Application of phytobacterium plantarum GOLDGUT-HNU082 in improving intestinal health of middle-aged and elderly people

Abstract

The invention discloses an application of plant lactobacillus GOLDGUT-HNU082 in improving intestinal health of middle-aged and elderly people, and relates to the technical field of microorganisms, and the preservation number of the plant lactobacillus GOLDGUT-HNU082 is CGMCC (China General Microbiological Culture Collection Center) NO.33075. The invention further discloses a preparation method of the plant lactobacillus GOLDGUT-HNU082. According to the plant lactobacillus GOLDGUT-HNU082 disclosed by the invention, the intestinal health condition of middle-aged and old people is remarkably improved, the GMWI2 index is improved, and the flora abundance of beneficial bacteria is improved; and the traditional Chinese medicine composition has the effect of reducing the high fasting blood glucose level of a subject, can improve related indexes of the liver function of the subject, and provides more resources for promoting the health of the aged.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and more specifically to application of Lactobacillus plantarum GOLDGUT-HNU082 in improving the intestinal health of middle-aged and elderly people. Background Art

[0002] As my country has officially entered a moderately aging society, promoting the health of the elderly has become an important national strategy. There are more than 180 million elderly people in my country suffering from various chronic diseases such as hypertension and diabetes. With the increase of age, the physiological metabolic function and immune function of various tissues and organs of the middle-aged and elderly people gradually decline, and the risk of viral or bacterial infection increases significantly. Although some middle-aged and elderly people do not show obvious lesions in clinical indicators, their health status may still turn to sub-health, mainly due to the potential hidden impact of intestinal health.

[0003] The human gastrointestinal tract is home to a large number of diverse microorganisms, collectively known as intestinal flora. Many studies have pointed out that intestinal flora is related to the health of middle-aged and elderly people, and its balance and diversity can affect the human immune system, metabolism, and inflammatory response. Intestinal flora imbalance leads to intestinal mucosal barrier damage and increased intestinal inflammatory factors, which in turn leads to major chronic diseases of the immune system, metabolic system, digestive system, and other elderly diseases.

[0004] The Gut Microbial Health Index (GMWI2) assesses health status based on species-level taxonomy of fecal metagenomic samples. The GMWI2 index is determined by comparing the relative abundance of two groups of microbial species associated with good and poor health, and has been reported to be able to distinguish between healthy and unhealthy intestinal groups. GMWI2 has also been applied to four recently published longitudinal intestinal microbiome studies to demonstrate its wide applicability in intestinal health tracking. These studies cover multiple aspects such as fecal microbiota transplantation (FMT) treatment, dietary intervention, antibiotic treatment, and the effects of prebiotic oligosaccharides on intestinal microbial communities. The results show that GMWI2 can reflect changes in intestinal health status earlier than traditional diversity indicators, providing a valuable tool for evaluating the recovery of the intestinal microbiome after acute illness and the effects of dietary fiber on intestinal health.

[0005] Although there is an increasing number of studies on intestinal flora, few studies use specific indicators to measure the overall health of the intestinal flora, and there are also limited microbial resources that can clearly improve the intestinal microbial health index.

[0006] Therefore, discovering more microbial resources that improve intestinal health and promote aging health is an urgent problem that technicians in this field need to solve. Summary of the invention

[0007] In view of this, the present invention provides the use of Lactobacillus plantarum GOLDGUT-HNU082 for improving the intestinal health of middle-aged and elderly people.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] Application of Lactobacillus plantarum GOLDGUT-HNU082 in improving intestinal health, wherein the deposit number of the Lactobacillus plantarum GOLDGUT-HNU082 is CGMCCNO.33075.

[0010] Another object of the present invention is to provide an application of Lactobacillus plantarum GOLDGUT-HNU082 in the preparation of a bacterial agent for improving intestinal health, wherein the deposit number of the Lactobacillus plantarum GOLDGUT-HNU082 is CGMCC NO.33075.

[0011] Another object of the present invention is to provide an application of a bacterial agent containing Lactobacillus plantarum GOLDGUT-HNU082 in the preparation of a product for improving intestinal health, wherein the deposit number of the Lactobacillus plantarum GOLDGUT-HNU082 is CGMCCNO.33075.

[0012] Another object of the present invention is to provide an application of Lactobacillus plantarum GOLDGUT-HNU082 in the preparation of a product for improving GMWI2, wherein the deposit number of the Lactobacillus plantarum GOLDGUT-HNU082 is CGMCC NO.33075.

[0013] Another object of the present invention is to provide the use of Lactobacillus plantarum GOLDGUT-HNU082 in the preparation of a blood sugar lowering product, wherein the deposit number of the Lactobacillus plantarum GOLDGUT-HNU082 is CGMCC NO.33075.

[0014] Another object of the present invention is to provide the use of Lactobacillus plantarum GOLDGUT-HNU082 in the preparation of a product for reducing serum total protein, wherein the deposit number of the Lactobacillus plantarum GOLDGUT-HNU082 is CGMCC NO.33075.

[0015] Beneficial effects: The Lactobacillus plantarum GOLDGUT-HNU082 of the present invention significantly improves the intestinal health status of the middle-aged and elderly groups, improves the GMWI2 index, and increases the abundance of beneficial bacteria; it also has a reducing effect on subjects with high fasting blood sugar values, and can improve the subjects' liver function-related indicators. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0017] Figure 1 This is the colony morphology of Lactobacillus plantarum GOLDGUT-HNU082.

[0018] Figure 2 This is the whole genome comparison result of Lactobacillus plantarum GOLDGUT-HNU082.

[0019] Figure 3 These are the test results of serum biochemical indicators before and after taking Lactobacillus plantarum GOLDGUT-HNU082.

[0020] Figure 4 Changes in intestinal health index before and after taking Lactobacillus plantarum GOLDGUT-HNU082 (A), Shannon index and Simpson index of intestinal microbiome (B), and principal coordinate analysis results of Bray-Curtis distance (C).

[0021] Figure 5 Gut microbiome annotation results at the phylum level, genus level (A), and species level (B, C) before and after administration of Lactobacillus plantarum GOLDGUT-HNU082.

[0022] Figure 6 The results of metabolic pathway analysis (A) and correlation analysis (B) before and after taking Lactobacillus plantarum GOLDGUT-HNU082. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Example 1

[0025] Isolation, Screening and Identification of Lactobacillus plantarum GOLDGUT-HNU082

[0026] 1. More than 30 fermented food samples were collected from farmers' markets in various counties and cities in Hainan Province, including Wanning, Wenchang, Wuzhishan, Baisha, and Lingshui. The collected fermented food samples were diluted tenfold in sterilized saline, and then a 10-fold dilution method was used to prepare a gradient of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Sample diluent, 0.1mL of 10 -3 , 10 -4 , 10 -5 The gradient samples were spread on MRS agar medium and cultured anaerobically at 37°C for 48 hours. After multiple separations, streaking, purification, culture and identification, 5 strains of Lactobacillus plantarum were screened out. The 5 strains were cultured in MRS liquid for 24 hours, and the OD value and viable count were measured. Finally, a strain of Lactobacillus plantarum was screened out and named GOLDGUT-HNU082. After the purified GOLDGUT-HNU082 strain was inoculated in MRS broth medium for activation for 24 hours, it was spread on MRS agar medium. After culturing at 37°C for 24 hours, the colonies were white, with a diameter of 0.1-0.2 cm, a smooth, moist and raised surface, and neat edges (see Appendix Figure 1 ). When observed under an optical microscope, the bacteria are rod-shaped.

[0027] 2. Identification information: The whole genome of GOLDGUT-HNU082 strain was sequenced. The sequence obtained by whole genome sequencing was used for genome alignment. The genome similarity analysis was performed using the online analysis platform Galaxy (https: / / usegalaxy.org / ), and the software used was FastANI (https: / / github.com / ParBLiSS / FastANI). The analysis results showed that the ANI value of the whole genome sequence of GOLDGUT-HNU082 and Lactiplantibacillus plantarum SRCM100442 was 99.0642 (see Appendix Figure 2 ).

[0028] 3. The strain is deposited in the China General Microbiological Culture Collection Center (CGMCC), the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC NO.33075, the deposit date is December 16, 2024, and the taxonomic name is Lactiplantibacillus plantarum.

[0029] Example 2

[0030] Lactobacillus plantarum GOLDGUT-HNU082 improves intestinal health

[0031] 1. Subject recruitment

[0032] We included 14 healthy middle-aged and elderly subjects (40-71 years old). All participants underwent a comprehensive physical examination. Exclusion criteria included: 1. Those with a history or family history of colon cancer, celiac disease, and inflammatory bowel disease; 2. Those who had been diagnosed with organic intestinal diseases by colonoscopy; 3. Those who were preparing for pregnancy recently (including men and women), pregnant or lactating women; 4. Those who were allergic to samples or ingredients; 5. Those who had taken probiotics and probiotic-related preparations in the past 2 weeks; 6. Those who had taken anti-anxiety, anti-depression and other psychotropic drugs in the past 2 weeks; 7. Those who had been diagnosed with myocardial infarction, cerebral infarction, malignant tumors and other serious diseases that the researchers considered unsuitable for inclusion; 8. Subjects with major mental illnesses, difficulty controlling their actions, and unable to cooperate; 9. Subjects who were illiterate, unable to understand informed consent, and unable to sign the informed consent form by themselves.

[0033] 2. Lactobacillus plantarum GOLDGUT-HNU082 intervention

[0034] The subjects were asked to take 2 g of Lactobacillus plantarum GOLDGUT-HNU082 (containing 1.0×10 10 CFU / g viable bacteria). Participants were required to provide stool and serum samples before taking probiotics (T0 period) and after 60 days of taking probiotics (T1 period). There were no dietary restrictions during the intervention period.

[0035] 3. Clinical indicator testing

[0036] (1) Blood samples were collected from all participants after fasting for 12 hours, and serum biochemical indicators (fasting blood glucose, mean corpuscular hemoglobin content, mean corpuscular volume, and serum total protein) were measured using a fully automatic chemiluminescence analyzer.

[0037] (2) Fecal samples from all participants were stored in sterile containers at -80°C. Bacterial DNA was extracted from the samples using the magnetic bead method. DNA quality was assessed by 0.8% agarose gel electrophoresis, and the absorbance ratio at 260 nm and 280 nm was measured spectrophotometrically. Shotgun metagenomic sequencing was performed using the Illumina novaseq 6000 platform. Paired-end reads of 150 bp were generated. The raw sequencing reads were quality filtered and sheared to remove the host genome. For metagenomic species annotation, MetaPhlan4 software (v4.0.6) was used to perform qualitative and quantitative analysis of bacterial species to obtain relative abundance at the species level.

[0038] (3) Based on the fecal metagenomic data, the intestinal library index of each sample was calculated using the gmwi2 software (v1.6), that is, the intestinal health status of the host was predicted by the intestinal microbiome classification profile. Specifically, potential human genome contaminants (base sequences mapped to GRCh38 / hg38) were first removed using the Bowtie2 software (v2.4.4). Then, the over-represented sequences in each metagenomic sample and the possible Illumina universal adapter sequences were identified and extracted using FastQC (v0.11.8). Next, the adapter sequences were spliced ​​and quality filtered using the Trimmomatic software (v0.39). At the same time, low-quality bases before and after each read (Phred quality score <3) were removed, and modified reads with a length of less than 60 bp were discarded. Finally, the Lasso-penalized model was used to train the metagenomic dataset to predict disease status based on binary presence / absence classification features. The best performing parameters were selected in each outer loop training, and the parameters used for the GMWI2 model were finally selected.

[0039] Note: All statistical analyses in this invention were performed using R software (v4.3.3, https: / / www.r-project.org / ). The alpha diversity of intestinal microorganisms was calculated using the "vegan" package, and the beta diversity was evaluated based on the Bray-curtis distance. Differential intestinal microorganisms were determined by t-test and visualized using the "pheatmap" package in R software. Differences in clinical indicators were performed using t-tests. GraphPadPrism (v8.0.2) statistical software was used to prepare histograms for analysis, and values ​​were expressed as mean ± SEM. The significance of the difference was calculated using the t-test, *P<0.05, **P<0.01, ***P<0.001. Correlation analysis was based on the Spearman rank correlation coefficient and visualized using the "pheatmap" package in R software.

[0040] 4. Results

[0041] (1) Improve blood indicators: The test results of serum biochemical indicators are shown in the attached Figure 3, it can be seen that the serum biochemical indicators of the subjects were healthier after taking Lactobacillus plantarum GOLDGUT-HNU082 than before taking it. Specifically, the reference range of fasting blood glucose is 3.9-7.8mmol / L. The results show that for volunteers within the reference range, the fasting blood glucose results do not fluctuate much. For volunteers with high fasting blood glucose, blood glucose is significantly reduced after taking Lactobacillus plantarum GOLDGUT-HNU082, reaching or approaching normal values; serum total protein is an indicator related to liver function, and its reference range is 60-80g / L. The results show that the overall serum total protein of the volunteers is high or at the high end of the normal range. After taking Lactobacillus plantarum GOLDGUT-HNU082, the serum total protein is significantly reduced; the reference range of the mean red blood cell hemoglobin content is 27-34pg. For volunteers within the reference range, the results do not fluctuate much. For volunteers with low this indicator, the mean red blood cell hemoglobin content increases after taking Lactobacillus plantarum GOLDGUT-HNU082 and approaches normal values; the reference range of the mean red blood cell volume is 80-100fL. After taking Lactobacillus plantarum GOLDGUT-HNU082, the mean red blood cell volume is significantly increased within the normal range. Taking Lactobacillus plantarum GOLDGUT-HNU082 can reduce the fasting blood sugar levels of subjects with high fasting blood sugar levels and improve the liver function of the subjects.

[0042] (2) Improvement of intestinal flora: Although the results of α and β diversity analysis showed that the richness and structure of the intestinal microbiota did not change significantly after supplementation with Lactobacillus plantarum GOLDGUT-HNU082 (see Appendix Figure 4 -B and Figure 4 -C), but by comparing the abundance of the bacterial phyla and the top 10 bacterial genera in the entire intestinal microbiome before and after supplementation with Lactobacillus plantarum GOLDGUT-HNU082 (see Appendix Figure 5-A), found that the abundance of phylum-level bacteria reported to be beneficial to the human body, including Actinobacteria, Bacteroidetes and CandidatusSaccharibacteria, increased, while the abundance of Cyanobacteria, Firmicutes and Synergistetes decreased. Among them, Actinobacteria is known to produce a variety of secondary metabolites, including antibiotics, anticancer agents and immunosuppressants; Bacteroidetes is considered to be the main degrader of polysaccharides; Candidatus Saccharibacteria has been found to have immunomodulatory activity. At the genus level, the abundance of Blautia, Fusobacterium, GGB3256, Ligilactobacillus, Prevotella and Romboutsia increased, and the abundance of Bacteroides, Faecalibacterium, Megamonas and Phocaeicola decreased. Studies have shown that among the upregulated bacteria, Blautia has the ability to regulate host health and alleviate metabolic syndrome, and has probiotic properties; Ligilactobacillus murinus is widely involved in host intestinal metabolism and immune activities; Romboutsia can improve obesity by regulating intestinal microbiota and lipid metabolism; and Megamonas, among the downregulated bacteria, is enriched in obese subjects. On this basis, we screened out the differential strains before and after supplementation with Lactobacillus plantarum GOLDGUT-HNU082, and found that it significantly enriched beneficial bacteria in the human intestine, such as Bifidobacterium animalis and Romboutsia timonensis (see Appendix). Figure 5 -B and 5-C). In addition, the relative abundance of some harmful species, such as Escherichia coli and Streptococcus mitis, decreased significantly after supplementation with Lactobacillus plantarum GOLDGUT-HNU082. In addition, we screened the metabolic pathways associated with the differential bacteria and found that the metabolic pathways related to the production of short-chain fatty acids, L-lysine fermentation to acetate and butyrate, and the pathways related to sugar and lipid metabolism ability, phospholipase, TCA cycle IV, fatty acid biosynthesis superpathway II, xylan degradation II, and NAD repair pathway V were enriched after supplementation with Lactobacillus plantarum GOLDGUT-HNU082 (see Appendix Figure 6 -A).

[0043] (3) Improve the health of the human intestine: To evaluate the health of the human intestine, we use GMWI2 to illustrate the health of the human intestine and quantify the balance between health-related microorganisms and disease-related microorganisms. GMWI is a reliable and biologically explainable health status prediction indicator based on the classification map of the intestinal microbiome. The higher the value, the better the health of the human body. The results showed that the GMWI2 of the population increased significantly by 54.14% after supplementing with Lactobacillus plantarum GOLDGUT-HNU082 (see Appendix). Figure 4 -A).

[0044] (4) GMWI2 is correlated with intestinal flora and clinical indicators: We quantified the interaction between Lactobacillus plantarum GOLDGUT-HNU082 in the subjects' intestines and clinical indicators and GMWI2 through their Spearman correlation coefficients. Correlation analysis showed that Lactobacillus plantarum GOLDGUT-HNU082 was positively correlated with GMWI2, and the GMWI2 index was negatively correlated with the subjects' blood glucose level, total protein level, and red blood cell level. The color is proportional to the strength of the correlation (see Appendix Figure 6 -B).

[0045] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0046] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of Lactobacillus plantarum GOLDGUT-HNU082 to improve intestinal health, characterized in that: The deposit number of the Lactobacillus plantarum GOLDGUT-HNU082 is CGMCC NO.33075.

2. Application of Lactobacillus plantarum GOLDGUT-HNU082 in the preparation of a bacterial agent for improving intestinal health, characterized in that: The deposit number of the Lactobacillus plantarum GOLDGUT-HNU082 is CGMCC NO.33075.

3. The use of a bacterial agent containing Lactobacillus plantarum GOLDGUT-HNU082 in the preparation of a product for improving intestinal health, characterized in that: The deposit number of the Lactobacillus plantarum GOLDGUT-HNU082 is CGMCC NO.33075.

4. Application of Lactobacillus plantarum GOLDGUT-HNU082 in the preparation of a product for improving GMWI2, characterized in that: The deposit number of the Lactobacillus plantarum GOLDGUT-HNU082 is CGMCC NO.33075.

5. Application of Lactobacillus plantarum GOLDGUT-HNU082 in the preparation of a product for lowering blood sugar, characterized in that: The deposit number of the Lactobacillus plantarum GOLDGUT-HNU082 is CGMCC NO.33075.

6. Application of Lactobacillus plantarum GOLDGUT-HNU082 in the preparation of a product for reducing serum total protein, characterized in that: The deposit number of the Lactobacillus plantarum GOLDGUT-HNU082 is CGMCC NO.33075.

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