Intestinal microorganism detection method based on polyphenol correlation analysis

Abstract

The invention relates to an intestinal microorganism detection method based on polyphenol correlation analysis. Comprising the following steps: S1, constructing a polyphenol standard curve; s2, measuring and calculating the polyphenol content in the blood sample according to the polyphenol standard curve; s3, calculating the relative abundance of the first batch of intestinal microorganisms by using a fitting formula according to the polyphenol content; and S4, according to the relative abundance of the first batch of enteric microorganisms, calculating the relative abundance of the second batch of enteric microorganisms by adopting a fitting formula. The detection method provided by the invention is simple, convenient and rapid, excrement sampling is not needed, the relative abundance of polyphenols in blood and microorganisms in intestinal tracts can be detected only by sampling a blood sample, the relative abundance of other common intestinal microorganisms can be obtained through the relative abundance of the microorganisms, and the problems that sampling is difficult and the detection time is short when the intestinal microorganisms are detected are solved. The time cost and the economic cost are saved, and the method can be widely applied to various situations in which the polyphenol and the intestinal microorganisms need to be rapidly detected.

Description

Technical Field

[0001] The present invention relates to an intestinal microorganism detection method based on correlation analysis of polyphenols in blood, which mainly estimates the relative abundance of certain bacteria in the human intestinal microorganisms by establishing a correlation relationship with the polyphenol content in human blood, and belongs to the field of biology and new medicine technology. Background Art

[0002] Intestinal microorganisms can also be called intestinal flora, which exist in large numbers in the human intestine. According to the number of culturable bacteria, the intestinal flora can be divided into dominant intestinal flora and secondary flora according to the number. The dominant flora exists in large numbers in the intestine and plays an important role in the life activities of the host, which will greatly affect the function of the entire flora. The secondary flora is mainly aerobic bacteria and facultative anaerobic bacteria, which are highly mobile and potentially pathogenic. Most of them belong to foreign flora and transient flora. Intestinal microorganisms are closely related to human health. Normal and healthy intestinal flora can improve human resistance. In addition, it has anti-aging, immune, detoxification, and anti-tumor effects. Many common chronic diseases of the human body, such as obesity, inflammatory bowel disease, depression, etc., are closely related to intestinal microorganisms. Therefore, detection and monitoring of the status of intestinal microorganisms can effectively prevent related diseases.

[0003] At present, the detection and monitoring of intestinal microorganisms are mostly carried out through 16s DNA sequencing of human fecal samples, which requires the samplers, testers and the tested to bear great psychological pressure. At the same time, this sampling method also has problems such as difficulty in fecal sampling, difficulty in grasping the sampling timing, and poor sample quality.

[0004] Polyphenols are a group of food bioactive compounds widely distributed in many everyday foods, such as fruits, vegetables, seeds, and beverages, and have been extensively studied in various diseases, such as cardiovascular disease, neurodegenerative diseases, cancer, and intestinal inflammatory diseases, and more recently in neurodevelopmental disorders, namely ASD and Down syndrome. Increasing evidence suggests that dietary polyphenols are also able to modulate the gut microbiota, which is important in ASD because these patients have dysbiosis in the gut microbiota. For example, citrus polyphenols have been reported to inhibit the growth of Helicobacter pylori in vitro, while red wine polyphenols have been shown to modulate the human gut microbiota, increasing the abundance of bacterial groups that are beneficial to health, namely Bifidobacterium, Bacteroidetes, and Enterococcus.

[0005] However, in real life, the bioavailability of polyphenols is low (about 5% to 10% is absorbed by the small intestine). The bioavailability of these compounds strictly depends on the enzymatic mechanism of intestinal microorganisms. Therefore, there may be a close relationship between intestinal microorganisms and polyphenols. For example, some intestinal microorganisms can metabolize polyphenols, such as Bifidobacterium, Bacteroidetes and Firmicutes. The reason why polyphenols are beneficial to the human body may be that some intestinal microorganisms can metabolize them into more bioavailable metabolites.

[0006] For example, several animal studies have shown that taking catechins, anthocyanins, and proanthocyanidins can promote the growth of Lactobacillus, Bifidobacterium, Akkermansia, Roseburia, and Faecalibacterium. In many cases, these prebiotic effects are accompanied by increased production of short-chain fatty acids (such as butyrate) and reduced concentrations of inflammatory mediators. Other phenolic compounds, such as lignans and stilbenes, increase the abundance of Lactobacillus and Bifidobacterium in animal models. Dietary polyphenols and their metabolites change the composition of intestinal microorganisms by stimulating the growth of beneficial bacteria while inhibiting pathogenic bacteria, thereby having a positive effect on promoting intestinal health. Polyphenols in oolong tea, black tea, and green tea have been shown to significantly affect the microbial composition by increasing Bifidobacterium and Lactobacillus. At the same time, it increases the production of short-chain fatty acids and reduces the production of Prevotella and Bacteroides.

[0007] The bacteria in the intestine are important factors affecting the intestinal microecology. These bacteria influence and restrict each other throughout the intestine. The relationships between these bacteria mainly include mutualism, commensalism, parasitism, predation, partial commensalism and competition. The interactions will have positive, negative and neutral effects on the participants. Various influences are intertwined to form a complex co-occurrence network. Therefore, in the intestinal microecology, bacteria that account for a relatively large proportion will promote the growth of bacteria that have a positive effect on them and inhibit the growth of bacteria that have a negative effect on them.

[0008] Correlation analysis refers to the analysis of two or more correlated variable elements to measure the degree of correlation between the two variable factors. Correlation analysis can only be performed if there is a certain connection or probability between the correlated elements. Currently, polyphenols and intestinal microorganisms are only tested separately, which is costly, and few researchers have combined the two. Summary of the invention

[0009] In view of the deficiencies of the prior art, the present invention provides an intestinal microbial detection method based on correlation analysis of polyphenols in the blood. The purpose is to reduce the diagnostic cost and provide another detection method for people who are not convenient to provide stool samples for intestinal microbial detection. The present invention performs a correlation analysis on the polyphenols and intestinal microorganisms that affect each other, calculates the correlation coefficient, and simultaneously fits the polyphenols with strong correlation with the intestinal microbial abundance to obtain a fitting formula. The intestinal microbial status can be determined quickly and accurately through the polyphenol content in the blood, and then the status of other common intestinal microorganisms can be obtained from the intestinal microbial status, so as to have an overall and comprehensive judgment on the intestinal microorganisms.

[0010] The technical solution of the present invention is as follows:

[0011] A method for detecting intestinal microorganisms based on correlation analysis of polyphenols in blood comprises the following steps:

[0012] S1: Construction of polyphenol standard curve;

[0013] S2: Based on the polyphenol standard curve constructed in S1, the polyphenol content in the blood sample was determined and calculated using the solid phase extraction / LC-MS / MS method;

[0014] S3: Based on the polyphenol content in the blood sample measured in S2, the relative abundance of the first batch of intestinal microorganisms was calculated using the fitting formula;

[0015] S4: Based on the relative abundance of the first batch of intestinal microorganisms obtained in S3, the relative abundance of the second batch of intestinal microorganisms is calculated using a fitting formula.

[0016] Preferably according to the present invention, in S1, the polyphenols are resveratrol, epigallocatechin gallate, (+)-catechin, proanthocyanidin, quercetin, sinapinic acid, epicatechin, oolong tea catechin, and dihydroquercetin.

[0017] Preferably according to the present invention, in S1, the polyphenol standard curve is:

[0018] Resveratrol: y = 8039.40023x + 5.23341e5;

[0019] Epigallocatechin gallate: y = 5.234524e4x + 4.69114e5;

[0020] (+)-catechin: y=6.105224e5x+3.12113e4;

[0021] Proanthocyanidins: y = 6.55241e4x + 2.45213e5;

[0022] Quercetin: y = 4.21132e4x + 3.12241e5;

[0023] Sinapic acid: y = 6.045462e4x + 2.45674e4;

[0024] Epicatechin: y = 57401.88677x + 6.672e4;

[0025] Oolong tea catechins: y = 4.24151e4x + 1.77652e5;

[0026] Dihydroquercetin: y=57121.80057x+6.601e4;

[0027] Wherein, y is the peak area and x is the polyphenol content (μg / L).

[0028] Preferably according to the present invention, in S3, the first batch of intestinal microorganisms are Enterococcus, Lactobacillus, Bacteroides, Bacillus, Eckermannella, Blautia, and Eubacterium.

[0029] Preferably, according to the present invention, in S3, the fitting formula is as follows:

[0030] The fitting formula for resveratrol and Enterococcus is: y = 0.00203x + 0.000101;

[0031] The fitting formula for resveratrol and Eckermansia is: y = 0.1263ln(x) + 0.01001;

[0032] The fitting formula of epigallocatechin gallate and Bacteroides is:

[0033] The fitting formula for (+)-catechin and Eckermannia is:

[0034] The fitting formula for proanthocyanidins and Bacteroides is: y = 0.5603ln(x) + 0.52;

[0035] The fitting formula for quercetin and Bacillus was: y = 0.254x-0.0012;

[0036] The fitting formula for quercetin and Eubacterium was: y = 0.001565x-0.0003;

[0037] The fitting formula for sinapinic acid and Lactobacillus is: y = 0.0062x + 0.0001;

[0038] The fitting formula for sinapinic acid and Eckermansia is: y = 0.01002x + 0.007;

[0039] The fitting formula for sinapinic acid and Blautia is: y = 0.001565x + 0.0003;

[0040] The fitting formula for epicatechin and Bacteroides is: y = 0.0032x + 0.0203;

[0041] The fitting formula of oolong tea catechins and Bacillus is: y = 0.204x-0.0302;

[0042] The fitting formula of oolong tea catechins and Eubacterium is: y = 0.094x-0.0012;

[0043] The fitting formula for dihydroquercetin and Lactobacillus is: y = 0.0014x + 0.0032;

[0044] Among them, y is the relative abundance of the first batch of intestinal microorganisms at the genus level, and x is the polyphenol content in the blood (mg / L).

[0045] Preferably according to the present invention, in S4, the second batch of intestinal microorganisms are Bifidobacterium, Lactococcus, Bacteroides, Ruminococcus, and Clostridium.

[0046] Preferably, according to the present invention, in S4, the fitting formula is as follows:

[0047] The fitting formula for Bifidobacterium and Lactobacillus is: y = 0.60236x + 0.00003;

[0048] The fitting formula for Lactococcus and Lactobacillus is: y = 0.82096x + 0.00001;

[0049] The fitting formula for Bacteroides and Bacteroides is: y = 0.30025x + 0.00001;

[0050] The fitting formula for Ruminococcus and Enterococcus is: y = 1.2334x + 0.00002;

[0051] The fitting formula for Clostridium and Akkermansia is: y = 0.12244x + 0.00122;

[0052] Among them, y is the relative abundance of the second batch of intestinal microorganisms at the genus level, and x is the relative abundance of the first batch of intestinal microorganisms at the genus level.

[0053] According to a preferred technical solution of the present invention, the intestinal microorganism detection method based on the correlation analysis of polyphenols in blood is as follows:

[0054] (1) Accurately weigh 0.001 mg of each of resveratrol, epigallocatechin gallate, (+)-catechin, proanthocyanidin, quercetin, sinapinic acid, epicatechin, oolong tea catechin, and dihydroquercetin standard products in a 10 ml volumetric flask, and dissolve them in 50% methanol-formic acid solution to prepare a mixed polyphenol standard solution with a polyphenol concentration of 1 mg / L;

[0055] (2) adding the mixed polyphenol standard solution prepared in step (1) to 2 ml of blank human plasma matrix to obtain a mixed standard solution with gradient concentrations; the gradient concentrations are 500 μg / L, 250 μg / L, 150 μg / L, 100 μg / L, 50 μg / L, 20 μg / L, 10 μg / L, 5 μg / L, 2.5 μg / L, 1 μg / L, 0.5 μg / L, 0.25 μg / L, and 0.1 μg / L;

[0056] (3) Add 5 ml of an aqueous solution containing 5% EDTA to the mixed standard solution of gradient concentration obtained in step (2), shake for 2 minutes, centrifuge at 4000 r / min for 3 minutes, and take the supernatant; then add anhydrous sodium sulfate twice the weight of the supernatant to the supernatant, mix well and add to a 10 ml solid phase extraction column, rinse the impurities in the column with 6 ml of hexane, elute with 6 ml of ether at a flow rate of 1 ml / min, collect the eluate, evaporate it in a 50°C water bath to nearly dryness, add a methanol-water mixed solution with a volume ratio of 8:2, let it stand for 1 minute to adjust the volume, and obtain a purified mixed standard solution of gradient concentration;

[0057] (4) injecting the gradient concentration mixed standard solution purified in step (3) into a high performance liquid chromatography-tandem mass spectrometer for detection, using the sample concentration as the ordinate and the peak area as the abscissa to construct a polyphenol standard curve;

[0058] The conditions of the liquid chromatography are as follows:

[0059] Column: Xbridge TM Phenyl HPLC column 2.1mm×150mm×3.5μm, connected to XTerra @ Guard column; column temperature 40℃, flow rate 50μl / min, acquisition time 10min, injection volume 3μl; mobile phase A is water; mobile phase B is acetonitrile; mobile phase C is 20mmol / L ammonium formate; mobile phase D is methanol;

[0060] Gradient elution program:

[0061]

[0062] The mass spectrometry source parameters of the tandem mass spectrometer are as follows: monitoring mode: MRM, GS1: 43 psi; GS2: 48 psi, curtain gas: 30 psi, collision gas: 7 psi, ion spray voltage: 5500 V, focusing voltage: 22 V, temperature: 233° C.;

[0063] The polyphenol standard curve is:

[0064] Resveratrol: y = 8039.40023x + 5.23341e5;

[0065] Epigallocatechin gallate: y = 5.234524e4x + 4.69114e5;

[0066] (+)-catechin: y=6.105224e5x+3.12113e4;

[0067] Proanthocyanidins: y = 6.55241e4x + 2.45213e5;

[0068] Quercetin: y = 4.21132e4x + 3.12241e5;

[0069] Sinapic acid: y = 6.045462e4x + 2.45674e4;

[0070] Epicatechin: y = 57401.88677x + 6.672e4;

[0071] Oolong tea catechins: y = 4.24151e4x + 1.77652e5;

[0072] Dihydroquercetin: y=57121.80057x+6.601e4;

[0073] Where y is the peak area, x is the polyphenol content (μg / L);

[0074] (5) collecting blood from the test subject, purifying it according to the method described in step (3), and injecting it into a high performance liquid chromatography-tandem mass spectrometer for detection, and calculating the content of resveratrol, epigallocatechin gallate, (+)-catechin, proanthocyanidin, quercetin, sinapinic acid, epicatechin, oolong tea catechin, and dihydroquercetin in the blood sample according to the polyphenol standard curve constructed in step (4);

[0075] (6) Calculating the relative abundance of the first batch of intestinal microorganisms using a fitting formula based on the contents of resveratrol, epigallocatechin gallate, (+)-catechin, proanthocyanidin, quercetin, sinapinic acid, epicatechin, oolong tea catechin, and dihydroquercetin determined in step (5);

[0076] The fitting formula is as follows:

[0077] The fitting formula for resveratrol and Enterococcus is: y = 0.00203x + 0.000101;

[0078] The fitting formula for resveratrol and Eckermansia is: y = 0.1263x + 0.01001;

[0079] The fitting formula for epigallocatechin gallate and Bacteroides is: y = 0.09665x + 0.02112;

[0080] The fitting formula for (+)-catechin and Eckermansia is: y = 0.00063x + 0.00016;

[0081] The fitting formula for proanthocyanidins and Bacteroides is: y = 0.5603x + 0.0052;

[0082] The fitting formula for quercetin and Bacillus was: y = 0.254x-0.0012;

[0083] The fitting formula for quercetin and Eubacterium was: y = 0.001565x-0.0003;

[0084] The fitting formula for sinapinic acid and Lactobacillus is: y = 0.0062x + 0.0001;

[0085] The fitting formula for sinapinic acid and Eckermansia is: y = 0.01002x + 0.007;

[0086] The fitting formula for sinapinic acid and Blautia is: y = 0.001565x + 0.0003;

[0087] The fitting formula for epicatechin and Bacteroides is: y = 0.0032x + 0.0203;

[0088] The fitting formula of oolong tea catechins and Bacillus is: y = 0.204x-0.0302;

[0089] The fitting formula of oolong tea catechins and Eubacterium is: y = 0.094x-0.0012;

[0090] The fitting formula for dihydroquercetin and Lactobacillus is: y = 0.0014x + 0.0032;

[0091] Among them, y is the relative abundance of the first batch of intestinal microorganisms at the genus level, and x is the polyphenol content in blood (mg / L);

[0092] (7) Calculating the relative abundance of the second batch of intestinal microorganisms using a fitting formula based on the relative abundance of the first batch of intestinal microorganisms calculated in step (6);

[0093] The fitting formula is as follows:

[0094] The fitting formula for Bifidobacterium and Lactobacillus is: y = 0.60236x + 0.00003;

[0095] The fitting formula for Lactococcus and Lactobacillus is: y = 0.82096x + 0.00001;

[0096] The fitting formula for Bacteroides and Bacteroides is: y = 0.30025x + 0.00001;

[0097] The fitting formula for Ruminococcus and Enterococcus is: y = 1.2334x + 0.00002;

[0098] The fitting formula for Clostridium and Akkermansia is: y = 0.12244x + 0.00122;

[0099] Among them, y is the relative abundance of the second batch of intestinal microorganisms at the genus level, and x is the relative abundance of the first batch of intestinal microorganisms at the genus level.

[0100] The beneficial effects of the present invention are:

[0101] The present invention analyzes the correlation between the polyphenol content in the blood and the intestinal microorganisms according to the second-generation high-throughput sequencing results of 1,000 children's intestinal microorganisms and the mass spectrometry detection results of the polyphenol content in the blood, and obtains the correlation fitting formula between the polyphenol content in the blood and the intestinal microorganisms. Then, according to the fitting formula, the abundance of Enterococcus, Lactobacillus, Bacteroides, Bacillus, Eckermanella, Blautia, and Eubacterium in the intestinal tract is calculated according to the content of resveratrol, epigallocatechin gallate, (+)-catechin, proanthocyanidin, quercetin, sinapinic acid, epicatechin, oolong tea catechin, and dihydroquercetin in the blood. At the same time, according to the relative abundance of the above-mentioned intestinal microorganisms, the relative abundance of other microorganisms in the intestinal tract is calculated with reference to the fitting formula between the intestinal microorganisms.

[0102] The intestinal microorganism detection method based on the correlation analysis of polyphenols in blood provided by the present invention is simple and rapid, and does not require fecal sampling. Only by taking a blood sample, the relative abundance of polyphenols in the blood and microorganisms in the intestine can be detected, which solves the problem of sampling difficulty and detection difficulty when detecting intestinal microorganisms, saves time and economic costs, and can not only obtain intestinal microorganisms related to polyphenols, but also obtain other microorganisms that play an important role in the intestine, and conduct a more comprehensive detection of the intestinal microbial state. It can be widely used in various situations where rapid detection of polyphenols and intestinal microorganisms is required, and effectively overcomes the problems of fecal sampling difficulty, sampling timing difficulty, poor sample quality, etc. in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 is a flow chart for the determination of the correlation between polyphenols and intestinal microorganisms. DETAILED DESCRIPTION

[0104] The present invention is further described below by means of specific examples, but is not limited thereto.

[0105] Instrument: AB Sciex 4500 triple quadrupole liquid spectrometer.

[0106] The human plasma blank matrix used in the present invention is obtained as follows: 4% bovine serum albumin solution (dissolved in physiological saline).

[0107] The solid phase extraction column used in the present invention is prepared as follows: take a clean acid burette with a length of 13 cm, plug the bottom with about 0.02 g of glass wool, add 0.15 g of PSA, 0.3 g of Florisil, and 0.45 g of Silica to the upper layer in sequence, and tap it tightly after adding each layer of material to make a solid phase extraction column.

[0108] Example 1: Determination of the fitting formula for polyphenols and intestinal microorganisms

[0109] 1. Acquisition of blood polyphenol content data

[0110] (1) Construction of polyphenol standard curve

[0111] ① Accurately weigh 0.001 mg of each of resveratrol, epigallocatechin gallate, (+)-catechin, proanthocyanidin, quercetin, sinapinic acid, epicatechin, oolong tea catechin, and dihydroquercetin standards in a 10 ml volumetric flask, and dissolve them in 50% methanol-formic acid solution to prepare a mixed polyphenol standard solution with a polyphenol concentration of 1 mg / L;

[0112] ②, add the mixed polyphenol standard solution prepared in step (1) to 2 ml of human plasma blank matrix to obtain a mixed standard solution with gradient concentrations; the gradient concentrations are 500 μg / L, 250 μg / L, 150 μg / L, 100 μg / L, 50 μg / L, 20 μg / L, 10 μg / L, 5 μg / L, 2.5 μg / L, 1 μg / L, 0.5 μg / L, 0. .25μg / L, 0.1μg / L; that is, the gradient concentrations of the above 9 polyphenols in the mixed standard solution are 500μg / L, 250μg / L, 150μg / L, 100μg / L, 50μg / L, 20μg / L, 10μg / L, 5μg / L, 2.5μg / L, 1μg / L, 0.5μg / L, 0.25μg / L, 0.1μg / L;

[0113] ③. Add 5 ml of an aqueous solution containing 5% EDTA to the mixed standard solution of gradient concentration obtained in step (2), shake for 2 min, centrifuge at 4000 r / min for 3 min, and take the supernatant; then add anhydrous sodium sulfate twice the weight of the supernatant to the supernatant, mix well and add to a 10 ml solid phase extraction column, rinse the impurities in the column with 6 ml of hexane, elute with 6 ml of ether at a flow rate of 1 ml / min, collect the eluate, evaporate it in a 50°C water bath to nearly dryness, add a methanol-water mixed solution with a volume ratio of 8:2, let stand for 1 min to adjust the volume, and obtain a purified mixed standard solution of gradient concentration;

[0114] ④, injecting the purified gradient concentration mixed standard solution obtained in step ③ into a high performance liquid chromatography-tandem mass spectrometer for detection, and constructing a polyphenol standard curve with sample concentration as the ordinate and peak area as the abscissa;

[0115] The conditions of the liquid chromatography are as follows:

[0116] Column: Xbridge TM Phenyl HPLC column 2.1mm×150mm×3.5μm, connected to XTerra @ Guard column; column temperature 40℃, flow rate 50μl / min, acquisition time 10min, injection volume 3μl; mobile phase A is water; mobile phase B is acetonitrile; mobile phase C is 20mmol / L ammonium formate; mobile phase D is methanol;

[0117] The gradient elution program is shown in Table 1:

[0118] Table 1

[0119]

[0120] The mass spectrometry source parameters of the tandem mass spectrometer are as follows: monitoring mode: MRM, GS1: 43 psi; GS2: 48 psi, curtain gas: 30 psi, collision gas: 7 psi, ion spray voltage: 5500 V, focusing voltage: 22 V, temperature: 233° C.;

[0121] The polyphenol standard curve is:

[0122] Resveratrol: y = 8039.40023x + 5.23341e5;

[0123] Epigallocatechin gallate: y = 5.234524e4x + 4.69114e5;

[0124] (+)-catechin: y=6.105224e5x+3.12113e4;

[0125] Proanthocyanidins: y = 6.55241e4x + 2.45213e5;

[0126] Quercetin: y = 4.21132e4x + 3.12241e5;

[0127] Sinapic acid: y = 6.045462e4x + 2.45674e4;

[0128] Epicatechin: y = 57401.88677x + 6.672e4;

[0129] Oolong tea catechins: y = 4.24151e4x + 1.77652e5;

[0130] Dihydroquercetin: y=57121.80057x+6.601e4;

[0131] Wherein, y is the peak area and x is the polyphenol content (μg / L).

[0132] (2) Obtaining data

[0133] 1000 typical healthy children in Shandong area were selected for blood sampling, and the blood samples were properly stored in a -80°C refrigerator; then the blood samples were purified according to the method described in step ③ and injected into a high performance liquid chromatography-tandem mass spectrometer, and detected according to the conditions described in step ④, and the contents of resveratrol, epigallocatechin gallate, (+)-catechin, proanthocyanidins, quercetin, sinapinic acid, epicatechin, oolong tea catechins, and dihydroquercetin in the blood samples were calculated based on the constructed polyphenol standard curve.

[0134] 2. Acquisition of relative abundance data of intestinal microorganisms in feces

[0135] (1) Obtain feces from 1,000 typical healthy children in Shandong Province from the same batch as the blood samples in step 1 above, and mark them one by one with the blood samples, and then use the solarbio fecal microbial DNA extraction kit to extract microbial DNA from the fecal samples;

[0136] Specifically: 100 mg of fecal sample was weighed, 500 μL of SA solution was added, placed on ice for 10 min, and centrifuged at 12000 g for 1 min;

[0137] The supernatant was discarded, and 400 μL of SB solution, 20 μL of 10 mg / mL RNase A, and 20 μL of 10 mg / mL proteinase K were added to the precipitate, vortexed for 30 s, incubated at 65 °C for 30-60 min, inverted several times, and centrifuged at 12000 g for 2 min;

[0138] The supernatant was placed in a 1.5 mL centrifuge tube, 400 μL of SC solution was added, and the tube was centrifuged at 12,000 g for 2 min. The supernatant was transferred to a filter column (the filter column was placed in a collection tube), and the tube was centrifuged at 12,000 rpm for 1 min.

[0139] Pipette the filtrate into the DNA adsorption column, let it stand at room temperature for 2 minutes, and centrifuge at 12000g for 1 minute (can be repeated once);

[0140] Discard the waste liquid in the collection tube, add 500 μL of rinse solution to the adsorption column, and centrifuge at 12000 rpm for 1 min (repeat once);

[0141] Discard the waste liquid in the collection tube, put the adsorption column back into the collection tube, and centrifuge at 12000 rpm for 1 min;

[0142] After drying the adsorption column at room temperature, place the adsorption column in a new centrifuge tube, add 100 μL of the eluent preheated at 65°C, and centrifuge at 12000 rpm for 1 min.

[0143] Add the liquid in the centrifuge tube back into the adsorption column and centrifuge at 12000 rpm for 1 min. The centrifuge tube contains the fecal microbial DNA solution.

[0144] (2) The fecal microbial DNA solution extracted in step (1) was sent to Novogene Sequencing Company for sequencing. After the sequencing results were obtained, the sequencing results were annotated with species classification using the dada2 package and the ggplot2 package in the R software to form an ASV table. The sequencing results were flattened using the vegan package. The relative abundance of species was calculated and analyzed using the MicrobiotaProcess, ggplot2, and phyloseq packages to obtain the relative abundance data of common intestinal microorganisms.

[0145] 3. Correlation analysis between relative abundance of intestinal microorganisms and polyphenol content

[0146] (1) Based on the obtained data on polyphenol content in blood and relative abundance of intestinal microorganisms in feces, the corrplot package, Hmisc package, and ggplot2 package in R software were used to calculate the correlation coefficients between the abundance of Enterococcus, Lactobacillus, Bacteroides, Bacillus, Eckermannella, Blautia, and Eubacterium microorganisms in feces and the content of resveratrol, epigallocatechin gallate, (+)-catechin, proanthocyanidins, quercetin, sinapinic acid, epicatechin, oolong tea catechins, and dihydroquercetin in blood. The results are shown in Table 2.

[0147] Table 2. Pearson correlation coefficient between polyphenols and intestinal microorganisms

[0148]

[0149] (2) Selecting polyphenols with strong correlation and intestinal microorganisms from the correlation coefficient table of intestinal microorganisms and polyphenols and calculating the correlation formula for each pair, using the BestFitM package in the R software to analyze the most suitable fitting parameter model for each pair of polyphenols and intestinal microorganism content; calculating the Pearson correlation coefficient between each intestinal microorganism and polyphenol according to the most suitable model parameters; comparing the Pearson correlation coefficients and selecting polyphenols and intestinal microorganisms with strong correlation (Pearson correlation coefficient>0.4); the calculation formula of the Pearson correlation coefficient is as follows:

[0150]

[0151] Among them, X i and Y i is the i-th pair of observations in the sample data. In the present invention, X is the polyphenol content in the blood, and Y is the relative abundance of microorganisms in the intestine at the genus level. and are the means of X and Y, respectively, and r is the Pearson correlation coefficient.

[0152] If there is a correlation between intestinal microorganisms and multiple polyphenols at the genus level, then the correlation formulas are established respectively; when determining the relative abundance of intestinal microorganisms through the correlation formula, multiple correlation formulas are used for calculation respectively, and the relative abundance of intestinal microorganisms refers to the average value of the calculation results;

[0153] (3) According to the Pearson correlation coefficient, resveratrol has a high correlation with Enterococcus and Eckermannia; epigallocatechin gallate has a high correlation with Bacteroides; (+)-catechin has a high correlation with Eckermannia; proanthocyanidins have a high correlation with Bacteroides; quercetin has a high correlation with Bacillus and Eubacterium; sinapinic acid has a high correlation with Lactobacillus, Eckermannia, and Blautia; epicatechin has a high correlation with Bacteroides; oolong tea catechin has a high correlation with Bacillus and Eubacterium; dihydroquercetin has a high correlation with Lactobacillus. The polyphenol content with high correlation and the intestinal microbial content were subjected to pairwise fitting analysis, and the fitting formula was obtained, as shown in Table 3.

[0154] Table 3. Fitting formulas for key intestinal microorganisms and polyphenols

[0155]

[0156]

[0157] The fitting formula is as follows:

[0158] The fitting formula for resveratrol and Enterococcus is: y = 0.00203x + 0.000101;

[0159] The fitting formula for resveratrol and Eckermansia is: y = 0.1263x + 0.01001;

[0160] The fitting formula between epigallocatechin gallate and Bacteroides is: y=0.09665x+0.02112; the fitting formula between (+)-catechin and Eckermannia is: y=0.00063x+0.00016;

[0161] The fitting formula for proanthocyanidins and Bacteroides is: y = 0.5603x + 0.0052;

[0162] The fitting formula for quercetin and Bacillus was: y = 0.254x-0.0012;

[0163] The fitting formula for quercetin and Eubacterium was: y = 0.001565x-0.0003;

[0164] The fitting formula for sinapinic acid and Lactobacillus is: y = 0.0062x + 0.0001;

[0165] The fitting formula for sinapinic acid and Eckermansia is: y = 0.01002x + 0.007;

[0166] The fitting formula for sinapinic acid and Blautia is: y = 0.001565x + 0.0003;

[0167] The fitting formula for epicatechin and Bacteroides is: y = 0.0032x + 0.0203;

[0168] The fitting formula of oolong tea catechins and Bacillus is: y = 0.204x-0.0302;

[0169] The fitting formula of oolong tea catechins and Eubacterium is: y = 0.094x-0.0012;

[0170] The fitting formula for dihydroquercetin and Lactobacillus is: y = 0.0014x + 0.0032;

[0171] Where y is the relative abundance of intestinal microorganisms at the genus level, and x is the polyphenol content in blood (mg / L)

[0172] Example 2: Accuracy determination of the intestinal microorganism and polyphenol fitting formula of the present invention

[0173] In this embodiment, the relative abundance of intestinal microorganisms determined by the correlation relationship fitting formula was compared with the relative abundance of intestinal microorganisms obtained by sequencing and 16s sequencing result analysis to determine the accuracy of the method of inferring the relative abundance of intestinal microorganisms through blood polyphenol content using the correlation relationship and fitting formula.

[0174] 1. Randomly obtain 20 blood samples from typical people in Shandong area, and then process the blood samples according to the method described in Example 1, and combine the polyphenol standard curve constructed in Example 1 to determine and calculate the contents of resveratrol, epigallocatechin gallate, (+)-catechin, proanthocyanidin, quercetin, sinapinic acid, epicatechin, oolong tea catechin, and dihydroquercetin in the 20 blood samples.

[0175] Then, according to the fitting formula obtained in Table 3 of Example 1, the relative abundance of Enterococcus, Lactobacillus, Bacteroides, Bacillus, Eckermansia, Blautia, and Eubacterium microorganisms in the intestine was calculated using the fitting formula based on the polyphenol content in the blood sample.

[0176] 2. Obtain 20 stool samples from a typical population in Shandong Province from the same batch as the blood samples in step 1 above, and mark them one by one with the blood samples, and then obtain the relative abundance of Enterococcus, Lactobacillus, Bacteroides, Bacillus, Eckermanella, Blautia, and Eubacterium microorganisms in the stool by sequencing according to the method described in Example 1.

[0177] 3. The deviation between the results of the method of extracting DNA from feces to determine the relative abundance of intestinal microorganisms and the method of calculating the relative abundance of intestinal microorganisms through polyphenol content based on the fitting formula was calculated. The results are shown in Table 4.

[0178] Table 4. Results and standard deviations of two intestinal microbial detection methods

[0179]

[0180]

[0181] As can be seen from Table 4, based on the fitting formula, the method of the present invention is used to measure the relative abundance of intestinal microorganisms at the genus level described in the table by the polyphenol content in the blood, and the relative error is within 13.2% compared with the result of 16s sequencing, especially for Lactobacillus, Bacillus, and Eubacterium, the relative error is within 10%, and the relative error of Eubacterium is within 5%, indicating that the present invention does not require fecal sampling, and only by taking blood samples, the detection of polyphenols in the blood and the relative abundance of microorganisms in the intestine can be achieved, and the detection results of Lactobacillus, Bacillus, and Eubacterium are more accurate.

[0182] Example 3: Determination of fitting formulas for different microorganisms in the intestine

[0183] In this example, a correlation analysis of intestinal microorganisms at the genus level was performed based on the first batch of intestinal microbial relative abundance data obtained in Example 1.

[0184] 1. The corrplot package, Hmisc package, and ggplot2 package in R software were used to calculate the correlation coefficient between the relative abundance of the first batch of intestinal microorganisms Lactobacillus, Bacteroides, Enterococcus, and Akkermansia in feces and the relative abundance of the second batch of intestinal microorganisms Bifidobacterium, Lactococcus, Bacteroides, Clostridium, and Ruminococcus. The results are shown in Table 5.

[0185] Table 5. Pearson correlation coefficient of relative abundance of the first batch of intestinal microorganisms and the second batch of intestinal microorganisms

[0186]

[0187] 2. The first batch of intestinal microorganisms and the second batch of intestinal microorganisms with strong correlation were selected from the Pearson correlation coefficients of the relative abundance of different intestinal microorganisms to calculate the correlation formula in pairs, and the BestFitM package in the R software was used to analyze the optimal fitting parameter model for each pair of intestinal microorganism content; the Pearson correlation coefficient between each intestinal microorganism was calculated according to the optimal model parameters; the Pearson correlation coefficient was compared to select the intestinal microorganism with strong correlation (Pearson correlation coefficient>0.4); the calculation formula of the Pearson correlation coefficient is as follows:

[0188]

[0189] Among them, X i and Y i is the i-th pair of observations in the sample data, in the present invention, X is the first batch of intestinal microorganisms with known relative abundance, Y is the relative abundance of the second batch of intestinal microorganisms in the intestine at the genus level, and are the means of X and Y, respectively, and r is the Pearson correlation coefficient.

[0190] If there are multiple correlation relationships between the first batch of intestinal microorganisms and the second batch of intestinal microorganisms at the genus level, correlation formulas are established respectively; when determining the relative abundance of the second batch of intestinal microorganisms through the correlation formula, multiple correlation formulas are used for calculation respectively, and the relative abundance of intestinal microorganisms refers to the average value of the calculation results;

[0191] (3) From the Pearson correlation coefficient, it can be seen that Lactobacillus and Bifidobacterium, Lactobacillus and Lactococcus, Bacteroides and Bacteroides, Enterococcus and Ruminococcus, Akkermansia and Clostridium are highly correlated. The first batch of intestinal microorganisms with high correlation and the second batch of intestinal microorganisms were subjected to pairwise fitting analysis to obtain the fitting formula, as shown in Table 6.

[0192] Table 6. Fitting formulas for the first batch of intestinal microorganisms and the second batch of intestinal microorganisms

[0193]

[0194]

[0195] The fitting formula is as follows:

[0196] The fitting formula for Bifidobacterium and Lactobacillus is: y = 0.60236x + 0.00003;

[0197] The fitting formula for Lactococcus and Lactobacillus is: y = 0.82096x + 0.00001;

[0198] The fitting formula for Bacteroides and Bacteroides is: y = 0.30025x + 0.00001;

[0199] The fitting formula for Ruminococcus and Enterococcus is: y = 1.2334x + 0.00002;

[0200] The fitting formula for Clostridium and Akkermansia is: y = 0.12244x + 0.00122;

[0201] Among them, y is the relative abundance of the second batch of intestinal microorganisms at the genus level, and x is the relative abundance of the first batch of intestinal microorganisms at the genus level.

[0202] Example 4: Accuracy determination of the intestinal microbial fitting formulas of the present invention

[0203] In this embodiment, the relative abundance of intestinal microorganisms determined by the correlation relationship fitting formula was compared with the relative abundance of intestinal microorganisms obtained by sequencing and 16s sequencing result analysis to determine the accuracy of the method of inferring the relative abundance of the second batch of intestinal microorganisms by the relative abundance of the first batch of intestinal microorganisms using the correlation relationship and fitting formula.

[0204] 1. Randomly obtain 20 blood samples from a typical population in Shandong Province, and then process the blood samples according to the method described in Example 1. Combined with the polyphenol standard curve constructed in Example 1, determine and calculate the contents of acetic acid, propionic acid, butyric acid, valeric acid, and caproic acid in the 20 blood samples.

[0205] Then, according to the fitting formula obtained in Table 3 of Example 1, the relative abundance of the first batch of intestinal microorganisms Lactobacillus, Bacteroides, Enterococcus, and Akkermansia in feces was calculated using the fitting formula based on the content of polyphenols in the blood sample.

[0206] 2. Obtain 20 stool samples from a typical population in Shandong Province from the same batch as the blood samples in step 1 above, and mark them one by one with the blood samples. Then, according to the method described in Example 1, obtain the relative abundance of the second batch of intestinal microorganisms Bifidobacterium, Lactococcus, Bacteroides, Ruminococcus, and Clostridium in the stool by sequencing.

[0207] 3. According to the fitting formula obtained in Table 6 in Example 3, the relative abundance of the second batch of intestinal microorganisms Bifidobacterium, Lactococcus, Bacteroides, Ruminococcus and Clostridium was calculated from the relative abundance of the first batch of intestinal microorganisms Lactobacillus, Bacteroides, Enterococcus and Akkermansia calculated in step 1.

[0208] 4. Compare the deviations of the results of the two methods: determining the relative abundance of intestinal microorganisms by extracting DNA from feces and calculating the relative abundance of the second batch of intestinal microorganisms based on the fitting formula using the relative abundance content of the first batch of intestinal microorganisms. The results are shown in Table 7.

[0209] Table 7. Results and standard deviations of two intestinal microbial detection methods

[0210]

[0211] As can be seen from Table 7, based on the fitting formula, the method of the present invention is used to measure the relative abundance of intestinal microorganisms at the genus level described in the table by the polyphenol content in the blood, and the relative error is within 15% compared with the result of 16s sequencing, especially for Lactococcus, Bacteroides and Clostridium, the relative error is within 10%, indicating that the present invention does not require fecal sampling, and only by taking blood samples, the detection of polyphenols in the blood and the relative abundance of microorganisms in the intestine can be achieved, and the relative abundance of other intestinal microorganisms can be calculated by the relative abundance of known intestinal microorganisms in the intestine.

Claims

1. A method for detecting intestinal microorganisms based on correlation analysis of polyphenols in blood, characterized in that: The steps include: S1: Construction of polyphenol standard curve; S2: Based on the polyphenol standard curve constructed in S1, the polyphenol content in the blood sample was determined and calculated using the solid phase extraction / LC-MS / MS method; S3: Based on the polyphenol content in the blood samples measured in S2, the relative abundance of the first batch of intestinal microorganisms was calculated using the fitting formula; S4: Based on the relative abundance of the first batch of intestinal microorganisms obtained in S3, the relative abundance of the second batch of intestinal microorganisms is calculated using a fitting formula.

2. The intestinal microorganism detection method based on blood polyphenol correlation analysis according to claim 1, characterized in that: In S1, the polyphenols are resveratrol, epigallocatechin gallate, (+)-catechin, proanthocyanidin, quercetin, sinapinic acid, epicatechin, oolong tea catechin, and dihydroquercetin.

3. The intestinal microorganism detection method based on blood polyphenol correlation analysis according to claim 1, characterized in that: In S1, the polyphenol standard curve is: Resveratrol: y = 8039.40023x + 5.23341e5; Epigallocatechin gallate: y = 5.234524e4x + 4.69114e5; (+)-catechin: y=6.105224e5x+3.12113e4; Proanthocyanidins: y = 6.55241e4x + 2.45213e5; Quercetin: y = 4.21132e4x + 3.12241e5; Sinapic acid: y = 6.045462e4x + 2.45674e4; Epicatechin: y = 57401.88677x + 6.672e4; Oolong tea catechins: y = 4.24151e4x + 1.77652e5; Dihydroquercetin: y=57121.80057x+6.601e4; Wherein, y is the peak area and x is the polyphenol content (μg / L).

4. The intestinal microorganism detection method based on blood polyphenol correlation analysis according to claim 1, characterized in that: In S3, the first batch of intestinal microorganisms are Enterococcus, Lactobacillus, Bacteroides, Bacillus, Eckermannella, Blautia, and Eubacterium.

5. The intestinal microorganism detection method based on polyphenol correlation analysis in blood according to claim 1, characterized in that ,, in S3, the fitting formula is as follows: The fitting formula for resveratrol and Enterococcus is: y = 0.00203x + 0.000101; The fitting formula for resveratrol and Eckermansia is: y = 0.1263ln(x) + 0.01001; The fitting formula of epigallocatechin gallate and Bacteroides is: The fitting formula for (+)-catechin and Eckermannia is: The fitting formula for proanthocyanidins and Bacteroides is: y = 0.5603ln(x) + 0.52; The fitting formula for quercetin and Bacillus was: y = 0.254x-0.0012; The fitting formula for quercetin and Eubacterium was: y = 0.001565x-0.0003; The fitting formula for sinapinic acid and Lactobacillus is: y = 0.0062x + 0.0001; The fitting formula for sinapinic acid and Eckermansia is: y = 0.01002x + 0.007; The fitting formula for sinapinic acid and Blautia is: y = 0.001565x + 0.0003; The fitting formula for epicatechin and Bacteroides is: y = 0.0032x + 0.0203; The fitting formula of oolong tea catechins and Bacillus is: y = 0.204x-0.0302; The fitting formula of oolong tea catechins and Eubacterium is: y = 0.094x-0.0012; The fitting formula for dihydroquercetin and Lactobacillus is: y = 0.0014x + 0.0032; Among them, y is the relative abundance of intestinal microorganisms at the genus level, and x is the polyphenol content in blood (mg / L).

6. The intestinal microorganism detection method based on blood polyphenol correlation analysis according to claim 1, characterized in that: In S4, the second batch of intestinal microorganisms are Bifidobacterium, Lactococcus, Bacteroides, Ruminococcus, and Clostridium.

7. The intestinal microorganism detection method based on blood polyphenol correlation analysis according to claim 1, characterized in that: In S4, the fitting formula is as follows: The fitting formula for Bifidobacterium and Lactobacillus is: y = 0.60236x + 0.00003; The fitting formula for Lactococcus and Lactobacillus is: y = 0.82096x + 0.00001; The fitting formula for Bacteroides and Bacteroides is: y = 0.30025x + 0.00001; The fitting formula for Ruminococcus and Enterococcus is: y = 1.2334x + 0.00002; The fitting formula for Clostridium and Akkermansia is: y = 0.12244x + 0.00122; Among them, y is the relative abundance of the second batch of intestinal microorganisms at the genus level, and x is the relative abundance of the first batch of intestinal microorganisms at the genus level.

8. The intestinal microorganism detection method based on blood polyphenol correlation analysis according to claim 1, characterized in that: The intestinal microorganism detection method based on the correlation analysis of polyphenols in blood is specifically as follows: (1) Accurately weigh 0.001 mg of each of resveratrol, epigallocatechin gallate, (+)-catechin, proanthocyanidin, quercetin, sinapinic acid, epicatechin, oolong tea catechin, and dihydroquercetin standard products in a 10 ml volumetric flask, and dissolve them in 50% methanol-formic acid solution to prepare a mixed polyphenol standard solution with a polyphenol concentration of 1 mg / L; (2) adding the mixed polyphenol standard solution prepared in step (1) to 2 ml of blank human plasma matrix to obtain a mixed standard solution with gradient concentrations; the gradient concentrations are 500 μg / L, 250 μg / L, 150 μg / L, 100 μg / L, 50 μg / L, 20 μg / L, 10 μg / L, 5 μg / L, 2.5 μg / L, 1 μg / L, 0.5 μg / L, 0.25 μg / L, and 0.1 μg / L; (3) Add 5 ml of an aqueous solution containing 5% EDTA to the mixed standard solution of gradient concentration obtained in step (2), shake for 2 minutes, centrifuge at 4000 r / min for 3 minutes, and take the supernatant; then add anhydrous sodium sulfate twice the weight of the supernatant to the supernatant, mix well and add to a 10 ml solid phase extraction column, rinse the impurities in the column with 6 ml of hexane, elute with 6 ml of ether at a flow rate of 1 ml / min, collect the eluate, evaporate it in a 50°C water bath to nearly dryness, add a methanol-water mixed solution with a volume ratio of 8:2, let it stand for 1 minute to adjust the volume, and obtain a purified mixed standard solution of gradient concentration; (4) injecting the gradient concentration mixed standard solution purified in step (3) into a high performance liquid chromatography-tandem mass spectrometer for detection, using the sample concentration as the ordinate and the peak area as the abscissa to construct a polyphenol standard curve; The conditions of the liquid chromatography are as follows: Column: Xbridge TM Phenyl HPLC column 2.1mm×150mm×3.5μm, connected to XTerra @ Guard column; column temperature 40℃, flow rate 50μl / min, acquisition time 10min, injection volume 3μl; mobile phase A is water; mobile phase B is acetonitrile; mobile phase C is 20mmol / L ammonium formate; mobile phase D is methanol; Gradient elution program: The mass spectrometry source parameters of the tandem mass spectrometer are as follows: monitoring mode: MRM, GS1: 43 psi; GS2: 48 psi, curtain gas: 30 psi, collision gas: 7 psi, ion spray voltage: 5500 V, focusing voltage: 22 V, temperature: 233° C.; The polyphenol standard curve is: Resveratrol: y = 8039.40023x + 5.23341e5; Epigallocatechin gallate: y = 5.234524e4x + 4.69114e5; (+)-catechin: y=6.105224e5x+3.12113e4; Proanthocyanidins: y = 6.55241e4x + 2.45213e5; Quercetin: y = 4.21132e4x + 3.12241e5; Sinapic acid: y = 6.045462e4x + 2.45674e4; Epicatechin: y = 57401.88677x + 6.672e4; Oolong tea catechins: y = 4.24151e4x + 1.77652e5; Dihydroquercetin: y=57121.80057x+6.601e4; Where y is the peak area, x is the polyphenol content (μg / L); (5) collecting blood from the test subject, purifying it according to the method described in step (3), and injecting it into a high performance liquid chromatography-tandem mass spectrometer for detection, and calculating the content of resveratrol, epigallocatechin gallate, (+)-catechin, proanthocyanidin, quercetin, sinapinic acid, epicatechin, oolong tea catechin, and dihydroquercetin in the blood sample according to the polyphenol standard curve constructed in step (4); (6) Calculating the relative abundance of the first batch of intestinal microorganisms using a fitting formula based on the contents of resveratrol, epigallocatechin gallate, (+)-catechin, proanthocyanidin, quercetin, sinapinic acid, epicatechin, oolong tea catechin, and dihydroquercetin determined in step (5); The fitting formula is as follows: The fitting formula for resveratrol and Enterococcus is: y = 0.00203x + 0.000101; The fitting formula for resveratrol and Eckermansia is: y = 0.1263x + 0.01001; The fitting formula for epigallocatechin gallate and Bacteroides is: y = 0.09665x + 0.02112; The fitting formula for (+)-catechin and Eckermansia is: y = 0.00063x + 0.00016; The fitting formula for proanthocyanidins and Bacteroides is: y = 0.5603x + 0.0052; The fitting formula for quercetin and Bacillus was: y = 0.254x-0.0012; The fitting formula for quercetin and Eubacterium was: y = 0.001565x-0.0003; The fitting formula for sinapinic acid and Lactobacillus is: y = 0.0062x + 0.0001; The fitting formula for sinapinic acid and Eckermansia is: y = 0.01002x + 0.007; The fitting formula for sinapinic acid and Blautia is: y = 0.001565x + 0.0003; The fitting formula for epicatechin and Bacteroides is: y = 0.0032x + 0.0203; The fitting formula of oolong tea catechins and Bacillus is: y = 0.204x-0.0302; The fitting formula of oolong tea catechins and Eubacterium is: y = 0.094x-0.0012; The fitting formula for dihydroquercetin and Lactobacillus is: y = 0.0014x + 0.0032; Among them, y is the relative abundance of intestinal microorganisms at the genus level, and x is the polyphenol content in blood (mg / L); (7) Calculating the relative abundance of the second batch of intestinal microorganisms using a fitting formula based on the relative abundance of the first batch of intestinal microorganisms calculated in step (6); The fitting formula is as follows: The fitting formula for Bifidobacterium and Lactobacillus is: y = 0.60236x + 0.00003; The fitting formula for Lactococcus and Lactobacillus is: y = 0.82096x + 0.00001; The fitting formula for Bacteroides and Bacteroides is: y = 0.30025x + 0.00001; The fitting formula for Ruminococcus and Enterococcus is: y = 1.2334x + 0.00002; The fitting formula for Clostridium and Akkermansia is: y = 0.12244x + 0.00122; Among them, y is the relative abundance of the second batch of intestinal microorganisms at the genus level, and x is the relative abundance of the first batch of intestinal microorganisms at the genus level.

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