Extract of Bacteroides monotheca capsular polysaccharide and its application in promoting the proliferation of lactic acid bacteria

Abstract

The present invention discloses a Bacteroides monomorpha capsule polysaccharide extract and its application in promoting the proliferation of lactic acid bacteria, wherein the Bacteroides monomorpha extract is a Bacteroides monomorpha capsule polysaccharide. The present invention discovers a Bacteroides monomorpha capsule polysaccharide for the first time, and verifies through in vivo and in vitro tests that the Bacteroides monomorpha capsule polysaccharide has the effect of promoting the proliferation of lactic acid bacteria, and is safe and effective, without toxic side effects, so as to further achieve the purpose of in vitro large-scale propagation or large-scale production of lactic acid bacteria, improve the yield and output efficiency of lactic acid bacteria, or based on its in vivo propagation effect, it is used to increase the number of lactic acid bacteria in the gastrointestinal tract of the subject, thereby achieving health care effects such as promoting digestion and absorption, and achieving the effect of maintaining human health.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Bacteroides monomorpha capsule polysaccharide extract and an application thereof in promoting the proliferation of lactic acid bacteria. Background Art

[0002] Probiotics are defined by the International Scientific Association of Probiotics and Prebiotics as "live microorganisms that confer a health benefit to the host when administered in adequate amounts". Compared with other commonly used dietary supplements, probiotics offer unique market quality requirements because they need to be live and have highly diverse microorganisms themselves. With the development of the probiotics market, the concepts of probiotics, prebiotics, synbiotics and postbiotics have been continuously proposed. Postbiotics are a general term for the metabolites of probiotics after processing, including bacteria and metabolites. Studies have confirmed that screened postbiotics are better than original live bacteria in enhancing immunity, and they still retain a high degree of physiological activity even after being exposed to high temperatures or treated with gastrointestinal digestive juices. Therefore, the development of new postbiotics has important market value.

[0003] Capsular polysaccharide (CPS) is a polysaccharide polymer commonly found on the surface of bacteria. It is fixed to the outer peptidoglycan of the cell wall by covalent bonding and is an important component for bacteria to exert their physiological functions.

[0004] Lactic acid bacteria (LAB) is a general term for bacteria that can produce a large amount of lactic acid using fermentable carbohydrates. It is one of the most widely used probiotics in the market. It is widely distributed in nature and has rich species diversity, including at least 18 genera and more than 200 species. Except for a few, most of them are essential bacteria in the human body and have important physiological functions, and are widely present in the human intestine. Typical lactic acid bacteria include Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus reuteri and Lactobacillus rhamnosus. Related studies have shown that natural polysaccharides such as inulin can increase the abundance of probiotics in vivo and in vitro. However, unlike substances such as inulin, there has been no research report on the role of new postbiotics such as strain capsular polysaccharides on probiotics. Therefore, whether it can play a role in the proliferation of lactic acid bacteria in vivo and in vitro is of great significance for the development of probiotic compounds.

[0005] Bacteroides monomorpha is a Gram-negative rod-shaped bacterium in the genus Bacteroides, which is derived from human and pig fecal flora and can also be isolated from human clinical samples. However, there are few reports on the related research of this bacterium and its extracts. Summary of the invention

[0006] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the present invention provides a Bacteroides monomorpha extract and its use in promoting the proliferation of lactic acid bacteria. In the present invention, it is discovered for the first time that a Bacteroides monomorpha extract, especially a Bacteroides monomorpha capsular polysaccharide, can effectively promote the growth and proliferation of lactic acid bacteria in both in vivo and in vitro environments, thereby achieving the purpose of large-scale in vitro propagation or large-scale production of lactic acid bacteria, and improving the yield and output efficiency of lactic acid bacteria. And based on its in vivo propagation effect, it can also increase the number of gastrointestinal lactic acid bacteria in the subject, thereby achieving health care effects such as promoting digestion and absorption, and maintaining human health.

[0007] The first aspect of the present invention provides a Bacteroides monomorpha capsular polysaccharide, which is composed of arabinose, galactose, glucose, galacturonic acid and glucuronic acid, and the molar ratio of arabinose, galactose, glucose, galacturonic acid and glucuronic acid is 0.51:0.25:300.19:1.95:1.72.

[0008] In the present invention, the inventors did not find any polysaccharide identical to the Bacteroides monomorpha capsular polysaccharide in the present invention in the prior art through database comparison. In addition, the types and proportions of monosaccharide compositions in the polysaccharides obtained from the existing Bacteroides genus (including Bacteroides fragilis) are different from those of the Bacteroides monomorpha capsular polysaccharide in the present invention, so it may be a bacterial extracellular heteropolysaccharide with a new structure.

[0009] In some embodiments of the present invention, the relative molecular mass of the Bacteroides monomorpha capsular polysaccharide is 1.819×10 4 Da.

[0010] In some embodiments of the present invention, the total sugar content of the Bacteroides monotheca capsular polysaccharide is 99.83%.

[0011] In some embodiments of the present invention, the Bacteroides monomorpha capsular polysaccharide is -1 There is strong absorption of hydroxyl groups.

[0012] In some embodiments of the present invention, the Bacteroides monotheca capsular polysaccharide is derived from Bacteroides monotheca ATCC8492.

[0013] The second aspect of the present invention provides a method for extracting Bacteroides monomorpha capsular polysaccharide, comprising the following steps:

[0014] (1) Bacteroides monothecae was repeatedly frozen and thawed, then ultrasonically disrupted, the supernatant was collected by centrifugation, concentrated under reduced pressure, and then precipitated with alcohol;

[0015] (2) taking the alcohol precipitate for deproteinization, ultrafiltration washing, and chromatography using ion exchange cellulose, eluting with NaCl, and collecting the eluate of the 0 mol / L peak to obtain the Bacteroides monomorpha capsular polysaccharide.

[0016] In some embodiments of the present invention, the Bacteroides monomorpha capsular polysaccharide is the Bacteroides monomorpha capsular polysaccharide described in the first aspect of the present invention.

[0017] In some embodiments of the present invention, the deproteinization treatment comprises deproteinization using papain and Sevag reagent.

[0018] In some embodiments of the present invention, the deproteinization treatment is: firstly enzymatic hydrolysis with papain, and then further deproteinization with Sevag reagent.

[0019] In some embodiments of the present invention, the ultrafiltration uses a 3KD ultrafiltration tube.

[0020] In some embodiments of the present invention, the ion exchange cellulose is DEAE cellulose-52 ion exchange cellulose.

[0021] In some embodiments of the present invention, in the chromatography, the eluent used is a NaCl solution.

[0022] In some embodiments of the present invention, the elution concentrations of the NaCl solution are 0, 0.05, 0.1, 0.3, and 0.5 mol / L, respectively.

[0023] In some embodiments of the present invention, the extraction method further comprises further purification using a dextran gel chromatography column.

[0024] In some embodiments of the present invention, the eluent of the dextran gel chromatography is a 0.1 mol / L NaCl solution.

[0025] The third aspect of the present invention provides the use of a Bacteroides monotheca extract as the sole active substance in promoting the growth or proliferation of lactic acid bacteria.

[0026] In some embodiments of the present invention, the Bacteroides monomorpha extract is Bacteroides monomorpha capsular polysaccharide.

[0027] In some embodiments of the present invention, the lactic acid bacteria include Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus reuteri and Lactobacillus rhamnosus.

[0028] In the present invention, the inventors confirmed through in vitro experiments that the capsular polysaccharide of Bacteroides monotheca has a proliferation-promoting effect on lactic acid bacteria, among which it has a significant promoting or stimulating effect on the growth of Lactobacillus acidophilus, Lactobacillus casei and Lactobacillus plantarum in the in vitro environment, while the proliferation-promoting effect on Lactobacillus reuteri and Lactobacillus rhamnosus is relatively small.

[0029] A fourth aspect of the present invention provides use of a Bacteroides monotheca extract in preparing a product for improving the abundance of lactic acid bacteria in an in vivo environment.

[0030] In some embodiments of the present invention, the Bacteroides monomorpha extract is Bacteroides monomorpha capsular polysaccharide.

[0031] In some embodiments of the present invention, the lactic acid bacteria include Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus reuteri and Lactobacillus rhamnosus.

[0032] In some embodiments of the present invention, the products include feed, feed additives, food, food additives and medicines.

[0033] In some embodiments of the present invention, the product further comprises auxiliary materials acceptable in feed, food or pharmacy.

[0034] In some embodiments of the present invention, the auxiliary materials include, but are not limited to: solvents, emulsifiers, softeners, antioxidants, preservatives, chelating agents, pH regulators, thickeners, penetration enhancers, and sunscreens.

[0035] In some embodiments of the present invention, the product is applicable to animals, and the animals are selected from humans, cats, cows, sheep, pigs, dogs, chickens, ducks, geese, rabbits, and mice; preferably, the animals are humans.

[0036] In some embodiments of the present invention, the content of Bacteroides monotheca extract in the product is 0.1-100 wt %.

[0037] In some embodiments of the present invention, the content of the Bacteroides monocytogenes extract is measured by the mass of bacteria per L of bacterial liquid (culture medium). In some embodiments of the present invention, the content of the Bacteroides monocytogenes extract is 0.1-10 mg bacteria / L culture medium.

[0038] In some embodiments of the present invention, the content of Bacteroides monomorpha extract in the product is 5 mg bacteria / L culture medium.

[0039] In some embodiments of the present invention, the in vivo environment is a gastrointestinal environment.

[0040] In the present invention, the inventors confirmed through in vivo experiments on mice that the capsular polysaccharide of Bacteroides monotheca has an in vivo proliferation-promoting effect on lactic acid bacteria, especially for Lactobacillus acidophilus and Lactobacillus casei, and has a certain promoting effect on Lactobacillus reuteri and Lactobacillus rhamnosus, but the effect is not very significant.

[0041] The beneficial effects of the present invention are:

[0042] The present invention discovers a Bacteroides monoxene capsular polysaccharide for the first time, and verifies through in vivo and in vitro tests that the Bacteroides monoxene capsular polysaccharide has the effect of promoting the proliferation of lactic acid bacteria, and is safe, effective, and has no toxic side effects. Therefore, it can further achieve the purpose of large-scale in vitro propagation or large-scale production of lactic acid bacteria, improve the yield and output efficiency of lactic acid bacteria, or based on its in vivo propagation effect, it can be used to increase the number of lactic acid bacteria in the gastrointestinal tract of the subject, thereby achieving health care effects such as promoting digestion and absorption, and achieving the effect of maintaining human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the gradient elution curve of different components of crude capsular polysaccharide of Bacteroides monotheca after DEAE cellulose-52 ion exchange cellulose chromatography using NaCl solution as eluent.

[0044] Figure 2 This is the elution curve after DEAE cellulose-52 ion exchange cellulose chromatography followed by further purification using Sephacryl S-300HR dextran gel.

[0045] Figure 3 This is the infrared spectrum of Bacteroides monocytogenes capsular polysaccharide BU-CPS-1B.

[0046] Figure 4 This is the UV spectrum of Bacteroides monocytogenes capsular polysaccharide BU-CPS-1B.

[0047] Figure 5 This is the liquid chromatogram of the mixed monosaccharide standard PMP derivatization.

[0048] Figure 6 This is the monosaccharide derivatization liquid chromatogram of Bacteroides monotheca capsular polysaccharide BU-CPS-1B.

[0049] Figure 7 Figure 3 is the effect of Bacteroides monomorpha capsular polysaccharide BU-CPS-1B on the proliferation of lactic acid strains in vitro, where A to E correspond to Lactobacillus acidophilus CIP 76.13, Lactobacillus casei ATCC 393, Lactobacillus plantarum DSM 10667, Lactobacillus rhamnosus JSM1136 and Lactobacillus reuteri JCM 1112, respectively.

[0050] Figure 8 This is the effect of Bacteroides monocytogenes capsular polysaccharide BU-CPS-1B on the body weight of mice.

[0051] Fig. 9 This is the effect of Bacteroides monocytogenes capsular polysaccharide BU-CPS-1B on the growth of lactic acid strains in the intestine of mice. DETAILED DESCRIPTION

[0052] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0053] In the following examples, the commercial standard strain Bacteroides monomorpha ATCC 8492 was selected as the Bacteroides monomorpha. The Lactobacillus acidophilus used was Lactobacillus acidophilus CIP 76.13. The Lactobacillus casei used was Lactobacillus casei ATCC 393. The Lactobacillus plantarum used was Lactobacillus plantarum DSM 10667. The Lactobacillus reuteri used was Lactobacillus reuteri JCM 1112. The Lactobacillus rhamnosus used was Lactobacillus rhamnosus JSM1136.

[0054] Cultivation and identification of lactic acid strains

[0055] In the clean bench, wipe the cryovials containing Bacteroides monotheca ATCC 8492, Lactobacillus acidophilus CIP 76.13, Lactobacillus casei ATCC 393, Lactobacillus plantarum DSM 10667, Lactobacillus reuteri JCM1112 and Lactobacillus rhamnosus JSM1136 strains with cotton wool soaked in 75% alcohol, dry the alcohol, untie the wrapped sealing film, heat the tube mouth with an alcohol lamp, open the cryovial, add 500 μL of sterile water to the strain freeze-dried powder, dissolve and mix, pick the bacterial solution with a sterile inoculation loop and streak it on the Columbia blood plate, and culture it at 37°C under anaerobic conditions for 48 hours. After 48 hours, pick a single colony from the streaked plate and culture it on a new blood plate, and culture it at 37°C under anaerobic conditions for 48 hours.

[0056] Bacterial genomic DNA extraction: Use a bacterial genomic DNA extraction kit (Tiangen Biochemical Technology) to extract the genomic DNA of each strain obtained by culture. Refer to the instructions for specific operations. After obtaining the DNA, its DNA concentration and purity are tested. The OD260 / OD280 ratio is considered qualified in the range of 1.7-1.9.

[0057] PCR amplification: PCR amplification is performed using qualified DNA as a template.

[0058] The PCR amplification system is shown in Table 1.

[0059] Table 1 PCR amplification system

[0060] Components content Taq PCR Master Mix 12.5μL DNA template 0.5μL Upstream primer (F) 1μL Downstream primer (R) 1μL Sterile water 10μL Total volume 25μL

[0061] Among them, the upstream primer is 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 1);

[0062] The downstream primer is 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2).

[0063] The PCR amplification procedure was: 94°C pre-denaturation for 4 min; 94°C denaturation for 1 min, 55°C annealing for 1 min, 72°C extension for 1.5 min, 30 cycles; 72°C extension for 10 min. The amplified product was commissioned to Shanghai Sangon Biotechnology Co., Ltd. for 16S rRNA sequencing to confirm the correct strain.

[0064] Extraction and purification of capsular polysaccharide from Bacteroides monotheca

[0065] The confirmed Bacteroides monocytogenes ATCC 8492 was cultured in BHI liquid medium at 37°C for 24 hours. After activation for two generations, it was inoculated in BHI liquid medium at an inoculum size of 2% (v / v) for expansion culture at 37°C for 48 hours.

[0066] After 48 hours of cultivation, centrifuge the fermentation liquid at 4°C and 4500rpm for 30 minutes to obtain the bacteria (precipitate), and wash it twice with PBS. Repeat the freezing and thawing of the bacterial precipitate 10 times. Then add 10 times the volume of ddH2O for ultrasonic disruption for 20 minutes, with an interval of 30 seconds. Centrifuge at 4500rpm for 30 minutes and collect the supernatant. Repeat the above ultrasonic and centrifugation steps for the precipitate. Collect all the supernatants, concentrate under reduced pressure to 1 / 5 of the original volume, add pre-cooled 95% ethanol while stirring to make the final ethanol concentration in the solution 80%, and keep at 4°C overnight. Take it out the next day and centrifuge it at 4000rpm for 30 minutes to obtain the crude extract of Bacteroides monomorpha capsular polysaccharide after alcohol precipitation.

[0067] The crude extract of Bacteroides monomorpha capsular polysaccharide was deproteinized. The crude extract of Bacteroides monomorpha capsular polysaccharide was deproteinized by papain combined with Sevag method. The specific operation was as follows: the crude extract of Bacteroides monomorpha capsular polysaccharide was dissolved with an appropriate amount of ultrapure water, and then papain was added to make the final concentration of papain in the solution 1 mg / mL. The solution was placed in a water bath for 1 h under stirring at 60 ° C, and boiled at 100 ° C for 15 min to fully inactivate the papain. Centrifuge at 4000 rpm for 30 min, take the supernatant, add 1 / 4 volume of Sevag reagent (chloroform: n-butanol = 4:1), vortex for 10 min, and centrifuge at 4000 rpm for 20 min. Repeat the operation of the water layer until the white milky substance between the water layer and the chloroform layer is removed. Collect the water layer, concentrate under reduced pressure to remove the residual organic reagent, and lyophilize to obtain the deproteinized solid form of Bacteroides monomorpha capsular polysaccharide.

[0068] The solid Bacteroides monomorpha capsular polysaccharide was reconstituted with ultrapure water, ultrafiltered and washed using a 3KD ultrafiltration tube, and centrifuged at 4000 rpm, which was repeated three times. A small amount of liquid was retained for resuspending and freeze-drying to obtain a crude extract of Bacteroides monomorpha capsular polysaccharide.

[0069] The crude extract of Bacteroides monomorpha crude capsule polysaccharide was purified using DEAE cellulose-52 ion exchange cellulose chromatography column. Weigh 20g of DEAE Cellulose 52 ion exchange cellulose in a glass beaker, add enough ultrapure water to swell until the volume remains unchanged, first treat with 0.5mol / L NaOH solution for 1h, then wash repeatedly with ultrapure water until neutral, then treat with 0.5mol / L HCl solution for 1h, and wash with ultrapure water until neutral. The column size is 2.6×30cm. After cleaning, fix it vertically on the iron frame, add 1 / 3 column volume of ultrapure water, and open the liquid outlet. Then slowly pour the filler into the column along the glass rod, let it settle naturally, and gently tap the column with a soft rod to remove bubbles, then repeatedly add filler (DEAEcellulose-52 ion exchange cellulose) until it is 5cm away from the top of the column, stop loading the column, connect a constant flow pump, and balance with ultrapure water at a flow rate of 1.0mL / min. Weigh 50 mg of crude extract of Bacteroides monomorpha capsule polysaccharide and fully dissolve it in 10 mL of ultrapure water. After centrifugation at 4500 rpm for 15 min, take the supernatant and filter it with a 0.45 μm filter membrane. Load the sample, turn on the constant flow pump and start elution. The eluents are 0, 0.05, 0.1, 0.3, and 0.5 mol / L NaCl solutions, respectively, with a flow rate of 1.0 mL / min, and 8 mL is collected in each tube. The collected sample solution is detected by sulfuric acid phenol method at 490 nm to detect the absorbance, and the elution curve is drawn. According to the elution curve, the components under the peak of 0 mol / L NaCl solution are collected. Then the collected eluents are concentrated and washed with 3 kD ultrafiltration tubes, and then freeze-dried to obtain BU-CPS-1, which is placed in a dryer for storage.

[0070] Sephacryl S-300HR dextran gel was further purified. Sephacryl S-300HR is a pre-treated filler, stored in 20% ethanol, and washed with ultrapure water before use. The size of the chromatography column is 1.6×90cm. After cleaning, it is fixed vertically on the iron frame, 1 / 3 column volume of ultrapure water is added, and the liquid outlet is opened. Then slowly pour the filler into the chromatography column along the glass rod, let it settle naturally, and gently tap the column with a soft rod to remove bubbles. Then repeatedly add filler to the appropriate height. After the filler liquid level remains calm, connect a constant flow pump and rinse the dextran gel column with 5 times the column volume of 0.1mol / L NaCl solution to remove the residual ethanol in the filler and further compact the gel column filler. Then weigh 50mg of each component purified by DEAE 52, fully dissolve it in 10mL ultrapure water, centrifuge at 4500rpm for 15min, take the supernatant and filter it with a 0.45μm filter membrane. Use the filtrate as the sample, turn on the constant flow pump to start elution. The eluent is 0.1mol / L NaCl solution, the flow rate is 0.5mL / min, and 4mL is collected in each tube. The collected sample liquid is detected by the sulfuric acid phenol method, and an elution curve is drawn. According to the elution curve, the eluate under the elution peak is collected. The eluate is concentrated, dialyzed, and freeze-dried to obtain the purified Bacteroides monomorpha capsular polysaccharide (BU-CPS-1B). Among them, in this embodiment, the BU-CPS-1B component is selected as the Bacteroides monomorpha capsular polysaccharide for the following detection. The peak segment distribution of the BU-CPS-1B component is 16-32, and the yield is 84%. The gradient elution curves corresponding to the components of BU-CPS-1B are shown as follows. Figure 1 and 2 shown.

[0071] Take an appropriate amount of dried BU-CPS-1B and press it into sheets. Use ATR-FTIR to perform infrared spectrum test at wave number 4000-400cm -1 Infrared scanning within range.

[0072] The results are as follows Figure 3 As shown, infrared determination of BU-CPS-1B at 3367cm -1 There is strong absorption of hydroxyl groups.

[0073] Use ultraviolet spectroscopy to analyze whether the purified polysaccharide sample contains nucleic acids and proteins. The specific operation is: use ultrapure water to prepare BU-CPS-1B into a 1mg / mL solution, perform ultraviolet full wavelength scanning in the 190-400nm wavelength region, and observe whether there are absorption peaks at 260nm and 280nm to determine whether the purified polysaccharide sample contains nucleic acids and proteins.

[0074] The results are as follows Figure 4As shown, UV detection found no protein and nucleic acid absorption peaks at 260 / 280, indicating that the purified polysaccharide sample did not contain nucleic acids and proteins.

[0075] Using glucose as the standard, the total sugar content of BU-CPS-1B was determined by the phenol-sulfuric acid method. The total sugar content in the sample can be calculated based on the standard curve:

[0076]

[0077] Wherein, C is the concentration calculated according to the standard curve, M is the actual weighed mass, and V is the total volume of the extract.

[0078] The specific operation is as follows: weigh 2-5 mg of sample, add 1 mL of sterile water to dissolve, ultrasonically extract for 10 min, centrifuge at 12000 rpm for 10 min. Take 0.4 mL of supernatant, dilute with ddH2O and mix with phenol and concentrated sulfuric acid. After the reaction is completed, measure the absorbance at 490 nm and calculate the total sugar content based on the obtained glucose standard curve.

[0079] The glucose standard curve obtained by the sulfuric acid phenol method is y = 2.81x + 0.0551, R 2 = 0.9986. After the calculation, it was found that the total sugar content of BU-CPS-1B was 99.83%, and its sugar purity was high.

[0080] Gel Permeation Chromatography (GPC) was used to detect the relative molecular weight of the polysaccharide of BU-CPS-1B and to test its purity. The specific operation was as follows: first, different molecular weights of dextran standards were injected successively, and their retention times (TR) were recorded. A standard curve was drawn with the TR of each dextran standard as the horizontal coordinate and the logarithm of the corresponding molecular weight (Mw) as the vertical coordinate, and the regression equation of Lg (Mw) and TR was obtained, specifically: lg (Mw) = -1.0977 x TR + 12.271, and the correlation coefficient R 2 =0.994. The chromatographic conditions were as follows: the HPLC was Shimadzu LC-20AT, the chromatographic column was PolySep-GFC-P 4000 chromatographic column (Phenomenex, 300×7.8 mm), the detector was an evaporative light scattering detector (ELSD), the detector was set at 60°C, the gain value was 10, the column temperature was 35°C, ultrapure water was used as the mobile phase, the flow rate was 1.0 mL / min, and the injection volume was 20 μL for detection.

[0081] The relative molecular mass of BU-CPS-1B was determined by HPGPC method. According to the linear regression equation, the retention time TR = 7.59 min was substituted into the calculation to obtain the relative molecular mass of BU-CPS-1B as 1.819 × 10 4 Da.

[0082] The monosaccharide composition of BU-CPS-1B was identified. The monosaccharide composition of BU-CPS-1B was determined by 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization-high performance liquid chromatography. The specific steps are as follows: weigh 5 mg of BU-CPS-1B in a stoppered reaction tube, add 2 mL of trifluoroacetic acid (TFA), seal it, and hydrolyze it in an oil bath at 135°C for 3 hours. After the hydrolysis is complete, cool it. Add 5 mL of methanol and concentrate it under reduced pressure until it is spin-dried. Repeat the process 3 times to remove the residual TFA. Then add 800 μL of deionized water to dissolve it. Take 100 μL of the completely hydrolyzed polysaccharide solution, add 100 μL of 0.5 mol / L PMP methanol solution and 0.3 mol / L NaOH solution, mix well, place it in a 70°C water bath for reaction for 30 minutes, and add 105 μL of 0.3 mol / L HCl solution after cooling. Add 200 μL of ultrapure water to dilute. Then add 600 μL of chloroform solution, vortex mix and centrifuge (10000 rpm, 15 min), discard the lower layer of chloroform, repeat 3 times to remove residual PMP. Collect the water layer and filter it through a 0.45 μm filter membrane and use HPLC for detection. The chromatographic conditions are as follows: the chromatographic system uses the Thermo ICS5000 ion chromatography system (ICS5000, Thermo Fisher Scientific, USA), and the monosaccharide components are analyzed and detected using an electrochemical detector. The chromatographic column is Dionex TM CarboPac TMPA20 (150*3.0mm, 10μm) liquid chromatography column. The injection volume is 5μL. Mobile phase A (H2O), mobile phase B (0.1M NaOH), mobile phase C (0.1M NaOH, 0.2M NaAc), flow rate 0.5ml / min; column temperature was 30℃; elution gradient: 0min, phase A / phase B / phase C (95:5:0, V / V); 26min, phase A / phase B / phase C (85:5:10, V / V); 42min, phase A / phase B / phase C (85:5:10, V / V); 42.1min, phase A / phase B / phase C (60:0:40, V / V); 52min, phase A / phase B / phase C (60:40:0, V / V); 52.1min, phase A / phase B / phase C (95:5:0, V / V); 60min, phase A / phase B / phase C (95:5:0, V / V). By comparing the elution time and peak area of ​​each standard monosaccharide (after derivatization treatment), the BU-CPS-1B component was qualitatively and quantitatively analyzed to obtain the monosaccharide composition and molar ratio of BU-CPS-1.

[0083] The results are as follows Figure 5 and Figure 6 shown.

[0084] The ion chromatogram of capsular polysaccharide ( Figure 5 ) and the peak diagram of the derivatization product of mixed monosaccharide standard ( Figure 6 ), according to the retention time, it can be known that the monomorphic Bacteroides capsular polysaccharide BU-CPS-1B is mainly composed of arabinose, galactose, glucose, galacturonic acid and glucuronic acid, and the molar ratio of arabinose, galactose, glucose, galacturonic acid and glucuronic acid is 0.51:0.25:300.19:1.95:1.72. And through database comparison, it was found that the monosaccharide composition of BU-CPS-1B in the present invention is different from the types and proportions of the monosaccharide composition in the polysaccharides obtained from the existing Bacteroides genus (including Bacteroides fragilis), and it is speculated that it may be a new structure of bacterial extracellular heteropolysaccharide.

[0085] Effect of Bacteroides monotheca capsular polysaccharide on the proliferation of lactic acid bacteria in vitro

[0086] (1) Preparation of Bacteroides monomorpha capsular polysaccharide BU-CPS-1B solution:

[0087] Take 100 mg of the purified Bacteroides monotheca capsular polysaccharide BU-CPS-1B obtained in the above example, inoculate it into MRS medium (liquid) according to the amount of 0.2%, 1% and 2% (V / V), and filter it with a 0.22 μm microporous filter membrane to obtain the Bacteroides monotheca capsular polysaccharide BU-CPS-1B solution for in vitro proliferation experiment.

[0088] (2) Effect of Bacteroides monocytogenes capsular polysaccharide BU-CPS-1B on the proliferation of lactic acid bacteria in vitro:

[0089] Take the lactic acid bacteria (Lactobacillus acidophilus CIP 76.13, Lactobacillus casei ATCC 393, Lactobacillus plantarum DSM 10667, Lactobacillus reuteri JCM 1112 and Lactobacillus rhamnosus JSM1136) revived in the above-mentioned embodiment, and dilute to OD600=0.2 with PBS after passage three generations. After further dilution 5 times, plate is plated, and equal volumes of Bacteroides monomorpha capsular polysaccharide BU-CPS-1 solution of different concentrations (0%, 0.1%, 0.5% and 1%) are added. Place in an anaerobic incubator, detect the OD600 of bacteria at different time points (0, 12, 24, 36 and 48h) and draw a growth curve.

[0090] The results are as follows Figure 7 shown.

[0091] In order to explore the effect of the Bacteroides monomorpha capsular polysaccharide in the embodiments of the present invention on the growth of lactic acid bacteria, the five most widely used representative strains of lactic acid bacteria were selected, including Lactobacillus acidophilus CIP 76.13, Lactobacillus casei ATCC 393, Lactobacillus plantarum DSM 10667, Lactobacillus reuteri JCM 1112 and Lactobacillus rhamnosus JSM1136. Under anaerobic conditions, the effect of adding 0.1%. 0.5% and 1% concentrations of Bacteroides monomorpha capsular polysaccharides on the growth of different lactic acid strains was observed. The results showed that with the increase of time and concentration, Bacteroides monomorpha capsular polysaccharides had a significant promoting or stimulating effect on the growth of Lactobacillus acidophilus, Lactobacillus casei and Lactobacillus plantarum, while the promoting effect on the proliferation of Lactobacillus reuteri and Lactobacillus rhamnosus was relatively small. The in vivo proliferation effect of Bacteroides monomorpha capsular polysaccharides on lactic acid bacteria

[0092] Fifteen six-week-old female C57BL / 6 mice were randomly divided into two groups (7-8 mice in each group) according to body weight after one week of adaptive feeding: a normal control group (Control) and a Bacteroides monotheca capsular polysaccharide group (BU-CPS). The normal group was gavaged with 200 μL PBS, and the BU-CPS group was gavaged with 200 μL of 1 mg / mL Bacteroides monotheca capsular polysaccharide BU-CPS-1B solution every day.

[0093] The weight changes of mice during the experiment were also recorded.

[0094] On the day the experiment ended, mouse feces were collected, and total DNA (as a DNA template) was obtained using a fecal genomic DNA extraction kit (Novozyme), followed by quantitative determination using a qPCR system.

[0095] The qPCR amplification system is shown in Table 2.

[0096] Table 2 qPCR amplification system

[0097]

[0098]

[0099] Among them, the selection of upstream and downstream primers is shown in Table 3.

[0100] Table 3 Primer sequences corresponding to each lactic acid bacteria

[0101]

[0102] The qPCR amplification program was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, and 45 cycles; the melting curve program was set to 95°C for 1 s; 65°C for 15 s, sample collection for 15 times, and maintenance at 95°C.

[0103] The results are as follows Figure 8-Figure 9 shown.

[0104] In order to explore the safety of Bacteroides monotheca capsular polysaccharides, the inventors used Bacteroides monotheca capsular polysaccharides to gavage mice and observed their weight changes for 9 consecutive days. The results showed that after gavage with Bacteroides monotheca capsular polysaccharides, the weight of mice remained stable and had a certain upward trend, indicating that Bacteroides monotheca capsular polysaccharides had no obvious toxic side effects and could benefit the growth and development of animals to a certain extent.

[0105] In addition, in order to explore the effect of Bacteroides monomorpha capsular polysaccharides on the growth of lactic acid bacteria in animals, the effect of gavage of Bacteroides monomorpha capsular polysaccharides on the relative abundance of lactic acid bacteria and lactic acid strains in vivo was explored by quantitatively measuring the microbial specific genes in mouse feces. The results show that Bacteroides monomorpha capsular polysaccharides can stimulate the growth of lactic acid bacteria in vivo, especially for Lactobacillus acidophilus and Lactobacillus casei. It has a very significant growth-promoting effect, while it has a certain promoting effect on Lactobacillus reuteri and Lactobacillus rhamnosus, but the effect is not very significant. As for Lactobacillus plantarum, since it was not detected in the feces, its actual in vivo effect cannot be determined.

[0106] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A Bacteroides monomorpha capsular polysaccharide, characterized in that The Bacteroides monomorpha capsule polysaccharide is composed of arabinose, galactose, glucose, galacturonic acid and glucuronic acid, and the molar ratio of arabinose, galactose, glucose, galacturonic acid and glucuronic acid is 0.51:0.25:300.19:1.95:1.72; The Bacteroides monomorpha capsular polysaccharide is extracted by an extraction method comprising the following steps: (1) After repeated freezing and thawing, the Bacteroides monocytogenes was ultrasonically disrupted, the supernatant was collected by centrifugation, concentrated under reduced pressure, and then precipitated with alcohol; (2) Deproteinizing the precipitate from the alcohol, washing it with ultrafiltration, and then performing chromatography on ion exchange cellulose, eluting it with NaCl, and collecting the eluate in the 0 mol / L peak to obtain the Bacteroides monomorpha capsular polysaccharide; The deproteinization treatment comprises: firstly performing enzymatic hydrolysis with papain, and then further removing the protein with Sevag reagent; The ultrafiltration uses a 3 KD ultrafiltration tube; The ion exchange cellulose is DEAE cellulose-52 ion exchange cellulose; The extraction method further comprises using a dextran gel chromatography column for further purification; The dextran gel is Sephacryl S-300 HR dextran gel; The eluent of the dextran gel chromatography is 0.1 mol / L NaCl solution, the flow rate is 0.5 mL / min, 4 mL is collected in each tube, and the collection peak segment distribution is 16-32.

2. The method for extracting Bacteroides monomorpha capsular polysaccharide according to claim 1, comprising the following steps: (1) After repeated freezing and thawing, the Bacteroides monocytogenes was ultrasonically disrupted, the supernatant was collected by centrifugation, concentrated under reduced pressure, and then precipitated with alcohol; (2) Deproteinizing the precipitate from the alcohol, washing it with ultrafiltration, and then performing chromatography on ion exchange cellulose, eluting it with NaCl, and collecting the eluate in the 0 mol / L peak to obtain the Bacteroides monomorpha capsular polysaccharide; The deproteinization treatment comprises: firstly performing enzymatic hydrolysis with papain, and then further removing the protein with Sevag reagent; The extraction method further comprises using a dextran gel chromatography column for further purification; The dextran gel is Sephacryl S-300 HR dextran gel; The eluent of the dextran gel chromatography was 0.1 mol / L NaCl solution, the flow rate was 0.5 mL / min, 4 mL was collected in each tube, and the collection peak segment distribution was 16-32.

3. Use of a Bacteroides monotheca extract as the sole active ingredient in promoting the growth or proliferation of lactic acid bacteria; the extract is the Bacteroides monotheca capsular polysaccharide as described in claim 1.

4. Use of a Bacteroides monotheca extract in the preparation of a product for improving the abundance of lactic acid bacteria in an in vivo environment; the extract is the Bacteroides monotheca capsular polysaccharide according to claim 1.

5. The use according to claim 3 or 4, characterized in that: The lactic acid bacteria include Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus reuteri and Lactobacillus rhamnosus.

6. The use according to claim 4, characterized in that: The products include feed, feed additives, food, food additives and medicines.

7. The use according to claim 6, characterized in that: The product also includes auxiliary materials acceptable in feed, food or pharmacy.

8. The use according to claim 4, characterized in that: The content of the Bacteroides monomorpha extract in the product is 0.1-100wt%.

9. The use according to claim 4, characterized in that: The in vivo environment is the gastrointestinal tract environment.

Images