CN117987315A - Zhang Wenhong bacteroides strain and application thereof in degradation preparation of hyaluronic acid oligosaccharide - Google Patents
Zhang Wenhong bacteroides strain and application thereof in degradation preparation of hyaluronic acid oligosaccharide Download PDFInfo
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Abstract
The invention relates to the field of degradation and preparation of active oligosaccharides, in particular to a Zhang Wenhong bacteroides strain and application thereof in degradation and preparation of hyaluronic acid oligosaccharides. Zhang Wenhong Bacteroides bacterial strain is specifically Zhang Wenhong Bacteroides bacterial strain D2-16 (Bacteroides zhangwenhongii D2-16), the bacterial strain is separated from healthy human excrement and is preserved in China Center for Type Culture Collection (CCTCC) NO: M20232720. The strain can degrade hyaluronic acid into unsaturated hyaluronic acid oligosaccharide with the polymerization degree of mainly 4.
Description
Technical Field
The invention relates to the field of degradation and preparation of active oligosaccharides, in particular to a Zhang Wenhong bacteroides strain and application thereof in degradation and preparation of hyaluronic acid oligosaccharides.
Background
Hyaluronic Acid (HA), commonly known as Hyaluronic acid, is a linear glycosaminoglycan. Hyaluronic acid is formed by alternately connecting disaccharide repeating units consisting of D-glucuronic acid and N-acetylamino-D-glucose (DOI: 10.3390/biom 10111525). Hyaluronic acid is present in joint synovial fluid and extracellular matrix of various tissues, plays an important role in tissue remodeling, inflammation and cancer formation, and has been widely used in the fields of medicine, food, medical care, and the like (DOI: 10.1016/j. High molecular weight hyaluronic acid has a strong water-retaining effect, but lacks immune activity; low molecular weight hyaluronic acid, in particular hyaluronic acid oligosaccharides, have pro-angiogenic and wound repair promoting (DOI:
10.3390/ijms 20153722), inhibiting tumor cell multidrug resistance (DOI: 10.1038/bjc.2014.332), etc. Studies have shown that hyaluronan tetrasaccharide can promote epidermal differentiation by enhancing CD44 gene expression, regulation of CD44 phosphoprotein, and human keratinocyte differentiation (DOI: 10.1111/ics.12105). In addition, the unsaturated hyaluronic acid oligosaccharide has an inhibitory effect on proliferation of human laryngeal cancer cells, medulloblastoma, human breast cancer cells and colon adenocarcinoma cells, and also has an antioxidant effect (DOI: 10.1016/j.carbpol.2010.06.042).
The current methods for preparing hyaluronic acid oligosaccharides mainly comprise a physical method, a chemical method and a biological enzyme method. The physical degradation method mainly degrades the hyaluronic acid with large molecular weight by ultrasonic wave and heating, the molecular weight distribution of the hyaluronic acid obtained by the method is narrow, and the hyaluronic acid is difficult to reach below 10kDa (DOI: 10.1016/j. Biotechadiv.2007.07.001); the chemical degradation method mainly uses strong acid and alkali to hydrolyze, but the product structure is easy to be destroyed, such as the six-membered ring in the monosaccharide is broken, thereby affecting the biological activity of the hyaluronic acid oligosaccharide (DOI: 10.1006/abbi.1997.9970). Although the bioenzyme method can produce hyaluronic acid oligosaccharide with specific polymerization degree (China patent application number: CN 115386608B), the cost of the current hyaluronidase is high, and the market application is limited to a certain extent. In recent years, the preparation of hyaluronic acid oligosaccharides by microbial fermentation degradation has received increasing attention. In 2023, we found that bacteroides salesii derived from intestinal tracts of healthy human bodies can be degraded to prepare hyaluronic acid oligosaccharides (Chinese patent application No. CN 202310238846.8), and the hyaluronic acid oligosaccharides produced by degradation are mainly octasaccharides.
Bacteroides has an important role in the degradation metabolism of complex carbohydrates (DOI: 10.3389/fmib.2022.1033355), and although Bacteroides salvinsis can degrade to prepare hyaluronic acid oligosaccharides, the degree of polymerization of oligosaccharides produced by degradation is high (Chinese patent application No. CN 202310238846.8). In addition, whether other Bacteroides exist or not to degrade and prepare hyaluronic acid oligosaccharides with specific molecular weight has not been studied. The hyaluronic acid tetraose has good and obvious effects of enhancing immunity and promoting the differentiation of epidermal cells (DOI: 10.1016/j. Carbpol.2021.118699; DOI: 10.1111/ics.12105), so that the preparation of hyaluronic acid oligosaccharide mainly comprising tetraose by microbial degradation has important application prospect. Zhang Wenhong Bacteroides is a normal symbiotic bacterium in the intestinal tract of healthy humans (DOI: 10.1099/ijsem.0.004772), however whether it can be used for degradation to produce hyaluronic acid oligosaccharides has not been reported so far. The study shows for the first time that Zhang Wenhong bacteroides (Bacteroides zhangwenhongii) D2-16 which is a source of intestinal tracts of healthy human bodies can degrade hyaluronic acid into unsaturated oligosaccharides with the polymerization degree of mainly 4. The research provides a new idea for preparing the hyaluronic acid oligosaccharide by microbial fermentation degradation, and has wide market application prospect.
Disclosure of Invention
Based on the defects in the prior art, the invention discloses a Zhang Wenhong bacteroides strain and application thereof in degradation preparation of hyaluronic acid oligosaccharides.
In a first aspect of the invention, a Zhang Wenhong bacteroides strain is provided, the Zhang Wenhong bacteroides strain is specifically a Zhang Wenhong bacteroides strain D2-16 (Bacteroides zhangwenhongii D2-16), the strain source is separated from healthy human excrement and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20232720.
Further, the strain D2-16 is isolated from healthy human feces.
Further, the DNA sequence of the strain D2-16 is shown as SEQ ID NO. 1.
In a second aspect, the invention provides an application of Zhang Wenhong bacteroides strain D2-16 in degradation preparation of hyaluronic acid oligosaccharide.
Further, the molecular weight of hyaluronic acid degraded by the Zhang Wenhong bacteroides strain D2-16 is 40-2000 kDa.
Further, the method for applying Zhang Wenhong bacteroides strain D2-16 in degradation preparation of hyaluronic acid oligosaccharide comprises the following steps: inoculating seed solution obtained by carrying out activation culture on Zhang Wenhong bacteroides strain D2-16 into a hyaluronic acid culture medium for anaerobic fermentation culture, and detecting degradation of hyaluronic acid by the strain by a thin layer chromatography method.
Further, the final concentration composition of the hyaluronic acid culture medium is as follows: 6-10 g/L of hyaluronic acid, 2-4 g/L of tryptone, 2-4 g/L of peptone, 2-4 g/L of yeast extract, 0.4-0.6 g/L of mucin, 0.3-0.5 g/L of No. 3 bile salt, 0.7-0.9 g/L of cysteine hydrochloride, 0.04-0.06 g/L of heme, 0.8-1.2 mL/L of Tween, 4-5 g/L of sodium chloride, 2-3 g/L of potassium chloride, 4-5 g/L of magnesium chloride, 0.1-0.3 g/L of calcium chloride, 0.3-0.5 g/L of monopotassium phosphate and 1-3 mL/L of trace elements, wherein the solvent is distilled water, and the pH value is 6.4-6.5.
Further, the specific application steps are as follows: step 1) seed culture: inoculating Zhang Wenhong bacteroides strain D2-16 into a seed culture medium, and culturing for 1-4 days at 25-42 ℃ to obtain seed liquid; the seed culture medium is characterized in that hyaluronic acid in the hyaluronic acid culture medium is replaced by 6-10 g/L glucose, and other components are the same as those of the hyaluronic acid culture medium; step 2) fermentation culture: inoculating the seed liquid obtained in the step 1) into a hyaluronic acid culture medium in an inoculum size of 1-10% of the volume ratio, culturing at 25-42 ℃, and tracking degradation of hyaluronic acid by thin layer chromatography.
Further, the hyaluronic acid oligosaccharides prepared by the degradation are unsaturated disaccharides, saturated trisaccharides, unsaturated tetrasaccharides, unsaturated hexasaccharides and unsaturated octasaccharides.
Advantages and beneficial effects of the invention
The bacterial strain Zhang Wenhong of bacteroides provided by the invention is D2-16, and can degrade hyaluronic acid with molecular weight of 40-2000 kDa into unsaturated hyaluronic acid oligosaccharide with polymerization degree of mainly 4.
Drawings
FIG. 1 is a thin layer chromatography of the degradation of HA by TLC detection Zhang Wenhong of Bacteroides strain D2-16, wherein Con is a non-inoculated sample and 1,2, 3 are 3 parallel samples;
fig. 2 is a graph showing the relative HA consumption by the Zhang Wenhong bacteroides strain D2-16 degradation fermentation HA, wherein the relative HA consumption at 0h is 100%, which indicates a very significant difference (P < 0.01) compared to the 0h group, and the relative HA consumption at 0h group, which indicates a very significant difference (P < 0.001) compared to the 0h group. Statistical test is carried out by using a Student t-test, and the data display mode is mean+/-SEM;
FIG. 3 shows the relative amounts of unsaturated tetraose produced by the fermentation HA degradation by the Zhang Wenhong Bacteroides strain D2-16, wherein the relative amounts of udp4 at 72h are 100%, indicating a very significant difference (P < 0.01) compared to group 0 h. Statistical test is carried out by using a Student t-test, and the data display mode is mean+/-SEM;
Fig. 4 shows the relative amounts of unsaturated hexasaccharides produced by the fermentation of HA by the Zhang Wenhong bacteroides strain D2-16, wherein the relative amounts of udp6 at 48h are 100%, indicating a significant difference (P < 0.05) compared to group 0h, and indicating a significant difference (P < 0.01) compared to group 0h. Statistical test is carried out by using a Student t-test, and the data display mode is mean+/-SEM;
FIG. 5 is a total ion mass spectrum of sample fluid obtained by MS detection inoculation Zhang Wenhong of Bacteroides strain D2-16 and fermentation for 48h, wherein the letters indicate the degree of polymerization of different oligosaccharides (dp, wherein dp is saturated oligosaccharide and udp is unsaturated oligosaccharide);
FIG. 6 is a graph showing the mass spectrum of unsaturated disaccharides from a sample solution obtained by fermenting a sample solution obtained by inoculating Zhang Wenhong Bacteroides strain D2-16 for 48 hours in MS detection, wherein the letters indicate the degree of polymerization of different oligosaccharides (the udp is an unsaturated oligosaccharide);
FIG. 7 is a saturated trisaccharide spectrum of a sample solution obtained by inoculating Zhang Wenhong Bacteroides strain D2-16 and fermenting for 48 hours in MS detection, wherein letters indicate the polymerization degree of different oligosaccharides (dp is saturated oligosaccharide);
FIG. 8 is a graph of the mass spectrum of unsaturated tetrasaccharides of sample fluid obtained by MS detection and inoculation of Zhang Wenhong Bacteroides strain D2-16 for 48h fermentation, wherein the letters indicate the degree of polymerization of the different oligosaccharides (the udp is an unsaturated oligosaccharide);
FIG. 9 is a graph of the mass spectrum of unsaturated hexasaccharides of sample liquid obtained by fermenting Zhang Wenhong Bacteroides strain D2-16 for 48h after MS detection, wherein the letters indicate the degree of polymerization of different oligosaccharides (the udp is an unsaturated oligosaccharide);
FIG. 10 is a graph of the mass spectrum of unsaturated octasaccharides from sample liquid obtained by fermenting Zhang Wenhong Bacteroides strain D2-16 for 48h in MS detection, wherein the letters indicate the degree of polymerization of the different oligosaccharides (the udp is an unsaturated oligosaccharide).
Detailed Description
Biological preservation description:
The invention provides Zhang Wenhong bacteroides bacterial strain D2-16, with the preservation number: CCTCC NO: M20232720; classification naming: a bacteroides species, zhang Wenhong bacteroides species; latin Wen Xueming: bacteroides zhangwenhongii D2 to 16; the strain is preserved in China center for type microbiological culture collection, with a preservation date of 2023, 12 months and 29 days, and a preservation address of eight-path 299-No. Wuhan university in Wuhan district of Wuhan, hubei province.
The Zhang Wenhong bacteroides strain D2-16 is derived from healthy human excrement, and the sequence of the 16S rDNA is shown in a sequence table SEQ ID NO. 1.
The invention will be further described with reference to the drawings and the detailed description, but the scope of the invention is not limited thereto.
Example 1.
Isolation and identification of Strain D2-16
(1) Preparation of culture Medium
The VI-algin oligosaccharide culture medium is prepared, and the specific components are as follows: alginate oligosaccharides (molecular weight of 3 kDa) 4g/L, tryptone 3g/L, peptone 3g/L, yeast extract 3g/L, mucin 0.5g/L, bile salt number 3 0.4g/L, cysteine hydrochloride 0.8g/L, heme 0.05g/L, tween 80 1mL/L, sodium chloride 4.5g/L, potassium chloride 2.5g/L, magnesium chloride 4.5g/L, calcium chloride 0.2g/L, potassium dihydrogen phosphate 0.4g/L, trace elements 2mL/L, distilled water as solvent, pH value 6.4-6.5, and filling the culture medium into an anaerobic vial, and sterilizing by nitrogen gas.
In the invention, the final concentration composition of the trace elements is as follows ：MgSO4·7H2O 3.0g/L、CaCl2·2H2O0.1g/L、MnCl2·4H2O 0.32g/L、FeSO4·7H2O 0.1g/L、CoSO4·7H2O 0.18g/L、ZnSO4·7H2O 0.18g/L、CuSO4·5H2O 0.01g/L、NiCl2·6H2O 0.092g/L.
(2) Pretreatment of feces
Fresh feces of 1 volunteer was taken, and a 20% (wt/vol) suspension was prepared with PBS (pH 7.0), thoroughly mixed, and then filtered through a metal sieve having a diameter of 2mm to remove large food particles, thereby obtaining a feces PBS solution.
(3) Inoculating culture
The obtained fecal PBS solution is inoculated into an anaerobic small bottle after high-temperature sterilization, and is cultured for 48 hours at 37 ℃ for preliminary enrichment culture. The culture broth was applied by gradient dilution, with plates of VI-AOS broth plus 1.2wt% agar final concentration. After the plate is placed in an anaerobic workstation for culturing for 48 hours at 37 ℃, single colony is picked up and subjected to purification culture in VI-AOS plate culture medium for two times, and then single colony is continuously picked up and cultured in VI-AOS liquid culture medium for 48 hours at 37 ℃.
(4) 16S rDNA sequence analysis
Extraction of DNA: the strain D2-16 obtained in the step (3) was subjected to DNA extraction using the fecal analysis kit (Cat No. 51604) of QIAGEN, germany. The resultant DNA was subjected to 16S rDNA full-length amplification. The experimental conditions and primer sequences for specific amplification are as follows:
primer sequence:
27F(5’-CAGAGTTTGATCCTGGCT-3’)
1492R(5’-AGGAGGTGATCCAGCCGCA-3’
Amplification system: 25. Mu.L of the reaction system, 100ng of DNA template, 2.5. Mu.L of 10 XPCR Buffer, 0.5. Mu.L of dNTP Mix (10 mM), 0.5. Mu.L of each of 10. Mu.L of upstream and downstream primers, 0.2. Mu.L of Taq enzyme (5U/. Mu.L) and the mixture was supplemented with deionized water to 25. Mu.L.
Amplification conditions: the pre-deformation is kept at 94 ℃ for 5min, circulated at 94 ℃ for 35s, kept at 72 ℃ for 1min, operated for 35 cycles, and extended for 8min.
The PCR product was purified and sent to the Shanghai Biotechnology (Shanghai, china) Co., ltd for DNA sequencing, as shown in SEQ ID No.1, and the sequencing result was submitted to NCBI database for Blast comparison. The comparison result shows that the homology of the strain with the Zhang Wenhong bacteroides (Bacteroides zhangwenhongii) is 98.33 percent, and the strain D2-16 is identified as Zhang Wenhong bacteroides (Bacteroides zhangwenhongii) according to the comparison of 16S rDNA, and is named Zhang Wenhong bacteroides Bacteroides zhangwenhongii D2-16.
Example 2.
Degradation of hyaluronic acid by Bacteroides Zhang Wenhong Strain D2-16
Hyaluronic acid is commercially available, and has a molecular weight of 40 to 2000kDa, and other reagents and the like are commercially available. The VI-HA culture medium is prepared, and the specific components are as follows: hyaluronic acid (HA, molecular weight is 1610 kDa) 8g/L, tryptone 3g/L, peptone 3g/L, yeast extract 3g/L, mucin 0.5g/L, no. 3 bile salt 0.4g/L, cysteine hydrochloride 0.8g/L, heme 0.05g/L, tween 80 1mL/L, sodium chloride 4.5g/L, potassium chloride 2.5g/L, magnesium chloride 4.5g/L, calcium chloride 0.2g/L, potassium dihydrogen phosphate 0.4g/L, trace elements 2mL/L, distilled water as a solvent, pH value is 6.4-6.5, and the culture medium is poured into an anaerobic vial and sterilized by filling nitrogen. The trace elements were the same as in example 1.
And (3) carrying out activation culture on the Zhang Wenhong bacteroides strain D2-16 to obtain a seed solution, and adopting an activation method conventional in the field. The seed solution was inoculated into VI-HA liquid medium at 1% by volume and fermentation was performed in an anaerobic incubator (CO 2:H2:N2 = 1:1:8) at 37 ℃. Samples were taken at 24h,48h,72h and 96h during fermentation, and the degradation conditions, degradation product structure and degradation capacity were analyzed, and 3 groups of experiments were arranged in parallel.
(1) Degradation conditions
After centrifugation of the samples by thin layer chromatography, 0.6 μl of the supernatant was spotted on a silica gel plate and the reaction samples were subjected to formic acid with 0h as reference: n-butanol: spreading in water (volume of 6:3:1) developing agent, drying, repeating spreading twice, soaking in special color developing agent (lichen phenol-sulfuric acid solution), drying, and heating at 120deg.C for 3min for developing. As can be seen from FIG. 1, zhang Wenhong Bacteroides strain D2-16 can degrade hyaluronic acid, and after 48 hours of reaction, hyaluronic acid is degraded and hyaluronic acid oligosaccharides are produced.
Wherein, the relative content of hyaluronic acid in the process of degrading hyaluronic acid by Zhang Wenhong bacteroides strain D2-16 is shown in figure 2 and table 1.
TABLE 1 relative content of hyaluronic acid
(2) Zhang Wenhong relative content of oligosaccharides produced by fermentation of hyaluronic acid by Bacteroides strain D2-16.
The relative amounts of unsaturated tetraose are shown in fig. 3 and table 2.
TABLE 2 relative content of hyaluronic acid unsaturated tetraose
The relative content of hyaluronic acid unsaturated hexasaccharide is shown in fig. 4 and table 3.
TABLE 3 relative content of hyaluronic acid unsaturated hexasaccharide
(3) MS analysis of degradation end product Structure
1ML Zhang Wenhong of Bacteroides bacterial strain D2-16 is taken and cultured in VI-HA liquid culture medium for 48 hours to obtain fermentation broth. 500. Mu.L of the fermentation broth was desalted using a Mini gravity desalting column (G-10, sephadex G-10 packing), the desalted sample was dried and reconstituted with 100. Mu.L of deionized water. The structure of the degradation products was analyzed by MS (for specific methods, reference: DOI:10.1016/j. Carbpol. 2021.118313). 1mL of the fermentation broth is concentrated by centrifugation to obtain 100 mu L of concentrated sample liquid, 100 mu L of acetonitrile is added, vortex mixing is carried out, centrifugation is carried out at 12000rpm for 10min, and the supernatant is taken for analyzing the structure of degradation products. The results in FIG. 5 show that hyaluronic acid is degraded by Zhang Wenhong Bacteroides strain D2-16 to produce a large amount of oligosaccharides, and it can be seen that the unsaturated tetraose is the majority. From FIGS. 6, 7, 8, 9 and 10, it can be seen that Zhang Wenhong Bacteroides strain D2-16 ferments hyaluronic acid to produce hyaluronic acid unsaturated disaccharides, saturated trisaccharides, unsaturated tetrasaccharides, unsaturated hexasaccharides and unsaturated octasaccharides.
Claims (9)
1. A Zhang Wenhong bacteroides bacterial strain D2-16 is characterized in that the Zhang Wenhong bacteroides bacterial strain is named Bacteroides zhangwenhongii D-16 and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20232720.
2. A Zhang Wenhong bacteroides strain D2-16 according to claim 1, wherein said strain D2-16 is isolated from healthy human faeces.
3. The Zhang Wenhong bacteroides strain D2-16 according to claim 1, wherein the DNA sequence of said strain D2-16 is shown in SEQ ID NO. 1.
4. Use of the Zhang Wenhong bacteroides strain D2-16 according to claim 1 for the preparation of hyaluronic acid oligosaccharides by degradation.
5. The use of Zhang Wenhong Bacteroides strain D2-16 according to claim 4 in the preparation of hyaluronic acid oligosaccharides by degradation, wherein the molecular weight of hyaluronic acid is 40-2000 kDa.
6. The use of Zhang Wenhong bacteroides strain D2-16 according to claim 4 for the preparation of hyaluronic acid oligosaccharides by degradation, wherein the method of use is as follows: inoculating seed solution obtained by carrying out activation culture on Zhang Wenhong bacteroides strain D2-16 into a hyaluronic acid culture medium for anaerobic fermentation culture, and detecting degradation of hyaluronic acid by the strain by a thin layer chromatography method.
7. The use of Zhang Wenhong bacteroides strain D2-16 according to claim 6 for the preparation of hyaluronic acid oligosaccharides by degradation, wherein the final concentration composition of the hyaluronic acid medium is: 6 to 10g/L of hyaluronic acid, 2 to 4g/L of tryptone, 2 to 4g/L of peptone, 2 to 4g/L of yeast extract, 0.4 to 0.6g/L of mucin, 0.3 to 0.5g/L of No. 3 bile salt, 0.7 to 0.9g/L of cysteine hydrochloride, 0.04 to 0.06g/L of heme, 0.8 to 1.2mL/L of Tween, 4 to 5g/L of sodium chloride, 2 to 3g/L of potassium chloride, 4 to 5g/L of magnesium chloride and 0.1 to 0.3 of calcium chloride
G/L, 0.3-0.5 g/L of monopotassium phosphate, 1-3 mL/L of trace elements, distilled water as solvent and pH value of 6.4-6.5.
8. The use of a Zhang Wenhong bacteroides strain according to claim 6 for the degradation preparation of hyaluronic acid oligosaccharides, wherein the application steps are specifically: step 1) seed culture: inoculating Zhang Wenhong bacteroides strain D2-16 into a seed culture medium, and culturing for 1-4 days at 25-42 ℃ to obtain seed liquid; the seed culture medium is characterized in that hyaluronic acid in the hyaluronic acid culture medium is replaced by 6-10 g/L glucose, and other components are the same as those of the hyaluronic acid culture medium; step 2) fermentation culture: inoculating the seed liquid obtained in the step 1) into a hyaluronic acid culture medium in an inoculum size of 1-10% of the volume ratio, culturing at 25-42 ℃, and tracking degradation of hyaluronic acid by thin layer chromatography.
9. The use of Zhang Wenhong Bacteroides strain D2-16 according to claim 4 in the degradation of hyaluronic acid oligosaccharides, wherein the hyaluronic acid oligosaccharides produced by the degradation are unsaturated disaccharides, saturated trisaccharides, unsaturated tetrasaccharides, unsaturated hexasaccharides and unsaturated octasaccharides.
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