CN107177648A - A kind of chitosan oligosaccharide enzymatic production process - Google Patents
A kind of chitosan oligosaccharide enzymatic production process Download PDFInfo
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- CN107177648A CN107177648A CN201710518893.2A CN201710518893A CN107177648A CN 107177648 A CN107177648 A CN 107177648A CN 201710518893 A CN201710518893 A CN 201710518893A CN 107177648 A CN107177648 A CN 107177648A
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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Abstract
The invention discloses a kind of chitosan oligosaccharide enzymatic production process, comprise the following steps：Chitosan is added in acetum by a, is configured to chitosan solution；B adds hydrogenperoxide steam generator into chitosan solution, stirs, and carries out degradation reaction, degradation solution is obtained after the completion of degraded；Degradation solution is cooled to room temperature by c, is then adjusted pH value to 6.2 6.5, is added β glucuroides, carries out first step enzymolysis, obtain first step enzymolysis liquid；D adds α Isosorbide-5-Nitrae glucose hydrolysis enzymes into first step enzymolysis liquid again, carries out second step enzymolysis, obtains second step enzymolysis liquid；Second step enzymolysis liquid pH value is adjusted to 6 6.5 by e, then is carried out successively after coarse filtration, ultrafiltration, nanofiltration, spray drying, sterilization, obtains finished product chitosan oligosaccharide.Present invention greatly enhances the yield of chitosan oligosaccharide, and final obtained chitosan oligosaccharide molecular weight product can be made to be less than 1500, size is relatively uniform, and product is free of monose, effectively raises the quality of chitosan oligosaccharide product.
Description
Technical field
The present invention relates to a kind of chitosan oligosaccharide production technology, a kind of chitosan oligosaccharide enzymatic production process is concretely related to.
Background technology
Chitosan oligosaccharide is called Chitosan poly oligosaccharide, chitosan oligomer, is 2~20 by chitosan through the obtained a kind of degree of polymerization of degrading
Between oligosaccharide product, molecular weight≤3200Da, be it is water-soluble preferably, the low molecule volume production that function is big, bioactivity is high
Product.It has the unexistent higher solubility of chitosan, and water is dissolved in entirely, easily many unique work(such as absorbs by organism
Can, it act as 14 times of chitosan.Chitosan oligosaccharide is unique positively charged cation basic amine group oligosaccharide in nature, is
Physical property cellulose.
Research has shown that：Chitosan oligosaccharide, which has, improves immune, suppresses cancerous swelling cell growth, promotes liver and spleen antibody to be formed, promotes calcium
And the absorption of mineral matter, breed the human body profitable strains such as Bifidobacterium, lactic acid bacteria, reducing blood lipid, hypotensive, hypoglycemic, regulation courage
Sterol, fat-reducing prevents the functions such as adult disease, can be applied to the fields such as medicine, functional food.Chitosan oligosaccharide can substantially eliminate people
Body Atomic oxygen radical anion, activates body cell, and anti-aging suppresses skin surface harmful bacteria and grown, performance of keeping humidity is excellent, is
The basic material of household chemicals field.Chitosan oligosaccharide not only possesses water solubility, easy to use, and suppresses the spoilage organisms impact of performance significantly,
Have both a variety of functions, be the wholefood antisepsis antistaling agent of function admirable.
The preparation method of chitosan oligosaccharide has physical degradation methods (sonication, gamma Rays degraded), glycosyl transfer anti-
Should, chemical method (Acid hydrolysis, oxidizing process) and enzyme process (degraded of selectivity enzyme, non-specific enzyme are degraded) etc..Wherein more commonly use
Be enzymatic isolation method, such as the degraded of selectivity enzyme can use chitinase, chitosan enzyme and lysozyme, but the degraded of selectivity enzyme is limited
It is higher in cost, it is unfavorable for large-scale production.Non-specific enzyme degraded can solve the problem of selectivity enzyme process cost is high, but existing
Non-specific enzyme degraded is bad to the degradation effect of chitosan such as using commercial lipases, protease, pectase, needs
In further lifting.
The content of the invention
Based on above-mentioned technical problem, the present invention provides a kind of chitosan oligosaccharide enzymatic production process.
The adopted technical solution is that：
A kind of chitosan oligosaccharide enzymatic production process, comprises the following steps：
Chitosan is added in acetum by a, is configured to chitosan solution, and the mass percent of chitosan solution is dense
Spend for 15%-20%；
Hydrogenperoxide steam generator is added in chitosan solution made from b to step a, is stirred, degradation reaction, control is carried out
Degradation temperature processed is 60-80 DEG C, and degradation time is 4-6 hours, and degradation solution is obtained after the completion of degraded；
The obtained degradation solutions of step b are cooled to room temperature by c, then add saturated sodium bicarbonate solution adjust pH value to
6.2-6.5, adds beta-glucosidase, carries out first step enzymolysis, and controlled enzymatic hydrolysis reaction temperature is 35-37 DEG C, enzymolysis time
For 3-5 hours, first step enzymolysis liquid is obtained；
α-Isosorbide-5-Nitrae-glucose hydrolysis enzyme is added in first step enzymolysis liquid made from d to step c, second step enzymolysis, control is carried out
Enzyme digestion reaction temperature processed is 40-42 DEG C, and enzymolysis time is 4-6 hours, obtains second step enzymolysis liquid；
E using food sodium hydroxide adjust second step enzymolysis liquid pH value to 6-6.5, then successively carry out coarse filtration, ultrafiltration,
After nanofiltration, spray drying, sterilization, finished product chitosan oligosaccharide is obtained.
It is preferred that, the mass percent concentration of the acetum is 1%-2%.
It is preferred that, the mass percent concentration of the hydrogenperoxide steam generator is 30%-40%, and chitosan is molten with hydrogen peroxide
The mass ratio of liquid is 1: 0.1-0.5.
It is preferred that, the addition of the beta-glucosidase is the 1%-1.2% of chitosan mass.
It is preferred that, the addition of the α-Isosorbide-5-Nitrae-glucose hydrolysis enzyme is the 0.03%-0.05% of chitosan mass.
It is preferred that, ultrafilter and collecting and filtering apparatus is respectively adopted in the ultrafiltration and nanofiltration, and spray drying temperature is 160 DEG C~180
℃。
The method have the benefit that：
The present invention first carries out degradation treatment using hydrogenperoxide steam generator to chitosan, then using beta-glucosidase and α-
Isosorbide-5-Nitrae-glucose hydrolysis enzyme coordinates carries out stepwise discretization to degradation solution, while coordinating the essence to each raw material dosage and enzymatic hydrolysis condition etc.
Fidelity is determined, and not only can carry out efficient selective degradation to chitosan, drastically increase the yield of chitosan oligosaccharide, Er Qieke
Final obtained chitosan oligosaccharide molecular weight product is set to be less than 1500, size is relatively uniform, and product is free of monose, effectively raises
The quality of chitosan oligosaccharide product, meets the requirement of the industrialization large-scale production of chitosan oligosaccharide product.
Embodiment
Embodiment 1
Chitosan is added in acetum by a, is configured to chitosan solution, and the mass percent of chitosan solution is dense
Spend for 15%；The mass percent concentration of acetum is 1%.
Hydrogenperoxide steam generator, the mass percent of hydrogenperoxide steam generator are added in chitosan solution made from b to step a
Concentration is 40%, and the mass ratio of chitosan and hydrogenperoxide steam generator is 1: 0.5；Stir, carry out degradation reaction, control degraded
Temperature is 80 DEG C, and degradation time is 4 hours, and degradation solution is obtained after the completion of degraded.
The obtained degradation solutions of step b are cooled to room temperature by c, then add saturated sodium bicarbonate solution adjust pH value to
6.2, beta-glucosidase is added, first step enzymolysis is carried out, the addition of beta-glucosidase is the 1% of chitosan mass；
Controlled enzymatic hydrolysis reaction temperature is 37 DEG C, and enzymolysis time is 3 hours, obtains first step enzymolysis liquid.
Addition α-Isosorbide-5-Nitrae-glucose hydrolysis enzyme in first step enzymolysis liquid made from d to step c, progress second step enzymolysis, α-
The addition of 1,4- glucose hydrolysis enzymes is the 0.03% of chitosan mass；Controlled enzymatic hydrolysis reaction temperature is 42 DEG C, enzymolysis time
For 4 hours, second step enzymolysis liquid is obtained.
E adjusts second step enzymolysis liquid pH value to 6.5 using food sodium hydroxide, then carries out coarse filtration, ultrafiltration successively, receives
After filter, spray drying, sterilization, finished product chitosan oligosaccharide is obtained.
Ultrafilter and collecting and filtering apparatus is respectively adopted in above-mentioned ultrafiltration and nanofiltration, and spray drying temperature is 160 DEG C~180 DEG C.
Analyze after testing, it is as a result as follows：Gained chitosan oligosaccharide outward appearance is light yellow, granularity 60-70 mesh, moisture ＜ 8.8%, ash
Divide 0.68%, insoluble matter ＜ 0.3%, molecular weight ＜ 1500, lead ＜ 5ppm, arsenic ＜ 1ppm, total number of bacteria 603/g, Escherichia coli
Do not detect, pathogenic bacteria do not detect, chitosan oligosaccharide yield (mass ratio of chitosan oligosaccharide and chitosan) 83%.
Embodiment 2
Chitosan is added in acetum by a, is configured to chitosan solution, and the mass percent of chitosan solution is dense
Spend for 20%；The mass percent concentration of acetum is 2%.
Hydrogenperoxide steam generator, the mass percent of hydrogenperoxide steam generator are added in chitosan solution made from b to step a
Concentration is 35%, and the mass ratio of chitosan and hydrogenperoxide steam generator is 1: 0.3；Stir, carry out degradation reaction, control degraded
Temperature is 60 DEG C, and degradation time is 4 hours, and degradation solution is obtained after the completion of degraded.
The obtained degradation solutions of step b are cooled to room temperature by c, then add saturated sodium bicarbonate solution adjust pH value to
6.5, beta-glucosidase is added, first step enzymolysis is carried out, the addition of beta-glucosidase is chitosan mass
1.2%；Controlled enzymatic hydrolysis reaction temperature is 35 DEG C, and enzymolysis time is 5 hours, obtains first step enzymolysis liquid.
Addition α-Isosorbide-5-Nitrae-glucose hydrolysis enzyme in first step enzymolysis liquid made from d to step c, progress second step enzymolysis, α-
The addition of 1,4- glucose hydrolysis enzymes is the 0.05% of chitosan mass；Controlled enzymatic hydrolysis reaction temperature is 40 DEG C, enzymolysis time
For 6 hours, second step enzymolysis liquid is obtained.
E using food sodium hydroxide adjust second step enzymolysis liquid pH value to 6, then successively carry out coarse filtration, ultrafiltration, nanofiltration,
After spray drying, sterilization, finished product chitosan oligosaccharide is obtained.
Ultrafilter and collecting and filtering apparatus is respectively adopted in above-mentioned ultrafiltration and nanofiltration, and spray drying temperature is 160 DEG C~180 DEG C.
Analyze after testing, it is as a result as follows：Gained chitosan oligosaccharide outward appearance is light yellow, granularity 60-70 mesh, moisture ＜ 8.8%, ash
Divide 0.68%, insoluble matter ＜ 0.3%, molecular weight ＜ 1500, lead ＜ 5ppm, arsenic ＜ 1ppm, total number of bacteria 621/g, Escherichia coli
Do not detect, pathogenic bacteria do not detect, chitosan oligosaccharide yield (mass ratio of chitosan oligosaccharide and chitosan) 85%.
Embodiment 3
Chitosan is added in acetum by a, is configured to chitosan solution, and the mass percent of chitosan solution is dense
Spend for 18%；The mass percent concentration of acetum is 1.5%.
Hydrogenperoxide steam generator, the mass percent of hydrogenperoxide steam generator are added in chitosan solution made from b to step a
Concentration is 40%, and the mass ratio of chitosan and hydrogenperoxide steam generator is 1: 0.3；Stir, carry out degradation reaction, control degraded
Temperature is 70 DEG C, and degradation time is 4 hours, and degradation solution is obtained after the completion of degraded.
The obtained degradation solutions of step b are cooled to room temperature by c, then add saturated sodium bicarbonate solution adjust pH value to
6.3, beta-glucosidase is added, first step enzymolysis is carried out, the addition of beta-glucosidase is chitosan mass
1.2%；Controlled enzymatic hydrolysis reaction temperature is 36 DEG C, and enzymolysis time is 4 hours, obtains first step enzymolysis liquid.
Addition α-Isosorbide-5-Nitrae-glucose hydrolysis enzyme in first step enzymolysis liquid made from d to step c, progress second step enzymolysis, α-
The addition of 1,4- glucose hydrolysis enzymes is the 0.04% of chitosan mass；Controlled enzymatic hydrolysis reaction temperature is 41 DEG C, enzymolysis time
For 5 hours, second step enzymolysis liquid is obtained.
E using food sodium hydroxide adjust second step enzymolysis liquid pH value to 6, then successively carry out coarse filtration, ultrafiltration, nanofiltration,
After spray drying, sterilization, finished product chitosan oligosaccharide is obtained.
Ultrafilter and collecting and filtering apparatus is respectively adopted in above-mentioned ultrafiltration and nanofiltration, and spray drying temperature is 160 DEG C~180 DEG C.
Analyze after testing, it is as a result as follows：Gained chitosan oligosaccharide outward appearance is light yellow, granularity 60-70 mesh, moisture ＜ 8.8%, ash
Divide 0.68%, insoluble matter ＜ 0.3%, molecular weight ＜ 1500, lead ＜ 5ppm, arsenic ＜ 1ppm, total number of bacteria 621/g, Escherichia coli
Do not detect, pathogenic bacteria do not detect, chitosan oligosaccharide yield (mass ratio of chitosan oligosaccharide and chitosan) 89%.
The present invention uses beta-glucosidase and α-Isosorbide-5-Nitrae-glucose hydrolysis enzyme to chitosan stepwise discretization, and to enzyme dosage
And the term restriction such as hydrolysis temperature, the yield of chitosan oligosaccharide is drastically increased, and make final obtained chitosan oligosaccharide molecular weight product
Less than 1500, the quality of chitosan oligosaccharide product is effectively raised.Need to carry out such as on enzyme dosage in the present invention and reaction temperature
Lower explanation：When optimum temperature and optimal enzyme content are matched, enzymatic reaction is most fast, too high or too low for temperature all to become reaction
Slowly, it is not that temperature more high molecular collision is fiercer, too high temperature can change the structure of enzyme molecule, or even inactivate enzyme,
Reaction rate is caused to decline, with the increase of enzyme amount, the content of reduced sugar no longer rises, and causes chitosan oligosaccharide yield also no longer to increase
Plus, therefore, in order to obtain higher chitosan oligosaccharide yield, it is necessary to which selection enzyme meticulously is combined and suitable enzyme amount.
Claims (6)
1. a kind of chitosan oligosaccharide enzymatic production process, it is characterised in that comprise the following steps：
Chitosan is added in acetum by a, is configured to chitosan solution, and the mass percent concentration of chitosan solution is
15%-20%；
Hydrogenperoxide steam generator is added in chitosan solution made from b to step a, is stirred, degradation reaction, control degraded is carried out
Temperature is 60-80 DEG C, and degradation time is 4-6 hours, and degradation solution is obtained after the completion of degraded；
The obtained degradation solutions of step b are cooled to room temperature by c, are then added saturated sodium bicarbonate solution and are adjusted pH value to 6.2-6.5,
Beta-glucosidase is added, first step enzymolysis is carried out, controlled enzymatic hydrolysis reaction temperature is 35-37 DEG C, and enzymolysis time is that 3-5 is small
When, obtain first step enzymolysis liquid；
α-Isosorbide-5-Nitrae-glucose hydrolysis enzyme is added in first step enzymolysis liquid made from d to step c, second step enzymolysis is carried out, enzyme is controlled
It is 40-42 DEG C to solve reaction temperature, and enzymolysis time is 4-6 hours, obtains second step enzymolysis liquid；
E using food sodium hydroxide adjust second step enzymolysis liquid pH value to 6-6.5, then successively carry out coarse filtration, ultrafiltration, nanofiltration,
After spray drying, sterilization, finished product chitosan oligosaccharide is obtained.
2. a kind of chitosan oligosaccharide enzymatic production process according to claim 1, it is characterised in that：The quality of the acetum
Percent concentration is 1%-2%.
3. a kind of chitosan oligosaccharide enzymatic production process according to claim 1, it is characterised in that：The hydrogenperoxide steam generator
Mass percent concentration is 30%-40%, and the mass ratio of chitosan and hydrogenperoxide steam generator is 1: 0.1-0.5.
4. a kind of chitosan oligosaccharide enzymatic production process according to claim 1, it is characterised in that：The beta-glucosidase
Addition is the 1%-1.2% of chitosan mass.
5. a kind of chitosan oligosaccharide enzymatic production process according to claim 1, it is characterised in that：α -1,4- the G/Ws
The addition for solving enzyme is the 0.03%-0.05% of chitosan mass.
6. a kind of chitosan oligosaccharide enzymatic production process according to claim 1, it is characterised in that：Ultrafiltration and the nanofiltration difference
Using ultrafilter and collecting and filtering apparatus, spray drying temperature is 160 DEG C~180 DEG C.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108559763A (en) * | 2018-03-29 | 2018-09-21 | 江南大学 | A kind of preparation method improving chitosan oligosaccharide degree of polymerization 3-6 sugared contents |
| CN108588150A (en) * | 2018-04-27 | 2018-09-28 | 厦门大学 | Batch feeding-oxidation pre-treatment auxiliary enzymes hydrolyzing chitosan prepares chitosan oligosaccharide method |
| CN109438103A (en) * | 2019-01-10 | 2019-03-08 | 江苏心实肥业集团有限公司 | A kind of chitosan oligosaccharide crop nutrition spray fertilizer and preparation method thereof |
| CN110387392A (en) * | 2019-08-12 | 2019-10-29 | 青岛博智汇力生物科技有限公司 | A kind of preparation method of specific aggregation degree chitosan oligosaccharide and its application on cosmetics |
| CN111393488A (en) * | 2020-03-06 | 2020-07-10 | 安徽科博瑞环境科技有限公司 | Method for purifying and concentrating water-soluble chitosan oligosaccharide |
| CN112273552A (en) * | 2020-11-26 | 2021-01-29 | 福建傲农生物科技集团股份有限公司 | Feed additive and preparation method and application thereof |
| CN112608958A (en) * | 2020-12-17 | 2021-04-06 | 黄河三角洲京博化工研究院有限公司 | Chitosan oligosaccharide preparation method and weight-losing tablets |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108559763A (en) * | 2018-03-29 | 2018-09-21 | 江南大学 | A kind of preparation method improving chitosan oligosaccharide degree of polymerization 3-6 sugared contents |
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| CN108588150A (en) * | 2018-04-27 | 2018-09-28 | 厦门大学 | Batch feeding-oxidation pre-treatment auxiliary enzymes hydrolyzing chitosan prepares chitosan oligosaccharide method |
| CN109438103A (en) * | 2019-01-10 | 2019-03-08 | 江苏心实肥业集团有限公司 | A kind of chitosan oligosaccharide crop nutrition spray fertilizer and preparation method thereof |
| CN110387392A (en) * | 2019-08-12 | 2019-10-29 | 青岛博智汇力生物科技有限公司 | A kind of preparation method of specific aggregation degree chitosan oligosaccharide and its application on cosmetics |
| CN111393488A (en) * | 2020-03-06 | 2020-07-10 | 安徽科博瑞环境科技有限公司 | Method for purifying and concentrating water-soluble chitosan oligosaccharide |
| CN112273552A (en) * | 2020-11-26 | 2021-01-29 | 福建傲农生物科技集团股份有限公司 | Feed additive and preparation method and application thereof |
| CN112608958A (en) * | 2020-12-17 | 2021-04-06 | 黄河三角洲京博化工研究院有限公司 | Chitosan oligosaccharide preparation method and weight-losing tablets |
| CN112608958B (en) * | 2020-12-17 | 2022-12-02 | 黄河三角洲京博化工研究院有限公司 | Chitosan oligosaccharide preparation method and weight-losing tablets |
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Application publication date: 20170919 |