CN113699197B - Process for extracting cyperus esculentus oligosaccharide by enzyme method - Google Patents

Process for extracting cyperus esculentus oligosaccharide by enzyme method Download PDF

Info

Publication number
CN113699197B
CN113699197B CN202111019573.5A CN202111019573A CN113699197B CN 113699197 B CN113699197 B CN 113699197B CN 202111019573 A CN202111019573 A CN 202111019573A CN 113699197 B CN113699197 B CN 113699197B
Authority
CN
China
Prior art keywords
oligosaccharide
solution
cyperus esculentus
mass ratio
extracting
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.)
Active
Application number
CN202111019573.5A
Other languages
Chinese (zh)
Other versions
CN113699197A (en
Inventor
张铁华
李晨飞
张�杰
于亚莉
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jilin University
Original Assignee
Jilin University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Jilin University filed Critical Jilin University
Priority to CN202111019573.5A priority Critical patent/CN113699197B/en
Publication of CN113699197A publication Critical patent/CN113699197A/en
Application granted granted Critical
Publication of CN113699197B publication Critical patent/CN113699197B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C12P19/14—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/18—Multi-enzyme systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/2488—Mannanases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01015—Polygalacturonase (3.2.1.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
    • C12Y402/02002—Pectate lyase (4.2.2.2)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Inorganic Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

The invention discloses a process for extracting cyperus esculentus oligosaccharide by an enzymatic method, which takes cyperus esculentus bean pulp as a raw material to prepare pretreated slurry, firstly, the pretreated slurry is adjusted to be alkaline, protein is removed, then, polysaccharide is obtained by ultrasonic extraction under an acidic condition, and then, the polysaccharide is degraded into oligosaccharide by complex enzyme enzymolysis under the conditions of a magnetic field and a pulse electric field. The method realizes the resource recycling of the cyperus bean pulp, has high oligosaccharide yield and low average molecular weight, and has excellent application and popularization prospects.

Description

Process for extracting cyperus esculentus oligosaccharide by enzyme method
Technical Field
The invention relates to the technical field of cyperus esculentus processing, in particular to a process for extracting cyperus esculentus oligosaccharide by an enzyme method.
Background
The cyperus esculentus, also called underground walnuts and tiger nuts, has strong growth adaptability, is a novel multipurpose crop integrating oil collection, grain, grazing and feeding with high quality, high yield and high comprehensive utilization value, and is also an ornamental plant for beautifying and greening the environment. Cyperus esculentus has been successfully and widely planted in tropical, subtropical and temperate regions. The cyperus esculentus does not contain gluten protein, is not easy to cause allergy, and is a high-quality raw material for producing formula food and special diet.
The cyperus esculentus is rich in nutrition and comprises: 20-30% of grease, 25-40% of starch, 15-20% of sugar, about 8% of crude protein and more than 80% of unsaturated fatty acid, wherein 65% of oleic acid and 15% of linoleic acid are rich in vitamin E and various trace mineral elements.
The cyperus esculentus is usually used for oil extraction, but the cyperus esculentus contains abundant starch, and the cyperus esculentus bean pulp generated after oil extraction has high starch content and high application value, and the direct waste is undoubtedly huge waste. The extraction of other ingredients of cyperus esculentus is currently utilized relatively rarely.
The oligosaccharide is a novel functional carbohydrate source and is widely applied to the fields of food, health care products, medicines, feed additives and the like. The oligosaccharide can improve the micro-ecological environment in human body, regulate the gastrointestinal function, and is beneficial to the proliferation of probiotics and the growth inhibition of putrefying bacteria; meanwhile, the composition has the function of promoting the development of the intestinal mucosa immune system and also has the function of promoting the whole body immune system outside the intestinal tract; the oligosaccharide can also improve the activity of intestinal mucosa cells and increase the mineral absorption capacity of the intestinal tract by adjusting the microecological balance of intestinal flora. If the cyperus esculentus bean pulp is used for oligosaccharide extraction, the cyperus esculentus bean pulp resource can be recycled, and the application value is very high.
Patent application CN111909282A discloses a method for extracting cyperus esculentus polysaccharide, which comprises the steps of crushing puffed cyperus esculentus, extracting soluble total sugar by ultrasonic wave, and precipitating with ethanol to obtain cyperus esculentus polysaccharide. The patent application takes the cyperus esculentus as a raw material, and if the cyperus esculentus is moved to the treatment of the cyperus esculentus bean pulp, the extraction effect is poor, the existing cyperus esculentus bean pulp resource can not be recycled, and the cyperus esculentus is not further processed into oligosaccharide products.
Disclosure of Invention
The invention aims to provide a process for extracting cyperus esculentus oligosaccharide by an enzymatic method, which realizes the resource recycling of cyperus esculentus bean pulp, and has high oligosaccharide yield and low average molecular weight.
In order to achieve the purpose, the invention is realized by the following scheme:
a process for extracting cyperus esculentus oligosaccharides by an enzymatic method comprises the following specific steps:
(1) Crushing cyperus bean pulp, performing extrusion puffing treatment to prepare bean pulp powder, adding the bean pulp powder into water, uniformly stirring to obtain slurry, and performing high-speed shearing and microwave treatment to obtain pretreated slurry;
(2) Adjusting the pretreated slurry to be alkaline, stirring, centrifuging to obtain precipitate so as to remove protein, adding the precipitate into distilled water, adjusting the pH to be between 4 and 6, performing ultrasonic extraction, centrifuging to obtain supernatant, concentrating, precipitating with ethanol, centrifuging, and drying to obtain polysaccharide;
(3) Then adding polysaccharide into distilled water, dispersing uniformly by ultrasonic waves, adding complex enzyme, carrying out enzymolysis under the conditions of a magnetic field and a pulse electric field, and carrying out post-treatment to obtain oligosaccharide;
the complex enzyme is prepared by the following method: firstly, amylase, cellulase, pectinase and mannase are mixed according to a mass ratio of 1:0.1 to 0.2: 0.02-0.03: 0.01 to 0.02 of the complex enzyme is dissolved in disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution to prepare mixed enzyme solution, then chitosan acetic acid solution and the mixed enzyme solution are mixed to prepare premixed solution, then the premixed solution is slowly dripped into corn oil containing emulsifier, stirred and emulsified, and then glutaraldehyde and layered titanate nano materials are added, cross-linking reaction is carried out, concentration and drying are carried out, thus obtaining the complex enzyme.
Preferably, in the step (1), the cyperus bean pulp is crushed to 80-100 meshes, and the mass ratio of the cyperus bean pulp powder to water is 1:5 to 7.
Preferably, in the step (1), the high-speed shearing process conditions are as follows: 20000-30000 r/min high speed shearing for 5-7 min.
Preferably, in the step (1), the process conditions of the microwave treatment are as follows: microwave treatment is carried out for 2-3 minutes at 300-500W.
Preferably, in the step (2), the specific method for removing the protein is as follows: adjusting the pH of the pretreated slurry to be 8-10, stirring the pretreated slurry for 40-50 minutes at 50-60 ℃, and centrifuging the pretreated slurry to obtain precipitate.
Preferably, in the step (2), the mass ratio of the precipitate to the distilled water is 1: 5-7, the technological conditions of ultrasonic extraction are as follows: under the condition of 40-50 ℃, the ultrasonic vibration treatment is carried out for 5-6 hours at 400-600W.
Preferably, in the step (2), the supernatant is concentrated to 1/4-1/3 of the original volume to obtain a concentrated solution, then 0.6-0.8 times of volume of absolute ethyl alcohol is added into the concentrated solution while stirring, the concentrated solution is kept stand for 12-15 hours at 0-4 ℃, centrifuged for 10-12 minutes at 5000-7000 r/min, and dried to obtain the product.
Preferably, in the step (3), the mass ratio of the polysaccharide to the distilled water to the complex enzyme is 1: 8-10: 1 to 2.
Preferably, in the step (3), the enzymolysis process conditions are as follows: the magnetic field intensity is 200-300 mT, the pulse electric field intensity is 15-25 kV/cm, and the enzymolysis is carried out for 3-5 hours at 35-40 ℃.
Preferably, in the step (3), the post-treatment method comprises the following specific steps: inactivating enzyme at 95-98 ℃ for 15-20 minutes, performing ultrafiltration treatment by using a filter membrane with the molecular weight cutoff of 10kDa, performing nanofiltration by using a filter membrane with the molecular weight cutoff of 1000Da, removing inorganic salt, glucose, sucrose and other small molecular substances, concentrating, and performing spray drying to obtain the oligosaccharide.
Further preferably, the working frequency of ultrafiltration is 26Hz, and the liquid inlet pressure is 0.5MPa; the operating frequency of nanofiltration is 40Hz, and the liquid inlet pressure is 2MPa.
Preferably, the mass ratio of the amylase to the buffer solution is 2-3: 100, the concentration of the buffer solution is 0.2mol/L, and the pH value is 5.9.
Preferably, the mass ratio of the chitosan acetic acid solution to the mixed enzyme solution is 1: 0.3-0.5, the chitosan acetic acid solution is obtained by adding chitosan into acetic acid solution with mass concentration of 5% which is 65-75 times of the weight of the chitosan, and oscillating the mixture by ultrasonic waves until the chitosan is completely dissolved.
Preferably, the mass ratio of the premixed liquid to the emulsifier to the corn oil to the glutaraldehyde to the layered titanate nano-material is 1:0.1 to 0.2:4 to 5: 0.04-0.05: 0.008 to 0.01 percent, and the emulsifier is span 80.
Preferably, the process conditions of the crosslinking reaction are as follows: stirring and reacting for 2-3 hours at 60-70 ℃.
Preferably, the layered titanate nano-material is prepared by the following method in parts by weight: firstly, 1 part of isopropyl titanate is dispersed in 9-11 parts of 8-9 mol/L sodium hydroxide solution by ultrasonic wave, then the obtained product is transferred to a hydrothermal reaction kettle, hydrothermal reaction is carried out for 20-22 hours at 160-180 ℃, and then the obtained product is obtained by filtering, washing with deionized water and drying.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention takes cyperus bean pulp as raw material to prepare pretreated slurry, firstly the pretreated slurry is adjusted to be alkaline, protein is removed, then polysaccharide is obtained by ultrasonic extraction under acidic condition, and then the polysaccharide is degraded into oligosaccharide by compound enzyme enzymolysis under magnetic field and pulse electric field condition. The method realizes the resource recycling of the cyperus bean pulp, has high oligosaccharide yield and low average molecular weight, and has excellent application and popularization prospects.
(2) The preparation method of the pretreatment slurry comprises the following steps: crushing cyperus bean pulp, performing extrusion puffing treatment to prepare bean pulp powder, adding the bean pulp powder into water, uniformly stirring to obtain slurry, and performing high-speed shearing and microwave treatment to obtain the cyperus bean pulp. The extrusion and expansion treatment can fully destroy the structure of the soybean meal, promote the release of the components contained in the soybean meal and provide a good basis for subsequent extraction and separation. After the bean pulp powder is added into water to prepare slurry, high-speed shearing and microwave treatment are sequentially carried out, so that the bean pulp particles in the pretreated slurry are very small, the full extraction of the cyperus bean pulp is facilitated, and the oligosaccharide yield is improved.
(3) The degradation steps of the polysaccharide are very critical, the enzymolysis is promoted by utilizing the combined action of the compound enzyme under the conditions of the magnetic field and the pulse electric field, the yield of the oligosaccharide is greatly improved, and the average molecular weight of the oligosaccharide is reduced. The complex enzyme is prepared by dissolving amylase, cellulase, pectinase and mannase in a specific ratio in a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution to prepare a mixed enzyme solution, mixing a chitosan acetic acid solution with the mixed enzyme solution to prepare a premixed solution, slowly dropwise adding the premixed solution into corn oil containing an emulsifier, stirring and emulsifying, adding glutaraldehyde and a layered titanate nano material, performing cross-linking reaction, concentrating and drying. Along with the proceeding of glutaraldehyde crosslinking reaction, the chitosan microsphere modified by the layered titanate nano material is generated, the specific surface area is large, the loading effect is achieved, the contact area of enzyme and polysaccharide is increased, the enzymolysis is promoted, the oligosaccharide yield is further improved, and the average molecular weight of oligosaccharide is reduced.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1
A process for extracting cyperus esculentus oligosaccharide by an enzymatic method comprises the following specific steps:
(1) Crushing cyperus bean pulp to 80 meshes, performing extrusion puffing treatment to obtain 100g of bean pulp powder, adding the bean pulp powder into 700g of water, uniformly stirring to obtain slurry, and performing high-speed shearing and microwave treatment to obtain pretreated slurry;
(2) Adjusting the pretreatment slurry to be alkaline, stirring, centrifuging to obtain precipitate to remove protein, adding the precipitate into distilled water, adjusting pH =4, performing ultrasonic extraction, centrifuging to obtain supernatant, concentrating, precipitating with ethanol, centrifuging, and drying to obtain polysaccharide;
(3) Then adding polysaccharide into distilled water, dispersing uniformly by ultrasonic waves, adding complex enzyme, carrying out enzymolysis under the conditions of a magnetic field and a pulse electric field, and carrying out post-treatment to obtain oligosaccharide;
the complex enzyme is prepared by the following method: firstly dissolving 1g of amylase, 0.2g of cellulase, 0.02g of pectinase and 0.02g of mannase in disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution to prepare mixed enzyme solution, then mixing chitosan acetic acid solution with the mixed enzyme solution to prepare premixed solution, then slowly dripping the premixed solution into corn oil containing an emulsifier, stirring and emulsifying, then adding glutaraldehyde and layered titanate nano materials, carrying out cross-linking reaction, concentrating and drying to obtain the complex enzyme.
In the step (1), the process conditions of high-speed shearing are as follows: 20000 rpm high shear for 7 minutes.
In the step (1), the process conditions of the microwave treatment are as follows: and (3) treating the mixture by using a microwave at 300W for 3 minutes.
In the step (2), the specific method for removing the protein comprises the following steps: the pretreatment slurry was adjusted to pH =8, treated with stirring at 60 ℃ for 40 minutes, and centrifuged to take the precipitate.
In the step (2), the mass ratio of the precipitate to the distilled water is 1:7, the technological conditions of ultrasonic extraction are as follows: the ultrasonic vibration treatment was carried out at 600W for 5 hours at 40 ℃.
And (2) concentrating the supernatant to 1/3 of the original volume to obtain a concentrated solution, adding 0.6 volume of absolute ethyl alcohol into the concentrated solution while stirring, standing at 4 ℃ for 12 hours, centrifuging at 7000 rpm for 10 minutes, and drying.
In the step (3), the mass ratio of the polysaccharide to the distilled water to the complex enzyme is 1:10:1.
in the step (3), the enzymolysis process conditions are as follows: the magnetic field intensity is 300mT, the pulse electric field intensity is 15kV/cm, and the enzymolysis is carried out for 3 hours at 40 ℃.
In the step (3), the post-treatment method comprises the following specific steps: inactivating enzyme at 98 ℃ for 15 minutes, performing ultrafiltration treatment by using a filter membrane with the molecular weight cutoff of 10kDa, performing nanofiltration by using a filter membrane with the molecular weight cutoff of 1000Da, removing inorganic salt, glucose, sucrose and other small molecular substances, concentrating, and performing spray drying to obtain the oligosaccharide.
The working frequency of ultrafiltration is 26Hz, and the liquid inlet pressure is 0.5MPa; the operating frequency of nanofiltration is 40Hz, and the liquid inlet pressure is 2MPa.
The mass ratio of the amylase to the buffer solution is 3:100, the concentration of the buffer solution is 0.2mol/L, and the pH value is 5.9.
The mass ratio of the chitosan acetic acid solution to the mixed enzyme solution is 1:0.3, the chitosan acetic acid solution is obtained by adding chitosan into acetic acid solution with mass concentration of 5% and weight of 75 times of the chitosan and carrying out ultrasonic oscillation until the chitosan is completely dissolved.
The process conditions of the crosslinking reaction are as follows: the reaction was stirred at 60 ℃ for 3 hours.
The layered titanate nano material is prepared by the following method: firstly, 1g of isopropyl titanate is dispersed in 9g of sodium hydroxide solution 9mol/L by ultrasonic waves, then the isopropyl titanate is transferred to a hydrothermal reaction kettle to carry out hydrothermal reaction for 22 hours at 160 ℃, and then the isopropyl titanate is obtained by filtering, washing with deionized water and drying.
Example 2
A process for extracting cyperus esculentus oligosaccharide by an enzymatic method comprises the following specific steps:
(1) Crushing cyperus bean pulp to 100 meshes, performing extrusion and puffing treatment to obtain 100g of bean pulp powder, adding the bean pulp powder into 500g of water, uniformly stirring to obtain slurry, and performing high-speed shearing and microwave treatment to obtain pretreated slurry;
(2) Adjusting the pretreatment slurry to be alkaline, stirring, centrifuging to obtain precipitate to remove protein, adding the precipitate into distilled water, adjusting pH =6, performing ultrasonic extraction, centrifuging to obtain supernatant, concentrating, precipitating with ethanol, centrifuging, and drying to obtain polysaccharide;
(3) Then adding polysaccharide into distilled water, dispersing uniformly by ultrasonic waves, adding complex enzyme, carrying out enzymolysis under the conditions of a magnetic field and a pulse electric field, and carrying out post-treatment to obtain oligosaccharide;
the complex enzyme is prepared by the following method: firstly dissolving 1g of amylase, 0.1g of cellulase, 0.03g of pectinase and 0.01g of mannase in disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution to prepare mixed enzyme solution, then mixing chitosan acetic acid solution with the mixed enzyme solution to prepare premixed solution, then slowly dripping the premixed solution into corn oil containing an emulsifier, stirring and emulsifying, then adding glutaraldehyde and layered titanate nano materials, carrying out cross-linking reaction, concentrating and drying to obtain the complex enzyme.
In the step (1), the process conditions of high-speed shearing are as follows: 30000 rpm high shear for 5 minutes.
In the step (1), the process conditions of the microwave treatment are as follows: microwave treatment at 500W for 2 minutes.
In the step (2), the specific method for removing the protein comprises the following steps: the pretreatment slurry was adjusted to pH =10, treated with stirring at 50 ℃ for 50 minutes, and centrifuged to take the precipitate.
In the step (2), the mass ratio of the precipitate to the distilled water is 1:5, the technological conditions of ultrasonic extraction are as follows: the ultrasonic vibration treatment was carried out at 400W for 6 hours at 50 ℃.
And (2) concentrating the supernatant to 1/4 of the original volume to obtain a concentrated solution, adding 0.8 times of absolute ethyl alcohol into the concentrated solution while stirring, standing at 0 ℃ for 15 hours, centrifuging at 5000 rpm for 12 minutes, and drying.
In the step (3), the mass ratio of the polysaccharide to the distilled water to the complex enzyme is 1:8:2.
in the step (3), the enzymolysis process conditions are as follows: the enzymolysis is carried out for 5 hours at 35 ℃ under the conditions of the magnetic field intensity of 200mT and the pulse electric field intensity of 25 kV/cm.
In the step (3), the post-treatment method comprises the following specific steps: inactivating enzyme at 95 ℃ for 20 minutes, performing ultrafiltration treatment by using a filter membrane with the molecular weight cutoff of 10kDa, performing nanofiltration by using a filter membrane with the molecular weight cutoff of 1000Da, removing inorganic salt, glucose, sucrose and other small molecular substances, concentrating, and performing spray drying to obtain the oligosaccharide.
The working frequency of ultrafiltration is 26Hz, and the liquid inlet pressure is 0.5MPa; the operating frequency of nanofiltration is 40Hz, and the liquid inlet pressure is 2MPa.
The mass ratio of the amylase to the buffer solution is 2:100, the concentration of the buffer solution is 0.2mol/L, and the pH value is 5.9.
The mass ratio of the chitosan acetic acid solution to the mixed enzyme solution is 1:0.5, the chitosan acetic acid solution is obtained by adding chitosan into acetic acid solution with mass concentration of 5% which is 65 times of the weight of chitosan and carrying out ultrasonic oscillation until the chitosan is completely dissolved.
The mass ratio of the premixed liquid to the emulsifier to the corn oil to the glutaraldehyde to the layered titanate nano material is 1:0.2:4:0.05:0.008, and the emulsifier is span 80.
The process conditions of the crosslinking reaction are as follows: the reaction was stirred at 70 ℃ for 2 hours.
The layered titanate nano material is prepared by the following method: firstly, 1g of isopropyl titanate is dispersed in 11g of 8mol/L sodium hydroxide solution by ultrasonic wave, then the isopropyl titanate is transferred into a hydrothermal reaction kettle, hydrothermal reaction is carried out for 20 hours at 180 ℃, and the isopropyl titanate is obtained by filtering, washing with deionized water and drying.
Example 3
A process for extracting cyperus esculentus oligosaccharide by an enzymatic method comprises the following specific steps:
(1) Firstly crushing cyperus bean pulp into 90 meshes, carrying out extrusion puffing treatment to obtain 100g of bean pulp powder, then adding the bean pulp powder into 600g of water, stirring and uniformly mixing to obtain slurry, and carrying out high-speed shearing and microwave treatment to obtain pretreated slurry;
(2) Adjusting the pretreatment slurry to be alkaline, stirring, centrifuging to obtain precipitate so as to remove protein, adding the precipitate into distilled water, adjusting the pH to be =5, performing ultrasonic extraction, centrifuging to obtain supernatant, concentrating, performing alcohol precipitation, centrifuging, and drying to obtain polysaccharide;
(3) Then adding polysaccharide into distilled water, dispersing uniformly by ultrasonic waves, adding complex enzyme, carrying out enzymolysis under the conditions of a magnetic field and a pulse electric field, and carrying out post-treatment to obtain oligosaccharide;
the complex enzyme is prepared by the following method: firstly, dissolving 1g of amylase, 0.15g of cellulase, 0.025g of pectinase and 0.015g of mannase in disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution to prepare mixed enzyme solution, then mixing chitosan acetic acid solution with the mixed enzyme solution to prepare premixed solution, then slowly dripping the premixed solution into corn oil containing an emulsifier, stirring and emulsifying, then adding glutaraldehyde and layered titanate nano materials, carrying out cross-linking reaction, concentrating and drying to obtain the complex enzyme.
In the step (1), the process conditions of high-speed shearing are as follows: 30000 rpm high shear for 6 minutes.
In the step (1), the process conditions of the microwave treatment are as follows: and (5) performing 400W microwave treatment for 2.5 minutes.
In the step (2), the specific method for removing the protein comprises the following steps: the pre-treatment slurry was adjusted to pH =9, treated with stirring at 55 ℃ for 45 minutes, and centrifuged to take the precipitate.
In the step (2), the mass ratio of the precipitate to the distilled water is 1:6, the process conditions of ultrasonic extraction are as follows: the mixture was subjected to 500W ultrasonic vibration treatment at 45 ℃ for 5.5 hours.
And (2) concentrating the supernatant to 1/3 of the original volume to obtain a concentrated solution, adding 0.7 volume of absolute ethyl alcohol into the concentrated solution while stirring, standing at 2 ℃ for 14 hours, centrifuging at 6000 rpm for 11 minutes, and drying.
In the step (3), the mass ratio of the polysaccharide to the distilled water to the complex enzyme is 1:9:1.5.
in the step (3), the enzymolysis process conditions are as follows: the magnetic field intensity is 300mT, the pulse electric field intensity is 20kV/cm, and the enzymolysis is carried out for 4 hours at 38 ℃.
In the step (3), the post-treatment method comprises the following specific steps: inactivating enzyme at 97 ℃ for 18 minutes, performing ultrafiltration treatment by using a filter membrane with the molecular weight cutoff of 10kDa, performing nanofiltration by using a filter membrane with the molecular weight cutoff of 1000Da, removing inorganic salt, glucose, sucrose and other small molecular substances, concentrating, and performing spray drying to obtain the oligosaccharide.
The working frequency of ultrafiltration is 26Hz, and the liquid inlet pressure is 0.5MPa; the operating frequency of nanofiltration is 40Hz, and the liquid inlet pressure is 2MPa.
The mass ratio of the amylase to the buffer solution is 2.5:100, the concentration of the buffer solution is 0.2mol/L, and the pH value is 5.9.
The mass ratio of the chitosan acetic acid solution to the mixed enzyme solution is 1:0.4, the chitosan acetic acid solution is obtained by adding chitosan into acetic acid solution with the mass concentration of 5 percent, which is 70 times of the weight of chitosan, and carrying out ultrasonic oscillation until the chitosan is completely dissolved.
The mass ratio of the premixed liquid to the emulsifying agent to the corn oil to the glutaraldehyde to the layered titanate nano material is 1:0.15:4.5:0.045:0.009 and the emulsifier is span 80.
The process conditions of the crosslinking reaction are as follows: the reaction was stirred at 65 ℃ for 2.5 hours.
The layered titanate nano material is prepared by the following method: firstly, 1g of isopropyl titanate is ultrasonically dispersed in 10g of 8.5mol/L sodium hydroxide solution, then the solution is transferred into a hydrothermal reaction kettle, hydrothermal reaction is carried out for 21 hours at the temperature of 170 ℃, and then the product is obtained through filtering, washing by deionized water and drying.
Comparative example 1
A process for extracting cyperus esculentus oligosaccharide by an enzymatic method comprises the following specific steps:
(1) Crushing cyperus bean pulp into 80 meshes to obtain 100g of bean pulp powder, adding the bean pulp powder into 700g of water, stirring and uniformly mixing to obtain slurry, and shearing at a high speed to obtain pretreated slurry;
(2) Adjusting the pretreatment slurry to be alkaline, stirring, centrifuging to obtain precipitate to remove protein, adding the precipitate into distilled water, adjusting pH =4, performing ultrasonic extraction, centrifuging to obtain supernatant, concentrating, precipitating with ethanol, centrifuging, and drying to obtain polysaccharide;
(3) Then adding polysaccharide into distilled water, dispersing uniformly by ultrasonic waves, adding complex enzyme, carrying out enzymolysis under the conditions of a magnetic field and a pulse electric field, and carrying out post-treatment to obtain oligosaccharide;
the complex enzyme is prepared by the following method: firstly, dissolving 1g of amylase, 0.2g of cellulase, 0.02g of pectinase and 0.02g of mannase in disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution to prepare mixed enzyme solution, then mixing chitosan acetic acid solution with the mixed enzyme solution to prepare premixed solution, then slowly dripping the premixed solution into corn oil containing an emulsifier, stirring and emulsifying, then adding glutaraldehyde and layered titanate nano materials, carrying out cross-linking reaction, concentrating and drying to obtain the complex enzyme.
In the step (1), the process conditions of high-speed shearing are as follows: 20000 rpm high shear for 7 minutes.
In the step (2), the specific method for removing the protein comprises the following steps: the pretreatment slurry was adjusted to pH =8, treated with stirring at 60 ℃ for 40 minutes, and centrifuged to take out the precipitate.
In the step (2), the mass ratio of the precipitate to the distilled water is 1:7, the process conditions of ultrasonic extraction are as follows: ultrasonic vibration treatment was carried out at 600W for 5 hours at 40 ℃.
And (2) concentrating the supernatant to 1/3 of the original volume to obtain a concentrated solution, adding 0.6 volume of absolute ethyl alcohol into the concentrated solution while stirring, standing at 4 ℃ for 12 hours, centrifuging at 7000 rpm for 10 minutes, and drying.
In the step (3), the mass ratio of the polysaccharide to the distilled water to the complex enzyme is 1:10:1.
in the step (3), the enzymolysis process conditions are as follows: the magnetic field intensity is 300mT, the pulse electric field intensity is 15kV/cm, and the enzymolysis is carried out for 3 hours at 40 ℃.
In the step (3), the post-treatment method comprises the following specific steps: inactivating enzyme at 98 ℃ for 15 minutes, performing ultrafiltration treatment by using a filter membrane with the molecular weight cutoff of 10kDa, performing nanofiltration by using a filter membrane with the molecular weight cutoff of 1000Da, removing inorganic salt, glucose, sucrose and other small molecular substances, concentrating, and performing spray drying to obtain the oligosaccharide.
The working frequency of ultrafiltration is 26Hz, and the liquid inlet pressure is 0.5MPa; the operating frequency of nanofiltration is 40Hz, and the liquid inlet pressure is 2MPa.
The mass ratio of the amylase to the buffer solution is 3:100, the concentration of the buffer solution is 0.2mol/L, and the pH value is 5.9.
The mass ratio of the chitosan acetic acid solution to the mixed enzyme solution is 1:0.3, the chitosan acetic acid solution is obtained by adding chitosan into acetic acid solution with mass concentration of 5% and weight of 75 times of the chitosan and carrying out ultrasonic oscillation until the chitosan is completely dissolved.
The mass ratio of the premixed liquid to the emulsifier to the corn oil to the glutaraldehyde to the layered titanate nano material is 1:0.1:5:0.04:0.01, and the emulsifier is span 80.
The process conditions of the crosslinking reaction are as follows: the reaction was stirred at 60 ℃ for 3 hours.
The layered titanate nano material is prepared by the following method: firstly, 1g of isopropyl titanate is dispersed in 9g of sodium hydroxide solution 9mol/L by ultrasonic waves, then the isopropyl titanate is transferred to a hydrothermal reaction kettle to carry out hydrothermal reaction for 22 hours at 160 ℃, and then the isopropyl titanate is obtained by filtering, washing with deionized water and drying.
Comparative example 2
A process for extracting cyperus esculentus oligosaccharide by an enzymatic method comprises the following specific steps:
(1) Crushing cyperus bean pulp to 80 meshes, performing extrusion puffing treatment to obtain 100g of bean pulp powder, adding the bean pulp powder into 700g of water, uniformly stirring to obtain slurry, and performing high-speed shearing and microwave treatment to obtain pretreated slurry;
(2) Adjusting the pretreatment slurry to be alkaline, stirring, centrifuging to obtain precipitate to remove protein, adding the precipitate into distilled water, adjusting pH =4, performing ultrasonic extraction, centrifuging to obtain supernatant, concentrating, precipitating with ethanol, centrifuging, and drying to obtain polysaccharide;
(3) Then adding polysaccharide into distilled water, dispersing uniformly by ultrasonic waves, adding complex enzyme, carrying out enzymolysis under the condition of a pulse electric field, and carrying out post-treatment to obtain oligosaccharide;
the complex enzyme is prepared by the following method: firstly, dissolving 1g of amylase, 0.2g of cellulase, 0.02g of pectinase and 0.02g of mannase in disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution to prepare mixed enzyme solution, then mixing chitosan acetic acid solution with the mixed enzyme solution to prepare premixed solution, then slowly dripping the premixed solution into corn oil containing an emulsifier, stirring and emulsifying, then adding glutaraldehyde and layered titanate nano materials, carrying out cross-linking reaction, concentrating and drying to obtain the complex enzyme.
In the step (1), the process conditions of high-speed shearing are as follows: 20000 rpm high shear for 7 minutes.
In the step (1), the process conditions of the microwave treatment are as follows: microwave treatment at 300W for 3 minutes.
In the step (2), the specific method for removing the protein comprises the following steps: the pretreatment slurry was adjusted to pH =8, treated with stirring at 60 ℃ for 40 minutes, and centrifuged to take out the precipitate.
In the step (2), the mass ratio of the precipitate to the distilled water is 1:7, the technological conditions of ultrasonic extraction are as follows: ultrasonic vibration treatment was carried out at 600W for 5 hours at 40 ℃.
And (2) concentrating the supernatant to 1/3 of the original volume to obtain a concentrated solution, adding 0.6 volume of absolute ethyl alcohol into the concentrated solution while stirring, standing at 4 ℃ for 12 hours, centrifuging at 7000 rpm for 10 minutes, and drying.
In the step (3), the mass ratio of the polysaccharide to the distilled water to the complex enzyme is 1:10:1.
in the step (3), the enzymolysis process conditions are as follows: the intensity of the pulse electric field is 15kV/cm, and the enzymolysis is carried out for 3 hours at 40 ℃.
In the step (3), the post-treatment method comprises the following specific steps: inactivating enzyme at 98 ℃ for 15 minutes, performing ultrafiltration treatment by using a filter membrane with the molecular weight cutoff of 10kDa, performing nanofiltration by using a filter membrane with the molecular weight cutoff of 1000Da, removing inorganic salt, glucose, sucrose and other small molecular substances, concentrating, and performing spray drying to obtain the oligosaccharide.
The working frequency of ultrafiltration is 26Hz, and the liquid inlet pressure is 0.5MPa; the operating frequency of nanofiltration is 40Hz, and the liquid inlet pressure is 2MPa.
The mass ratio of the amylase to the buffer solution is 3:100, the concentration of the buffer solution is 0.2mol/L, and the pH value is 5.9.
The mass ratio of the chitosan acetic acid solution to the mixed enzyme solution is 1:0.3, the chitosan acetic acid solution is obtained by adding chitosan into acetic acid solution with mass concentration of 5% and weight of 75 times of the chitosan and carrying out ultrasonic oscillation until the chitosan is completely dissolved.
The mass ratio of the premixed liquid to the emulsifying agent to the corn oil to the glutaraldehyde to the layered titanate nano material is 1:0.1:5:0.04:0.01, and the emulsifier is span 80.
The process conditions of the crosslinking reaction are as follows: the reaction was stirred at 60 ℃ for 3 hours.
The layered titanate nano material is prepared by the following method: firstly, 1g of isopropyl titanate is dispersed in 9g of sodium hydroxide solution 9mol/L by ultrasonic waves, then the isopropyl titanate is transferred to a hydrothermal reaction kettle to carry out hydrothermal reaction for 22 hours at 160 ℃, and then the isopropyl titanate is obtained by filtering, washing with deionized water and drying.
Comparative example 3
A process for extracting cyperus esculentus oligosaccharide by an enzymatic method comprises the following specific steps:
(1) Crushing cyperus bean pulp to 80 meshes, performing extrusion puffing treatment to obtain 100g of bean pulp powder, adding the bean pulp powder into 700g of water, uniformly stirring to obtain slurry, and performing high-speed shearing and microwave treatment to obtain pretreated slurry;
(2) Adjusting the pretreatment slurry to be alkaline, stirring, centrifuging to obtain precipitate so as to remove protein, adding the precipitate into distilled water, adjusting the pH to be =4, performing ultrasonic extraction, centrifuging to obtain supernatant, concentrating, performing alcohol precipitation, centrifuging, and drying to obtain polysaccharide;
(3) Then adding polysaccharide into distilled water, dispersing uniformly by ultrasonic waves, adding complex enzyme, carrying out enzymolysis under the conditions of a magnetic field and a pulse electric field, and carrying out post-treatment to obtain oligosaccharide;
the complex enzyme is prepared by the following method: firstly, dissolving 1g of amylase, 0.2g of cellulase, 0.02g of pectinase and 0.02g of mannase in a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution to prepare a mixed enzyme solution, then mixing a chitosan acetic acid solution with the mixed enzyme solution to prepare a premixed solution, then slowly dripping the premixed solution into corn oil containing an emulsifier, stirring and emulsifying, then adding glutaraldehyde, carrying out a cross-linking reaction, concentrating and drying to obtain the complex enzyme.
In the step (1), the process conditions of high-speed shearing are as follows: 20000 rpm high shear for 7 minutes.
In the step (1), the process conditions of the microwave treatment are as follows: microwave treatment at 300W for 3 minutes.
In the step (2), the specific method for removing the protein comprises the following steps: the pretreatment slurry was adjusted to pH =8, treated with stirring at 60 ℃ for 40 minutes, and centrifuged to take out the precipitate.
In the step (2), the mass ratio of the precipitate to the distilled water is 1:7, the technological conditions of ultrasonic extraction are as follows: the ultrasonic vibration treatment was carried out at 600W for 5 hours at 40 ℃.
And (2) concentrating the supernatant to 1/3 of the original volume to obtain a concentrated solution, adding 0.6 volume of absolute ethyl alcohol into the concentrated solution while stirring, standing at 4 ℃ for 12 hours, centrifuging at 7000 rpm for 10 minutes, and drying.
In the step (3), the mass ratio of the polysaccharide to the distilled water to the complex enzyme is 1:10:1.
in the step (3), the enzymolysis process conditions are as follows: the magnetic field intensity is 300mT, the pulse electric field intensity is 15kV/cm, and the enzymolysis is carried out for 3 hours at 40 ℃.
In the step (3), the post-treatment method comprises the following specific steps: inactivating enzyme at 98 ℃ for 15 minutes, performing ultrafiltration treatment by using a filter membrane with the molecular weight cutoff of 10kDa, performing nanofiltration by using a filter membrane with the molecular weight cutoff of 1000Da, removing inorganic salt, glucose, sucrose and other small molecular substances, concentrating, and performing spray drying to obtain the oligosaccharide.
The working frequency of ultrafiltration is 26Hz, and the liquid inlet pressure is 0.5MPa; the operating frequency of nanofiltration is 40Hz, and the liquid inlet pressure is 2MPa.
The mass ratio of the amylase to the buffer solution is 3:100, the concentration of the buffer was 0.2mol/L, and the pH was 5.9.
The mass ratio of the chitosan acetic acid solution to the mixed enzyme solution is 1:0.3, the chitosan acetic acid solution is obtained by adding chitosan into acetic acid solution with mass concentration of 5 percent which is 75 times of the weight of the chitosan and oscillating the chitosan by ultrasonic waves until the chitosan is completely dissolved.
The mass ratio of the premixed liquid to the emulsifier to the corn oil to the glutaraldehyde is 1:0.1:5:0.04 and the emulsifier is span 80.
The process conditions of the crosslinking reaction are as follows: the reaction was stirred at 60 ℃ for 3 hours.
Comparative example 4
A process for extracting cyperus esculentus oligosaccharide by an enzymatic method comprises the following specific steps:
(1) Crushing cyperus bean pulp to 80 meshes, performing extrusion puffing treatment to obtain 100g of bean pulp powder, adding the bean pulp powder into 700g of water, uniformly stirring to obtain slurry, and performing high-speed shearing and microwave treatment to obtain pretreated slurry;
(2) Adjusting the pretreatment slurry to be alkaline, stirring, centrifuging to obtain precipitate to remove protein, adding the precipitate into distilled water, adjusting pH =4, performing ultrasonic extraction, centrifuging to obtain supernatant, concentrating, precipitating with ethanol, centrifuging, and drying to obtain polysaccharide;
(3) Then adding polysaccharide into distilled water, dispersing uniformly by ultrasonic waves, adding complex enzyme, carrying out enzymolysis under the conditions of a magnetic field and a pulse electric field, and carrying out post-treatment to obtain oligosaccharide;
wherein the complex enzyme is prepared by uniformly mixing 1g of amylase, 0.2g of cellulase, 0.02g of pectinase and 0.02g of mannase.
In the step (1), the process conditions of high-speed shearing are as follows: 20000 rpm high shear for 7 minutes.
In the step (1), the process conditions of the microwave treatment are as follows: microwave treatment at 300W for 3 minutes.
In the step (2), the specific method for removing the protein comprises the following steps: the pretreatment slurry was adjusted to pH =8, treated with stirring at 60 ℃ for 40 minutes, and centrifuged to take out the precipitate.
In the step (2), the mass ratio of the precipitate to the distilled water is 1:7, the technological conditions of ultrasonic extraction are as follows: ultrasonic vibration treatment was carried out at 600W for 5 hours at 40 ℃.
And (2) concentrating the supernatant to 1/3 of the original volume to obtain a concentrated solution, adding 0.6 volume of absolute ethyl alcohol into the concentrated solution while stirring, standing at 4 ℃ for 12 hours, centrifuging at 7000 rpm for 10 minutes, and drying.
In the step (3), the mass ratio of the polysaccharide to the distilled water to the complex enzyme is 1:10:1.
in the step (3), the enzymolysis process conditions are as follows: the magnetic field intensity is 300mT, the pulse electric field intensity is 15kV/cm, and the enzymolysis is carried out for 3 hours at 40 ℃.
In the step (3), the post-treatment method comprises the following specific steps: inactivating enzyme at 98 ℃ for 15 minutes, performing ultrafiltration treatment by using a filter membrane with the molecular weight cutoff of 10kDa, performing nanofiltration by using a filter membrane with the molecular weight cutoff of 1000Da, removing inorganic salt, glucose, sucrose and other small molecular substances, concentrating, and performing spray drying to obtain the oligosaccharide.
The working frequency of ultrafiltration is 26Hz, and the liquid inlet pressure is 0.5MPa; the operating frequency of nanofiltration is 40Hz, and the liquid inlet pressure is 2MPa.
The weight m0 of the cyperus bean pulp in examples 1 to 3 and comparative examples 1 to 4 (the weight m0 of the cyperus bean pulp is 100 g) was recorded, the units of the weight m1, m0 and m1 of the obtained oligosaccharide were g, and the oligosaccharide yield (%) = m1/m0 × 100% was calculated.
The average molecular weight of the resulting oligosaccharide was measured by a digital rotational viscometer, and the results are shown in Table 1.
TABLE 1 oligosaccharide yield and average molecular weight
Oligosaccharide weight (g) Oligosaccharide yield (%) Average molecular weight of oligosaccharide (Da)
Example 1 42.7 42.7 5100
Example 2 39.5 39.5 5300
Example 3 40.2 40.2 4800
Comparative example 1 30.1 30.1 7400
Comparative example 2 37.2 37.2 6300
Comparative example 3 33.4 33.4 6900
Comparative example 4 28.6 28.6 14200
As can be seen from table 1, the methods of examples 1 to 3 greatly improved the yield of oligosaccharides (close to the starch content in the cyperus bean pulp, that is, the present invention achieved sufficient degradation of starch in the cyperus bean pulp to convert into oligosaccharides), and the average molecular weight of the obtained oligosaccharides was small.
Comparative example 1 in the preparation of the pre-treated slurry, the extrusion puffing treatment and the microwave treatment steps are omitted, comparative example 2 in the enzymolysis is omitted, comparative example 3 in the preparation of the complex enzyme is omitted, the complex enzyme of comparative example 4 adopts a direct mixing method, resulting in low oligosaccharide yield, and the average molecular weight of the obtained oligosaccharide is large, which indicates that the micronization treatment of the cyperus bean pulp and the composition of the complex enzyme jointly promote the dissociation and degradation of polysaccharide in the cyperus bean pulp, thereby improving the oligosaccharide yield and reducing the average molecular weight of the oligosaccharide.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims (10)

1. A process for extracting cyperus esculentus oligosaccharide by an enzymatic method is characterized by comprising the following specific steps:
(1) Crushing cyperus bean pulp, performing extrusion puffing treatment to prepare bean pulp powder, adding the bean pulp powder into water, uniformly stirring to obtain slurry, and performing high-speed shearing and microwave treatment to obtain pretreated slurry;
(2) Adjusting the pretreated slurry to be alkaline, stirring, centrifuging to obtain precipitate so as to remove protein, adding the precipitate into distilled water, adjusting the pH to be between 4 and 6, performing ultrasonic extraction, centrifuging to obtain supernatant, concentrating, precipitating with ethanol, centrifuging, and drying to obtain polysaccharide;
(3) Then adding polysaccharide into distilled water, dispersing uniformly by ultrasonic waves, adding complex enzyme, carrying out enzymolysis under the conditions of a magnetic field and a pulse electric field, and carrying out post-treatment to obtain oligosaccharide;
the complex enzyme is prepared by the following method: firstly, amylase, cellulase, pectinase and mannase are mixed according to a mass ratio of 1:0.1 to 0.2: 0.02-0.03: 0.01 to 0.02 of the complex enzyme is dissolved in disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution to prepare mixed enzyme solution, then chitosan acetic acid solution and the mixed enzyme solution are mixed to prepare premixed solution, then the premixed solution is slowly dripped into corn oil containing emulsifier, stirred and emulsified, and then glutaraldehyde and layered titanate nano material are added, cross-linking reaction, concentration and drying are carried out, thus obtaining the complex enzyme.
2. The process for extracting cyperus esculentus oligosaccharides by using the enzymatic method according to claim 1, wherein in the step (1), cyperus esculentus meal is crushed to 80-100 meshes, and the mass ratio of the cyperus esculentus meal to water is 1:5 to 7.
3. The process for extracting cyperus esculentus oligosaccharides by using the enzymatic method according to claim 1, wherein in the step (1), the process conditions of high-speed shearing are as follows: 20000-30000 r/min high speed shearing for 5-7 min.
4. The process for extracting cyperus esculentus oligosaccharide by using the enzymatic method according to claim 1, wherein the microwave treatment in the step (1) is carried out under the following process conditions: microwave treatment is carried out for 2-3 minutes at 300-500W.
5. The process for extracting cyperus esculentus oligosaccharides by using the enzymatic method according to claim 1, wherein in the step (2), the mass ratio of the precipitate to the distilled water is 1: 5-7, the technological conditions of ultrasonic extraction are as follows: under the condition of 40-50 ℃, the ultrasonic vibration treatment is carried out for 5-6 hours at 400-600W.
6. The process for extracting cyperus esculentus oligosaccharide by using the enzymatic method according to claim 1, wherein in the step (2), the supernatant is concentrated to 1/4-1/3 of the original volume to obtain a concentrated solution, absolute ethyl alcohol with the volume 0.6-0.8 times of that of the concentrated solution is added into the concentrated solution while stirring, the concentrated solution is kept stand at 0-4 ℃ for 12-15 hours, centrifuged at 5000-7000 rpm for 10-12 minutes, and dried to obtain the cyperus esculentus oligosaccharide.
7. The process for extracting cyperus esculentus oligosaccharide by using the enzymatic method according to claim 1, wherein the enzymatic hydrolysis in step (3) is carried out under the following process conditions: the magnetic field intensity is 200-300 mT, the pulse electric field intensity is 15-25 kV/cm, and the enzymolysis is carried out for 3-5 hours at the temperature of 35-40 ℃.
8. The process for extracting cyperus esculentus oligosaccharide by using the enzymatic method according to claim 1, wherein the mass ratio of amylase to buffer solution is 2-3: 100, the concentration of the buffer solution is 0.2mol/L, and the pH value is 5.9.
9. The process for extracting cyperus esculentus oligosaccharide by using the enzymatic method according to claim 1, wherein the mass ratio of the chitosan acetic acid solution to the mixed enzyme solution is 1:0.3 to 0.5, the chitosan acetic acid solution is obtained by adding chitosan into acetic acid solution with mass concentration of 5 percent, which is 65 to 75 times of the weight of the chitosan, and oscillating the mixture by ultrasonic waves until the chitosan is completely dissolved.
10. The process for extracting cyperus esculentus oligosaccharides by using the enzymatic method according to claim 1, wherein the mass ratio of the premixed liquid to the emulsifying agent to the corn oil to the glutaraldehyde to the layered titanate nano-materials is 1:0.1 to 0.2:4 to 5: 0.04-0.05: 0.008-0.01, and span 80 is used as the emulsifier.
CN202111019573.5A 2021-09-01 2021-09-01 Process for extracting cyperus esculentus oligosaccharide by enzyme method Active CN113699197B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111019573.5A CN113699197B (en) 2021-09-01 2021-09-01 Process for extracting cyperus esculentus oligosaccharide by enzyme method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111019573.5A CN113699197B (en) 2021-09-01 2021-09-01 Process for extracting cyperus esculentus oligosaccharide by enzyme method

Publications (2)

Publication Number Publication Date
CN113699197A CN113699197A (en) 2021-11-26
CN113699197B true CN113699197B (en) 2023-04-07

Family

ID=78658688

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111019573.5A Active CN113699197B (en) 2021-09-01 2021-09-01 Process for extracting cyperus esculentus oligosaccharide by enzyme method

Country Status (1)

Country Link
CN (1) CN113699197B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116212446A (en) * 2023-03-27 2023-06-06 吉林大学 Novel method for preparing degreased oil meal by utilizing glycerol

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102234335A (en) * 2010-04-21 2011-11-09 长春大学 Production technology for continuously extracting starch, oil and sugar from cyperus esculentus
CN103589766A (en) * 2013-11-22 2014-02-19 东北农业大学 Method for simultaneously extracting soybean peptide and soybean oligosaccharide from aqueous phase produced through aqueous enzymatic method
WO2015192247A1 (en) * 2014-06-21 2015-12-23 The Royal Institution For The Advancement Of Learning/Mcgill University Enzymatic production of prebiotic oligosaccharides from potato pulp
CN105779524A (en) * 2016-03-15 2016-07-20 常州市蓝勖化工有限公司 Preparation method for extracting soybean oligosaccharides from soybean meal
CN108359701A (en) * 2018-02-28 2018-08-03 浙江天草生物科技股份有限公司 A method of mannan-oligosaccharides being prepared from coffee bean with enzymatic isolation method
CN108359700A (en) * 2018-02-28 2018-08-03 亿利耐雀生物科技有限公司 A kind of enzymolysis prepares the method and its application of sand sagebrush (Artemisia filifolia) oligosaccharide
CN111909282A (en) * 2019-05-07 2020-11-10 中国农业机械化科学研究院 Method for extracting cyperus esculentus polysaccharide
CN113201564A (en) * 2021-06-10 2021-08-03 内蒙古大学 Method for saccharifying cyperus esculentus dregs

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102234335A (en) * 2010-04-21 2011-11-09 长春大学 Production technology for continuously extracting starch, oil and sugar from cyperus esculentus
CN103589766A (en) * 2013-11-22 2014-02-19 东北农业大学 Method for simultaneously extracting soybean peptide and soybean oligosaccharide from aqueous phase produced through aqueous enzymatic method
WO2015192247A1 (en) * 2014-06-21 2015-12-23 The Royal Institution For The Advancement Of Learning/Mcgill University Enzymatic production of prebiotic oligosaccharides from potato pulp
CN105779524A (en) * 2016-03-15 2016-07-20 常州市蓝勖化工有限公司 Preparation method for extracting soybean oligosaccharides from soybean meal
CN108359701A (en) * 2018-02-28 2018-08-03 浙江天草生物科技股份有限公司 A method of mannan-oligosaccharides being prepared from coffee bean with enzymatic isolation method
CN108359700A (en) * 2018-02-28 2018-08-03 亿利耐雀生物科技有限公司 A kind of enzymolysis prepares the method and its application of sand sagebrush (Artemisia filifolia) oligosaccharide
CN111909282A (en) * 2019-05-07 2020-11-10 中国农业机械化科学研究院 Method for extracting cyperus esculentus polysaccharide
CN113201564A (en) * 2021-06-10 2021-08-03 内蒙古大学 Method for saccharifying cyperus esculentus dregs

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Zhenle Yang et al.Sucrose metabolism in developing oil-rich tubers of Cyperus esculentus: comparative transcriptome analysis.BMC Plant Biol .2018,第19卷(第19期),全文. *
田晓琳 ; 张国权 ; .响应面法优化油莎豆粕低聚糖的提取工艺.食品工业.2020,(第03期),全文. *

Also Published As

Publication number Publication date
CN113699197A (en) 2021-11-26

Similar Documents

Publication Publication Date Title
CN103749954B (en) Method for preparing microcrystalline cellulose
CN106387924B (en) Method for extracting suaeda salsa dietary fiber
EP3094734B1 (en) Process for fractionation of oligosaccharides from agri-waste
CA2710504A1 (en) Process for producing saccharide
KR101904019B1 (en) Alginic acid materialization method using waste seaweed and enzymes
CN101077197A (en) Technology for preparing functional dietary fiber through waste bamboo shell enzymolysis
CN113699197B (en) Process for extracting cyperus esculentus oligosaccharide by enzyme method
CN107279743A (en) A kind of barley young leaf green juice powder and preparation method thereof
CN1952162A (en) Method for improving straw enzymolysis efficiency by synergistic pretreatment of Steam Explosion and ultra micro-pulverization
CN114568709B (en) Extraction method for improving water-soluble dietary fiber content of square bamboo shoots
CN113100447A (en) Preparation method and application of micron-sized emulsified citrus peel dietary fiber
CN109111497A (en) A kind of method for producing of peony seeds albumen
CN109170922B (en) Preparation method of wheat bran soluble dietary fiber
CN108034688A (en) The preparation process of rice bran polysaccharide
CN113208120B (en) Preparation method and application of Pickering emulsifying nano citrus peel dietary fiber
CN111150069A (en) Method for preparing asparagus byproduct dietary fiber by adopting enzymolysis method and spray drying
CN101797036B (en) Preparation method of canna edulis ker soluble dietary fiber
CN111700279B (en) Preparation method of wheat bran dietary fiber
CN108997446A (en) The extraction process of small molecule fucoidin and the application in cosmetics
CN101665534B (en) Preparation method of concentrated protein of cottonseeds
CN116284462A (en) Method for efficiently preparing high-crystallinity V-type dextrin
CN102212595A (en) Preparation method and application of water-soluble nano grain polysaccharide
CN114045316A (en) Modified cereal dietary fiber and preparation method thereof
CN111378709A (en) Preparation method of corn polypeptide-selenium chelate
CN112029011A (en) Green process for extracting ultralow-viscosity low-M/G-value sodium alginate from kelp

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant