CN109136307A - A kind of method and application thereof preparing chitosan oligosaccharide with glusulase - Google Patents
A kind of method and application thereof preparing chitosan oligosaccharide with glusulase Download PDFInfo
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- CN109136307A CN109136307A CN201811039866.8A CN201811039866A CN109136307A CN 109136307 A CN109136307 A CN 109136307A CN 201811039866 A CN201811039866 A CN 201811039866A CN 109136307 A CN109136307 A CN 109136307A
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- chitosan oligosaccharide
- glusulase
- chitosan
- oligosaccharide
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- RQFQJYYMBWVMQG-IXDPLRRUSA-N chitotriose Chemical compound O[C@@H]1[C@@H](N)[C@H](O)O[C@H](CO)[C@H]1O[C@H]1[C@H](N)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)[C@@H](CO)O1 RQFQJYYMBWVMQG-IXDPLRRUSA-N 0.000 title claims abstract description 101
- 238000000034 method Methods 0.000 title claims abstract description 47
- 108010087005 glusulase Proteins 0.000 title claims abstract description 40
- 229920001661 Chitosan Polymers 0.000 claims abstract description 47
- 108090000790 Enzymes Proteins 0.000 claims abstract description 41
- 102000004190 Enzymes Human genes 0.000 claims abstract description 41
- 230000007071 enzymatic hydrolysis Effects 0.000 claims abstract description 17
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims abstract description 17
- 239000000758 substrate Substances 0.000 claims abstract description 15
- 201000009794 Idiopathic Pulmonary Fibrosis Diseases 0.000 claims abstract description 14
- 208000036971 interstitial lung disease 2 Diseases 0.000 claims abstract description 14
- 239000000243 solution Substances 0.000 claims description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 239000000047 product Substances 0.000 claims description 15
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 239000012141 concentrate Substances 0.000 claims description 10
- 239000012153 distilled water Substances 0.000 claims description 9
- 239000004627 regenerated cellulose Substances 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 7
- 239000007974 sodium acetate buffer Substances 0.000 claims description 7
- 241000237858 Gastropoda Species 0.000 claims description 6
- 238000000502 dialysis Methods 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 5
- 239000002994 raw material Substances 0.000 claims description 5
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 5
- 238000004821 distillation Methods 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
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- 238000006460 hydrolysis reaction Methods 0.000 abstract description 18
- 238000004108 freeze drying Methods 0.000 abstract description 5
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- ISWRGOKTTBVCFA-UHFFFAOYSA-N pirfenidone Chemical compound C1=C(C)C=CC(=O)N1C1=CC=CC=C1 ISWRGOKTTBVCFA-UHFFFAOYSA-N 0.000 description 7
- CHVZQMAANSUXJU-JJKGCWMISA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanamide;hydrochloride Chemical compound Cl.NC(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO CHVZQMAANSUXJU-JJKGCWMISA-N 0.000 description 6
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- 238000010438 heat treatment Methods 0.000 description 3
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- 238000006116 polymerization reaction Methods 0.000 description 3
- 208000005069 pulmonary fibrosis Diseases 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- 238000000859 sublimation Methods 0.000 description 3
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- 206010002091 Anaesthesia Diseases 0.000 description 2
- 108010059892 Cellulase Proteins 0.000 description 2
- 241000588921 Enterobacteriaceae Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
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- 235000013305 food Nutrition 0.000 description 2
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- 238000005303 weighing Methods 0.000 description 2
- WCWOEQFAYSXBRK-GASJEMHNSA-N (3r,4s,5s,6r)-2-amino-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound NC1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O WCWOEQFAYSXBRK-GASJEMHNSA-N 0.000 description 1
- 241000208340 Araliaceae Species 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- 125000003047 N-acetyl group Chemical group 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- YBCVMFKXIKNREZ-UHFFFAOYSA-N acoh acetic acid Chemical compound CC(O)=O.CC(O)=O YBCVMFKXIKNREZ-UHFFFAOYSA-N 0.000 description 1
- 231100000570 acute poisoning Toxicity 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- MSWZFWKMSRAUBD-QZABAPFNSA-N beta-D-glucosamine Chemical compound N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-QZABAPFNSA-N 0.000 description 1
- 229960001561 bleomycin Drugs 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000011449 brick Substances 0.000 description 1
- 238000011095 buffer preparation Methods 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000002612 cardiopulmonary effect Effects 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000006196 deacetylation Effects 0.000 description 1
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- 230000002538 fungal effect Effects 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000003694 hair properties Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
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- 229920002521 macromolecule Polymers 0.000 description 1
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- 238000005457 optimization Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229960001922 sodium perborate Drugs 0.000 description 1
- YKLJGMBLPUQQOI-UHFFFAOYSA-M sodium;oxidooxy(oxo)borane Chemical compound [Na+].[O-]OB=O YKLJGMBLPUQQOI-UHFFFAOYSA-M 0.000 description 1
- 235000000891 standard diet Nutrition 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/716—Glucans
- A61K31/722—Chitin, chitosan
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12P19/14—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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Pulmonology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
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- Polysaccharides And Polysaccharide Derivatives (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to oligosaccharide preparation technical fields, and in particular to a kind of method and its application that chitosan oligosaccharide is prepared with glusulase.The method provided by the invention for preparing chitosan oligosaccharide with glusulase, the specific process conditions such as enzymolysis time, hydrolysis temperature, concentration of substrate, enzyme bottom mass ratio to glusulase enzymatic hydrolysis chitosan are explored and are optimized, improve enzymolysis efficiency, in conjunction with freeze-drying, maintain the character of chitosan oligosaccharide, convenient for product preservation and further use, the mass production for preparing chitosan oligosaccharide for glusulase provides foundation.Chitosan oligosaccharide produced by the present invention can also significantly improve the fibrocyte activation of idiopathic pulmonary fibrosis rat model, have good improvement result to idiopathic pulmonary fibrosis, be conducive to the rehabilitation of idiopathic pulmonary fibrosis.
Description
Technical field
The invention belongs to oligosaccharide preparation technical fields, and in particular to it is a kind of with glusulase prepare chitosan oligosaccharide method and its
Purposes.
Background technique
Chitosan is also known as acetyl chitin, is the compound that chitin takes off N- acetyl group, has biodegradability, safety
Property, good bio-compatibility.But chitosan is long-chain macromolecule, and poorly water-soluble under physiological condition, solution viscosity is big, makes its work
It is very limited for the application of biological effector agent.Chitosan oligosaccharide is its catabolite, refers to Glucosamine of the degree of polymerization less than 10
Polymer, relative molecular mass is small, good water solubility, is easily dispersed and is absorbed, and has many biologies similar with chitosan
The chitosan oligosaccharide that property, the especially degree of polymerization are 5 and 6 is learned, has anti-oxidant, antitumor, Immune-enhancing effect, activation enterobacteriaceae etc. living
Property, when treating tumour in vivo, do not cause the rapid decline of acute poisoning and weight, toxic side effect is smaller, suitable for facing
Bed treatment, also can be used as food additives and health care product, has a wide range of applications in food, medicine and cosmetic field.
Mainly there are 3 kinds currently with the method that chitosan prepares chitosan oligosaccharide: physical degradation methods, chemical degradation method and enzymatic hydrolysis
Method.Physical degradation methods are mainly degraded and the chemical bond in chitosan molecule in radiative process to that fracture occur, such as ultrasound
Wave method, microwave irradiation etc., yield is lower, production cost is relatively high, realizes that industrialization difficulty is larger.Chemical degradation method is main
Including acid hydrolyzation, hydrogen peroxide and sodium perborate oxidation degradation method etc., such as acid hydrolyzation customary salt acid degradation chitosan, although being suitble to
Industrialized production, but the research of the depolymerization and acetylization reaction according to EinbuAslak et al. for chitin in hydrochloric acid is sent out
Existing acid hydrolyzation products obtained therefrom monosaccharide is more, and chitosan oligosaccharide content is low, and bioactivity is difficult to embody, and needs in preparation process a large amount of
Chemical reagent would seriously pollute the environment.
Enzymatic isolation method mainly has specificity enzymatic hydrolysis and non-specific enzymatic hydrolysis, although the former is strong to the degradation specificity of chitosan,
But the enzymatic productivity for obtaining chitosan enzyme bacterial strain at present is lower, and limited source.Non-specific enzyme, such as Chinese patent literature
A kind of method preparing chitosan oligosaccharide using papain freeze-drying disclosed in 201711093783.2, can be fast in a short time
Speed reduces chitosan viscosity, obtains more chitosan oligosaccharide, but gained chitosan oligosaccharide average molecular weight is too small, may be to product
Bioactivity has an impact.It can be seen that often viscosity is lower for product, and oligosaccharides contains when a kind of non-specific enzyme is used alone preparing chitosan oligosaccharide
Amount is few, and monosaccharide is more, and product quality is bad or degradation reaction low efficiency, it is difficult to practical application, it is even more noteworthy, single-minded
Enzyme is expensive, is difficult to be commercialized for property.
In recent years, chitosan oligosaccharide is prepared as research hotspot using a variety of non-specific enzyme combined degradation chitosans.Such as albumen
On the one hand the use in conjunction such as enzyme, cellulase can quickly reduce the viscosity of reactant chitosan, on the other hand can obtain a large amount of raw
The chitosan oligosaccharide that the high degree of polymerization of object activity is 3~7, but this method must use a variety of enzymes, and the proportion between each enzyme is mutually assisted
Tune effect is different, and obtained chitosan oligosaccharide molecular weight is different, though it can obtain determining the chitosan oligosaccharide of molecular weight with composite degradation method, it should
Method very complicated is commercially produced using being restricted, and the stability and quality of resulting chitosan oligosaccharide are also limited, and reduce shell
The bioactivity of oligosaccharides and with value.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for preparing chitosan oligosaccharide with glusulase, exist mainly for the prior art
The problem of: physical degradation methods higher cost and low yield, chemical degradation method step is various, and products obtained therefrom monosaccharide is more, chitosan oligosaccharide
Content is low and a large amount of chemical reagent pollutes the environment, and when preparing chitosan oligosaccharide using common enzymatic isolation method, the drop of each list enzyme
It is limited, expensive to solve inefficient and certain specificity enzyme sources, whens several non-specific enzymes of use in conjunction must be taken into consideration again
Interact degradation problem, causes to be restricted when selecting enzyme.The method letter provided by the invention that chitosan oligosaccharide is prepared with glusulase
Just, quickly, environmental pollution is small, and gained chitosan oligosaccharide character is good, molecular weight is ideal.
In order to solve the above-mentioned technical problem, the technical scheme is that
A method of chitosan oligosaccharide is prepared with glusulase, comprising the following steps:
S1 takes chitosan raw material drying to constant weight, is dissolved in acetic acid-sodium acetate buffer solution, stirring and dissolving is configured to matter
Measure the chitosan solution that concentration is 1.0%；
The snail enzyme solutions that mass concentration is 1.0%, the glusulase are added into chitosan solution obtained by step S1 by S2
It is 1:10 with chitosan substrate mass ratio, after digesting 2h~3h in 35~45 DEG C of water bath, 100 DEG C of 5~15min of water-bath go out
It is living, it is cooled to room temperature, continuously stirs, the NaOH solution that mass concentration is 5% is added dropwise, adjust pH value to 4~6, filter is collected in filtering
Liquid；
It is 3000Da regenerated cellulose bag filter, outer layer that filtrate obtained by step S2 is packed into internal layer equipped with molecular cut off by S3
It is surrounded by the dialysis apparatus that molecular cut off is 1000Da regenerated cellulose bag filter, is dialysed in distilled water for 24 hours, every 8h is changed once
Dialyzate continuously collects dialyzate, by the dialyzate of collection in heating concentration on electric furnace, obtains concentrate；
Gained concentrate in step S3 is laid in pallet by S4, with a thickness of 4 ± 1mm, is put into precooling in refrigerator, until
It when condenser temperature is down to -50 DEG C~-60 DEG C, is put into drying sublimation device, vacuum drying distillation, to temperature recovery to 28 DEG C
When, it takes out, collects dry powder to obtain the final product.
Further, the acetic acid-sodium acetate buffer solution concentration in the step S1 is 0.2mol/L, pH value 4.5.
Further, the acetic acid-acetic acid for the 0.2mol/L that 1% snail enzyme solutions in the step S2 are 4.5 by pH value
Sodium buffer preparation forms.
Further, the hydrolysis temperature in the step S2 is 40 DEG C.
Further, the enzymolysis time in the step S2 is 2.5h.
Further, the chitosan oligosaccharide molecular weight obtained in the step S3 is less than 1000Da and 1000Da~3000Da.
Further, the precooling in the step S4 is program cooling, and temperature per minute reduces by 1 DEG C, temperature is reduced to-
56℃。
In addition, the present invention also provides the chitosan oligosaccharides that the method for preparing chitosan oligosaccharide with glusulase is prepared to make
The standby purposes improved in idiopathic pulmonary fibrosis drug or health care product.
Chitosan oligosaccharide is that one kind by 2-acetylamino-2-deoxy-D-glucose and D- Glucosamine passes through β-Isosorbide-5-Nitrae glucosides key connection
At natural cationic high molecular polymer, be by shrimp, crab, insect shell and fungal cell wall in chitin through concentrated base
Product obtained from deacetylate after processing.There are the glycosidic bonds of 4 seed types in chitosan straight-chain molecular structure:
GlcNAc-GlcNAc, GlcNAc-GlcN, GlcN-GlcN and GlcN-GlcNAc.Glusulase is that one kind contains cellulase, fruit
The mixed enzyme of more than the 20 kinds of enzymes such as glue enzyme, protease uses the hydrolysis result of specificity enzyme or non-specific enzyme compared with the prior art,
Degradation of chitosan is that the permanent molecular weight of product quality is less than 1000Da and molecular weight is by natural glusulase provided by the invention
The chitosan oligosaccharide of 1000~3000Da.
The present invention is dropped chitosan oligosaccharide concentrate with program in precooling using chitosan oligosaccharide concentrate made from glusulase
Warm therapy reduces by 1 DEG C per minute, until -56 DEG C, resulting chitosan oligosaccharide powder be it is faint yellow, quality is fine and smooth, and molecular structure stabilized can
Long-term preservation.
The present invention is few less than 1000Da shell with the chitosan oligosaccharide molecular weight that the method that glusulase prepares chitosan oligosaccharide is prepared
The average viscosity of sugar is 1.46/mPa.s, and the chitosan oligosaccharide average viscosity that molecular weight is 1000Da~3000Da is 1.81/mPa.s,
The viscosity for being 40.04/mPa.s much smaller than raw materials of chitosan.The chitosan oligosaccharide that the present invention obtains has anti-oxidant, antitumor, raising
Immunity and activation enterobacteriaceae isoreactivity, have a wide range of applications in food, medicine and cosmetic field, additionally by testing me
It has also been found that, chitosan oligosaccharide prepared by the present invention have the function of improvement idiopathic pulmonary fibrosis, be conducive to property pulmonary fibrosis pending
The rehabilitation of patient.
Compared with prior art, the invention has the following advantages:
(1) method provided by the invention for preparing chitosan oligosaccharide with glusulase is easy, quick, safe, is suitble to industrialization production,
With good commercial introduction practicability and value.
(2) method provided by the invention for preparing chitosan oligosaccharide with glusulase has obtained molecular weight less than 1000Da and molecular weight
For the chitosan oligosaccharide of 1000Da~3000Da, by testing it was found that chitosan oligosaccharide produced by the present invention can significantly improve spy
The lung fibroblast activation of hair property pulmonary fibrosis in rats, has good improvement result to idiopathic pulmonary fibrosis, favorably
In the rehabilitation of idiopathic pulmonary fibrosis.
(3) freeze-drying that present invention combination uses can preferably keep the physical behavior of chitosan oligosaccharide, while advantageous
In the holding of chitosan oligosaccharide physiological activity, convenient for product preservation and further use.
(4) enzymolysis time, hydrolysis temperature, concentration of substrate, enzyme bottom mass ratio etc. of the present invention to glusulase enzymatic hydrolysis chitosan
Specific process conditions are explored and are optimized, and improve enzymolysis efficiency, and combine freeze-drying, have carried out a series of test
Research and screening, the mass production for preparing chitosan oligosaccharide for glusulase provide foundation.
Detailed description of the invention
The spectral scan map of Fig. 1 aminoglucose hydrochloride standard serial solution；
Fig. 2 aminoglucose hydrochloride standard curve；
Fig. 3 enzymolysis time-Changes of Reducing Sugar Content curve；
Fig. 4 hydrolysis temperature-Changes of Reducing Sugar Content curve；
Fig. 5 concentration of substrate-Changes of Reducing Sugar Content curve；
Fig. 6 enzyme bottom mass ratio-Changes of Reducing Sugar Content curve.
Specific embodiment
The following describes the present invention further through the description of specific embodiments, but it is to limit of the invention that this, which is not,
System, those skilled in the art's basic thought according to the present invention can make various modifications or improvements, but without departing from this
The basic thought of invention, it is within the scope of the present invention.
Chitosan lot number used in the present invention: 121211A, model: deacetylation 95%, producer: Shandong aokang biology
Science and Technology Ltd.)；Glusulase (BR), producer: Shanghai Yuan Ye Biotechnology Co., Ltd；(the retention of regenerated cellulose bag filter
Molecular weight 1000Da and 3000Da), producer: Town in Shanghai spectrum experiment Science and Technology Co., Ltd..
Embodiment 1, a kind of method for preparing chitosan oligosaccharide with glusulase
S1 takes chitosan raw material drying to be dissolved in the 0.2mol/L acetic acid-sodium acetate buffer solution of pH value 4.5 to constant weight,
Stirring and dissolving is configured to the chitosan solution that mass concentration is 1.0%；
The snail enzyme solutions that mass concentration is 1.0%, the glusulase are added into chitosan solution obtained by step S1 by S2
It is 1:10 with chitosan substrate mass ratio, after digesting 2.5h in 40 DEG C of water baths, 100 DEG C of water-bath 10min inactivations are cooled to room
Temperature continuously stirs, and the NaOH solution that mass concentration is 5% is added dropwise, and adjusts pH value to 4.5, filtrate is collected in filtering；
It is 3000Da regenerated cellulose bag filter, outer layer that filtrate obtained by step S2 is packed into internal layer equipped with molecular cut off by S3
It is surrounded by the dialysis apparatus that molecular cut off is 1000Da regenerated cellulose bag filter, is dialysed in distilled water for 24 hours, every 8h is changed once
Dialyzate continuously collects dialyzate, by the dialyzate of collection in heating concentration on electric furnace, obtains concentrate；
Gained concentrate in step S3 is laid in pallet by S4, with a thickness of 4 ± 1mm, is put into precooling in refrigerator, until
When condenser temperature is down to -56 DEG C, it is put into drying sublimation device, vacuum drying distillation is taken out when temperature recovery is to 28 DEG C,
It is less than 1000Da and molecular weight up to molecular weight for the chitosan oligosaccharide dry powder of 1000Da~3000Da.
Embodiment 2, a kind of method for preparing chitosan oligosaccharide with glusulase
S1 takes chitosan raw material drying to be dissolved in the 0.2mol/L acetic acid-sodium acetate buffer solution of pH value 4.5 to constant weight,
Stirring and dissolving is configured to the chitosan solution that mass concentration is 1.0%；
The snail enzyme solutions that mass concentration is 1.0%, the glusulase are added into chitosan solution obtained by step S1 by S2
It is 1:10 with chitosan substrate mass ratio, after digesting 2h in 40 DEG C of water baths, 100 DEG C of water-bath 15min inactivations are cooled to room
Temperature continuously stirs, and the NaOH solution that mass concentration is 5% is added dropwise, and adjusts pH value to 6, filtrate is collected in filtering；
It is 3000Da regenerated cellulose bag filter, outer layer that filtrate obtained by step S2 is packed into internal layer equipped with molecular cut off by S3
It is surrounded by the dialysis apparatus that molecular cut off is 1000Da regenerated cellulose bag filter, is dialysed in distilled water for 24 hours, every 8h is changed once
Dialyzate continuously collects dialyzate, by the dialyzate of collection in heating concentration on electric furnace, obtains concentrate；
Gained concentrate in step S3 is laid in pallet by S4, with a thickness of 4 ± 1mm, is put into precooling in refrigerator, until
When condenser temperature is down to -60 DEG C, it is put into drying sublimation device, vacuum drying distillation is taken out when temperature recovery is to 25 DEG C,
It is less than 1000Da and molecular weight up to molecular weight for the chitosan oligosaccharide dry powder of 1000Da~3000Da.
Test example one, glusulase enzymatic hydrolysis prepare the factors influencing of chitosan oligosaccharide
1. enzymolysis time experiment of single factor
It is 1.0% in fixed chitosan mass concentration, enzyme bottom mass ratio is 10%, and hydrolysis temperature is 50 DEG C, pH value 4.5
Under conditions of, it is to comment by content of reducing sugar after measurement reaction when selection enzymolysis time is respectively 0.5h, 1h, 2h, 4h, 6h, 8h
Valence index investigates influence of the enzymolysis time to content of reducing sugar.The experimental result of influence of the enzymolysis time to content of reducing sugar is such as
Shown in Fig. 3.From the figure 3, it may be seen that other enzymatic hydrolysis conditions are constant, chitosan mass concentration is 1.0%, and enzyme bottom mass ratio is 10%, enzyme
Temperature 50 C is solved, content of reducing sugar is maximum when digesting 2h under conditions of pH4.5, then with the growth of enzymolysis time, reduced sugar
It tends to be steady again after content decline.After experiment of single factor interpretation of result, select enzymolysis time 1h, 2h and 4h as orthogonal reality
Three levels testing investigate influence of the enzymolysis time to chitosan oligosaccharide content.
2. hydrolysis temperature experiment of single factor
It is 1.0% in fixed chitosan mass concentration, enzyme bottom mass ratio is 10%, enzymolysis time 2h, and pH value is 4.5
Under the conditions of, when selection hydrolysis temperature is respectively 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C and 70 DEG C, by being restored after measurement reaction
Sugared content is evaluation index, investigates influence of the hydrolysis temperature to content of reducing sugar.Influence of the hydrolysis temperature to content of reducing sugar
Experimental result is as shown in Figure 4.As shown in Figure 4, other enzymatic hydrolysis conditions are constant, and chitosan mass concentration is 1.0%, enzyme bottom mass ratio
It is 10%, content of reducing sugar is maximum when hydrolysis temperature is 40 DEG C under conditions of enzymolysis time 2h, pH4.5, then with enzymatic hydrolysis
The increase of temperature, content of reducing sugar sharply decline.After experiment of single factor interpretation of result, 30 DEG C, 40 DEG C of hydrolysis temperature are selected
Influence of the hydrolysis temperature to chitosan oligosaccharide content is investigated with 50 DEG C of three levels for orthogonal experiment.
3. concentration of substrate experiment of single factor
It is 10% in immobilized enzyme bottom mass ratio, hydrolysis temperature is 40 DEG C, enzymolysis time 2h, under conditions of pH value is 4.5,
It is anti-by measuring when selection concentration of substrate (chitosan mass concentration) is respectively 0.5%, 1%, 1.5%, 2%, 2.5% and 3%
Content of reducing sugar is evaluation index after answering, and investigates influence of the concentration of substrate to content of reducing sugar.Concentration of substrate is to content of reducing sugar
Influence experimental result it is as shown in Figure 5.As shown in Figure 5, other enzymatic hydrolysis conditions are constant, and enzyme bottom mass ratio is 10%, enzymatic hydrolysis temperature
40 DEG C, enzymolysis time 2h of degree, content of reducing sugar is maximum when concentration of substrate is 1% under conditions of pH value is 4.5, then the bottom of with
The increase of object concentration after content of reducing sugar slowly declines and tends to be steady.
4. the influence that enzyme substrate amount compares reaction
It is 1.0% in fixed chitosan mass concentration, hydrolysis temperature is 40 DEG C, enzymolysis time 2h, the condition that pH is 4.5
Under, when choosing enzyme substrate amount than being respectively 5%, 10%, 15%, 20%, 25% and 30%, contained by reduced sugar after measurement reaction
Amount is evaluation index, investigates the influence that enzyme substrate amount compares content of reducing sugar.Enzyme substrate amount compares the influence of content of reducing sugar
Experimental result is as shown in Figure 6.It will be appreciated from fig. 6 that other enzymatic hydrolysis conditions are constant, chitosan mass concentration is 1.0%, hydrolysis temperature 40
DEG C, under conditions of enzymolysis time 2h, pH4.5, with the increase of enzyme bottom mass ratio, content of reducing sugar is in 10% enzyme bottom mass ratio
Preceding rapid raising, content of reducing sugar tends to be steady after 10%, changes unobvious.After experiment of single factor interpretation of result, choosing
Enzyme bottom mass ratio 5%, 10% and 15% is selected as three levels of orthogonal experiment to investigate enzymolysis time to chitosan oligosaccharide content
It influences.
5. the preferred preparation method of orthogonal test
According in above-mentioned 1-4 as a result, temperature, zymolyte mass ratio (glusulase and chitosan mass ratio) are chosen, when enzymatic hydrolysis
Between be used as variable, carry out L9 (3^4) orthogonal test, using content of reducing sugar as index optimization prescription, orthogonal experiments such as table 1
It is shown.
Table 1:L9 (3^4) range analysis of orthogonal experiment
Note: K1、K2、K3Horizontal the sum of the experimental result of each factor is respectively tested every time；R1、R2、R3It is respectively each because
The average value of each horizontal result of element；RPoleFor the difference of the average value of each horizontal result of each factor.
As shown in Table 1, range analysis of orthogonal experiment is the results show that the i.e. influence reduced sugar of the corresponding factor of very poor maximum value contains
The biggest factor for measuring (chitosan oligosaccharide content) is enzyme bottom mass ratio, is secondly enzymolysis time, is influenced the smallest for hydrolysis temperature.It is optimal
Testing program be A2B2C2, i.e., hydrolysis temperature is 40 DEG C, enzyme bottom mass ratio is 10% and enzymolysis time is 2h.
Test example two, chitosan oligosaccharide assay
1, aminoglucose hydrochloride absorbing wavelength scans
Respectively at addition 0.00mL, 0.20mL, 0.40mL, 0.60mL, 0.80mL and 1.00mL mass in 10mL colorimetric cylinder
Concentration be 0.1% Glucosamine standard solution, then be separately added into 1.00mL, 0.80mL, 0.60mL, 0.40mL, 0.20mL and
The distilled water of 0.00mL, is separately added into 1mLDNS reagent later, and boiling water bath 10min colour developing divides after taking out cooled to room temperature
10mL is not settled to distilled water；
Spectral scan is carried out to aminoglucose hydrochloride with ultraviolet-visible spectrophotometer, as a result such as Fig. 1, curve by
Under to being above followed successively by 0.20mL, 0.40mL, 0.60mL, 0.80mL and 1.00mL aminoglucose hydrochloride standard solution DNS method
The curve of spectrum.As shown in Figure 1, the maximum absorption peak wavelength of aminoglucose hydrochloride series standard solution is 485nm.
2, aminoglucose hydrochloride Specification Curve of Increasing
Using No. 0 pipe as reference, wavelength 485nm measures various concentration gradient aminoglucose hydrochloride UV absorption intensity
(n=3), standard brick curve, such as Fig. 2 are drawn.
As shown in Figure 2, with Glucosamine concentration (mg/mL) for abscissa, absorbance value (mean value) is ordinate, is returned
Return curve, obtains regression equation y=10.041x-0.0162, R2=0.9987, it is linear good.
According to this standard curve, chitosan oligosaccharide content is indicated using the content of reduced sugar in enzymolysis liquid, to calculate enzymatic hydrolysis
Chitosan oligosaccharide content in solution.
3, chitosan oligosaccharide assay
The 500 μ L of chitosan oligosaccharide that Example 1 and 2 is prepared, is placed in 10mL colorimetric cylinder, adds distilled water to 1mL, then plus
Enter 1mLDNS reagent, it is ginseng with blank tube that cooled to room temperature is taken out in boiling water bath 10min colour developing, and distilled water is settled to 10mL
Than measuring absorbance value, calculating the content of chitosan oligosaccharide.The calculation of yield of chitosan oligosaccharide:
Chitosan oligosaccharide yield and correlation calculation result are as shown in table 2.
It is freeze-dried yield, as the chitosan oligosaccharide quality (m after weighing constant weighta) divided by the chitosan gross mass (m of addition0),
Multiplied by 100%.
In formula:
η %: freeze-drying yield
ma: the amount (mg) for the chitosan oligosaccharide that Enzymatic Hydrolysis of Chitosan generates
m0: the amount (mg) of chitosan
4, the Qualitive test of chitosan oligosaccharide product
It is water-soluble to be configured to 1% chitosan oligosaccharide with distilled water for the chitosan oligosaccharide product for weighing suitable 1000Da and 3000Da respectively
Liquid, then weigh and 1% aqueous solution is made less than 1000Da molecular weight chitosan oligosaccharide sample.It is measured respectively using digital rotational viscometer
Products obtained therefrom 1000Da~3000Da molecular weight chitosan oligosaccharide, less than 1000Da molecular weight chitosan oligosaccharide sample, 1% chitosan solution
Apparent viscosity.The measurement result three times of 1% chitosan solution apparent viscosity is respectively 39.85,40.27,39.99mPa.s, is averaged
Value is 40.04mPa.s, and the results are shown in Table 2 for chitosan oligosaccharide assay produced by the present invention.
5, reducing sugar method surveys the average molecular weight of chitosan oligosaccharide bulk pharmaceutical chemicals
The chitosan oligosaccharide of the molecular weight 1000Da and 1000~3000Da of chitosan oligosaccharide made from glusulase of the invention are taken respectively
Bulk pharmaceutical chemicals measure its average molecular weight with reducing sugar method.
6, test result
Chitosan oligosaccharide content, moisture and qualified products result such as table 2.
2. chitosan oligosaccharide qualification result of table
It can be seen that by 2 result of table, chitosan oligosaccharide made from present invention glusulase, after bag filter separates, obtained molecule
Chitosan oligosaccharide yield of the amount less than 1000Da and 1000Da~3000Da is between 21~26%, chitosan oligosaccharide weight made from this method
Renaturation is good (RSD < 5%), and obtained molecular weight is more equal less than the chitosan oligosaccharide Mass Distribution of 1000Da and 1000Da~3000Da
It is horizontal.
It is 1.4 that molecular weight made from present invention glusulase, which is less than 1000Da and the viscosity of 1000Da~3000Da chitosan oligosaccharide,
Between~1.9mPa.s, much smaller than the 40mPa.s of chitosan, meet chitosan oligosaccharide viscosity criterion.
Using the reducing sugar method measurement chitosan oligosaccharide of the invention made from glusulase the results show that the dialysis that the present invention uses
The chitosan oligosaccharide of molecular weight made from the method embodiment 1 less than 1000Da and 1000Da~3000Da and embodiment 2, measured reality
Border average molecular weight is 482 ± 12Da, 468 ± 20Da and 1382 ± 46Da, 1298 ± 53Da, illustrates that shell produced by the present invention is few
Glycan molecule amount is evenly distributed.
The influence test of test example three, chitosan oligosaccharide to idiopathic pulmonary fibrosis
1, subjects: healthy male SD rat 40 is chosen, weight is 250 ± 20g, by Zhongshan University experimental animal
The heart provides.
2, test material: chitosan oligosaccharide prepared by embodiment 1, pirfenidone capsule are purchased from the limited public affairs of Beijing Kang Dini medicine company
Department, national drug standard H20133376.
3, idiopathic pulmonary fibrosis Animal Model:
It selects 10 at random from 50 rats and is only used as control rats, remaining 3% concentration of rats by intraperitoneal injection is
The yellow Jackets of 30mg/kg, fixed mouse platform of lying on the back after anesthesia, routine disinfection drape after neck preserved skin, cut skin after successively
Blunt separation exposes tracheae, be pierced into the syringe diagonal of 1mL it is intratracheal, to intrapulmonary fast injection bleomycin solution 0.2~
0.3ml holds up mouse plate immediately, after the 2min that rolls, sterilizes skin closure wound again.
4, test method:
Successful 40 rats will be modeled and be randomly divided into control group, model group, pirfenidone group, 1 group of test, each group administration
It measures as follows:
Control group: the isometric physiological saline of stomach-filling；
Model group: the isometric physiological saline of stomach-filling；
Pirfenidone group: the pirfenidone of stomach-filling 10mg/kg；
Test chitosan oligosaccharide (physiological saline solution) prepared by 1 group: stomach-filling 10ml/kg embodiment 1；
Rat free water during test, standard diet.Measurement rat body weight is primary weekly.After on-test administration
4th week execution rat.Rat is put to death after yellow Jackets anesthesia using femoral artery depletion method rat lies on the back and is fixed on mouse platform
On, it opens chest and appears cardiopulmonary, cleaned after removing lungs with physiological saline, weighed in and lung weight with electronic balance repeatedly, calculate lung
Coefficient (lung weight/weight × 100%) uses Serum Laminin (LN) content of kit detection rat.
5, test result:
5.1, influence of the chitosan oligosaccharide to idiopathic pulmonary fibrosis rat model paragonimus cyst and LN content prepared by embodiment 1 be such as
Shown in table 3.
Influence of the chitosan oligosaccharide of 3 embodiment 1 of table preparation to idiopathic pulmonary fibrosis rat model paragonimus cyst and LN content
| Group | Paragonimus cyst | LN content (pg/mL) |
| Control group | 0.44±0.023 | 80.86±10.50 |
| Model group | 0.57±0.04 | 138.64±16.33 |
| Pirfenidone group | 0.47±0.04* | 85.24±11.56** |
| Test 1 group | 0.49±0.06* | 101.78±12.14* |
Compared with model group,*P < 0.05,**P<0.01。
As shown in Table 3, compared with model group, 1 group of pirfenidone group and test can reduce idiopathic pulmonary fibrosis mould
The paragonimus cyst of type, difference has statistical significance, and the reducing effect for testing 1 group is suitable with pirfenidone group, meanwhile, test 1
The LN content of idiopathic pulmonary fibrosis model also can be significantly reduced in group, illustrates that low molecule chitosan oligosaccharide produced by the present invention sends out spy
Property pulmonary fibrosis have significant improvement result.
Claims (8)
1. a kind of method for preparing chitosan oligosaccharide with glusulase, which comprises the following steps:
S1 takes chitosan raw material drying to constant weight, is dissolved in acetic acid-sodium acetate buffer solution, it is dense to be configured to quality for stirring and dissolving
The chitosan solution that degree is 1.0%；
The snail enzyme solutions that mass concentration is 1.0%, the glusulase and shell are added into chitosan solution obtained by step S1 by S2
Glycan substrate mass ratio is 1:10, and after digesting 2h~3h in 35~45 DEG C of water bath, 100 DEG C of 5~15min of water-bath inactivations are cold
But it to room temperature, continuously stirs, the NaOH solution that mass concentration is 5% is added dropwise, adjust pH value to 4~6, filtrate is collected in filtering；
It is 3000Da regenerated cellulose bag filter that filtrate obtained by step S2 is packed into internal layer equipped with molecular cut off by S3, and outer layer is surrounded by
Molecular cut off is the dialysis apparatus of 1000Da regenerated cellulose bag filter, is dialysed in distilled water for 24 hours, every 8h changes primary dialysis
Liquid continuously collects dialyzate, and concentration obtains concentrate；
Gained concentrate in step S3 is laid in pallet by S4, with a thickness of 4 ± 1mm, is put into precooling in refrigerator, until condensation
When device temperature is down to -50 DEG C~-60 DEG C, vacuum drying distillation is taken out when temperature recovery is to 28 DEG C, collects dry powder to obtain the final product.
2. the method for preparing chitosan oligosaccharide with glusulase as described in claim 1, which is characterized in that the second in the step S1
Acid-sodium acetate buffer concentration is 0.2mol/L, pH value 4.5.
3. the method for preparing chitosan oligosaccharide with glusulase as described in claim 1, which is characterized in that 1% in the step S2
Acetic acid-the sodium acetate buffer solution for the 0.2mol/L that snail enzyme solutions are 4.5 by pH value is formulated.
4. the method for preparing chitosan oligosaccharide with glusulase as described in claim 1, which is characterized in that the enzymatic hydrolysis in the step S2
Temperature is 40 DEG C.
5. the method for preparing chitosan oligosaccharide with glusulase as described in claim 1, which is characterized in that the enzymatic hydrolysis in the step S2
Time is 2.5h.
6. the method for preparing chitosan oligosaccharide with glusulase as described in claim 1, which is characterized in that obtained in the step S3
Chitosan oligosaccharide molecular weight is less than 1000Da and 1000Da~3000Da.
7. the method for preparing chitosan oligosaccharide with glusulase as described in claim 1, which is characterized in that the pre-cooling in the step S4
Freezing is that program cools down, and temperature per minute reduces by 1 DEG C.
8. prepared by the chitosan oligosaccharide that the method for preparing chitosan oligosaccharide with glusulase as described in claim 1~7 is any is prepared
Improve the purposes in idiopathic pulmonary fibrosis drug or health care product.
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| WO2020048270A1 (en) * | 2018-09-06 | 2020-03-12 | 广东药科大学 | Method for preparing chitooligosaccharide using snailase and application thereof |
| CN113684234A (en) * | 2021-05-19 | 2021-11-23 | 广东海洋大学 | Chitosan oligosaccharide prepared by enzyme method and application thereof in food preservation |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020048270A1 (en) * | 2018-09-06 | 2020-03-12 | 广东药科大学 | Method for preparing chitooligosaccharide using snailase and application thereof |
| CN113684234A (en) * | 2021-05-19 | 2021-11-23 | 广东海洋大学 | Chitosan oligosaccharide prepared by enzyme method and application thereof in food preservation |
| CN113684234B (en) * | 2021-05-19 | 2023-05-23 | 广东海洋大学 | Enzymatic preparation of chitosan oligosaccharide and application thereof in food fresh-keeping |
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| CN109136307B (en) | 2021-08-13 |
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