Application of highly acetylated glucomannan in promoting proliferation of Bacteroides coprocola

Abstract

The invention discloses an application of highly acetylated glucomannan in promoting the proliferation of Bacteroides coprocola bacteria, the acetyl substitution degree of the highly acetylated glucomannan is 1.07 to 1.82, and the molecular weight of the highly acetylated glucomannan is 1112 to 1431 kDa. According to the application disclosed by the invention, excrement of prediabetic people is subjected to in-vitro fermentation through highly acetylated glucomannan, the fact that the highly acetylated glucomannan can be used for remarkably regulating the intestinal flora structure of the prediabetic people and remarkably enriching the proliferation of Bacteroides coprocola bacteria is found, and in a Bacteroides coprocola bacteria culture system, the highly acetylated glucomannan can be used for remarkably promoting the proliferation of the Bacteroides coprocola bacteria culture system.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the role of highly acetylated glucomannan in promoting... Bacteroides coprocola Applications in bacterial proliferation. Background Technology

[0002] Bacteroides is one of the resident bacteria in the human gut, playing an important role in maintaining the balance of the gut microbiota and participating in host metabolism. Bacteroides coprocola Bacteroides is a common gut bacterium belonging to the genus Bacteroides, primarily found in the gut microbiota of humans and animals. Recent studies have shown that gut microbiota dysbiosis is closely related to the development and progression of metabolic diseases such as prediabetes. Bacteroides coprocola As an important component of the gut microbiota, bacteria often exhibit abnormal fluctuations in abundance in the gut of people with prediabetes, suggesting that these bacteria may have a potential link with the host's metabolic health.

[0003] However, at present Bacteroides coprocola The cultivation and screening of microorganisms still face challenges. Traditional microbial culture media (such as GAM medium and BHI medium) use conventional carbon sources (such as glucose), which have poor selectivity and are difficult to specifically enrich targets from the complex gut microbiota. Bacteroides coprocola This results in a large workload and low efficiency in screening, and makes it difficult to obtain functional strains with specific substrate utilization capabilities.

[0004] Glucomannan is a natural polysaccharide widely found in plants such as konjac, exhibiting good biocompatibility and biodegradability. Acetylation modification is one of the important methods for polysaccharide modification; by adjusting the degree of acetylation, the physicochemical properties and biological activities of polysaccharides can be altered. However, to date, no studies have reported on the effects of acetylation gradient-regulated glucomannan on... Bacteroides coprocola The specific regulatory role of bacterial proliferation has not been applied to... Bacteroides coprocola In a highly efficient screening and culture system for bacteria. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, this invention provides a method for promoting the use of highly acetylated glucomannan. Bacteroides coprocola Applications in gut microbiota proliferation. Highly acetylated glucomannan can regulate the gut microbiota structure in individuals with prediabetes and significantly enrich gut microbiota. Bacteroides coprocola Bacterial proliferation, and in Bacteroides coprocola In bacterial culture systems, highly acetylated glucomannan can significantly promote bacterial proliferation.

[0006] The technical solution of the present invention is as follows: The first objective of this invention is to provide a highly acetylated glucomannan that promotes… Bacteroidescoprocola Applications in bacterial proliferation.

[0007] In one embodiment of the present invention, the degree of acetyl substitution of the highly acetylated glucomannan is 1.07-1.82, and the molecular weight is 1112-1431 kDa; Its preparation method includes the following steps: Adjust the pH of the glucomannan aqueous solution to 8-10, add acetic anhydride dropwise at room temperature while maintaining the pH of the reaction system at 8-10, and add hydrochloric acid to adjust the pH to neutral after the reaction is complete; The reaction product was precipitated in a gradient ethanol solution, filtered, washed, and freeze-dried under vacuum to obtain the highly acetylated glucomannan.

[0008] In one embodiment of the present invention, glucomannan is derived from konjac tubers, is a white powder with a particle size greater than 120 mesh, viscosity ≥ 30000 mPa·s, moisture content ≤ 10%, and transparency ≥ 50%.

[0009] In one embodiment of the present invention, the addition ratio of glucomannan to acetic anhydride is 1:14-22 g / mL.

[0010] In one embodiment of the present invention, a sodium hydroxide solution is used to adjust the pH, wherein the concentration of the sodium hydroxide solution is 1.5 mol / L or 10 mol / L.

[0011] In one embodiment of the present invention, the concentrations of the gradient ethanol solutions are 50%, 75%, 95%, and 100%, respectively.

[0012] In one embodiment of the present invention, the degree of acetyl substitution of the highly acetylated glucomannan is 1.82.

[0013] In one embodiment of the present invention, the method for preparing highly acetylated glucomannan includes the following steps: Mix 0.3 g g glucomannan with 30 mL distilled water, and adjust the pH to 8-10 with 1.5 mol / L NaOH solution; Acetic anhydride (4.6-6.6 mL) was added dropwise at room temperature, and the pH was adjusted to 8-10 with 10 mol / L NaOH solution. The mixture was then cooled, and the pH was neutralized with 10 mol / L HCl solution to terminate the reaction. The acetylated reaction mixture was precipitated using a gradient of ethanol solutions (50%, 75%, 95%, 100%), then filtered and washed twice with distilled water. The resulting product was freeze-dried under vacuum to obtain acetylated glucomannan powder.

[0014] In one embodiment of the present invention Bacteroides coprocolaDuring the bacterial culture process, the above-mentioned highly acetylated glucomannan was added to the culture medium.

[0015] In one embodiment of the present invention, highly acetylated glucomannan is used for cultivation. Bacteroides coprocola The only carbon source in the culture medium for bacteria.

[0016] In one embodiment of the present invention, promoting Bacteroides coprocola The method for bacterial proliferation is as follows: exist Bacteroides coprocola In the bacterial culture system, take Bacteroides coprocola The bacterial culture was inoculated at a rate of 2% into a culture medium with highly acetylated glucomannan as the sole carbon source and cultured anaerobicly at 37°C for 48 h in an anaerobic station. The volume concentrations of each gas in the anaerobic station were 80% N2, 10% CO2, and 10% H2.

[0017] In one embodiment of the present invention, the culture medium consists of: 5.0 g / L tryptone, 5.0 g / L peptone, 10.0 g / L yeast extract, 5.0 g / L ox heart extract, 5.0 g / L highly acetylated glucomannan, 2.0 g / L dipotassium hydrogen phosphate, 1 g / L Tween 80, 0.01 g / L calcium chloride dihydrate, 0.02 g / L magnesium sulfate heptahydrate, 0.04 g / L potassium dihydrogen phosphate, 0.4 g / L sodium bicarbonate, 0.08 g / L sodium chloride, 1.0 mg / L resazurin, 0.5 g / L L-cysteine ​​hydrochloride, 0.1 g / L vitamin K1, and 0.5 g / L heme chloride; the pH of the culture medium is 7.2 ± 0.2.

[0018] The beneficial technical effects of this invention are as follows: The high degree of acetylation of glucomannan in this invention promotes Bacteroides coprocola Its application in bacterial proliferation is specifically reflected in: (1) significantly enriching the gut microbiota of people with prediabetes. Bacteroides coprocola Bacteria; (2) Bacteroides coprocola In bacterial culture systems, highly acetylated glucomannan can significantly promote bacterial proliferation.

[0019] Therefore, highly acetylated glucomannan is useful in screening for substances that can utilize it. Bacteroides coprocola Its application in bacteria has enormous potential. Attached Figure Description

[0020] Figure 1 The effect of highly acetylated glucomannan on the gut microbiota structure in people with prediabetes; Figure 2 The effect of highly acetylated glucomannan on differential species of gut microbiota in individuals with prediabetes; Figure 3The effect of highly acetylated glucomannan on gut microbiota in people with prediabetes Bacteroides coprocola The effect of relative abundance of bacteria; Figure 4 For highly acetylated glucomannan Bacteroides coprocola The effect of the concentration of the fermentation supernatant. Detailed Implementation

[0021] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0022] The konjac glucomannan (product number: PN01482, content: 99%, specification: 1kg, appearance: off-white powder, viscosity: ≥ 30000 mPa·s, moisture: ≤ 10%, transparency: ≥ 50%, ash content: ≤ 3%) involved in the following examples was purchased from Bofeimei Co., Ltd.; β-D-pentaacetyl glucose was purchased from Beijing Kaisenlai Technology Co., Ltd.; acetic anhydride, hydroxylamine hydrochloride, NaOH, HCl, FeCl3, anhydrous ethanol, tryptone, peptone, yeast extract, sodium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, Tween 80, calcium chloride dihydrate, magnesium sulfate heptahydrate, sodium bicarbonate, resazurin, and L-cysteine ​​hydrochloride were purchased from Sinopharm Chemical Reagent Co., Ltd.; ox heart extract powder was purchased from Hongrun Baoshun Technology Co., Ltd.; mGAM medium (without glucose and soluble starch) was purchased from Qingdao Haibo Biotechnology Co., Ltd.; vitamin K1 and heme chloride were purchased from Qingdao Rishui Biotechnology Co., Ltd. Bacteroides coprocola The bacteria (BNCC 354141, DSM 17136) were purchased from Beina Biotechnology Co., Ltd.

[0023] The degree of acetyl substitution was determined by the hydroxylamine-ferric chloride colorimetric method: Accurately weigh 0.3180 g of β-D-pentaacetylglucose into a 50 mL volumetric flask, add 20.0 mL of anhydrous ethanol, dissolve in a water bath at 60 °C, and cool to room temperature; dilute to volume with pure water to obtain a β-D-pentaacetylglucose stock solution with an acetyl concentration of 3.507 mg / L, and store at 4 °C for later use. Accurately transfer 2.0, 4.0, 6.0, 8.0, 10.0, and 12 mL of the acetyl stock solution into 50 mL brown volumetric flasks, and dilute to volume with pure water. Pipette 5 mL of each standard solution into a 50 mL volumetric flask, add 5 mL of 0.1 mol / L hydroxylamine hydrochloride solution and 5 mL of 1.5 mol / L NaOH solution respectively, mix well, and let stand for 20 min; add 5 mL of 2.0 mol / L HCl to each, mix well, and let stand for 20 min; add 10.0 mL of 0.37 mol / L FeCl3 solution to each, dilute to volume with pure water, mix well, and let stand for 10 min. Measure the absorbance at 500 nm using a UV spectrophotometer. Use pure water instead of β-D-pentaacetyl glucose solution as a blank control. Plot a standard curve with the mass solubility of acetyl groups as the x-axis (C1) and the absorbance value as the y-axis (A1).

[0024] Dissolve 1 mg of dried glucomannan in a 50 mL volumetric flask, add pure water, and dilute to volume. Use 5.0 mL of the sample solution instead of the acetyl standard gradient solution to determine the degree of acetyl substitution in the sample using the method for establishing a standard curve. Calculate the acetyl content in the sample based on the regression equation, and then calculate the degree of acetyl substitution using the following formula:

[0025]

[0026] W1: Acetyl content (mg) in glucomannan; W2: Mass of glucomannan (mg); 162: The relative molecular weight of a single monosaccharide unit in glucomannan; 43: The relative molecular weight of the acetyl group; DS: The average number of acetyl substituents attached to each sugar unit in glucomannan, i.e., the degree of acetyl substitution.

[0027] Example 1 A method for preparing highly acetylated glucomannan includes the following steps: Mix 0.3 g g glucomannan with 30 mL distilled water, and adjust the pH to 8 with 1.5 mol / L NaOH solution; 4.6 mL of acetic anhydride was added dropwise at room temperature, and the pH was adjusted to 8 with 10 mol / L NaOH solution. The mixture was then cooled, and the pH was neutralized with 10 mol / L HCl solution to terminate the reaction. The acetylated reaction mixture was precipitated using a gradient of ethanol solutions (50%, 75%, 95%, 100%), then filtered and washed twice with distilled water. The resulting product was freeze-dried under vacuum to obtain acetylated glucomannan powder.

[0028] Example 2 Same as Example 1, except that the amount of acetic anhydride used is 6.6 mL.

[0029] Example 3 Same as Example 1, except that the pH of the NaOH solution is adjusted to 10 in both cases.

[0030] Example 4 Same as Example 1, except that the pH of the NaOH solution is adjusted to 9 in both cases.

[0031] The degree of acetyl substitution of the acetylated glucomannan powders prepared in Examples 1-2 is shown in Table 1 below.

[0032] Table 1

[0033] The acetylation degree of the acetylated glucomannan powder prepared according to Examples 1-2 was used to name the high-acetylated glucomannan as AcKGM-1.0 and AcKGM-1.8, respectively.

[0034] The culture medium formulations involved in the following examples are shown in Tables 2-3. Table 2 is the formulation of mGAM medium (containing no glucose or soluble starch, with highly acetylated glucomannan as the sole carbon source) for in vitro fermentation of feces from prediabetic individuals, and Table 3 is the formulation of mPYG medium (…). Bacteroides coprocola Formula for bacterial culture system (culture medium).

[0035] Table 2

[0036] Table 3

[0037] Test example: Test Example 1: The effect of highly acetylated glucomannan on the gut microbiota structure of people with prediabetes The specific steps are as follows: Four volunteers with prediabetes (2 males and 2 females) were selected. They were required to have abnormal fasting blood glucose (5.6 ≤ FBG < 6.9 mmol / L), have not received antibiotic treatment in the past 6 months, have no intestinal diseases, have no history of probiotic consumption, and have not used probiotic products or consumed alcohol in the past month. Fresh morning stool samples were collected from each of the four volunteers and collected in 35 mL stool sampling tubes. The tube caps were partially tightened and placed in a laboratory-specific self-sealing bag containing an anaerobic gas-generating bag to create an anaerobic environment. The samples were temporarily stored at 4°C, ensuring processing began within two hours.

[0038] Fecal samples were collected from four individuals, placed in ice packs, and transported to a sterile laminar flow hood in the laboratory for experimental use. 1.5 g of fecal sample from each individual was weighed and collected together. 42 mL of pre-prepared, sterilized, and pre-cooled (4°C) PBS solution was added, and the mixture was homogenized (vortexed) and filtered through four layers of gauze. The resulting filtrate was the fecal inoculum.

[0039] The formulation of the PBS solution is shown in Table 4.

[0040] Table 4

[0041] Weigh 4.1 g of mGAM medium (excluding glucose and soluble starch), dissolve it in 60 mL of distilled water, autoclave at 121℃ for 15 min, and cool to approximately 50℃. After passing the syringe through the membrane, add 0.1 g / L vitamin K1 and 0.5 g / L heme chloride. Divide the above medium into three groups: a blank control group (CK), and two experimental groups, AcKGM-1.0 and AcKGM-1.8, respectively, with 3.5 g / L AcKGM-1.0 and AcKGM-1.8 added. The experiment was conducted in parallel for 6 experiments. Specific groupings and compositions are shown in Table 5.

[0042] Table 5

[0043] The obtained fecal inoculum was inoculated into the culture medium at a volume ratio of 40% and cultured anaerobically at 37°C (80% N2, 10% CO2, 10% H2) for 24 h to obtain a fermentation broth containing intestinal microorganisms. The fermentation broth was then centrifuged to separate the supernatant and precipitate. The supernatant was then aliquoted into 5 mL centrifuge tubes and stored at -80°C.

[0044] The specific method is as follows: Fermentation precipitates were sent to the DNBSEQ-T7 platform of Beijing Novogene Co., Ltd. for metagenomic sequencing. For the raw data, Trimmomatic 0.39 software was first used for preprocessing to remove low-quality sequences. Then, Bowtie 0.7.17, Samtools 1.9, and Bedtools 2.30.0 were used to align the filtered sequences with the human reference genome (Homo sapiens genome assembly GRCh38, hg38) to remove host-derived genes. Finally, MetaPhlAn3 was used to annotate the high-quality sequences after quality control, resulting in a species abundance table. Beta diversity was calculated using NMDS (Non-metric Multidimensional Scaling) based on Bray-Curtis distance. Results are shown below. Figure 1 .

[0045] The results showed that the Stress value of 0.074 indicated good sorting quality, the F value of PERMANOVA was 34.322, and the P value was 0.001, which supported the statistical significance of the differences between groups. The CK group was significantly separated from the AcKGM-1.0 and AcKGM-1.8 groups in the NMDS space, indicating that the high acetylated glucomannan group can significantly change the gut microbiota structure of people with prediabetes. Test Example 2: Effects of Highly Acetylated Glucomannan on Differential Species of Gut Microbiota in Prediabetic Individuals The specific method is as follows: LEfSe intergroup difference analysis was used to reduce the dimensionality of the data, identify the most significant influencing factors, and assess the influence of differentially expressed species. The p-threshold for intergroup differences was set at 0.05, and the threshold for linear discriminant analysis (LDA) was set at 4. Species were considered differentially expressed if p < 0.05 and LDA > 3.5. Results are shown below. Figure 2 .

[0046] The results showed that, at the species level, the AcKGM-1.0 group had the following differences compared to the control group: Faecalibacterium prausnitzii , Bacteroides coprocola , Clostridium perfringens , Catenibacterium mitsuokai and Bacteroide uniformis The AcKGM-1.8 group showed the following differences in species: Bacteroides coprocola , Catenibacterium mitsuokai and Bacteroides uniformis This indicates that highly acetylated glucomannan significantly affects the gut microbiota in individuals with prediabetes.Bacteroides coprocola , Catenibacterium mitsuokai and Bacteroides uniformis .

[0047] Test Example 3: Effects of Highly Acetylated Glucomannan on Gut Microbiota in Prediabetic Individuals Bacteroides coprocola Effect of relative abundance of bacteria The specific method is as follows: Based on species abundance information obtained from metagenomics, the differences between groups Bacteroides coprocola A plot of the relative abundance of bacteria. Results are shown below. Figure 3 .

[0048] The results show: CK group Bacteroides coprocola The relative abundance of bacteria was 0.1751 ± 0.01710%, in the AcKGM-1.0 group. Bacteroides coprocola The relative abundance of bacteria was 1.056 ± 0.2265%, AcKGM-1.8 group Bacteroides coprocola The relative abundance of bacteria was 1.335 ± 0.1384%. Compared with the CK group, the AcKGM-1.0 group... Bacteroides coprocola The relative abundance of bacteria increased significantly to 1.056±0.2265% (p<0.01), with an increase rate of 83.42%; compared with the CK group, the AcKGM-1.8 group... Bacteroides coprocola The relative abundance of bacteria increased significantly to 1.335 ± 0.1384% (p < 0.001), with an increase rate of 86.88%. These experiments demonstrate that highly acetylated glucomannan can significantly enrich the gut microbiota of individuals with prediabetes. Bacteroides coprocola bacteria.

[0049] Test Example 4: Highly acetylated glucomannan on Bacteroides coprocola Effect of bacterial fermentation supernatant concentration The specific steps are as follows: To further verify the effectiveness of highly acetylated glucomannan (AcKGM-1.8) in screening for substances that can utilize it... Bacteroides coprocola Its application in bacteria has been carried out. Bacteroides coprocola Bacterial culture experiment: 2% inoculum was inoculated into medium without carbon source, medium with glucose as carbon source and medium with AcKGM-1.8 as carbon source, respectively, and cultured anaerobically at 37℃ for 48 h. The parallel experiment was set to 3.

[0050] The specific method is as follows: The bacterial culture was removed from the anaerobic station, mixed well, and its OD600 value was measured using an ELISA reader. The results are shown below. Figure 4 .

[0051] The results show: OD600 It can reflect the concentration of bacterial culture medium, thereby estimating bacterial proliferation. Compared to media without a carbon source, the medium using AcKGM-1.8 and glucose as carbon sources has a higher OD value. 600 The results of the above experiments indicate that highly acetylated glucomannan can significantly promote (p<0.01) the increase in glucomannan levels. Bacteroides coprocola The proliferation of bacteria.

[0052] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. Highly acetylated glucomannan promotes... Bacteroides coprocola Applications in bacterial proliferation.

2. The application according to claim 1, characterized in that, The degree of acetyl substitution of highly acetylated glucomannan is 1.07-1.82, and the molecular weight is 1112-1431 kDa; Its preparation method includes the following steps: Adjust the pH of the glucomannan aqueous solution to 8-10, add acetic anhydride dropwise at room temperature while maintaining the pH of the reaction system at 8-10, and add hydrochloric acid to adjust the pH to neutral after the reaction is complete; The reaction product was precipitated in a gradient ethanol solution, filtered, washed, and freeze-dried under vacuum to obtain the highly acetylated glucomannan.

3. The application according to claim 2, characterized in that, Glucomannan is derived from konjac tubers. It is a white powder with a particle size greater than 120 mesh, a viscosity of ≥30000 mPa·s, a moisture content of ≤10%, and a transparency of ≥50%.

4. The application according to claim 2, characterized in that, The ratio of glucomannan to acetic anhydride is 1:14-22 g / mL.

5. The application according to claim 2, characterized in that, The pH was adjusted using sodium hydroxide solution with a concentration of 1.5 mol / L or 10 mol / L.

6. The application according to claim 2, characterized in that, The concentrations of the gradient ethanol solutions were 50%, 75%, 95%, and 100%, respectively.

7. The application according to claim 2, characterized in that, The degree of acetyl substitution of the highly acetylated glucomannan is 1.

82.

8. The application according to claim 1, characterized in that, Highly acetylated glucomannan is used for cultivation Bacteroides coprocola The only carbon source in the culture medium for bacteria.

9. The application according to claim 1, characterized in that, Promote Bacteroides coprocola The method for bacterial proliferation is as follows: exist Bacteroides coprocola In the bacterial culture system, take Bacteroides coprocola The bacterial culture was inoculated at a rate of 2% into a culture medium with highly acetylated glucomannan as the sole carbon source and cultured anaerobicly at 37°C for 48 h in an anaerobic station. The volume concentrations of each gas in the anaerobic station were 80% N2, 10% CO2, and 10% H2.

10. The application according to claim 9, characterized in that, The culture medium consisted of: 5.0 g / L tryptone, 5.0 g / L peptone, 10.0 g / L yeast extract, 5.0 g / L ox heart extract, 5.0 g / L highly acetylated glucomannan, 2.0 g / L dipotassium hydrogen phosphate, 1 mL / L Tween 80, 0.01 g / L calcium chloride dihydrate, 0.02 g / L magnesium sulfate heptahydrate, 0.04 g / L potassium dihydrogen phosphate, 0.4 g / L sodium bicarbonate, 0.08 g / L sodium chloride, 1.0 mg / L resazurin, 0.5 g / L cysteine ​​hydrochloride, 0.1 g / L vitamin K1, and 0.5 g / L heme chloride; the pH of the culture medium was 7.2 ± 0.2.

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