CN115232773B - Bacillus licheniformis and application thereof in degradation of bletilla striata polysaccharide - Google Patents
Bacillus licheniformis and application thereof in degradation of bletilla striata polysaccharide Download PDFInfo
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- CN115232773B CN115232773B CN202210938151.6A CN202210938151A CN115232773B CN 115232773 B CN115232773 B CN 115232773B CN 202210938151 A CN202210938151 A CN 202210938151A CN 115232773 B CN115232773 B CN 115232773B
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12R2001/07—Bacillus
- C12R2001/10—Bacillus licheniformis
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Abstract
The application discloses bacillus licheniformis and application thereof in degradation of bletilla striata polysaccharide. The Bacillus licheniformis strain is named Bacillus licheniformis BJ2022, the preservation unit is China center for type culture Collection, and the preservation number is CCTCC NO: M2022816. When the bacillus licheniformis is used for degrading mannans, isomannans and mannooligosaccharides, beta-1, 4-D-mannoside bonds can be broken, oligosaccharide fragments can be generated, and bletilla polysaccharide can be effectively degraded into bletilla oligosaccharide. Compared with the physical method and the chemical method, the bacillus licheniformis provided by the application is used for degrading the bletilla polysaccharide and the various mannans, isomannans and mannooligosaccharides containing beta-1, 4-D-mannoside bonds, has high efficiency, is green and environment-friendly, and is suitable for large-scale preparation of the bletilla oligosaccharide and other oligosaccharides.
Description
Technical Field
The application relates to the technical field of degradation of bletilla polysaccharide, in particular to bacillus licheniformis and application thereof in degradation of bletilla polysaccharide.
Background
The bletilla striata is a traditional rare and precious traditional Chinese medicinal material in China, and has extremely high ornamental value, medicinal value and economic value. The composition of the compounds in the bletilla tuber is more than 150, wherein the bletilla polysaccharide is the most abundant substance in the dry tuber, and the content can reach 60 percent at most. Bletilla polysaccharide is a high molecular viscosity polysaccharide, the chemical components of the polysaccharide are glucomannan (including beta-glucose and beta-mannose), monosaccharide residues are connected by beta-1, 4-glycosidic bonds, and the molar ratio of the two is about 1:4, the molecular weight is 70000-600000 Da. The bletilla striata polysaccharide has the biological activities of resisting bacteria, promoting wound healing, resisting tumor, resisting fibrosis, resisting oxidation, resisting digestive tract ulcer and the like, and has wide application in the industries of food, cosmetics and the like due to no stimulation and no side effect. The bletilla striata polysaccharide has high viscosity and poor water solubility, and brings great inconvenience in subsequent processing and utilization. In view of the absolute ratio of polysaccharide in bletilla tuber, how to effectively reduce the viscosity of polysaccharide while maintaining the bioactivity of polysaccharide has become a major scientific problem and industrial bottleneck currently faced by bletilla industry.
Bletilla oligosaccharides (typically less than 30) with a smaller degree of polymerization have increased water solubility and decreased viscosity compared to bletilla polysaccharides, and are more biologically active when administered orally into the intestinal tract. At present, the method for degrading bletilla polysaccharide into oligosaccharide mainly comprises three major types of chemical (such as acid and alkali hydrolysis), physical (such as ultrasonic) and biological methods, but the chemical method has the defects of severe reaction conditions, difficult quality control, large environmental pollution and low degradation efficiency in the physical method, and the biological method can effectively avoid the problems.
The microbial degradation method is an emerging polysaccharide degradation technology developed in recent years, adopts special microorganisms to efficiently degrade polysaccharide into oligosaccharide, is environment-friendly compared with the traditional chemical and physical methods, and is particularly suitable for large-scale preparation of the oligosaccharide.
Therefore, finding a microorganism capable of efficiently degrading bletilla striata polysaccharide becomes one of the key methods for solving the technical problems.
Disclosure of Invention
In view of the above, the embodiment of the application discloses bacillus licheniformis and application thereof in degrading bletilla striata polysaccharide, so as to solve or alleviate the technical problems of the parts.
In a first aspect, the embodiment of the application discloses a bacillus licheniformis which is named Bacillus licheniformis BJ2022, the preservation unit is China center for type culture collection, the address is in eight paths 299 of Wuchang district of Wuhan, hubei province, the preservation number is CCTCC NO: M2022816, and the preservation date is 2022, 6 and 7.
In a second aspect, an embodiment of the application discloses a microbial inoculum applied to degradation of bletilla striata polysaccharide, wherein the microbial inoculum comprises bacillus licheniformis according to the first aspect.
Further, the microbial inoculum contains the living microbial cells of the bacillus licheniformis or the microbial inoculum contains the mixed microbial cells of the living microbial cells and the dead microbial cells of the bacillus licheniformis.
Preferably, when the microbial inoculum contains a mixture of live and dead microbial cells of the bacillus licheniformis, the number of live microbial cells is higher than the number of dead microbial cells.
More preferably, the microbial inoculum comprises viable bacteria of the bacillus licheniformis.
Further, the microbial inoculum is a liquid microbial inoculum and/or a solid microbial inoculum.
Preferably, the preparation form of the microbial inoculum is bacterial liquid or freeze-dried bacterial cells.
In a third aspect, embodiments of the present application disclose the use of the bacillus licheniformis and/or the microbial inoculum of the first aspect for degrading mannans;
wherein the polysaccharide comprises beta-1, 4-glycosidic linkages.
Preferably, the application comprises degradation of bletilla polysaccharide.
In a fourth aspect, the embodiments of the present application disclose the use of the bacillus licheniformis and/or the microbial inoculum of the first aspect for the fermentative preparation of bletilla oligosaccharides.
In a fifth aspect, embodiments of the present application disclose a kit comprising bacillus licheniformis as described in the first aspect or the microbial inoculum as described in the second aspect.
In a sixth aspect, embodiments of the present application disclose a method for screening bacillus licheniformis according to the first aspect, comprising:
taking a fecal sample and preparing an intestinal flora dilution;
enrichment and screening of strains;
separating and purifying bacillus licheniformis strains;
and identification of the strain.
Further, the preparation method of the intestinal flora diluent is a gradient centrifugation method.
Further, the method for separating the bacillus licheniformis strain is the only carbon source screening method.
Further, the identification mode of the strain is morphological feature verification and 16S rDNA sequencing identification.
Compared with the prior art, the application has at least one of the following beneficial effects:
the application relates to bacillus licheniformis and application thereof in degradation of bletilla polysaccharide. When the bacillus licheniformis is used for degrading mannans, isomannans and mannooligosaccharides, beta-1, 4-D-mannoside bonds can be broken, oligosaccharide fragments are generated, and bletilla polysaccharide contains a large amount of beta-1, 4-D-mannoside bonds. Compared with the physical method and the chemical method, the bacillus licheniformis provided by the application is used for degrading the bletilla polysaccharide and the various mannans, isomannans and mannooligosaccharides containing beta-1, 4-D-mannoside bonds, has high efficiency, is green and environment-friendly, and is suitable for large-scale preparation of the bletilla oligosaccharide and other oligosaccharides.
Drawings
Fig. 1 shows the gram stain (1000×) and colony photograph of bacillus licheniformis Bacillus licheniformis BJ2022 provided by the examples of the present application.
FIG. 2 is a measurement of fermentation process parameters of bletilla oligosaccharides provided in the examples of the awake application; wherein A is a bacillus licheniformis growth curve, B is a fermentation broth pH value change curve, C is a fermentation broth total sugar content change curve, and D is a fermentation broth reducing sugar content change curve.
FIG. 3 is a diagram of structural identification of bletilla striata oligosaccharides according to the examples of the present application; wherein A is the molecular weight of the bletilla oligosaccharide monosaccharide detected by HPGPC, and B, C is the composition of the two bletilla oligosaccharide monosaccharides detected by HPLC.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application. Reagents not specifically and individually described in this application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.
It should be noted that, the terms "first," "second," and the like in the description and the claims of the present invention and the above drawings are used for distinguishing similar objects, and are not necessarily used for describing a particular sequence or order, nor do they substantially limit the technical features that follow. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
In a first aspect, the embodiment of the application discloses a bacillus licheniformis, which is named Bacillus licheniformis BJ2022, and has a preservation number of CCTCC NO: M2022816 as a preservation unit of China center for type culture Collection.
The embodiment of the application discloses a screening method of bacillus licheniformis, which comprises the following specific steps:
1. preparation of intestinal flora bacterial liquid
The fecal sample collection site of the present example was martial arts, specifically 5 healthy volunteers (male: female=3:2). After taking 2g of feces from each volunteer, thoroughly shaking and mixing them with 20mL of anaerobic PBS (containing 0.5g/L of L-cysteine), centrifuging at 1000rpm for 5min, and removing food residues. The fecal suspension was centrifuged at 6000rpm for 5min and resuspended in 20mL of anaerobic PBS to obtain a 10% intestinal flora broth.
1. Culture and isolation of strains
Further diluting the intestinal flora bacterial solution into 10 according to a 10-fold dilution method -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 A dilution of the sample suspension;
selecting dilution of 10 -4 、10 -5 、10 -6 The diluted sample suspensions were pipetted with 1mL sterile respectively onto plates of bletilla polysaccharide carbon source Selection Medium (SM) which were incubated in an incubator at 37 ℃ for 24 hours to obtain colonies.
2. Separation and purification of bacillus licheniformis
Screening a flat colony with irregular edges, white color and rough surface wrinkles in the middle of about 3-4 mm on an SM flat plate;
culturing the screened bacterial colony by a nutrient broth culture medium (LB), and taking fresh bacterial liquid to obtain longbacillus which is 100 times-4.0 mu m, 0.8-1.0 mu m in width and blue-violet or purple in gram staining by an optical microscope;
and (5) scribing and purifying the screened bacterial colony on an LB plate, and then preserving the bacterial colony on a test tube inclined plane for standby.
3. Identification of Bacillus licheniformis Strain
The application extracts DNA from the strain screened by the application, and carries out 16SrDNA detection, and the detection result shows that the strain can be identified as bacillus licheniformis, is named Bacillus licheniformis BJ2022 and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2022816 in 6 months of 2022.
In a second aspect, embodiments of the present application provide a microbial inoculant comprising bacillus licheniformis (Bacillus licheniformis BJ 2022) as described above for use in degrading bletilla striata polysaccharides or mannans, isomannides and mannooligosaccharides comprising beta-1, 4-D-mannoside linkages.
In the present application, the concentration of Bacillus licheniformis in the microbial inoculum is not particularly limited, and may be specifically selected according to specific conditions.
In the present application, the microbial inoculum contains living and/or dead bacteria of the bacillus licheniformis. Preferably, the microbial inoculum contains a mixture of live and dead bacteria of the bacillus licheniformis (Bacillus licheniformis BJ 2022). More preferably, the microbial inoculum contains viable bacteria of the bacillus licheniformis (Bacillus licheniformis BJ 2022). When the microbial inoculum contains a mixture of live and dead microbial cells of the Bacillus licheniformis (Bacillus licheniformis BJ 2022), the number of live microbial cells is preferably higher than the number of dead microbial cells.
In the present application, the dosage form of the microbial inoculum is not particularly limited, and may be prepared into various dosage forms, such as liquid microbial inoculum and/or solid microbial inoculum, and corresponding excipients and the like may be added according to the intended use. Preferably, the preparation form of the microbial inoculum is bacterial liquid or freeze-dried bacterial cells. The addition of excipients to the bacterial agents in which the dosage forms are known to those skilled in the art is not described in detail herein.
In a third aspect, embodiments of the present application disclose the use of the bacillus licheniformis and/or the microbial inoculum of the first aspect in degrading polysaccharides;
wherein the polysaccharide comprises beta-1, 4-glycosidic linkages.
Preferably, the application comprises degradation of bletilla polysaccharide.
According to the application, the application of the bacillus licheniformis or the microbial inoculum to degrading polysaccharide is respectively verified, wherein the polysaccharide comprises bletilla polysaccharide, locust bean gum, guar gum, astragalus polysaccharide, rhizoma polygonati polysaccharide, xanthan gum, grifola frondosa polysaccharide, cellulose, konjac gum and pachyman.
Preparing polysaccharide solution of 2.5mg/mL, weighing 50mg polysaccharide (rhizoma bletilla polysaccharide, locust bean gum, guar gum, radix astragali polysaccharide, rhizoma Polygonati polysaccharide, xanthan gum, maitake Mushroom polysaccharide, cellulose, konjac gum, pachyman), and fixing volume to 20mL.
According to the application, the number of thalli in the bacillus licheniformis and/or the microbial inoculum is at least 10 7 CFU (number of cells degrading 50mg polysaccharide, if the mass of polysaccharide is changed, the number of cells is changed), preferably, the number of viable cells in the Bacillus licheniformis and/or the microbial inoculum is at least 10 7 CFU. In the present application, the number of cells can be measured according to GB 4789.2-94.
According to the present application, the degradation conditions include: the temperature was 35℃and the pH was 8 for 2h.
Experimental results: the results of degradation of different polysaccharides by bacillus licheniformis and/or the microbial agents are shown in table 1:
TABLE 1
As can be seen from Table 1, the Bacillus licheniformis and/or the microbial inoculum described herein have high activity on rhizoma bletillae polysaccharide and konjac gum, wherein the degradation rate of the rhizoma bletillae polysaccharide is slightly higher than that of the konjac gum.
Analysis: the bletilla polysaccharide is a high molecular viscous polysaccharide, the chemical components of the bletilla polysaccharide are glucomannan (including beta-glucose and beta-mannose), and monosaccharide residues are connected by beta-1, 4-glycosidic bonds; the konjac glucomannan is a polysaccharide formed by connecting D-mannose and D-glucose, and the ratio of the D-mannose to the D-glucose is 1:1.6, monosaccharides are also linked by β -1, 4-glycosidic linkages; the bacillus licheniformis and/or the microbial inoculum can effectively break beta-1, 4 glycosidic bonds, and the action substrates are mannans, glucomannans, galactomannans and the like, so that the bacillus licheniformis and/or the microbial inoculum has high degradation rate on bletilla polysaccharide and konjac glucomannan. Similarly, we can conclude that the Bacillus licheniformis and/or the microbial inoculum described herein have higher degradation rates for polysaccharides containing beta-1, 4 glycosidic linkages.
In a fourth aspect, the embodiments of the present application disclose the use of the bacillus licheniformis and/or the microbial inoculum of the first aspect for the fermentative preparation of bletilla oligosaccharides.
In the embodiment of the application, a method for preparing bletilla striata oligosaccharides by using bacillus licheniformis fermentation is provided, and the specific implementation steps are as follows:
(1) 30g of bletilla striata was taken and added to 1L of buffer (pH 7.5) (the solution comprised 0.2g (NH) 4 ) 2 SO 4 ，0.2g KH 2 PO 4 ，0.4g NaCl，0.04gmgSO 4 ) Soaking for 12h at low temperature. Pulping by using a wall breaking machine.
(2) Transferring the mixed solution obtained in the step (1) into a 5L fermentation tank, and sterilizing for 15min at 115 ℃.
(3) And (3) placing the sterilized fermentation tank in the step (2) on a full-automatic fermentation system, and inoculating the activated bacillus licheniformis bacteria liquid into the fermentation tank according to the proportion of 5%. The fermentation parameters were set as: 10% of dissolved oxygen, 100rpm of rotating speed and 37 ℃ of temperature, and ventilation fermentation for 72h. The change of OD600, pH, total sugar and reducing sugar of the culture medium was monitored periodically during the fermentation, and the results are shown in FIG. 2.
(4) And (3) taking out the fermentation liquor in the step (3), centrifuging at 8000rpm for 5min, concentrating the supernatant to a feed-liquid ratio of 1:5, adding absolute ethyl alcohol to an alcohol concentration of 80%, standing at 4 ℃ for 12h, and collecting the precipitate.
(5) And (3) re-dissolving the precipitate with distilled water, concentrating on a rotary evaporator, collecting all sugar solutions, and freeze-drying to obtain bletilla striata oligosaccharide powder.
Results: as shown in FIG. 3, the average molecular weight of the bletilla striata oligosaccharide is 3000Da, and the monosaccharide composition is glucose and mannose.
In a fifth aspect, embodiments of the present application disclose a kit comprising bacillus licheniformis and/or a microbial inoculum as described above.
From the above results, it is known that when the bacillus licheniformis provided by the application is applied to degradation of mannans, isomannans and mannooligosaccharides, beta-1, 4-D-mannosides can be broken to generate oligosaccharide fragments, and bletilla polysaccharide contains a large amount of beta-1, 4-D-mannosides. Compared with the physical method and the chemical method, the bacillus licheniformis provided by the application is used for degrading the bletilla polysaccharide and the various mannans, isomannans and mannooligosaccharides containing beta-1, 4-D-mannoside bonds, has high efficiency, is green and environment-friendly, and is suitable for large-scale preparation of the bletilla oligosaccharide and other oligosaccharides.
The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present application should be covered by the scope of the present application.
Claims (8)
1. Bacillus licheniformis, which is namedBacillus licheniformisBJ2022 is preserved in China center for type culture collection, and has an address of 299 th No. eight channels in Wuchang district of Wuhan, hubei province, a preservation number of CCTCC NO: M2022816, and a preservation date of 2022, 6 months and 7 days.
2. A microbial inoculant comprising the bacillus licheniformis of claim 1.
3. The microbial inoculum of claim 2, wherein the microbial inoculum comprises a mixture of live and dead microbial cells of the bacillus licheniformis.
4. The microbial agent according to claim 3, wherein the number of living microbial cells is higher than the number of dead microbial cells.
5. The microbial inoculum of any one of claims 2-4, wherein the microbial inoculum is a liquid microbial inoculum or a solid microbial inoculum.
6. Use of the bacillus licheniformis of claim 1 and/or the microbial inoculum of any one of claims 2-5 for degrading bletilla striata polysaccharides.
7. The use of the bacillus licheniformis of claim 1 and/or the microbial inoculum of any one of claims 2-5 in the fermentation preparation of bletilla striata oligosaccharides.
8. A kit comprising bacillus licheniformis of claim 1 or the microbial inoculum of any of claims 2-5.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101386824A (en) * | 2008-07-30 | 2009-03-18 | 黑龙江大学 | Engineering strain for producing mannanase |
| CN102071156A (en) * | 2009-12-22 | 2011-05-25 | 黑龙江大学 | Bacillus licheniformis capable of producing beta-mannanase |
| KR20110076337A (en) * | 2009-12-29 | 2011-07-06 | 경남과학기술대학교 산학협력단 | Cellulase-producing bacillus licheniformis cs48 and its culture fluid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101386824A (en) * | 2008-07-30 | 2009-03-18 | 黑龙江大学 | Engineering strain for producing mannanase |
| CN102071156A (en) * | 2009-12-22 | 2011-05-25 | 黑龙江大学 | Bacillus licheniformis capable of producing beta-mannanase |
| KR20110076337A (en) * | 2009-12-29 | 2011-07-06 | 경남과학기술대학교 산학협력단 | Cellulase-producing bacillus licheniformis cs48 and its culture fluid |
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| 地衣芽孢杆菌产β-甘露聚糖酶分批发酵动力学模型的建立;田雪;解鑫;周晓杭;葛菁萍;凌宏志;宋刚;平文祥;;中国农学通报(第18期);全文 * |
| 枯草芽孢杆菌群β-甘露聚糖酶基因克隆及同源性分析;刘勇;毛爱军;李辉;程池;;基因组学与应用生物学(第05期);全文 * |
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