Method for evaluating capacity of metabolic cereal non-starch polysaccharide of intestinal flora of diabetic patient

Abstract

The invention discloses a method for evaluating the ability of intestinal flora of a diabetic to metabolize cereal non-starch polysaccharide. The method comprises the following steps: firstly, establishing an in-vitro static digestion model, then collecting excrement of a type 2 diabetes patient, inoculating excrement flora to establish an in-vitro fermentation model, detecting the content of arabinose and xylose residues in fermentation liquor, and performing metagenome sequencing; statistical analysis is carried out, and the arabinose and xylose fermentation capacity of different individual excrement flora and the relation between the arabinose and xylose fermentation capacity and intestinal flora are determined; and finally, giving an individualized diet intervention scheme according to the capacity of fermenting non-starch polysaccharide in the excrement of the diabetic patient and the intestinal flora composition. According to the invention, the consumption amount of the in-vitro fermentation arabinose is taken as a specific index, and the clinical index, diet condition and intestinal flora composition of a subject are combined, so that precise nutrition intervention is carried out on a diabetic patient, glycolipid metabolism and intestinal flora disorder of the patient are improved, and the application value of the index is further verified.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and health management technology, specifically involving a method for establishing an assessment of the ability of gut microbiota from different fecal donors to metabolize non-starch polysaccharides using in vitro fermentation, and applying it to the selection of cereal staple foods for diabetic patients. Background Technology

[0002] Cereal foods, including rice and its products, wheat and its products, corn and its products, and all other grain products, are the primary food source for Chinese residents, largely reflecting the population's carbohydrate intake level and typically serving as a major source of energy and dietary fiber. Studies have confirmed that cereal foods play a crucial role in the prevention and dietary management of type 2 diabetes mellitus (T2DM). Consuming whole grains is an effective way to increase dietary fiber intake and reduce the risk of chronic non-communicable diseases, including type 2 diabetes.

[0003] The content and types of dietary fiber vary greatly among different types of grains. Most of the carbohydrates in refined grains are digestible, breaking down into monosaccharides (mainly glucose) in the small intestine before being absorbed and used for energy. Whole grains refer to grains that have not undergone refined processing or have only undergone simple physical treatment, retaining all the components of the whole grain. They are rich in nutrients such as dietary fiber, polyphenols, phytic acid, and vitamins. Non-starch polysaccharides are the main component of dietary fiber. They cannot be digested or are not completely digested in digestive fluids such as saliva, gastric juice, and intestinal juice. In the colon, they become substrates for intestinal flora and are selectively broken down and fermented. Arabicaxylan is the most abundant non-starch polysaccharide in most grains. As a functional polysaccharide, it promotes the growth of beneficial bacteria such as Bifidobacteria and Lactobacillus, and has various physiological functions such as promoting bowel movements, lowering blood sugar, and lowering blood lipids. It plays an important role in preventing chronic diseases and protecting human health.

[0004] The gut microbiota is hailed as a highly specific "living organ of the human body," with its composition, activity, and metabolites directly related to human health. Gut microbiota dysbiosis is a characteristic feature of type 2 diabetes, and targeting gut microbiota with cereal foods may be an effective strategy for improving type 2 diabetes. In vitro fermentation of gut microbiota is currently a major research focus. In vitro fermentation can simulate the gut environment, assess the impact of food components on gut microbiota, and provide a basis for the evaluation and development of health foods. Studies have found that gut microbiota from different fecal donors have varying abilities to process indigestible carbohydrates. Currently, the evaluation indicators for in vitro fermentation mainly focus on the diversity and compositional changes of gut microbiota and the content of short-chain fatty acids in its metabolites. There is a lack of specific indicators for evaluating the ability of gut microbiota from different fecal donors to metabolize non-starch polysaccharides, and methods for using in vitro evaluation indicators to guide staple food choices for diabetic patients. Summary of the Invention

[0005] The purpose of this invention is to provide a method for evaluating the ability of gut microbiota in diabetic patients to metabolize non-starch polysaccharides from cereals.

[0006] A method for assessing the ability of gut microbiota in diabetic patients to metabolize cereal non-starch polysaccharides comprises the following steps:

[0007] 1) Establish an in vitro static digestion model to simulate the digestion of grains in the oral cavity, stomach and small intestine. Dialyze and freeze-dry the final digestion products and mix them with the culture medium as fermentation substrate.

[0008] 2) Collect stool samples from patients with type 2 diabetes;

[0009] 3) Establish an in vitro fermentation model by inoculating fecal microbiota from diabetic patients, detect the content of arabinose and xylose residues in the fermentation broth, and perform metagenomic sequencing;

[0010] 4) Conduct statistical analysis to determine the ability of different individuals' fecal microbiota to ferment arabinose and xylose and their relationship with the composition of the gut microbiota;

[0011] 5) Based on the ability of diabetic patients to ferment non-starch polysaccharides in their feces and the composition of their gut microbiota, provide individualized dietary intervention plans.

[0012] Step 1) describes an in vitro static digestion method that uses a simple bag-shaped device to simulate oral chewing, including crushing and cutting. 10g of the sample is weighed and placed in a 250ml conical flask, and 10ml of oral simulated digestion solution is added. The mixture is then vortexed for 2 minutes to ensure homogeneity. 20ml of gastric simulated digestion solution is added, and the pH is adjusted to 3.0. Digestion is then carried out in a constant-temperature shaking incubator at 37℃ and 120r / min. After 2 hours of gastric digestion, intestinal digestion begins. 40ml of intestinal simulated digestion solution is added, and the pH is adjusted to 7.0. Digestion continues for 2 hours in a constant-temperature shaking incubator at 37℃ and 120r / min. At the end of intestinal digestion, a heat shock treatment at 100℃ for 5 minutes is performed to inhibit digestive enzyme activity. The sample is then dialyzed at 4℃ for 72 hours and then freeze-dried.

[0013] The oral cavity simulates digestion at pH 7.0 and contains 75 U / ml of α-amylase.

[0014] The simulated gastric digestive fluid has a pH of 3.0 and contains 2000 U / mL of pepsin.

[0015] The intestinal simulated digestive fluid has a pH of 7.0 and contains 100 U / mL of pancreatic enzymes.

[0016] Step 2) Collect the feces using the provided clean, dry paper container. Transfer 1.5-2.5g of fresh feces to a sterile tube using a sterile spoon and place it in an ice box. Transport the tube to the laboratory within 4 hours.

[0017] The fermentation broth formulation is as follows: 10 g / L tryptone, 2.5 g / L yeast extract, 1 g / L L-cysteine, 0.01 g / L heme, 0.9 g / L NaCl, 0.09 g / L CaCl2, 0.45 g / L KH2PO4, 0.09 g / L MgSO4·7H2O, 0.1 mg / L biotin, 0.1 mg / L cobalamin, 0.3 mg / L para-aminobenzoic acid, 0.5 mg / L folic acid, and 1.5 mg / L pyridoxine; the amount of freeze-dried cereal digest product powder added is 0.5% (w / v).

[0018] The in vitro fermentation process was as follows: feces and phosphate-buffered saline were vortexed at a ratio of 1:9 (w / v) for 10 min; the fecal slurry was filtered through sterile gauze and then mixed with the culture medium at a ratio of 1:10 (w / v); CO2 was introduced throughout the process to maintain an anaerobic environment, and the mixture was incubated at 37°C for 24 h; each sample was replicated in triplicate; the fermentation broth was centrifuged at 12,000 rpm at 4°C for 5 min, and the precipitate was collected for metagenomic sequencing.

[0019] The beneficial effects of this invention are as follows: This invention uses ion chromatography to detect the content of arabinose consumed in in vitro fermentation, evaluates the relationship between arabinose consumption and the F / B ratio, and with *Prevotella* spp., suggesting that this indicator may be related to the reduction of intestinal inflammation and polysaccharide degradation. This invention uses the consumption of arabinose in in vitro fermentation as a specific indicator, combined with the subjects' clinical indicators, dietary status, and gut microbiota composition, to provide precise nutritional intervention for diabetic patients, improving their glucose and lipid metabolism and gut microbiota dysbiosis, further validating the application value of this indicator. Attached Figure Description

[0020] Figure 1 This is an ion chromatogram of sugar residues.

[0021] Figure 2 The values ​​represent the arabinose and xylose consumed during in vitro fermentation with different substrates, **P<0.01.

[0022] Figure 3 A heatmap showing the correlation between sugar residue consumption during in vitro fermentation.

[0023] Figure 4 For intergroup comparisons of α-diversity, *P<0.05.

[0024] Figure 5 For intergroup comparisons of β-diversity.

[0025] Figure 6 Distribution of fermentation product microbial populations at the phylum level.

[0026] Figure 7This represents the distribution of fermentation product microbial populations at the genus level.

[0027] Figure 8 The results of metagenomic sequencing analysis of gut microbiota (Krona plot) are shown in Figure A, which represents the period before intervention, and Figure B represents the period after intervention.

[0028] Figure 9 The results of metagenomic sequencing analysis of gut microbiota (Krona plot) are shown in Figure A, which represents the period before intervention, and Figure B represents the period after intervention.

[0029] Figure 10 The results of metagenomic sequencing analysis of gut microbiota (Krona plot) are shown in Figure A, which represents the period before intervention, and Figure B represents the period after intervention. Detailed Implementation

[0030] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0031] Example 1

[0032] A simple bag-like device was used to simulate oral chewing, including crushing and cutting. 10g of the sample was weighed and placed in a 250ml Erlenmeyer flask. 10ml of simulated oral digestion solution was added, and the mixture was vortexed for 2 minutes to ensure homogeneity. 20ml of simulated gastric digestion solution was then added, and the pH was adjusted to 3.0. Digestion was carried out in a constant-temperature shaking incubator at 37℃ and 120 rpm. After 2 hours of gastric digestion, intestinal digestion began with 40ml of simulated intestinal digestion solution, and the pH was adjusted to 7. The sample was digested for 2 hours at 37°C and 120 r / min in a constant temperature shaking incubator. At the end of intestinal digestion, a heat shock treatment was performed at 100°C for 5 minutes to inhibit digestive enzyme activity. After dialysis at 4°C for 72 hours, the sample was freeze-dried. The oral simulated digestion solution had a pH of 7.0 and contained 75 U / mL α-amylase. The gastric simulated digestion solution had a pH of 3.0 and contained 2000 U / mL pepsin. The intestinal simulated digestion solution had a pH of 7.0 and contained 100 U / mL pancreatic enzyme.

[0033] The digestion products and digestion fluids of two types of grains (rice and tricolor brown rice) were freeze-dried to prepare a fermentation broth. The fermentation broth formulation was as follows: 10 g / L tryptone, 2.5 g / L yeast extract, 1 g / L L-cysteine, 0.01 g / L heme, 0.9 g / L NaCl, 0.09 g / L CaCl2, 0.45 g / L KH2PO4, 0.09 g / L MgSO4·7H2O, 0.1 mg / L biotin, 0.1 mg / L cobalamin, 0.3 mg / L para-aminobenzoic acid, 0.5 mg / L folic acid, and 1.5 mg / L pyridoxine; the freeze-dried grain digestion product powder was added at a concentration of 0.5% (w / v).

[0034] Fresh feces were collected from 20 volunteers, prepared into fecal slurry, and then mixed with culture medium containing only lyophilized digestive fluid powder (blank control group), or mixed with culture medium containing 0.5% (w / v) lyophilized cereal digestive products at a ratio of 1:10, and incubated at 37°C for 24 hours.

[0035] In vitro fermentation was performed as follows: feces and phosphate-buffered saline were mixed at a ratio of 1:9 (w / v) and vortexed for 10 min; the fecal slurry was filtered through sterile gauze and then mixed with culture medium at a ratio of 1:10 (w / v); CO2 was introduced throughout the process to maintain an anaerobic environment, and the mixture was incubated at 37°C for 24 h; each sample was replicated in triplicate; the fermentation broth was centrifuged at 12,000 rpm at 4°C for 5 min, and the precipitate was collected for metagenomic sequencing.

[0036] After freeze-drying, the fermentation broth was hydrolyzed with sulfuric acid. The sugar residue content was detected by ion chromatography using an anion exchange column (CarboPac PA1, Dionex), with a column temperature of 30℃, a flow rate of 1 mL / min, and 200 mm NaOH as the eluent for cleaning the column. The column was then equilibrated with 3 mm NaOH for 10 minutes before injection, and the sugar residues were detected by an integrating amperometric detector. Figure 1 The image shows the ion chromatogram of the in vitro fermentation products. Peaks 2 and 5 represent arabinose and xylose, respectively. The results indicate that the in vitro fermentation using tricolor brown rice as a substrate consumed significantly more arabinose and xylose than that using rice as a substrate. (See image) Figure 2 Furthermore, the consumption of arabinose and xylose on the same fermentation substrate showed a significant positive correlation, while the consumption of the same sugar residue on different fermentation substrates also showed a significant positive correlation, suggesting a correlation between the ability of the same fecal donor to ferment different cereal non-starch polysaccharides. (See [link to article]). Figure 3 .

[0037] The in vitro fermentation broth was centrifuged at 12,000 rpm and 4°C for 5 min, and the precipitate was used for metagenomic sequencing. SDS was used as the main lysis reagent, and zirconia beads were used for mechanical cell disruption to ensure complete lysis of bacterial cells and release of nucleic acids. Acetate reagent was added to remove PCR inhibitors and adjust the pH. Genomic DNA was adsorbed using DNA extraction magnetic beads. After three washes, DNA was eluted with DNA elution buffer to obtain fecal metagenomic DNA. Library construction and detection were performed, and sequencing was conducted using the Illumina HiSeq high-throughput sequencing platform.

[0038] Alpha diversity analysis showed that, compared with tricolor brown rice, in vitro fermentation using rice as a substrate significantly reduced the Evenness index, while there were no significant differences in the Simpson and Shannon indices. Figure 4 As shown. The PCoA results are as follows. Figure 5 As shown, PCo1 and PCo2 explained 30.45% and 15.74% of the overall variance, respectively. Diversity analysis suggests differences in the richness and diversity of gut microbiota among different fermentation substrates.

[0039] Figure 6 The relative abundance of taxa at the phylum level was shown. The dominant phyla in all samples were Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria. The ratio of Firmicutes to Bacteroidetes (F / B) was considered a potential biomarker of intestinal inflammation in patients with type 2 diabetes. The consumption of arabinose in the tricolor brown rice group was significantly negatively correlated with the F / B ratio (r = -0.527, P < 0.05).

[0040] Figure 7 The relative abundance of the top 15 taxa at the genus level was shown. Prevotella, which has the potential to degrade complex polysaccharides in mixed high-fiber diets, was positively correlated with diets rich in plant fiber and carbohydrates and was defined as a next-generation probiotic. The consumption of arabinose in the tricolor brown rice group was significantly positively correlated with the abundance of Prevotella (r = 0.642, P < 0.01).

[0041] Individualized dietary plans were provided based on the subjects' clinical indicators, dietary habits, gut microbiota composition, and ability to metabolize non-starch polysaccharides.

[0042] Subject a has type 2 diabetes with elevated fasting blood glucose. Their staple food is mainly pasta, and their intake of dairy products and coarse grains is low. There is an ecological imbalance in the gut microbiota, with a lack of Bacteroidetes and an excess of Proteobacteria, and a low ability to metabolize non-starch polysaccharides (in vitro fermentation arabinose consumption < Q1). Individualized dietary advice and whole grain staple foods (selecting whole grains with a low non-starch polysaccharide content) were given for 2 weeks. The fasting blood glucose decreased from 9.2 mmol / L to 7.6 mmol / L; Krona was used to visually display the species annotation results, and the results are as shown in Figure 8 A and B show that the relative abundance of Bacteroidetes increased from 5.2% to 15.8%, and the relative abundance of Proteobacteria decreased from 40.2% to 1.7%, indicating that the gut microbiota disorder was significantly improved.

[0043] Subject b has type 2 diabetes with elevated fasting blood glucose and elevated triglycerides. Their staple food is mainly pasta, and their intake of dairy products and coarse grains is low, while their intake of red meat is high. There is an ecological imbalance in the gut microbiota, with a lack of Bacteroidetes, and an excess of Firmicutes and Actinobacteria, and the ability to metabolize non-starch polysaccharides is roughly normal (Q2 < in vitro fermentation arabinose consumption < Q3). Individualized dietary advice and whole grain staple foods (selecting whole grains with a medium non-starch polysaccharide content) were given for 2 weeks. The fasting blood glucose decreased from 11.4 mmol / L to 9.3 mmol / L, and the triglycerides decreased from 2.49 mmol / L to 1.74 mmol / L; Krona was used to visually display the species annotation results, and the results are as shown in Figure 9 A and B show that the relative abundance of Bacteroidetes increased from 7.9% to 20.7%, the relative abundance of Actinobacteria decreased from 50.5% to 45.0%, and the relative abundance of Firmicutes decreased from 41.3% to 33.1%, suggesting that the gut microbiota disorder was significantly improved.

[0044] Subject c is pre-diabetic with a high insulin resistance index. Their staple food is mainly rice, and their intake of coarse grains is low, while their intake of red meat is high. The gut microbiota composition is basically normal, and the ability to metabolize non-starch polysaccharides is basically normal (in vitro fermentation arabinose consumption > Q3). Individualized dietary advice and whole grain staple foods (selecting whole grains with a high non-starch polysaccharide content) were given for 2 weeks. The insulin resistance index decreased from 34.52 to 14.13. Krona was used to visually display the species annotation results, and the results are as shown in Figure 10 A and B show that the relative abundance of Prevotella increased from 13.5% to 35.9%.

[0045] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for assessing the ability of gut microbiota in diabetic patients to metabolize cereal non-starch polysaccharides, characterized in that, Follow these steps: 1) Establish an in vitro static digestion model to simulate the digestion of grains in the oral cavity, stomach and small intestine. Dialyze and freeze-dry the final digestion products and mix them with the culture medium as fermentation substrate. 2) Collect stool samples from patients with type 2 diabetes; 3) Establish an in vitro fermentation model by inoculating fecal microbiota from diabetic patients, detect the content of arabinose and xylose residues in the fermentation broth, and perform metagenomic sequencing; 4) Conduct statistical analysis to determine the ability of different individuals' fecal microbiota to ferment arabinose and xylose and their relationship with the composition of the gut microbiota; 5) Based on the ability of diabetic patients to ferment non-starch polysaccharides in their feces and the composition of their gut microbiota, provide individualized dietary intervention plans.

2. The method for evaluating the ability of gut microbiota to metabolize cereal non-starch polysaccharides in diabetic patients according to claim 1, characterized in that, Step 1) describes an in vitro static digestion method that uses a simple bag-shaped device to simulate oral chewing, including crushing and cutting. 10g of the sample is weighed and placed in a 250ml conical flask, and 10ml of oral simulated digestion solution is added. The mixture is then vortexed for 2 minutes to ensure homogeneity. 20ml of gastric simulated digestion solution is added, and the pH is adjusted to 3.

0. Digestion is then carried out in a constant-temperature shaking incubator at 37℃ and 120r / min. After 2 hours of gastric digestion, intestinal digestion begins. 40ml of intestinal simulated digestion solution is added, and the pH is adjusted to 7.

0. Digestion continues for 2 hours in a constant-temperature shaking incubator at 37℃ and 120r / min. At the end of intestinal digestion, a heat shock treatment at 100℃ for 5 minutes is performed to inhibit digestive enzyme activity. The sample is then dialyzed at 4℃ for 72 hours and then freeze-dried.

3. The method for evaluating the ability of gut microbiota to metabolize cereal non-starch polysaccharides in diabetic patients according to claim 2, characterized in that, The oral cavity simulates digestion at pH 7.0 and contains 75 U / ml of α-amylase.

4. The method for evaluating the ability of gut microbiota to metabolize cereal non-starch polysaccharides in diabetic patients according to claim 2, characterized in that, The simulated gastric digestive fluid has a pH of 3.0 and contains 2000 U / mL of pepsin.

5. The method for assessing the ability of gut microbiota to metabolize cereal non-starch polysaccharides in diabetic patients according to claim 2, characterized in that, The intestinal simulated digestive fluid has a pH of 7.0 and contains 100 U / mL of pancreatic enzymes.

6. The method for assessing the ability of gut microbiota to metabolize cereal non-starch polysaccharides in diabetic patients according to claim 1, characterized in that, Step 2) Collect the feces using the provided clean, dry paper container. Transfer 1.5-2.5g of fresh feces to a sterile tube using a sterile spoon and place it in an ice box. Transport the tube to the laboratory within 4 hours.

7. The method for assessing the ability of gut microbiota to metabolize cereal non-starch polysaccharides in diabetic patients according to claim 1, characterized in that, The fermentation broth formulation is as follows: 10 g / L tryptone, 2.5 g / L yeast extract, 1 g / L L-cysteine, and 0.01 g / L heme. 0.9 g / L NaCl, 0.09 g / L CaCl2, 0.45 g / L KH2PO4, 0.09 g / L MgSO4·7H2O, 0.1 mg / L Biotin, 0.1 mg / L Cobalamin, 0.3 mg / L p-aminobenzoic acid, 0.5 mg / L folic acid, 1.5 mg / L pyridoxine; the addition amount of lyophilized cereal digest product powder is 0.5% (w / v).

8. The method for assessing the ability of gut microbiota to metabolize cereal non-starch polysaccharides in diabetic patients according to claim 1, characterized in that, The in vitro fermentation process was as follows: feces and phosphate-buffered saline were vortexed at a ratio of 1:9 (w / v) for 10 min; the fecal slurry was filtered through sterile gauze and then mixed with the culture medium at a ratio of 1:10 (w / v); CO2 was introduced throughout the process to maintain an anaerobic environment, and the mixture was incubated at 37°C for 24 h; each sample was replicated in triplicate; the fermentation broth was centrifuged at 12,000 rpm at 4°C for 5 min, and the precipitate was collected for metagenomic sequencing.

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