1:1; bifidobacterium animalis subsp. Lactis TYSOl, with CGMCC number 21255 as accession number; bifidobacterium animalis subsp. Lactis U9, with CGMCC number 11560 as an admission number. 2. Probiotic fermentation product prepared by a bifidobacterium animalis subsp. Lactis TYSOl and a bifidobacterium animalis subsp. Lactis U9, where the probiotic fermentation product is prepared by mixing the two strains in a ratio for regarding the number of viable cells of 1:1; bifidobacterium animalis subsp. Lactis TYSOl, with CGMCC number 21255 as accession number; bifidobacterium animalis subsp. Lactis U9, with CGMCC number 11560 as admission number;. 3. Product prepared from the probiotic composition according to con inclusion 1 or the probiotic fermentation product according to conclusion 2, where the product is one of the following: foodstuff, ge health product or medicine. 4. Product within the meaning of claim 3, in which the product is a probiotic tablet, probiotic powder, a probiotic capsule or a probi is an exotic dairy product. 5. Product within the meaning of claim 4, in which the probiotic dairy pro The product is probiotic stirred yogurt or yogurt. 6. Application of the probiotic composition in accordance with claim 1 or the probiotic fermentation product according to claim 2 at the be preparation of weight loss preparations. 7. Application pursuant to claim 6, in which the application one or more of the following is, (1) an application of Bifidobacterium animalis subsp. Lactis TY 801+U9 in the preparation of preparations for the reduction of body fat; (2) an application of Bifidobacterium animalis subsp. Lactis TY 801+U9 for the preparation of preparations for lowering the body Mass index (BMI) (3) the use of Bifidobacterium animalis subsp. Lactis TY 801+U9 in the preparation of preparations for lowering the blood lipid level; and (4) the use of Bifidobacterium animalis subsp. Lactis TY 801+U9 in the preparation of preparations for lowering the level of inflammatory factors, where the inflammatory factors include TNFd and CRP. 8. Application pursuant to claim 6, in which the application one or more of the following is, (1) an application of Bifidobacterium animalis subsp. Lactis TY 801+U9 in preparations for reducing fatty areas; and (2. an application of Bifidobacterium animalis subsp. Lactis TY 801+U9 in the preparation of preparations for lowering a low-density lipoprotein cholesterol level and / or a high density lipoprotein cholesterol level and / or a total cholesterol terol level and / or a triglyceride level. 9. Application pursuant to claim 6, in which the application an passing is for promoting the proliferation of bifidobac terium and / or blautia in the intestinal flora. Fig.1 Fig.2