CN102321153B - Preparation method of xin'ao glycoside peptide powdery solid - Google Patents
Preparation method of xin'ao glycoside peptide powdery solid Download PDFInfo
- Publication number
- CN102321153B CN102321153B CN 201110261822 CN201110261822A CN102321153B CN 102321153 B CN102321153 B CN 102321153B CN 201110261822 CN201110261822 CN 201110261822 CN 201110261822 A CN201110261822 A CN 201110261822A CN 102321153 B CN102321153 B CN 102321153B
- Authority
- CN
- China
- Prior art keywords
- fermented liquid
- understand
- xin
- peptide
- silica gel
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the technical fields of biology and chemistry, and concretely relates to a method for exacting xin'ao glycoside peptide, and especially relates to a method for separating and extracting xin'ao glycoside peptide from fermentation liquor. The preparation method for xin'ao glycoside peptide powdery solid comprises the following steps: (1) preparing crude paste: heating the fermentation liquor and sterilizing, then adding a filter aid and a flocculating agent, filtering, concentrating to obtain the crude paste; (2) purifying and refining: mixing the crude paste with column chromatography silicon gel, vacuum drying, crushing, sieving, performing a silicon gel column chromatography, vacuum distilling , recovering an elution solvent, concentrating and drying to obtain the xin'ao glycoside peptide powdery product.The invention has the advantages that the consumption of the organic solvent is small, the purity of the xin'ao glycoside peptide product is high, the industrialization of production is easy and the like.
Description
Affiliated field
The invention belongs to technical field of biochemical industry, be specifically related to a kind of extracting method of new glycosides peptide difficult to understand, particularly a kind of from fermented liquid the method for the new glycosides peptide difficult to understand of separation and Extraction.
Background technology
In recent years, China has researched and developed a series of Nucleoside Agricultural Antibiotics.As, have the nucleoside antibiotics Astromicin of cytidine skeleton, cytosintetidemycin, many antibiotic (polyoxin) contain Ningnanmycin, Yunnan mycin and the qingfengmeisu etc. of cytidine(C peptide class formation.Above Nucleoside Agricultural Antibiotics is by microbial fermentation and produces, and the amount of the nucleoside active matter that the microorganism strains fermentation produces is often very low, also have other a large amount of impurity and the by product that coexists with nucleoside active matter in fermented liquid in addition, if any microorganism cells, protein, pigment, other products of cellular metabolism and the last substratum that is finished etc., extracting the separation nucleoside active matter from fermented liquid is also the process of a more complicated.At present, the extraction that is derived from the Nucleoside Agricultural Antibiotics of microbial fermentation generation mainly contains two kinds of extracting method, and a kind of is ion-exchange techniques, comprises the acidifying of fermented liquid, solid-liquid separation, and ion exchange chromatography, concentrated etc.; Another kind method is the film extracting method, by employing microfiltration membrane micro-filtration, then ultrafiltration, nanofiltration and spraying drying etc.
This laboratory is in carrying out antibiotic research process, find that strain actinomycetes have the bioactive pyrimidine nucleoside peptide matters of broad-spectrum antimicrobial through the fermentation energy generation, especially the inhibition activity to gram-positive microorganism, plant virus is very strong, if any the activity of very strong anti-plant viral disease such as tobacco, capsicum, Tomato yellow mosaic virus etc.The pyrimidine nucleoside peptide class antimicrobial substance that the inventor names these actinomycetes to produce is Xin Aogan peptide (or new mycin difficult to understand), main component is a kind of new uridine peptide (1-uridylic-4 sarcosyl-seryl amino-1,4-dideoxy-beta d glucopyranosiduronic acid) (number of patent application: 200910060121.4), chemical structure is:
Present published new glycosides peptide extractive technique content difficult to understand (number of patent application: the technology of preparing that 200910060121.4) relates generally to the new glycosides peptide sample difficult to understand of trace (mg level), after comprising new glycosides peptide fermentation ends difficult to understand, by adding methyl alcohol or the ethanol of 2-4 times of fermentating liquid volume, collect new glycosides peptide difficult to understand from above-mentioned fermentation culture, separate with reversed-phase silica gel chromatography by the purification on normal-phase silica gel chromatography again, the glycosides peptide sample new difficult to understand that obtains, with the further separation and purification of high performance liquid chromatography, prepare the glycosides peptide new difficult to understand of trace.This extractive technique method steps is loaded down with trivial details, consumes a large amount of organic solvents, and extracting cycle is long, and loss of activity is large, is not suitable for the preparation of a large amount of new glycosides peptides difficult to understand.If extract new glycosides peptide difficult to understand by traditional ion-exchange techniques, what obtain is the product of paste; And extract with membrane technique, the disposable apparatus investment is very large.
Summary of the invention
In order to overcome the above problems, the invention provides a kind of from fermented liquid the novel method of the new glycosides peptide difficult to understand of separation and Extraction, realize a large amount of preparations of the new glycosides peptide powdery solid difficult to understand of high purity.The present invention comprises following steps: fermented liquid, sterilization, pre-treatment, concentrating under reduced pressure, column chromatography, concentrating under reduced pressure, drying, new glycosides peptide powdery product difficult to understand.
Fermented liquid in above-mentioned steps is by the actinomycetes that can produce new glycosides peptide difficult to understand or its genetic improvement bacterial strain, by cultivating in the level liquid substratum, as seed liquor; Will be in above-mentioned first step liquid nutrient medium cultured seed liquor be inoculated in the substratum that is fit to actinomycetes or its genetic improvement strain growth and produces new glycosides peptide difficult to understand and cultivate, 26 ℃-37 ℃ fermentation culture 2-5 days, after fermentation ends, collect fermented liquid;
Fermented liquid in above-mentioned steps first is heated to 60 ℃, keeps 10-60 minute, kills actinomycetes or its genetic improvement bacterial strain viable bacteria body in fermented liquid, then the fermented liquid temperature is down to below 30 ℃;
Pretreatment operation in above-mentioned steps is to add the flocculating aids of 0.1%-3.0% (grams per milliliter) in sterilization fermentation liquid, fully stir, placed 10-30 minute, the flocculation agent that adds 0.01%-0.1% (grams per milliliter), fully stir, be that 600-2000 purpose filter cloth filters with model, remove the solid substances such as thalline, cell debris and other protein.
Described flocculating aids can be activated carbon, perlite, kaolin or diatomite;
Described flocculation agent can be alum, poly aluminium chloride or ferric salt;
Pretreated fermented liquid in above-mentioned steps, after filtering, at vacuum tightness-0.08MPa to-0.09Mpa, 60～70 ℃ or 50～55 ℃ of bath temperatures; The thick cream of gained mixes with column chromatography silica gel (100～200 order), and its ratio is thick cream: silica gel=1: 1～2.5, stir, and vacuum-drying under 40 ℃ of conditions grinds, and crosses 100 mesh sieves.Silica gel with G100～200 is filled out post, its quality is 5～10 times that upper prop stirs material, carry out silica gel column chromatography, adopt chloroform: methyl alcohol=80～20: 20～80 binary eluents carry out gradient elution, lower column liquid gathers with thin layer chromatography analysis segmentation as a result, the lower Distillation recovery eluting solvent of decompression, the enriched material drying obtains new glycosides peptide powdery product difficult to understand.
The present invention changes into the binary eluent system with the silica gel column chromatography ternary eluent system in the extracting method of the disclosed new glycosides peptide difficult to understand of patent 200910060121.4, and removed reversed-phase silica gel chromatography and separated and the high-efficient liquid phase chromatogram purification step, have and with an organic solvent consume less, the glycosides peptide product purity new difficult to understand that obtains is high, is easy to the advantages such as industrialization.
Description of drawings
The process flow sheet of new glycosides peptide powdery products production difficult to understand.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment 1
Get the new glycosides peptide fermented liquid difficult to understand of 2L (10000 units of tiring), fermented liquid first is heated to 60 ℃, kept 30 minutes, kill actinomycetes or its genetic improvement bacterial strain viable bacteria body in fermented liquid, then the fermented liquid temperature is down to below 30 ℃, add 2.0% (grams per milliliter) super-cell to the sterilization after fermented liquid in, stir, placed 20 minutes, and added the alum of 0.05% (grams per milliliter), fully stir, with 600 order filter-cloth filterings, filtrate is concentrated under reduced pressure, and vacuum degree control is at-0.08MPa, and temperature is controlled at 60 ℃; The thick cream of gained mixes with column chromatography silica gel (100～200 order), and its ratio is thick cream: silica gel=1: 1.5, stir, and 40 ℃ of lower vacuum-dryings, grind, cross 100 mesh sieves.Silica gel with G100～200 is filled out post, its quality is 8 times of upper prop material, carry out silica gel column chromatography, adopt chloroform: methyl alcohol=80～20: 20～80 binary eluents carry out gradient elution, lower column liquid gathers with thin layer chromatography analysis segmentation as a result, the lower Distillation recovery eluting solvent of decompression, the enriched material drying obtains new glycosides peptide powdery product difficult to understand, high pressure liquid chromatographic analysis product purity 90.4%.
Embodiment 2
Get the new glycosides peptide fermented liquid difficult to understand of 2L (10000 units of tiring), fermented liquid first is heated to 60 ℃, kept 30 minutes, kill actinomycetes or its genetic improvement bacterial strain viable bacteria body in fermented liquid, then the fermented liquid temperature is down to below 30 ℃, add 2.0% (grams per milliliter) super-cell to the sterilization after fermented liquid in, stir, placed 20 minutes, and added the alum of 0.05% (grams per milliliter), fully stir, with 600 order filter-cloth filterings, filtrate is concentrated under reduced pressure, and vacuum degree control is in-0.085MPa left and right, and temperature is controlled at 60 ℃; The thick cream of gained mixes with column chromatography silica gel (100～200 order), and its ratio is thick cream: silica gel=1: 1.5, stir, and 40 ℃ of lower vacuum-dryings, grind, cross 100 mesh sieves.Silica gel with G100～200 is filled out post, its quality is 8 times of upper prop material, carry out silica gel column chromatography, adopt chloroform: methyl alcohol=80～20: 20～80 binary eluents carry out gradient elution, lower column liquid gathers with thin layer chromatography analysis segmentation as a result, the new glycosides peptide wash-out difficult to understand lower Distillation recovery eluting solvent that partly reduces pressure obtains enriched material; This enriched material carries out the secondary silica gel column chromatography again, silica gel of eluent employing obtains chloroform and the methyl alcohol same ratio Non-gradient elution of this component, the lower Distillation recovery eluting solvent of decompression, the enriched material drying obtains new glycosides peptide powdery product difficult to understand, high pressure liquid chromatographic analysis product purity 94.2%.
Embodiment 3
Get the new glycosides peptide fermented liquid difficult to understand of 2L (10000 units of tiring), fermented liquid first is heated to 60 ℃, kept 30 minutes, kill actinomycetes or its genetic improvement bacterial strain viable bacteria body in fermented liquid, then the fermented liquid temperature is down to below 30 ℃, add 2.0% (grams per milliliter) pearlite filtering aid to the sterilization after fermented liquid in, stir, placed 20 minutes, and added the alum of 0.05% (grams per milliliter), fully stir, with 600 order filter-cloth filterings, filtrate is concentrated under reduced pressure, and vacuum degree control is in-0.09MPa left and right, and temperature is controlled at 55 ℃; The thick cream of gained mixes with column chromatography silica gel (100～200 order), and its ratio is thick cream: silica gel=1: 1.5, stir, and 40 ℃ of lower vacuum-dryings, grind, cross 100 mesh sieves.Silica gel with G100～200 is filled out post, its quality is 8 times of upper prop material, carry out silica gel column chromatography, adopt chloroform: methyl alcohol=80～20: 20～80 binary eluents carry out gradient elution, lower column liquid gathers with thin layer chromatography analysis segmentation as a result, the lower Distillation recovery eluting solvent of decompression, the enriched material drying obtains new glycosides peptide powdery product difficult to understand, high pressure liquid chromatographic analysis product purity 91.1%.
Embodiment 4
Get the new glycosides peptide fermented liquid difficult to understand of 2L (10000 units of tiring), fermented liquid first is heated to 60 ℃, kept 30 minutes, kill actinomycetes or its genetic improvement bacterial strain viable bacteria body in fermented liquid, then the fermented liquid temperature is down to below 30 ℃, add 2.0% (grams per milliliter) pearlite filtering aid to the sterilization after fermented liquid in, stir, placed 20 minutes, and added the alum of 0.05% (grams per milliliter), fully stir, with 600 order filter-cloth filterings, filtrate is concentrated under reduced pressure, and vacuum degree control is in-0.09MPa left and right, and temperature is controlled at 55 ℃; The thick cream of gained mixes with column chromatography silica gel (100～200 order), and its ratio is thick cream: silica gel=1: 1.5, stir, and 40 ℃ of lower vacuum-dryings, grind, cross 100 mesh sieves.Silica gel with G100～200 is filled out post, its quality is 8 times of upper prop material, carry out silica gel column chromatography, adopt chloroform: methyl alcohol=80～20: 20～80 binary eluents carry out gradient elution, lower column liquid gathers with thin layer chromatography analysis segmentation as a result, the new glycosides peptide wash-out difficult to understand lower Distillation recovery eluting solvent that partly reduces pressure obtains enriched material; This enriched material carries out the secondary silica gel column chromatography again, silica gel of eluent employing obtains chloroform and the methyl alcohol same ratio Non-gradient elution of this component, the lower Distillation recovery eluting solvent of decompression, the enriched material drying obtains new glycosides peptide powdery product difficult to understand, high pressure liquid chromatographic analysis product purity 94.8%.
Claims (1)
1. the preparation method of a new glycosides peptide powdery solid difficult to understand is characterized in that: comprise the following steps:
(1) thick cream preparation: fermented liquid first is heated to 60 ℃, keeps 10-60 minute, then the fermented liquid temperature is down to below 30 ℃; Add perlite or diatomite, fully stir, placed 10-30 minute, adding alum again, fully stir, is that 600-2000 purpose filter cloth filters with model, filtrate at vacuum tightness-0.08MPa to-0.09MPa, concentrate to get thick cream under 60～70 ℃ or 50～55 ℃ conditions of bath temperature, described fermented liquid is produced by the actinomycete fermentation that produces new glycosides peptide difficult to understand, and the per-cent that the perlite that adds or diatomaceous quality gram account for the volume milliliter of fermented liquid is 0.1%-3.0%; The per-cent that the quality gram of the alum that adds accounts for the volume milliliter of fermented liquid is 0.01%-0.1%;
(2) purification refine: the thick cream of gained mixes with 100～200 purpose column chromatography silica gels, its ratio is thick cream: silica gel=1:1～2.5, stir, vacuum-drying under 40 ℃ of conditions, grind, cross 100 mesh sieves, silica gel with G100～200 is filled out post, its quality is 5～10 times that upper prop stirs material, carry out silica gel column chromatography, adopt chloroform: methyl alcohol=80～20:20～80 binary eluents carry out gradient elution, and lower column liquid gathers with thin layer chromatography analysis segmentation as a result, the lower Distillation recovery eluting solvent of decompression, the enriched material drying obtains new glycosides peptide powdery product difficult to understand.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110261822 CN102321153B (en) | 2011-09-06 | 2011-09-06 | Preparation method of xin'ao glycoside peptide powdery solid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110261822 CN102321153B (en) | 2011-09-06 | 2011-09-06 | Preparation method of xin'ao glycoside peptide powdery solid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102321153A CN102321153A (en) | 2012-01-18 |
| CN102321153B true CN102321153B (en) | 2013-06-05 |
Family
ID=45449010
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110261822 Expired - Fee Related CN102321153B (en) | 2011-09-06 | 2011-09-06 | Preparation method of xin'ao glycoside peptide powdery solid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102321153B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103965275B (en) * | 2014-05-27 | 2016-08-31 | 中国科学院成都生物研究所 | A kind of separating and extracting process of streptomycete fermentation metabolite newly mycin difficult to understand |
| CN110849766B (en) * | 2019-10-18 | 2022-03-01 | 中国石油天然气集团有限公司 | Method for correcting adsorbed gas content of shale isothermal adsorption experiment under low pressure |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1312813A (en) * | 1998-06-09 | 2001-09-12 | 宝酒造株式会社 | Therapeutic agents |
-
2011
- 2011-09-06 CN CN 201110261822 patent/CN102321153B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1312813A (en) * | 1998-06-09 | 2001-09-12 | 宝酒造株式会社 | Therapeutic agents |
Non-Patent Citations (5)
| Title |
|---|
| 人工培养蛹虫草子座中核苷类化合物的提取工艺研究;汪宇等;《中国药师》;20041231;第7卷(第12期);第930页第1栏最后1段 * |
| 新型生物杀菌剂新奥霉素防治烟草花叶病毒病的田间药效;冯振群，卢清;《现代农药》;20110831;第10卷(第4期);第50-52页 * |
| 新奥霉素发酵液的理化性质和提取方法研究;宁停波等;《湖南农业科学》;20110715(第13期);第23页第1栏第2段，第23页第2栏第1-2段，第24页第1栏第2、7段 * |
| 生物农药的研究和应用进展;邓洪渊等;《世界科技研究与发展》;20050228;第27卷(第1期);第76-80页 * |
| 葡萄灰孢霉代谢产物的分离及生物活性;周金燕等;《应用与环境生物学报》;20021028;第8卷(第5期);第532-534页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102321153A (en) | 2012-01-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101481715B (en) | Method for purifying tacrolimus through biological fermentation | |
| CN114409660B (en) | CPA type indole alkaloid compound and preparation method and application thereof | |
| CN102718843A (en) | Preparation method of single teicoplanin components | |
| JP6592500B2 (en) | Purification method of fidaxomycin | |
| CN114350722B (en) | Method for preparing genistein | |
| CN103772086B (en) | Pretreatment process for preparing fractions of marine microorganism small molecule metabolites | |
| CN113897406B (en) | Method for extracting and purifying salidroside from rhodiola rosea powder | |
| CN102321153B (en) | Preparation method of xin'ao glycoside peptide powdery solid | |
| CN109666051B (en) | Purification method of kasugamycin | |
| CN104846043A (en) | Process for separating and purifying fidaxomicin from fermentation broth | |
| CN103965275B (en) | A kind of separating and extracting process of streptomycete fermentation metabolite newly mycin difficult to understand | |
| CN104402976A (en) | Method for preparing enramycin refined powder | |
| CN103848900A (en) | Method for separating WF11899A from fermentation liquor and purifying | |
| CN103073624B (en) | A kind of preparation method of high purity cyclosporin A derivative | |
| CN112409426B (en) | Preparation method of sisomicin sulfate | |
| CN113912662A (en) | C in fermentation liquor21Method for purifying steroid compound | |
| CN101638401A (en) | Method for preparing high-purity danshinolic acid B | |
| CN108586435B (en) | Preparation method of Echinulin | |
| CN107686492A (en) | A kind of method of rhodioside in extraction purification zymotic fluid using macroporous absorbent resin | |
| CN113789357A (en) | Industrial preparation method for producing cordycepin and pentostatin by combined fermentation | |
| Zhao et al. | Coupling of spinosad fermentation and separation process via two-step macroporous resin adsorption method | |
| CN102952180B (en) | A kind of preparation method of high purity echinocandin B | |
| CN107904267B (en) | Method for synthesizing p-hydroxybenzaldehyde by adopting microbial transformation | |
| CN112390817B (en) | Method for salting out and extracting tacrolimus fermentation liquor | |
| CN115521877B (en) | Penicillium strain and application thereof in preparation of brefeldin A |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130605 Termination date: 20210906 |