Method for producing methyl glucuronic acid xylotriose and xylopentasaccharide
Through the composite bacteria system, beech xylan is degraded in the fermentation medium, and high-purity xytriose and xylenose are produced, which solves the problems of high production costs and complex processes of acidic oligosaccharides. Achieving low-cost and efficient product acquisition.
Patent Information
- Application Number
- CN202411104247.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-13
- Publication Date
- 2024-12-13
AI Technical Summary
The existing production methods of acidic xylosol rely on enzyme methods, which are costly and complex in technology, making it difficult to achieve efficient production of a single product, which limits its application in the fields of functional food, medicine and feed additives.
Using a complex bacteria system, including Bacillus amyloligosaccharide and E. coli, the degradation of beech xytriose and xylenose in the fermentation medium was used to produce xytriose methylglucuronate and xylenose, and a high-purity product was obtained by chromatographic purification technology.
It reduces production costs, simplifies the process flow, is suitable for industrial production, and does not produce harmful gases and wastewater, and improves the purity and yield of the product.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for producing methyl glucuronic acid xylotriose and xylopentaose. Background Art
[0002] Xylooligosaccharides are a type of prebiotics. They can effectively promote the proliferation of beneficial bacteria in the body and inhibit the growth of harmful bacteria, ultimately leading to a balance in the microecological flora in the body. They have the effects of enhancing immunity and promoting the absorption of nutrients, and have broad application prospects in the fields of medicine, food, and feed. Acidic xylooligosaccharides are xylooligosaccharides with methyl glucuronic acid side chains on the main chain of xylose connected by 1,4 glycosidic bonds. The latest research shows that methyl glucuronic acid modification can give xylooligosaccharides better antioxidant effects and increase the solubility of xylooligosaccharides. However, there are still no commercialized acidic xylooligosaccharides, which greatly limits their application in functional foods, medicines, and feed additives.
[0003] Bacillus has GH11 and GH30 xylanases. The former acts on methyl glucuronyl xylan to produce xylobiose and xylotriose that are easily absorbed and utilized by Bacillus, while the latter acts on methyl glucuronyl xylan to produce methyl glucuronyl oligoxylose that cannot be absorbed and utilized by Bacillus, in which the methyl glucuronyl group is connected to the penultimate xylose of the reducing end xylose with a 1,2 bond.
[0004] The production methods of acidic xylooligosaccharides in the literature are mainly based on enzymatic methods. The production cost of enzymes is high, and the enzymatic hydrolysis reaction is sensitive to factors such as temperature, pH, and salt concentration. Separating xylooligosaccharides from the enzymatic hydrolysis product requires graded alcohol precipitation to remove polysaccharides, which is costly and time-consuming. The separated xylooligosaccharides are generally a mixture with a degree of polymerization of 3 to 8 after mass spectrometry analysis. In order to obtain a single xylooligosaccharide, the synergistic action of multiple enzymes is generally required, and the process complexity is relatively high. Summary of the invention
[0005] The first aspect of the present invention aims to provide a composite bacteria.
[0006] The second aspect of the present invention aims to provide an application.
[0007] The third aspect of the present invention aims to provide a method.
[0008] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:
[0009] The first aspect of the present invention provides a composite bacteria, comprising Bacillus and xylose-utilizing bacteria.
[0010] Preferably, the Bacillus comprises Bacillus amyloliquefaciens PM415, with a deposit number of CGMCC No. 26918, which is used to produce methyl glucuronide xylotriose.
[0011] Preferably, the Bacillus includes Bacillus pumilus, with the deposit number of CMCCB63202, which can be used to produce methyl glucuronide xylopentose.
[0012] Preferably, the inoculation ratio of the Bacillus and the xylose utilizing bacteria is 1:500 to 1:50.
[0013] Preferably, the xylose-utilizing bacteria include lactic acid bacteria, Enterococcus faecalis, and Escherichia coli.
[0014] Preferably, the Escherichia coli includes Escherichia coli BL21 and Escherichia coli DH5α.
[0015] Preferably, the lactic acid bacteria include Lactobacillus furfurali.
[0016] The second aspect of the present invention provides the use of the composite bacteria in the preparation of methyl glucuronic acid xylotriose and / or xylopentaose.
[0017] The third aspect of the present invention provides a method for preparing methyl glucuronic acid xylotriose and / or xylopentaose, comprising inoculating the composite bacteria of the first aspect of the present invention into a fermentation medium and culturing the mixture.
[0018] The methyl glucuronyl xylotriose is 4-O-methyl glucuronyl xylotriose, and its PubChem CID is 91847053.
[0019] The methyl glucuronic acid xylopentose is 4-O-methyl glucuronic acid-xylopentose, and the structure is based on 4-O-methyl glucuronic acid-xylopentose, and has two more xylose groups on the main chain.
[0020] Preferably, the fermentation medium comprises a carbon source.
[0021] Preferably, the carbon source comprises xylan.
[0022] Preferably, the xylan comprises beech xylan.
[0023] Preferably, the mass percentage of the carbon source in the fermentation medium is 0.5% to 2%.
[0024] Preferably, the fermentation medium further comprises a nitrogen source and an inorganic salt.
[0025] Preferably, the nitrogen source comprises ammonium sulfate.
[0026] Preferably, the inorganic salt includes at least one of sodium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and magnesium chloride.
[0027] Preferably, the culture medium comprises, by mass fraction, 0.5-2% beech xylan, 0.1%-0.2% (NH4)2SO4, 0.06%-0.09% NaCl, 0.06%-0.1% KH2PO4, 0.01-0.09% K2HPO4, 0.002-0.005% MgCl2 and the balance water.
[0028] Preferably, the pH of the culture medium is 7.0-8.0.
[0029] Preferably, the culture temperature is 30-40°C.
[0030] Preferably, the culturing time is 20 to 90 hours.
[0031] Preferably, purification is further included after the culturing is completed.
[0032] Preferably, said purification comprises chromatographic purification.
[0033] Preferably, the chromatographic purification includes at least one of a reverse phase chromatography solid phase extraction column, a graphitized carbon column solid phase extraction column, and an ion exchange chromatography.
[0034] Preferably, the specific process of the reverse phase chromatography includes: using reverse phase chromatography to purify and remove polypeptides and proteins in the filtrate, first activating the C18 reverse phase column with 1-3 mL / 100 mg of methanol as adsorbent, then balancing the column with 1-3 mL / 100 mg of 5% acetic acid as adsorbent and discarding the filtrate, loading the supernatant and starting to collect the filtrate, washing and collecting the filtrate with 1-3 mL / 100 mg of 5% acetic acid as adsorbent, and finally eluting with 1-3 mL / 100 mg of 5% acetic acid as adsorbent and collecting the filtrate.
[0035] Further preferably, the filtrate obtained by reverse phase column chromatography is further subjected to graphitized carbon column chromatography. The purification steps are: firstly activate the graphitized carbon column with 3-5 mL / 100 mg of acetonitrile of the adsorbent, then balance the column with 3-5 mL / 100 mg of ultrapure water of the adsorbent, load the filtrate collected in the reverse phase chromatography, wash with 2-4 mL / 100 mg of ultrapure water of the adsorbent, and finally elute with 1-3 mL / 100 mg of 5% acetic acid of the adsorbent and collect the filtrate, so as to obtain methyl glucuronide xylotriose or methyl glucuronide xylopentaose with high purity.
[0036] Preferably, the purification further includes centrifugation and filtration steps.
[0037] Preferably, the centrifugation is specifically to centrifuge the fermentation broth at 5000-8000 g for 10-30 min.
[0038] Preferably, the pore size of the filter is 0.22-0.45 μm.
[0039] The beneficial effects of the present invention are:
[0040] The present invention provides a method for producing methyl glucuronyl xylotriose and xylopentose, wherein Bacillus amyloliquefaciens PM415 or Bacillus pumilus is fermented with Escherichia coli BL21 and purified. The method is to produce methyl glucuronyl xylotriose or methyl glucuronyl xylopentose in the process of degrading and utilizing beech xylan by Bacillus amyloliquefaciens PM415 or Bacillus pumilus, and then utilize Escherichia coli BL21 to metabolize and remove xylose in the product, and finally purify from the fermentation liquid to obtain methyl glucuronyl xylotriose or methyl glucuronyl xylopentose. The method has low production cost, short time consumption, does not require high temperature, high pressure and a large amount of energy input, does not require expensive raw materials and cumbersome process flow, is suitable for industrial production, does not produce harmful gases and wastewater, is environmentally friendly, has simple products and high yield, and facilitates subsequent separation and purification steps. BRIEF DESCRIPTION OF THE DRAWINGS
[0041] Figure 1 Schematic diagram of the structure of methyl glucuronyl xylotriose and methyl glucuronyl xylopentaose.
[0042] Figure 2 Schematic diagram of the product results obtained by single and mixed fermentation of Bacillus amyloliquefaciens PM415.
[0043] Figure 3 The figures are the results of producing methyl glucuronide xylotriose by strains with different inoculation ratios; A is the result of producing methyl glucuronide xylotriose by fermentation of Bacillus amyloliquefaciens PM415 with different inoculation ratios; B is the result of producing methyl glucuronide xylotriose by fermentation of Escherichia coli BL21 with different inoculation ratios.
[0044] Figure 4 These are the results of the combined fermentation of different xylose-utilizing bacteria with Bacillus amyloliquefaciens PM415 to produce methylglucuronide xylotriose.
[0045] Figure 5 Schematic diagram of the product results obtained by fermentation of single and mixed bacteria of Bacillus pumilus.
[0046] Figure 6 These are the results of producing methyl glucuronide xylopentose by strains at different inoculation ratios; A is the result of producing methyl glucuronide xylopentose by fermentation of Bacillus pumilus at different inoculation ratios; B is the result of producing methyl glucuronide xylopentose by fermentation of Escherichia coli BL21 at different inoculation ratios.
[0047] Figure 7 These are the results of the co-fermentation of different xylose-utilizing bacteria with Bacillus pumilus to produce methylglucuronic acid xylopentose.
[0048] Figure 8 These are the mass spectrometry analysis results of the products obtained from the combined fermentation of Bacillus amyloliquefaciens PM415 and Escherichia coli BL21.
[0049] Fig. 9 This is the mass spectrometry analysis result of the product obtained by the combined fermentation of Bacillus pumilus and Escherichia coli BL21. DETAILED DESCRIPTION
[0050] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.
[0051] Example 1 Production of methyl glucuronic acid xylotriose by mixed bacterial fermentation
[0052] 1. Experimental Materials
[0053] Bacillus amyloliquefaciens PM415 was deposited on March 28, 2023 at the General Microbiology Center of China Culture Collection Administration (CGMCC), address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, deposit number CGMCC No. 26918, and has been disclosed in the previous patent CN116875497A.
[0054] Escherichia coli BL21 was purchased from Shanghai Angyu Biotechnology Co., Ltd.
[0055] 2. Fermentation method
[0056] The frozen bacterial liquid of Bacillus amyloliquefaciens PM415 and Escherichia coli BL21 were spread on solid LB plates and activated at 37℃ overnight. On the next day, a single clone was picked from the activated bacterial plate and inoculated into 2mL liquid LB medium. The culture was cultured at 37℃ and 200r / min for 16 hours to obtain the activated bacterial liquid of Bacillus amyloliquefaciens PM415 and Escherichia coli BL21. 100μL of activated bacterial liquid of Bacillus amyloliquefaciens PM415 was drawn into 10mL fermentation medium (inoculation ratio 1:100), and after culturing at 200r / min and 37℃ for 24h, 100μL of activated bacterial liquid of Escherichia coli BL21 (inoculation ratio 1:100) was added, and the culture was continued at 200r / min and 37℃ for 24h. Centrifuge at 8000g for 10min, collect the supernatant and filter through a 0.22μm filter membrane, which can then be used for TLC experiments or further purified for mass spectrometry analysis.
[0057] Liquid LB medium: by mass fraction (g / 100mL), including 0.5% yeast powder, 1% tryptone, 1% sodium chloride, pH 7.0, the solvent is water, and the preparation method is to mix the components evenly and sterilize at 121°C for 20 minutes.
[0058] Solid LB medium: calculated by mass fraction (g / 100mL), including 0.5% yeast powder, 1% tryptone, 1% sodium chloride, 1.5% agar, pH 7.0, solvent is water, and the preparation method is to mix the components evenly and sterilize at 121°C for 20 minutes.
[0059] Fermentation medium: by mass fraction (g / 100 mL), including 1% beech xylan (Shanghai Yuanye Biotechnology Co., Ltd.), 0.2% (NH4)2SO4, 0.08% NaCl, 0.06% KH2PO4, 0.09% K2HPO 4、 0.002% MgCl2, pH 7.0-8.0, solvent is water, sterilize at 121℃ for 20min.
[0060] Example 2 Production of methyl glucuronide xylotriose by different PM415 inoculation ratios
[0061] This embodiment is substantially the same as embodiment 1, except that the inoculation ratio of Bacillus amyloliquefaciens PM415 is 1:50 and 1:500.
[0062] Example 3 Production of methyl glucuronide xylotriose by different BL21 inoculation ratios
[0063] This embodiment is substantially the same as embodiment 1, except that the inoculation ratios of Escherichia coli BL21 are 1:50 and 1:500.
[0064] Example 4 Co-production of methyl glucuronide xylotriose by different xylose-utilizing bacteria and PM415
[0065] This example is basically the same as Example 1, except that, in addition to Escherichia coli BL21, the effects of other xylose-utilizing bacteria, namely Enterococcus faecium (Guangdong Provincial Microbiological Culture Collection Center GDMCC No. 1.130) and Lactobacillus pyrenoidosa (China General Microbiological Culture Collection Center CGMCC No. 1.15605), on removing xylose were also compared.
[0066] Example 5 Production of methyl glucuronic acid xylopentose by mixed bacterial fermentation
[0067] This embodiment is basically the same as embodiment 1, except that Bacillus amyloliquefaciens PM415 is replaced by Bacillus pumilus.
[0068] Example 6 Production of methyl glucuronide xylopentose by different inoculation ratios of Bacillus pumilus
[0069] This embodiment is basically the same as embodiment 2, except that Bacillus amyloliquefaciens PM415 is replaced by Bacillus pumilus.
[0070] Example 7 Production of methyl glucuronide xylopentose by different BL21 inoculation ratios
[0071] This example is basically the same as Example 3, except that Bacillus amyloliquefaciens PM415 is replaced by Bacillus pumilus.
[0072] Example 8 Co-production of methyl glucuronide xylopentaose by different xylose-utilizing bacteria and Bacillus pumilus
[0073] This example is basically the same as Example 4, except that Bacillus amyloliquefaciens PM415 is replaced by Bacillus pumilus.
[0074] Comparative Example 1 Production of methyl glucuronide xylotriose by fermentation with Bacillus amyloliquefaciens
[0075] 1. Experimental Materials
[0076] The Bacillus amyloliquefaciens used in this comparative example is the same as that in Example 1.
[0077] 2. Fermentation method
[0078] Spread the frozen bacterial solution of Bacillus amyloliquefaciens PM415 on a solid LB plate and activate it at 37°C overnight. On the second day, pick a single clone from the activated bacterial plate and inoculate it into 2 mL of liquid LB medium. Culture it at 37°C and 200 r / min for 16 hours to obtain the activated bacterial solution of Bacillus amyloliquefaciens PM415. The same method can be used to obtain the activated bacterial solution of BL21.
[0079] 100 μL of activated bacterial liquid of Bacillus amyloliquefaciens PM415 was inoculated into 10 mL of fermentation medium to a final concentration of 100 CFU / mL, and cultured at 200 r / min and 37°C for 24 h. 100 μL of activated bacterial liquid of BL21 was inoculated and cultured at 200 r / min and 37°C for 24 h. The culture was centrifuged at 8000 g for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane.
[0080] Comparative Example 2 Blank Control
[0081] The fermentation medium was not inoculated with any bacterial liquid, cultured at 200 r / min and 37°C for 48 h, centrifuged at 8000 g for 10 min, and the liquid was collected and filtered through a 0.22 μm filter membrane.
[0082] Comparative Example 3 Production of methyl glucuronic acid xylopentose by fermentation with Bacillus pumilus
[0083] This comparative example is basically the same as comparative example 1, except that Bacillus amyloliquefaciens is replaced by Bacillus pumilus. Effect Example 1 Thin Layer Chromatography (TLC) Identification of Oligosaccharide Components in Filtrate
[0084] The filtrate samples obtained in Example 1 and Comparative Examples 1-2 were subjected to thin layer chromatography identification, and the standard substances were xylose (X1), xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentose (X5), and xylohexaose (X6). 6 μg of each standard substance and 10 μL of sample were spotted on a silica gel thin layer chromatography plate (Merck, item number 105729). The developing agent was n-butanol: acetic acid: water in a volume ratio of 2:1:1. The developing time was about 1 hour. The color development method was to blow dry, spray the color developer, and heat at 85°C for 10 minutes. The formula of the color developer was: 200 mL acetone, 4 mL aniline, 4 g diphenylamine, and 20 mL 85% phosphoric acid.
[0085] The structure of methyl glucuronide xylotriose is as follows Figure 1 As shown in A, the TLC results are as follows Figure 2 As shown, sample No. 3 is a blank control with no colored bands. Sample No. 4, i.e., the 24h fermentation broth of Bacillus amyloliquefaciens PM415, has two main bands, namely, xylose and methyl glucuronide xylotriose (predicted by the xylanase system). Sample No. 5, i.e., the fermentation broth obtained by co-culturing Bacillus amyloliquefaciens PM415 and Escherichia coli BL21 for 24h, lacks the band corresponding to xylose compared to sample No. 4, which is consistent with the characteristic that Escherichia coli can utilize xylose, and methyl glucuronide xylotriose can be purified from the sample later. Effect Example 2 Production of methyl glucuronide xylotriose at different PM415 inoculation ratios
[0086] The results of fermentation of methylglucuronic acid xylotriose by Bacillus amyloliquefaciens PM415 with different inoculation ratios are shown in Figure 2. Figure 3 As shown in A, the inoculation ratio as high as 1:50 does not affect the type of the final product. Although the inoculation ratio as low as 1:500 does not show the band of methyl glucuronide xylotriose at 24h, the band of the product can be seen after 48h of fermentation. Therefore, the fermentation inoculation ratio of PM415 can range from 1:500 to 1:50.
[0087] Effect Example 3 Production of methyl glucuronide xylotriose by different BL21 inoculation ratios
[0088] The results of fermentation of methylglucuronide xylotriose by Escherichia coli BL21 with different inoculation ratios are shown in Figure 3 As shown in B, the results obtained with the inoculation ratios of 1:50 and 1:100 are similar to those obtained in Example 1 ( Figure 2 ) are the same. Therefore, the fermentation inoculation ratio of BL21 can range from 1:500 to 1:50.
[0089] Effect Example 4 Production of methyl glucuronide xylotriose by fermentation with different xylose-utilizing bacteria
[0090] According to the method in Example 1, replacing Escherichia coli BL21 with Enterococcus faecium (Guangdong Provincial Microbiological Culture Collection Center GDMCC No. 1.130) or Lactobacillus granariensis (China General Microbiological Culture Collection Center CGMCC No. 1.15605) can also absorb xylose monosaccharide in the fermentation broth ( Figure 4 ). Therefore, the method of the present invention is also applicable to other xylose-utilizing bacteria including Enterococcus faecium and Lactobacillus cerebri.
[0091] Effect Example 5 Production of methyl glucuronic acid xylopentose by mixed bacterial fermentation
[0092] The filtrate samples obtained in Example 5 and Comparative Examples 2-3 were subjected to thin layer chromatography identification, and the standard substances were xylose (X1), xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentose (X5), and xylohexaose (X6). 6 μg of each standard substance and 10 μL of sample were spotted on a silica gel thin layer chromatography plate (Merck, item number 105729). The developing agent was n-butanol: acetic acid: water in a volume ratio of 2:1:1. The developing time was about 1 hour. The color development method was to blow dry, spray the color developer, and heat at 85°C for 10 minutes. The formula of the color developer was: 200 mL acetone, 4 mL aniline, 4 g diphenylamine, and 20 mL 85% phosphoric acid.
[0093] The structure of methyl glucuronide xylopentaose is as follows Figure 1 As shown in B, the TLC results are Figure 5As shown, Blank sample is a blank control without color band. Sample 103925 (24h) is the fermentation broth of Bacillus pumilus for 24h, and the position corresponding to X6 is speculated to be methyl glucuronide xylopentose (predicted by the xylanase system). Sample 103925+BL21 (48h) is the fermentation broth obtained by co-culturing Bacillus pumilus and Escherichia coli BL21 for 24h (total fermentation time 48h). Compared with sample 103925 (24h), the band corresponding to xylose is missing, which is consistent with the characteristic that Escherichia coli can utilize xylose. Methyl glucuronide xylopentose can be purified from this sample later.
[0094] Effect Example 6 Production of methyl glucuronic acid xylopentose by different inoculation ratios of Bacillus pumilus
[0095] The results of fermentation of methylglucuronic acid xylopentose by Bacillus pumilus with different inoculation ratios are shown in Figure 2. Figure 6 As shown in A, the inoculation ratio as high as 1:50 or as low as 1:500 does not affect the position of the band of the final product. Therefore, the fermentation inoculation ratio of Bacillus pumilus can range from 1:500 to 1:50.
[0096] Effect Example 7 Production of methyl glucuronide xylopentose by different BL21 inoculation ratios
[0097] The results of fermentation of methylglucuronic acid xylopentose by Escherichia coli BL21 with different inoculation ratios are shown in Figure 6 As shown in B, the results obtained with inoculation ratios of 1:50 and 1:500 are the same. Therefore, the fermentation inoculation ratio of BL21 can range from 1:500 to 1:50.
[0098] Effect Example 8 Production of methyl glucuronide xylopentose by different xylose-utilizing bacteria
[0099] According to the method in Example 5, replacing Escherichia coli BL21 with Enterococcus faecium (Guangdong Provincial Microbiological Culture Collection Center GDMCC No. 1.130) or Lactobacillus granariensis (China General Microbiological Culture Collection Center CGMCC No. 1.15605) can also absorb xylose monosaccharide in the fermentation broth ( Figure 7 ). Therefore, the method of the present invention is also applicable to other xylose-utilizing bacteria including Enterococcus faecium and Lactobacillus cerebri.
[0100] Effect Example 9 Purification and Identification of Methyl Glucuronide Xylotriose
[0101] 800 μL of the filtrate obtained in Example 1 was taken and purified with 100 mg of C18 adsorbent. First, the C18 reverse phase column was activated with 3 mL of methanol and the filtrate was discarded, then the column was balanced with 3 mL of 5% acetic acid and the filtrate was discarded, 800 μL of the filtrate was loaded and collected, and the filtrate was washed with 3 mL of 5% (v / v) acetic acid and the filtrate was collected, and finally eluted with 3 mL of 5% acetic acid and the filtrate was collected.
[0102] Prepare 100 mg of graphitized carbon, first activate the graphitized carbon column with 3 mL of acetonitrile, then balance the column with 3 mL of ultrapure water, load the filtrate collected in the previous step, wash with 4 mL of ultrapure water, and finally elute with 3 mL of 5% acetic acid and collect the filtrate to obtain methyl glucuronide xylotriose with high purity. After drying with nitrogen, weigh and obtain 4.5 mg of dry powder.
[0103] The purified methyl glucuronide xylotriose was prepared into a 10 mg / mL solution with deionized water and then subjected to ESI-MS detection using negative ion mode. Figure 8 As shown, the peak with m / z of 603.43 is methyl glucuronic acid xylotriose after losing hydrogen ions, and no other oligoxylose exists, and the purity is relatively high.
[0104] Effect Example 10 Purification and Identification of Methyl Glucuronide Xylopentaose
[0105] 800 μL of the filtrate obtained in Example 5 was taken and purified with 100 mg of C18 adsorbent. First, the C18 reverse phase column was activated with 3 mL of methanol and the filtrate was discarded, then the column was balanced with 3 mL of 5% acetic acid and the filtrate was discarded, 800 μL of the sample was loaded and the filtrate was collected, and the filtrate was washed with 3 mL of 5% (v / v) acetic acid and the filtrate was collected, and finally eluted with 3 mL of 5% acetic acid and the filtrate was collected.
[0106] Prepare 100 mg of graphitized carbon, first activate the graphitized carbon column with 3 mL of acetonitrile, then balance the column with 3 mL of ultrapure water, load the filtrate collected in the previous step, wash with 4 mL of ultrapure water, and finally elute with 3 mL of 5% acetic acid and collect the filtrate to obtain methyl glucuronide xylopentose with high purity. After drying with nitrogen, weigh and obtain 1 mg of dry powder.
[0107] The purified methyl glucuronide xylopentose was prepared into a 10 mg / mL solution with deionized water and then subjected to ESI-MS detection using negative ion mode. Fig. 9 As shown, the peak with m / z of 867.17 is methyl glucuronic acid xylopentose after losing hydrogen ions, and no other oligoxylose exists, and the purity is relatively high.
Claims
1. A composite bacteria comprising Bacillus and xylose-utilizing bacteria.
2. The composite bacteria according to claim 1, characterized in that: The xylose-utilizing bacteria include at least one of lactic acid bacteria, Enterococcus faecalis, and Escherichia coli; Preferably, the Escherichia coli includes at least one of Escherichia coli BL21 and Escherichia coli DH5α.
3. The composite bacteria according to claim 2, characterized in that: The Bacillus is at least one of Bacillus amyloliquefaciens and Bacillus pumilus.
4. Use of the composite bacteria according to any one of claims 1 to 3 in the preparation of methyl glucuronic acid xylotriose and / or xylopentaose.
5. A method for preparing methyl glucuronide xylotriose, comprising inoculating the Bacillus of claim 1 into a fermentation medium, pre-culturing, inoculating xylose utilizing bacteria, and culturing; Preferably, the pre-culture time is 16 to 32 hours.
6. The method according to claim 5, characterized in that: The fermentation medium comprises a carbon source; Preferably, the carbon source comprises xylan; Preferably, the xylan is beech xylan; Preferably, the mass percentage of the carbon source in the fermentation medium is 0.5% to 2%.
7. The method according to claim 6, characterized in that: The fermentation medium also includes a nitrogen source and an inorganic salt.
8. The method according to claim 7, characterized in that: The culture temperature is 30-40°C; and / or The culture time is 20 to 90 hours.
9. The method according to any one of claims 5 to 8, characterized in that: After the culture is completed, a purification step is also included.
10. The method according to claim 9, characterized in that: Said purification comprises chromatography purification; Preferably, the chromatographic purification comprises at least one of a reverse phase chromatography solid phase extraction column, a graphitized carbon column solid phase extraction column, and an ion exchange chromatography.