A method for constructing an intestinal flora of obese people in vitro and a method for screening functional components thereof

Abstract

The present application relates to the technical field of microorganism, and in particular to a method for constructing intestinal flora of obese people in vitro and a method for screening functional components thereof.The culture medium for constructing intestinal flora of obese people in vitro provided by the present application comprises the following components: 10-30 g / L of carbon source and nitrogen source, 10-20 g / L of inorganic salt, 0-1 g / L of amino acid, and 40-120 g / L of special growth factor; wherein the carbon source and nitrogen source comprise mucin and organic nitrogen source; and the special growth factor comprises polydextrose, horse blood and sheep blood. The culture medium can simulate the characteristics of the intestinal flora of obese people in a relatively short time and with relatively high accuracy, and can better ensure the accuracy and efficiency of the in-vitro screening of the functional components.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to an in vitro method for constructing gut microbiota in obese individuals and a method for screening its functional components. Background Technology

[0002] With economic development and improved living standards, obesity has become a significant factor affecting human health. To address this issue, developing foods and health products that promote weight loss and reduce body fat percentage has become a key focus for companies in related fields. Traditional methods for screening active ingredients that reduce body fat primarily rely on animal or clinical trials, which are not only time-consuming but also have a high failure rate, leading to increased product development costs.

[0003] Recent studies have found that differences in gut microbiota composition can affect the degree of obesity in humans. Among them, Firmicutes, Bacteroides, Bifidobacteria, Lactobacillus, and Akkermansia have the greatest impact on obesity. Some probiotics, prebiotics, and synbiotics have been shown to control body fat percentage and improve obesity by regulating the proportion of these microbiota. To achieve rapid screening of active ingredients for reducing body fat, an in vitro simulation system of gut microbiota in obese individuals has been developed based on research on the gut microbiota of obese people. This system enables in vitro screening of active ingredients with short cycles, high accuracy, and low cost, which is of great significance for meeting the development needs of products with body fat-reducing effects.

[0004] The human gut is an extremely complex environment. The gut microbiota contains multiple components, including microorganisms, intestinal epithelial cells, food residue, and metabolic waste, making accurate in vitro simulation extremely difficult. Based on existing research, developing static simulations targeting only the gut microbiota is relatively easier to achieve. Patent application CN113430242A discloses a method for screening plant extracts that can regulate gut microbiota based on fresh feces from healthy adults. This method uses an in vitro fecal fermentation system and microbial nucleic acid quantification technology to achieve rapid screening of plant extracts. However, this method targets the gut microbiota of healthy individuals, whose gut microbiota only involves different genera of Bifidobacteria, which is significantly different from the gut microbiota of obese individuals. Patent application CN108117991B discloses an in vitro gut microbiota simulation culture method that enables the screening and evaluation of different probiotics and prebiotics that improve the gut microbiota of constipated individuals and promote the secretion of beneficial metabolites. However, this method is for constipated and normal individuals and is not suitable for simulating the gut microbiota of obese individuals. Patent CN103740589B discloses a complete digestive dynamic simulation system from the stomach to the colon. This system can simulate not only the digestion process of food from the stomach to the intestines, but also the colonization of gut microbiota. Although the system is relatively complete, its process is complex and lengthy. Furthermore, the culture medium used is a conventional medium for normal individuals, making it unsuitable for the in vitro construction of gut microbiota in obese individuals. Currently, there are no known methods for the in vitro construction of gut microbiota and the in vitro screening of active ingredients specifically for obese individuals. Summary of the Invention

[0005] One of the objectives of this invention is to provide a method for constructing in vitro gut microbiota in obese individuals.

[0006] Another objective of this invention is to provide a method for screening the functional components of gut microbiota in obese individuals.

[0007] Due to differences in the composition and characteristics of gut microbiota, in vitro culture of gut microbiota in normal individuals or those with constipation mostly focuses on Bifidobacteria, Lactobacillus, Firmicutes, and Bacteroides. The in vitro culture methods for these bacteria are relatively well-established, and they constitute a large proportion of the gut microbiota, making in vitro culture simulation easier. However, Akkermansia, Lachnoclostridium, and Allobaculum, which are significantly associated with obesity, constitute a smaller proportion of the gut microbiota, making in vitro culture difficult with existing systems. Furthermore, in cross-culture of multiple bacterial groups, the different growth rates of each group and the potential complex interactions between them make it difficult to detect changes in the characteristic microbiota of obese individuals. In addition, constructing a gut microbiota in vitro requires not only ensuring the healthy growth of each bacterial group in vitro but also accurately mimicking their in vivo growth state. Therefore, constructing a gut microbiota in obese individuals in vitro presents significant challenges.

[0008] This invention targets the gut microbiota of obese individuals, developing a culture medium and method suitable for in vitro simulation of gut microbiota in this population. During the research and development process, this invention discovered that existing culture media used for in vitro construction of gut microbiota in normal or other populations are difficult to simulate the characteristics of gut microbiota in obese individuals. However, the optimized in vitro culture medium of this invention enables the gut microbiota of obese individuals to better simulate the in vivo state during in vitro culture, more accurately simulating the characteristics of gut microbiota in obese individuals, thereby improving the accuracy of screening for effective components.

[0009] Specifically, the present invention provides the following technical solutions:

[0010] In a first aspect, the present invention provides a culture medium for in vitro construction of gut microbiota in obese individuals, the culture medium comprising the following components: 10-30 g / L of carbon and nitrogen sources, 10-20 g / L of inorganic salts, 0.1-1 g / L of amino acids, and 40-120 g / L of specific growth factors;

[0011] The carbon and nitrogen sources include mucin and organic nitrogen sources;

[0012] The special growth factors include polydextrose, horse blood, and sheep blood.

[0013] This invention has discovered that adding a specific ratio of polydextrose, horse blood, and sheep blood to the in vitro culture medium can reasonably improve the in vitro growth rate of the gut microbiota in obese individuals, resulting in a better match between the in vitro cultured gut microbiota characteristics and those in vivo.

[0014] More preferably, the culture medium comprises the following components: 20-30 g / L of carbon and nitrogen sources, 15-20 g / L of inorganic salts, 0.5-1 g / L of amino acids, and 40-90 g / L of special growth factors.

[0015] Preferably, the mass ratio of horse blood to sheep blood in the culture medium is 1:(1-3).

[0016] The mass ratio of polydextrose to the total mass of horse blood and sheep blood is 1:(5-20). More preferably, it is 1:(5-16).

[0017] More preferably, in the culture medium, the concentration of polydextrose is 5-10 g / L, the concentration of horse blood is 10-30 g / L, and the concentration of sheep blood is 20-50 g / L.

[0018] In the above culture medium, the organic nitrogen source preferably includes yeast extract and peptone; the mass ratio of yeast extract, peptone and mucin is preferably 1:(0.5-2):(0.1-0.5).

[0019] Preferably, in the culture medium, the concentration of yeast extract is 1-3 g / L, the concentration of peptone is 1-3 g / L, and the concentration of mucin is 0.5-2 g / L.

[0020] The carbon and nitrogen sources in the culture medium also include carbohydrates, with a concentration of 10-20 g / L.

[0021] This invention discovers that, for the gut microbiota of obese individuals, using the carbon and nitrogen sources described above for in vitro culture can reasonably improve the in vitro growth rate of the gut microbiota in obese individuals, resulting in a better match between the in vitro cultured gut microbiota characteristics and those in vivo.

[0022] Preferably, the carbohydrate includes glucose.

[0023] In the above culture medium, the inorganic salts include dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and sodium bicarbonate in a mass ratio of 1:(1.5-4):(0.3-1.5).

[0024] Preferably, in the culture medium, the concentration of dipotassium hydrogen phosphate is 3-4 g / L, the concentration of potassium dihydrogen phosphate is 10-12 g / L, and the concentration of sodium bicarbonate is 1-3 g / L.

[0025] The amino acids in the above culture medium include cysteine.

[0026] Preferably, the culture medium further includes an emulsifier. The concentration of the emulsifier in the culture medium is preferably 1-3 mL / L.

[0027] More preferably, the emulsifier is Tween 80.

[0028] In a preferred embodiment of the present invention, the culture medium comprises the following components: dipotassium hydrogen phosphate 3-4 g / L, potassium dihydrogen phosphate 10-12 g / L, sodium bicarbonate 1-3 g / L, yeast extract 1-3 g / L, peptone 1-3 g / L, mucin 0.5-2 g / L, glucose 10-20 g / L, cysteine ​​0.3-1 g / L, polydextrose 5-10 g / L, horse blood 10-50 g / L, sheep blood 20-60 g / L, and Tween 80 1-3 mL / L.

[0029] In a preferred embodiment of the present invention, the culture medium comprises the following components: dipotassium hydrogen phosphate 3-4 g / L, potassium dihydrogen phosphate 10-12 g / L, sodium bicarbonate 2-3 g / L, yeast extract 2-3 g / L, peptone 2-3 g / L, mucin 0.5-1 g / L, glucose 15-20 g / L, cysteine ​​0.5-1 g / L, polydextrose 5-8 g / L, horse blood 10-30 g / L, sheep blood 20-50 g / L, and Tween 80 1-3 mL / L.

[0030] In this invention, the obese population refers to individuals with a BMI ≥ 28.

[0031] Secondly, the present invention provides the application of the culture medium described above in the in vitro construction of gut microbiota in obese individuals.

[0032] Thirdly, the present invention provides a method for constructing in vitro gut microbiota in obese individuals, the method comprising: pre-treating fecal samples from obese individuals and inoculating them into the culture medium described above for in vitro construction of gut microbiota in obese individuals for anaerobic culture.

[0033] Preferably, the anaerobic culture is carried out at 36-38℃ and 60-100 rpm.

[0034] The preferred anaerobic culture time is 5-10 hours, more preferably 5-7 hours.

[0035] The culture medium provided by this invention can enable the in vitro construction of gut microbiota in obese individuals in a short time, shortening the culture time and improving the construction efficiency.

[0036] The preferred method for pretreatment of fecal samples is as follows: after diluting the fecal sample with a diluent, homogenize and separate the solid and liquid components, and collect the liquid portion for inoculation.

[0037] Preferably, the diluent comprises the following components: 8-9 g / L dipotassium hydrogen phosphate, 6-7 g / L potassium dihydrogen phosphate, 0.08-0.12 g / L sodium mercaptoacetate, and 0.01-0.02 g / L sodium bisulfite.

[0038] The present invention has found that the above-mentioned diluent can effectively maintain the activity of gut microbiota in obese individuals, and allows them to adapt to the in vitro culture system more quickly after inoculation.

[0039] The preferred dilution ratio for the above-mentioned fecal samples is 5-15 times.

[0040] The preferred inoculation volume of the supernatant is 0.5-1 mL supernatant / 8 mL culture medium.

[0041] The above solid-liquid separation can be achieved by centrifugation to remove precipitates and recover the supernatant.

[0042] Fourthly, the present invention provides a primer set for detecting gut microbiota characteristics in obese individuals, the primer set comprising the primers shown in SEQ ID NO.1-16.

[0043] The primers mentioned above were designed to target the characteristic sequences of Bacteroidetes, Firmicutes, Bifidobacterium, Lactobacillus, Akkermansia, Clostridium, Allobaculum, and Lachnoclostridium in the gut microbiota of obese individuals, and can specifically and sensitively detect these microorganisms.

[0044] Fifthly, the present invention provides the application of the primer set described above in detecting gut microbiota characteristics in obese individuals.

[0045] In a sixth aspect, the present invention provides a method for screening the functional components of gut microbiota in obese individuals, the method comprising: mixing pretreated fecal samples from obese individuals, the culture medium used for in vitro construction of gut microbiota in obese individuals, and the components to be screened, and then performing anaerobic culture at 36-38°C and 60-100 rpm to obtain a culture medium, and extracting the total nucleic acid of the microbiota in the culture medium;

[0046] Using the total nucleic acid as a template, the phyla Bacteroidetes, Firmicutes, Lactobacillus, Bifidobacterium, Akkermansia, Clostridium tenuifolium, Allobaculum, and Lachnoclostridium were quantified.

[0047] The above-mentioned quantification of gut microbiota can be achieved by quantitative PCR using the primer set described above for detecting gut microbiota characteristics in obese individuals.

[0048] Alternatively, bacterial community quantification can be achieved through genome sequencing analysis. Specifically, Illumina MiSeq was used to obtain the proportional relationships and changes among bacterial communities via 16S rDNA sequencing.

[0049] Based on the above-mentioned detection of the contents of Bacteroidetes, Firmicutes, Lactobacillus, Bifidobacterium, Akermania, Clostridium nobile, Allobaculum, and Lachnoclostridium, the changes in the proportion of each microorganism in the gut microbiota compared with the group without the added component were analyzed, thereby evaluating the effect of the component to be screened on the gut microbiota of obese individuals.

[0050] Preferably, if, compared with the addition of the component to be screened, the gut microbiota undergoes at least one of the following changes: 1) a decrease in the Firmicutes / Bacteroidetes ratio, 2) an increase in the Akkermansia ratio, 3) an increase in the Lactobacillus ratio, 4) an increase in the Bifidobacterium ratio, 5) a decrease in the Allobaculum ratio, 6) a decrease in the Lachnoclostridium ratio, and (7) an increase in the Clostridium truncatum ratio, then the component to be screened is judged to have the activity of improving the gut microbiota of obese people.

[0051] Preferably, the PCR reaction program includes: 95℃ for 10 min, annealing at 60℃ for 30 s, amplification at 72℃ for 30 s, 40 cycles, thermal denaturation at 95℃ for 15 s, and melting curve temperature at 95℃.

[0052] The above screening methods may also include detecting the content of short-chain fatty acids such as formic acid, propionic acid, and acetic acid in the in vitro culture medium of intestinal flora, and screening for active ingredients by comparing the content and changes of specific short-chain fatty acids.

[0053] The beneficial effects of this invention are as follows: This invention focuses on the gut microbiota of obese individuals and develops an in vitro construction method to simulate the microbiota composition of obese individuals in vitro. The in vitro constructed culture medium of this invention can accurately simulate the microbiota characteristics of obese individuals within a shorter culture time, providing a foundation for the in vitro screening of active ingredients. This better ensures the accuracy of in vitro screening of active ingredients and achieves rapid and efficient screening of active ingredients. Attached Figure Description

[0054] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0055] Figure 1 This is the gut microbiota of an obese adult male population simulated by in vitro fermentation in Experimental Example 1 of the present invention.

[0056] Figure 2 This is the gut microbiota of an obese adult female population simulated by in vitro fermentation in Experimental Example 2 of the present invention.

[0057] Figure 3 In Experiment 3 of this invention, the in vitro fermentation was performed using the culture media of Example 1 and Comparative Examples 1-8 to simulate the gut microbiota of obese individuals. The obese control represents the results of direct detection of the gut microbiota of obese individuals without culture. Inulin represents the culture medium used in Comparative Example 1, polydextrose represents the culture medium used in Example 1, resistant dextrin represents the culture medium used in Comparative Example 2, apple fiber represents the culture medium used in Comparative Example 3, horse blood represents the culture medium used in Comparative Example 5, sheep blood represents the culture medium used in Comparative Example 4, fetal bovine serum represents the culture medium used in Comparative Example 6, pig liver extract represents the culture medium used in Comparative Example 7, and chicken heart extract represents the culture medium used in Comparative Example 8.

[0058] Figure 4 This study investigates the effects of different proportions of horse blood and sheep blood (Examples 2, 3, and Comparative Example 9) on the gut microbiota simulation effect in obese individuals in Experiment 3 of this invention.

[0059] Figure 5 This is the effect of xylooligosaccharides and galactooligosaccharides on the gut microbiota of obese individuals in Experimental Example 4 of the present invention.

[0060] Figure 6 This is the result of using an animal model in Experiment Example 4 of the present invention to verify the prebiotic and probiotic combination obtained by the screening method of the present invention. Detailed Implementation

[0061] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0062] Example 1: Culture medium for in vitro construction of gut microbiota in obese individuals (1)

[0063] This embodiment provides a culture medium for in vitro construction of gut microbiota in obese individuals, the composition of which is as follows:

[0064] Dipotassium hydrogen phosphate 3.5 g / L, potassium dihydrogen phosphate 10.9 g / L, sodium bicarbonate 2 g / L, yeast extract 2 g / L, peptone 2 g / L, mucin 1 g / L, cysteine ​​0.5 g / L, Tween 80 2 mL / L, glucose 15 g / L, polydextrose 5 g / L, horse blood 30 g / L, sheep blood 50 g / L.

[0065] The mucin was extracted from the small intestine of pigs. It was first prepared into a solution with a concentration of 20 mg / mL using 1M NaOH, and then added to the culture medium according to the dosage.

[0066] Example 2: Culture medium for in vitro construction of gut microbiota in obese individuals (2)

[0067] This embodiment provides a culture medium for in vitro construction of gut microbiota in obese individuals, the composition of which is as follows:

[0068] Dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 11.5g / L, sodium bicarbonate 3g / L, yeast extract 3g / L, peptone 1.5g / L, mucin 0.5g / L, cysteine ​​1g / L, Tween 80 1mL / L, glucose 20g / L, polydextrose 8g / L, horse blood 20g / L, sheep blood 20g / L.

[0069] The mucin was extracted from the small intestine of pigs. It was first prepared into a solution with a concentration of 20 mg / mL using 1M NaOH, and then added to the culture medium according to the dosage.

[0070] Example 3: Culture medium for in vitro construction of gut microbiota in obese individuals (3)

[0071] This embodiment provides a culture medium for in vitro construction of gut microbiota in obese individuals, the composition of which is as follows:

[0072] Dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 11.5g / L, sodium bicarbonate 3g / L, yeast extract 1g / L, peptone 2g / L, mucin 0.5g / L, cysteine ​​1g / L, Tween 80 1mL / L, glucose 20g / L, polydextrose 8g / L, horse blood 10g / L, sheep blood 30g / L.

[0073] The mucin was extracted from the small intestine of pigs. It was first prepared into a solution with a concentration of 20 mg / mL using 1M NaOH, and then added to the culture medium according to the dosage.

[0074] Comparative Example 1

[0075] This comparative example provides a culture medium for the in vitro construction of human intestinal flora, the only difference from Example 1 being that polydextrose is replaced with inulin.

[0076] Comparative Example 2

[0077] This comparative example provides a culture medium for the in vitro construction of human intestinal flora, the only difference from that in Example 1 being the replacement of polydextrose with resistant dextrin.

[0078] Comparative Example 3

[0079] This comparative example provides a culture medium for the in vitro construction of human intestinal flora, the only difference from Example 1 being that polydextrose is replaced with apple fiber.

[0080] Comparative Example 4

[0081] This comparative example provides a culture medium for the in vitro construction of human intestinal flora, the only difference from that in Example 1 being that horse blood is replaced with sheep blood.

[0082] Comparative Example 5

[0083] This comparative example provides a culture medium for the in vitro construction of human intestinal flora, the only difference from that in Example 1 being that sheep blood is replaced with horse blood.

[0084] Comparative Example 6

[0085] This comparative example provides a culture medium for the in vitro construction of human intestinal flora, the only difference from that in Example 1 being that horse blood is replaced with fetal bovine serum.

[0086] Comparative Example 7

[0087] This comparative example provides a culture medium for the in vitro construction of human intestinal flora, the only difference from that in Example 1 being that horse blood is replaced with pig liver extract.

[0088] Comparative Example 8

[0089] This comparative example provides a culture medium for the in vitro construction of human intestinal flora, the only difference from that in Example 1 being that horse blood is replaced with chicken heart extract.

[0090] Comparative Example 9

[0091] This comparative example provides a culture medium for the in vitro construction of human intestinal flora, the only difference from Example 2 being: 30 g / L horse blood and 10 g / L sheep blood.

[0092] Example 4: In vitro construction method of gut microbiota in obese individuals

[0093] 1. Preparation of intestinal flora simulation solution for obese individuals

[0094] Fresh fecal samples were collected from 3–10 obese Chinese adults with a BMI ≥ 28. 10g of fecal matter was collected from each subject, mixed thoroughly, and then diluted 10-fold with a sterile diluent (diluent formulation: 8.8g / L dipotassium hydrogen phosphate, 6.8g / L potassium dihydrogen phosphate, 0.1g / L sodium thioglycolate, 0.015g / L sodium bisulfite). The mixture was homogenized for 10 minutes. After homogenization, the sample was centrifuged at 500g for 2 minutes to remove larger precipitates. The supernatant was used as the gut microbiota simulator for obese individuals, and it needed to be freshly prepared for each experiment.

[0095] 2. Simulated fermentation

[0096] Take 8 mL of the culture medium used for in vitro construction of gut microbiota in obese individuals from Example 1 or 2, add 0.5–1 mL of microbiota simulation solution, mix well, and anaerobically culture at 37°C on a shaker for 6 hours at 90 rpm. After fermentation, take 1 mL of culture medium, centrifuge at 12000 rpm for 5 min to extract bacterial cells, extract total nucleic acid samples of the microbiota using a nucleic acid extraction kit, and store at -4°C.

[0097] 3. Quantitative analysis of characteristic microbiota in obese individuals

[0098] Standard sequence plasmids for the target bacterial populations were constructed according to the characteristic sequences shown in Table 1. Simultaneously, PCR primers for the corresponding bacterial populations were synthesized, and standard curves for each plasmid versus nucleic acid concentration were constructed using quantitative PCR, serving as the basis for quantification. Using the total nucleic acid sample extracted in step 2 as a template, the proportions of Bacteroidetes, Firmicutes, Lactobacillus, Bifidobacterium, Akkermansia (AKK), Clostridium tenuifolium, Allobaculum, and Lachnoclostridium were determined by absolute quantitative PCR using the primers shown in Table 1. PCR amplification conditions were as follows: 95℃ for 10 min, annealing at 60℃ for 30 s, amplification at 72℃ for 30 s, 40 cycles, heat denaturation at 95℃ for 15 s, and melting curve temperature at 95℃. The PCR amplification reaction system was as follows: a 20 μL system containing 0.5 μL upstream primer, 0.5 μL downstream primer, 1 μL template DNA, 8 μL ultrapure water, and 10 μL LSYBR Green I (Takara, Japan).

[0099] Table 1. Characteristic sequences and primer sequences of the target bacterial community

[0100]

[0101]

[0102] Example 5: Screening method for functional components of gut microbiota in obese individuals

[0103] 1. Preparation of intestinal flora simulation solution for obese individuals

[0104] Fresh fecal samples were collected from 3–10 obese Chinese adults with a BMI ≥ 28. 10g of fecal matter was collected from each subject, mixed thoroughly, and then diluted 10-fold with a sterile diluent (diluent formulation: 8.8g / L dipotassium hydrogen phosphate, 6.8g / L potassium dihydrogen phosphate, 0.1g / L sodium thioglycolate, 0.015g / L sodium bisulfite). The mixture was homogenized for 10 minutes. After homogenization, the sample was centrifuged at 500g for 2 minutes to remove larger precipitates. The supernatant was used as the gut microbiota simulator for obese individuals, and it needed to be freshly prepared for each experiment.

[0105] 2. Simulated fermentation

[0106] Take 8 mL of the culture medium used for in vitro construction of gut microbiota in obese individuals from Example 1 or 2, add 0.5–1 mL of microbiota mimicry solution, add the active ingredient to be tested, mix well, and anaerobically culture at 37°C on a shaker for 6 hours at 90 rpm. After fermentation, take 1 mL of culture medium, centrifuge at 12000 rpm for 5 min to extract bacterial cells, extract total nucleic acid samples of the microbiota using a nucleic acid extraction kit, and store at -4°C.

[0107] 3. Quantitative analysis of characteristic microbiota in obese individuals

[0108] Standard sequence plasmids for the target bacterial groups were constructed according to the characteristic sequences shown in Table 1. Simultaneously, PCR primers for the corresponding bacterial groups were synthesized, and standard curves for each plasmid and nucleic acid concentration were constructed using quantitative PCR to serve as the basis for quantification. Using the total nucleic acid sample extracted in step 2 as a template, the proportions of Bacteroidetes, Firmicutes, Lactobacillus, Bifidobacterium, Akkermansia (AKK), Clostridium tenuifolium, Allobaculum, and Lachnoclostridium were determined by absolute quantitative PCR using the primers shown in Table 1. PCR amplification conditions were as follows: 95℃ for 10 min, annealing at 60℃ for 30 s, amplification at 72℃ for 30 s, 40 cycles, heat denaturation at 95℃ for 15 s, and melting curve temperature at 95℃. The PCR amplification reaction system was as follows: a 20 μL system containing 1 μL upstream primer, 1 μL downstream primer, 1 μL template DNA, 7 μL ultrapure water, and 10 μL SYBR Green I (Takarra, Japan).

[0109] 4. Result Determination

[0110] The results showing that the gut microbiota of obese individuals is improved are manifested by a decrease in the proportion of Firmicutes / Bacteroidetes, an increase in the proportion of Akkermansia, an increase in the proportion of Lactobacillus, an increase in the proportion of Bifidobacterium, a decrease in the proportion of Allobaculum, a decrease in the proportion of Lachnoclostridium, and an increase in the proportion of Clostridium tenuifolium. The presence of one or more of these results indicates that the tested active ingredient has the activity of improving the gut microbiota of obese patients.

[0111] Example 1: In vitro construction of gut microbiota in obese adult men

[0112] The gut microbiota of obese adult men was constructed using the method provided in Example 4 (using the culture medium from Example 1 as the in vitro construction medium), as detailed below:

[0113] Fresh stool samples were collected from three obese adult Chinese men with BMIs of 33.7, 34.8, and 29.2 (obese group) and three normal-weight Chinese men with BMIs of 23.5, 23.8, and 22.1 (normal group). 10g of stool was collected from each subject. The stool samples were mixed thoroughly according to group, then diluted 10-fold with a sterile diluent (diluent formulation: 8.8g / L dipotassium hydrogen phosphate, 6.8g / L potassium dihydrogen phosphate, 0.1g / L sodium thioglycolate, 0.015g / L sodium bisulfite). The mixture was homogenized for 10 minutes, followed by centrifugation at 500g for 2 minutes to remove larger precipitates. The supernatant was used as the intestinal flora simulation solution, and it had to be freshly prepared for each experiment.

[0114] Take 8 mL of the culture medium from Example 1 for in vitro construction of gut microbiota in obese individuals, add 1 mL of microbiota simulation solution to each group, mix well, and culture anaerobically in a shaker at 37°C for 6 hours at 90 rpm. After fermentation, take 1 mL of culture medium, centrifuge at 12000 rpm for 5 min to extract bacterial cells, and extract total nucleic acid samples of the microbiota using a nucleic acid extraction kit, and store at -4°C.

[0115] Using the primers shown in Table 1, the proportions of Bacteroidetes, Firmicutes, Lactobacillus, Bifidobacterium, Akkermansia, Clostridium tenuifolium, Allobaculum, and Lachnoclostridium were determined by absolute quantitative PCR. The PCR amplification conditions were as follows: 95℃ for 10 min, annealing at 60℃ for 30 s, amplification at 72℃ for 30 s, 40 cycles, heat denaturation at 95℃ for 15 s, and melting curve temperature at 95℃. The PCR amplification reaction system was the same as that shown in Example 3.

[0116] Quantitative calculations are performed according to the formulas in Table 2, where x is the CQ value and y is the Log value of the total bacterial count, in CFU.

[0117] Table 2 Calculation Formulas for Quantitative PCR

[0118] Firmicutes y = -3.142x + 10.225 0.998 Bacteroidetes y = -3.889x + 31.15 0.999 Bifidobacterium y = -3.106x + 39.87 0.999 Lactobacillus y = -3.466x + 38.63 0.999 Akkermania y = -3.561x + 39.04 0.999 Clostridium perfringens Y = -6.388x + 12.631 0.999 Allobaculum Y = -5.871x + 43.522 0.999 Lachnoclostridium Y = -8.314x + 56.717 0.999

[0119] The gut microbiota composition was calculated using the formulas in Table 2. Differences in gut microbiota composition between normal individuals and obese men in in vitro culture systems were compared, and the results are as follows: Figure 1 As shown, obese individuals have a higher proportion of Firmicutes / Bacteroidetes, fewer Bifidobacteria and Lactobacillus, and fewer Akkermansia, while the proportion of Clostridium truncatum is reduced and the proportions of Allobaculum and Lachnoclostridium are increased. This is consistent with the currently reported characteristics of the gut microbiota composition in obese individuals, indicating that the in vitro constructed gut microbiota culture model has a similar simulation effect to the proportion of characteristic gut microbiota in obese individuals and can reflect the characteristics of the gut microbiota in obese individuals.

[0120] Experimental Example 2: In vitro construction of gut microbiota in obese adult women

[0121] The gut microbiota of obese adult women was constructed using the method provided in Example 4 (using the culture medium in Example 1 as the in vitro construction medium), as detailed below:

[0122] Fresh stool samples were collected from three obese adult women with BMIs of 28.5, 29.1, and 33.5, and from three women of normal weight with BMIs of 21.5, 22.6, and 23.1. 10g of stool was collected from each subject. The stool samples were mixed thoroughly according to their respective groups and then diluted 10-fold with a sterile diluent (the diluent consisted of 8.8g / L dipotassium hydrogen phosphate, 6.8g / L potassium dihydrogen phosphate, 0.1g / L sodium thioglycolate, and 0.015g / L sodium bisulfite). The mixture was homogenized for 10 minutes and then centrifuged at 500g for 2 minutes to remove larger precipitates. The supernatant was used as the intestinal flora simulation solution, and it had to be freshly prepared for each experiment.

[0123] Take 8 mL of the culture medium used in Example 2 for in vitro construction of gut microbiota in obese individuals, add 1 mL of microbiota simulation solution, mix well, and culture anaerobically in a shaker at 37°C for 6 hours at 90 rpm. After fermentation, take 1 mL of culture medium, centrifuge at 12000 rpm for 5 min to extract bacterial cells, and use a nucleic acid extraction kit to extract total nucleic acid samples of the microbiota. Store at -4°C.

[0124] Using the primers in Table 1, the proportions of Bacteroidetes, Firmicutes, Lactobacillus, Bifidobacterium, AKK bacteria, Clostridium tenuifolium, Allobaculum, and Lachnoclostridium were determined by absolute quantitative PCR. PCR amplification conditions: 95℃ for 10 min, annealing at 60℃ for 30 s, amplification at 72℃ for 30 s, 40 cycles, heat denaturation at 95℃ for 15 s, and melting curve temperature at 95℃. The PCR amplification reaction system was the same as shown in Example 3.

[0125] The formula in Table 3 is used to calculate the quantitative bacterial community.

[0126] Table 3 Calculation Formulas for Quantitative PCR

[0127] Firmicutes y = -3.156x + 31.64 0.999 Bacteroidetes y = -3.452x + 27.64 0.999 Bifidobacterium y = -3.286x + 33.18 0.999 Lactobacillus y = -3.599x + 40.31 0.999 Akkermania y = -3.531x + 38.24 0.999 Clostridium perfringens Y = -3.911x + 41.16 0.999 Allobaculum y = -5.35x + 65.22 0.999 Lachnoclostridium y = -6.71x + 48.39 0.999

[0128] The bacterial community content in different groups was calculated according to the formula in Table 3, and the results are as follows: Figure 2As shown, the results indicate that, compared with women of normal weight, obese individuals have a significantly higher proportion of Firmicutes / Bacteroidetes in their gut microbiota, while the proportions of Bifidobacterium, Lactobacillus, Akkermansia, and Clostridium nobile are significantly lower. At the same time, the proportions of Allobaculum and Lachnoclostridium are increased. These results are consistent with the gut microbiota characteristics of obese individuals in the literature.

[0129] Example 3: In vitro construction of gut microbiota in obese individuals

[0130] The gut microbiota of obese individuals were constructed using the culture media of Examples 1-3 and Comparative Examples 1-9, respectively. Except for the different culture media used, the other methods were the same as those in Example 4, and the details of the fecal samples were the same as those in Experiment 1.

[0131] The results are as follows Figure 3 As shown, the results indicate that, compared to the comparative culture medium, the culture media of Examples 1-3 promoted the growth of *Allobaculum*, *AKK*, *Lachnoclostridium*, and *Clostridium tenuifolium* more effectively without affecting the proportions of other bacterial groups. Replacing polydextrose with inulin and apple fiber increased the proportion of *Lactobacillus*, which is inconsistent with the characteristics of obese bacterial communities. Replacing polydextrose with resistant dextrin, inulin, and apple fiber resulted in weakened *Allobaculum* proliferation. Compared to other components, the combination of horse blood and sheep blood had the best effect on promoting the proliferation of *Lachnoclostridium*. Replacing horse blood with fetal bovine serum, porcine liver extract, and chicken heart extract significantly reduced the proliferation capacity of *Lachnoclostridium*. Replacing horse blood with fetal bovine serum and porcine liver extract significantly reduced the proportion of *AKK*, which is inconsistent with the characteristics of obese bacterial communities. Moreover, the effect of using horse blood or sheep blood alone in simulating bacterial communities was still inferior to the synergistic effect of the two.

[0132] The effects of different ratios of horse blood and sheep blood on gut microbiota culture are as follows: Figure 4 As shown, the gut microbiota of obese individuals without culture was used as the control group. The culture of gut microbiota in obese individuals after mixing horse blood and sheep blood at mass ratios of 3:1 (Comparative Example 9), 1:1 (Example 2), and 1:3 (Example 3) was compared. Among the gut microbiota cultured after mixing horse blood and sheep blood at ratios of 1:1 and 1:3, the proliferation-promoting effect of Lachnoclostridium was better than that of horse blood or sheep blood alone. The differences of other microbiota were not significant. The above results indicate that mixing horse blood and sheep blood at ratios of 1:1 to 1:3 can have a significant synergistic effect.

[0133] In conclusion, adding polydextrose and horse blood and sheep blood in a mass ratio of 1:1 to 1:3 to the culture medium helps to construct obese gut microbiota, which can better simulate the gut microbiota characteristics of obese people.

[0134] Experiment Example 4: Screening of functional components of gut microbiota in obese individuals

[0135] Using the method described in Example 4, in vitro screening of the active ingredients in the gut microbiota of obese individuals was conducted. Two prebiotics, xylooligosaccharides and galacooligosaccharides, were identified as capable of improving the gut microbiota in obese individuals. The screening results are as follows: Figure 5 As shown, the composition of Bifidobacterium lactis MN-Gup (disclosed in patent application CN111826299A, deposited on April 10, 2018 at the China General Microbiological Culture Collection Center, accession number CGMCCNo.15578, classified as Bifidobacterium animalis), with xylooligosaccharide and galactooligosaccharide respectively, can significantly increase the proportion of Bifidobacterium, Lactobacillus, Allobaculum, and Clostridium truncatum, but has no significant effect on the proportion of Firmicutes and Bacteroidetes, Akkermansia and Lachnoclostridium.

[0136] To further validate the functions of xylooligosaccharides (XOS) and galactooligosaccharides (GOS) using a rat obesity model, 25 six-week-old male SD rats were purchased and housed in an SPF-grade animal facility, fed standard diets and sterile water. After one week of acclimatization, they were randomly divided into five groups of five rats each. The control group was fed a normal diet, while the other groups underwent a nine-week high-fat diet to establish the obesity model. After nine weeks, three intervention groups received sample interventions: yogurt containing 50 billion CFU / 100g MN-Gup, synbiotic yogurt containing 50 billion CFU / 100g MN-Gup + 1.5% XOS, and synbiotic yogurt containing 50 billion CFU / 100g MN-Gup + 1.5% GOS. The intervention lasted for eight weeks, while the control group received the same volume of saline. The rat weight and food intake were recorded weekly. At the end of the intervention, the total weight and the weight of epididymal and perirenal fat were measured. Results are as follows: Figure 6 As shown, the combination of xylooligosaccharides, galactooligosaccharides and probiotics is significantly better than the effects of probiotics alone in improving body fat percentage, reducing epididymal fat and perirenal fat. This indicates that the in vitro screening method constructed in this invention can guide the screening of probiotics and prebiotics that have the effect of improving gut microbiota and reducing body fat in obese individuals.

[0137] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture medium for in vitro construction of gut microbiota in obese individuals, characterized in that, The culture medium consists of the following components: dipotassium hydrogen phosphate 3-4 g / L, potassium dihydrogen phosphate 10-12 g / L, sodium bicarbonate 1-3 g / L, yeast extract 1-3 g / L, peptone 1-3 g / L, mucin 0.5-2 g / L, glucose 10-20 g / L, cysteine ​​0.3-1 g / L, polydextrose 5-10 g / L, horse blood 10-50 g / L, sheep blood 20-60 g / L, and Tween 80 1-3 mL / L; In the culture medium, the mass ratio of horse blood to sheep blood is 1:(1-3). The ratio of polydextrose to the total mass of horse blood and sheep blood is 1:(5-20).

2. A method for constructing in vitro gut microbiota in obese individuals, characterized in that, The method includes: pre-treating fecal samples from obese individuals and then inoculating them into the culture medium for in vitro construction of intestinal flora in obese individuals as described in claim 1 for anaerobic culture.

3. The method according to claim 2, characterized in that, The anaerobic culture was carried out at 36-38℃ and 60-100 rpm.

4. The method according to claim 2 or 3, characterized in that, The pretreatment includes: diluting the fecal sample with a diluent, homogenizing and separating the solid and liquid components, and collecting the liquid portion for inoculation.

5. The method according to claim 4, characterized in that, The diluent comprises the following components: 8-9 g / L dipotassium hydrogen phosphate, 6-7 g / L potassium dihydrogen phosphate, 0.08-0.12 g / L sodium mercaptoacetate, and 0.01-0.02 g / L sodium bisulfite.

6. A method for screening the functional components of gut microbiota in obese individuals, characterized in that, The method includes: mixing pretreated fecal samples from obese individuals, the culture medium for in vitro construction of gut microbiota in obese individuals as described in claim 1, and the components to be screened, and then anaerobically culturing them at 36-38℃ and 60-100 rpm to obtain a culture medium, and extracting the total nucleic acid of the microbiota in the culture medium; Using the total nucleic acid as a template, the phyla Bacteroidetes, Firmicutes, Lactobacillus, Bifidobacterium, Akkermansia, Clostridium tenuifolium, Allobaculum, and Lachnoclostridium were quantified.

7. The method according to claim 6, characterized in that, The quantification was achieved by quantitative PCR using a primer set for detecting gut microbiota characteristics in obese individuals. The primer set contains the primers shown in SEQ ID NO.1-16.

8. The method according to claim 7, characterized in that, The PCR reaction procedure includes: 95℃ for 10 min, annealing at 60℃ for 30 s, amplification at 72℃ for 30 s, 40 cycles, heat denaturation at 95℃ for 15 s, and melting curve temperature at 95℃.

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