CN108504715A - A kind of drone pupae polypeptide extract - Google Patents
A kind of drone pupae polypeptide extract Download PDFInfo
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- CN108504715A CN108504715A CN201810130220.4A CN201810130220A CN108504715A CN 108504715 A CN108504715 A CN 108504715A CN 201810130220 A CN201810130220 A CN 201810130220A CN 108504715 A CN108504715 A CN 108504715A
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Abstract
The invention discloses a kind of drone pupae polypeptide extract, the method for preparing extractive the specific steps are：Drone pupae is crushed, sieving obtains drone pupae powder, and petroleum ether is then added, is heated to reflux to get drone pupae albumen；Distilled water is added into drone pupae albumen powder, sterilizes after mixing, is cooled to room temperature, then adjusts the pH of solution, is inoculated with composite bacteria, adds arabinose, fermented and cultured obtains zymotic fluid；Enzymolysis liquid is subjected to ultrafiltration through ultrafiltration membrane and obtains ultrafiltration enzymolysis liquid；Chitosan oligosaccharide is added into zymotic fluid, oscillation absorption, centrifugation, supernatant concentration, freeze-drying is drone pupae polypeptide.It has the beneficial effect that：For polypeptide of the present invention without bitter taste, of light color, safe and non-toxic, extraction efficiency, yield and purity are high, need not carry out the drone pupae polypeptide extract of de- suffering reason.
Description
Technical field
The present invention relates to technical field of biological extraction, more particularly to a kind of drone pupae polypeptide extract.
Background technology
China is most one of country of raising bees in the world, and honeybee has long cultivation history, abundant money in China
Source.China is long to the research history of the edible value of bee products, forms oneself unique research body in various aspects at present
System, and multiple product has been developed, but still waited in utilization there are many field.The unfertilized egg of queen bee production, holds sway over a region after hatching
Larva of bee, the pupa further developed into are known as drone pupae.Bee pupa contains abundant nutriment, contains someone in bee pupa protein
8 kinds of essential amino acids necessary to body, account for the 39.39% of total amino acid content, therefore are the protein sources of ideals of human being.Bee pupa
In also contain abundant mineral element, vitamin, hormone, enzyme and flavone compound, be a kind of good food resource.Food
It can be improved a poor appetite with bee pupa, improve sleep, build up health, improve immunity of organisms.
Peptide of the molecular structure between amino acid and protein is the segment of the structure and function with protein, tool
There is the protein of physiological function of the number in terms of necessarily to be realized by peptide.Peptide have the function of such numerous and complicated structure with by
The amino acid classes of peptide, different numbers is constituted to be determined with putting in order.Dipeptides and tripeptides are taken off by 2 or 3 amino acid
Water condensation made of, tetrapeptide, pentapeptide can and so on.It is, in general, that oligopeptides is that have amino acid number within 10 on peptide chain,
Polypeptide is exactly 10-50 amino acid number, and protein is 50 or more amino acid numbers.Small peptide or oligopeptide i.e. few
In peptide two, tripeptides.Polypeptide is a-amino acid with the compound that peptide chain links together and is formed, it is also protein hydrolysis
Intermediate product.Two amino acid molecular dehydrating condensations can form dipeptides, multiple amino acid molecular dehydrating condensations can be formed tripeptides,
Tetrapeptide, pentapeptide etc..The compound usually formed by 10~50 amino acid molecular dehydrating condensations can penetrate semi-permeable membrane polypeptide,
It is not precipitated by trichloroacetic acid and ammonium sulfate.Polypeptide is always an active area in nutrient research, from natural animal, is planted
Activity polypeptid substance is found in object, microorganism, develops new varieties on its basis, is always a direction of peptides research,
With diversified biological function, such as hormonal action, immunological regulation, anti-hypertension, norcholesterol and antitumaous effect, antioxygen
Change acts on, promote growth, adjust flavour of food products etc..The method of polypeptide is obtained other than being extracted directly from histocyte, may be used also
Using the certain albumen of the proteolytic enzymes hydrolize of some amino acid specificities, biologically active peptide material is obtained.For a long time
Since, the bee pupa protein product of domestic production is mainly bee pupa albumen powder.But it is only simply processed, or by fresh pupa or dry powder
Directly as material outlet, the technology content and added value of product are low, and the resource of most bee pupas is not opened effectively yet
Hair utilizes.Therefore, bee pupa polypeptide is obtained by protein raw material of bee pupa to be provided for the further development and utilization of bee product
With reference to.
The prior art such as Authorization Notice No. is the Chinese invention patent of CN103005466B, discloses a kind of drone pupae polypeptide
The application of extract, including the drone pupae polypeptide extract is as with the food for improving memory and/or anti-oxidation function
Product are prepared with the application in the food for improving memory and/or anti-oxidation function；Drone pupae polypeptide extract as with
Improve the health products of memory and/or anti-oxidation function or prepares in the health products for having and improving memory and/or anti-oxidation function
Using；Application of the drone pupae polypeptide extract in preparing the drug for having and improving memory and/or anti-oxidation function.The drone pupae
Polypeptide extract significantly improves the function of memory and preferable antioxidation, is used as or prepares with improvement note
Recall and/or the product of anti-oxidation function.But the content of polypeptide is not high in the drone pupae polypeptide extract, and have bitter taste.Invention Content
The purpose of the present invention is to provide a kind of no bitter tastes, of light color, safe and non-toxic, and extraction efficiency, yield and purity are high, are not required to
Carry out the drone pupae polypeptide extract of de- suffering reason.
The present invention in view of the above technology in the problem of mentioning, the technical solution taken is：
Microorganism used therefor of the present invention and its purchase enterprise are：The Xuzhou bio tech ltd Sai Fu.
A kind of drone pupae polypeptide extract, the preparation method of the drone pupae polypeptide extract, including pretreatment, drone pupae egg
White enzymolysis, ultrafiltration, separation, the specific steps are：
Pretreatment：Drone pupae is crushed, 100-200 mesh is crossed and sieves to obtain drone pupae powder, it is 1 then to press solid-liquid ratio:5-8（g/mL）To
It petroleum ether is added in drone pupae powder, is heated to reflux 5-6h to get drone pupae albumen, it is spare, since grease type is deposited in drone pupae
The effect of enzymolysis drone pupae albumen is being substantially reduced, while can also be aoxidized in enzymolysis liquid, is generating undesirable flavor and add
The color of deep enzymolysis liquid；Further, since also because of the effect of emulsification, cause to bring in later stage filtering, clarification enzymolysis liquid prodigious
Difficulty, so must ungrease treatment be carried out to drone pupae first, the step process method is at low cost, and degreasing effect is good；
Drone pupae proteolysis：It is 1 by solid-liquid ratio:20-25（g/mL）Distilled water is added into drone pupae albumen powder, is uniformly mixed
After sterilize, be cooled to room temperature, then adjust solution pH to 6.5-8.0, by inoculum concentration be 0.8-1.2% inoculation composite bacteria, then
The arabinose of composite bacteria total amount 0.02-0.5% is added, then fermented and cultured 25-30h must ferment under conditions of 25-35 DEG C
Liquid, spare, above-mentioned composite bacteria is that weight ratio is 1:0.3-0.4:Bacillus subtilis, aspergillus niger and the saccharomycete of 0.2-0.3,
Arabinose contains the D-arabinose of 0.1-1.5%, and the growth for existing for microorganism of the arabinose provides carbon source, can
Promote the growth metabolism of microorganism, improves microorganism yield of enzyme and increase, institute's protease production is made to be in higher level, shorten more
The extraction time of peptide, the yield for improving polypeptide, and the addition of D-arabinose can adjust the ratio of endopeptidase and peptide ending enzyme,
While improving the yield of polypeptide can also in modified amino acid molecule hydrophobic group effect, improve the flavor of polypeptide, the step
The rapid proteolytic enzyme protolysate matter generated using microorganism prepares polypeptide, and existing endopeptidase has peptide ending enzyme again in generated enzyme,
It can be cut simultaneously in peptide middle-of-chain and both ends, the polypeptide yield height of preparation, performance are good, while fermentation process can take off
Go bitter polypeptide, the polypeptide of preparation that need not carry out de- suffering reason, be produced into considerably reduce technological process, reduce
This, and the generation of fragranced substance is also had in fermentation process, the flavor and taste of polypeptide can be improved；
Ultrafiltration：Enzymolysis liquid is subjected to ultrafiltration through the ultrafiltration membrane that molecular cut off is 6-10kDa and obtains ultrafiltration enzymolysis liquid, it is spare, by
Complicated in the microorganism zymolyte comparison of ingredients of protein, the existing protein macromolecule not being fully hydrolyzed also has molecular weight big
Small not equal polypeptide and free amino acid, the step want the moment to ensure that inlet pressure is no more than 0.2MPa so that ultrafiltration can
It is smoothed out, ultrafiltration membrane ultrafiltration purification belongs to physics mode, does not introduce chemical reagent, preferably saves small molecular protein Like
Primary characteristic, and ultrafiltration has skimmed dairy enzymolysis liquid and preferably isolates and purifies effect；
Separation：It is that chitosan oligosaccharide is added into zymotic fluid by 0.1-0.2mg/mL by additive amount, absorption is vibrated under conditions of 30-40 DEG C
25-35min is subsequently placed in the centrifuge that rotating speed is 4000-6000r/min and centrifuges 8-15min, takes supernatant concentration 8-15
Times, freeze-drying is drone pupae polypeptide, which occurs mainly in the absorption of peptide the outer surface of microballoon, existing without interior diffusion
As, therefore reaction rate constant is big, time of equilibrium adsorption is short, improves the separative efficiency of polypeptide, and obtained drone pupae polypeptide is without hardship
It is taste, of light color, safe and non-toxic, have effects that enhance immunological regulation, antitumor, anti-oxidant, antifatigue and somatotrophic, can answer
For food, medicine, field of health care products.
Preferably, chitosan oligosaccharide is chitosan oligosaccharide magnetic particle in separating step, preparation method is：
Step 1：It is 1 by solid-liquid ratio:12-16（g/L）Chitosan is added to the water, its pH to 4.5-5.5 is adjusted, is then pressed enzyme
Amount is that complex enzyme is added in 5-7%, and the proportioning of papain and cellulase is 1 in complex enzyme:0.4-0.6 is 40- in temperature
4-6h is digested under conditions of 50 DEG C, is aided with hydrogen peroxide degradation in enzymolysis process, is then adjusted the pH value of enzymolysis liquid to neutrality, is sunk
Form sediment undegradable chitosan, supernatant concentration, vacuum drying obtain water-soluble chitosan oligosaccharide, the step using chitosan as raw material,
Chitosan oligosaccharide is prepared using double enzymolysis method joint hydrogen peroxide method degradation chitosan, the glucosides between GlcN and GlcNAC can be cut off
Key, for preferential degradation compared with long chain and the higher part of the degree of polymerization, production relative molecular weight is low and the chitosan oligosaccharide of narrowly distributing, should
Preparation method reaction condition is mild, easily controllable, free from environmental pollution, speed is fast；
Step 2：0.4-0.6 parts by weight chitosan oligosaccharides are dissolved in 18-22 parts by weight of deionized water, 0.2-0.3 parts by weight are added
Fe3O4 magnetic particles, be in ultrasonic power, under conditions of be ultrasonically treated 1-2min, be then added slowly with stirring 70-90 weights
Part atoleine and 3-5 parts by weight Tween 80s are measured, 20-40min is stirred under the conditions of 700-1000rpm, adds 4-6 parts by weight
Glutaraldehyde continues to stir 2-3h, then is placed at 50-70 DEG C and keeps the temperature 1.5-2.5h, and product petroleum ether, acetone, deionized water are anti-
After backwashing is washed, and is finally dried in vacuo at 35-45 DEG C to get chitosan oligosaccharide magnetic particle, in chitosan oligosaccharide structure on C2 there are one-NH2,
- OH there are one on the positions C3 is equatorial bond, is conducive to form complex with metal ion, which is prepared into magnetic
Grain, make it have magnetic responsiveness, can under external magnetic field from solution quick separating, can recycle.
Further preferably, it is that enzymolysis terminates preceding 50-70min that hydrogenperoxide steam generator, which is added the time, hydrogenperoxide steam generator it is dense
It is 8-12% to spend, and the volume ratio of H2O2 and enzyme solution is 1:6-10.The effect of the assistant degradation of hydrogen peroxide is good, and height is added too early
Concentration H2O2 solution can influence the vigor of enzyme, hinder the release of reduced sugar, and the condition can be such that hydrolysis result and degradation effect reaches
To best, while chitosan dissolves just in weak acid solution, and is not easy to form ammonium salt, and oxidation site is more, and degradation speed is fast so that
Molecular weight of product is low, and hydrogen peroxide degradation method is at low cost, easy to operate, while being influenced on the subsequent processing of product small.
For optimisation technique method, the measure taken further includes：Catechol containing 0.2-0.3mM in hydrogenperoxide steam generator
With 0.01-0.1mM ellagic acids, catechol and ellagic acid can enrich the functional group of chitosan oligosaccharide by chemical modification, improve it
Physico-chemical property improves the binding performance of itself and Fe3O4 magnetic particles so that chitosan oligosaccharide is successfully modified on Fe3O4 magnetic particles surface, together
When can protect the stability of amino in chitosan, hydroxyl isoreactivity group amino, avoid that agglomeration occurs between particle, improve
Chitosan oligosaccharide wraps up the leakproofness of Fe3O4 magnetic particles, and the final absorption property for improving chitosan oligosaccharide magnetic particle improves the separation of polypeptide
Efficiency, and it is avoided that the generation of monosaccharide, the yield of chitosan oligosaccharide is improved, extraction cost is reduced.
Compared with the prior art, the advantages of the present invention are as follows：1）Polypeptide extract of the present invention is without bitter taste, of light color, safe
It is nontoxic, have effects that enhance immunological regulation, antitumor, anti-oxidant, antifatigue and somatotrophic, can be applied to food, medicine,
Field of health care products；2）The extracting method simple possible of the polypeptide extract, extraction efficiency, yield and purity are high, are easy to industrialize
Large-scale production has good application value and market potential；2）The present invention utilizes the proteolytic enzyme protolysate that microorganism generates
Matter prepares polypeptide, and polypeptide yield is high, performance is good, need not carry out de- suffering reason, considerably reduce technological process, reduces life
Cost is produced, and has the generation of fragranced substance in fermentation process, improves the flavor and taste of active peptide.
Specific implementation mode
The present invention program is described further below by embodiment：
Embodiment 1：
A kind of drone pupae polypeptide extract, the preparation method of the drone pupae polypeptide extract, including pretreatment, drone pupae protease
Solution, ultrafiltration, separation, the specific steps are：
1）Pretreatment：Drone pupae is crushed, 200 mesh is crossed and sieves to obtain drone pupae powder, it is 1 then to press solid-liquid ratio:5（g/mL）To drone
Petroleum ether is added in pupa powder, is heated to reflux 6h to get drone pupae albumen, it is spare, due to the presence of grease type in drone pupae, significantly
The effect of enzymolysis drone pupae albumen is reduced, while can also be aoxidized in enzymolysis liquid, undesirable flavor is generated and deepens to digest
The color of liquid；Further, since also because of the effect of emulsification, cause to bring prodigious difficulty, institute in later stage filtering, clarification enzymolysis liquid
That must carry out ungrease treatment to drone pupae first, the step process method is at low cost, and degreasing effect is good；
2）Drone pupae proteolysis：It is 1 by solid-liquid ratio:20（g/mL）Distilled water is added into drone pupae albumen powder, is uniformly mixed
After sterilize, be cooled to room temperature, then adjust solution pH to 8.0, by inoculum concentration be 0.8% be inoculated with composite bacteria, add compound
The arabinose of strain total amount 0.5%, then fermented and cultured 30h obtains zymotic fluid under conditions of 25 DEG C, spare, above-mentioned compound bacteria
It is 1 that kind, which is weight ratio,:0.3:0.3 bacillus subtilis, aspergillus niger and saccharomycete, the D- that arabinose contains 0.1% are Arabic
The growth for existing for microorganism of sugar, the arabinose provides carbon source, can promote the growth metabolism of microorganism, improves microorganism
Yield of enzyme increases, and institute's protease production is made to be in higher level, shortens the extraction time of polypeptide, improves the yield of polypeptide, and
The addition of D-arabinose can adjust the ratio of endopeptidase and peptide ending enzyme, and ammonia can also be modified while improving the yield of polypeptide
The effect of hydrophobic group in base acid molecule, improves the flavor of polypeptide, which utilizes the protease hydrolytic egg that microorganism generates
White matter prepares polypeptide, and existing endopeptidase has peptide ending enzyme again in generated enzyme, can be carried out simultaneously in peptide middle-of-chain and both ends
Cutting, the polypeptide yield of preparation is high, performance is good, while fermentation process can slough bitter polypeptide, and the polypeptide of preparation need not be into
The de- suffering reason of row, to considerably reduce technological process, reduce production cost, and also has fragranced in fermentation process
The generation of substance can improve the flavor and taste of polypeptide；
3）Ultrafiltration：Enzymolysis liquid is subjected to ultrafiltration through the ultrafiltration membrane that molecular cut off is 10kDa and obtains ultrafiltration enzymolysis liquid, it is spare, by
Complicated in the microorganism zymolyte comparison of ingredients of protein, the existing protein macromolecule not being fully hydrolyzed also has molecular weight big
Small not equal polypeptide and free amino acid, the step want the moment to ensure that inlet pressure is no more than 0.2MPa so that ultrafiltration can
It is smoothed out, ultrafiltration membrane ultrafiltration purification belongs to physics mode, does not introduce chemical reagent, preferably saves small molecular protein Like
Primary characteristic, and ultrafiltration has skimmed dairy enzymolysis liquid and preferably isolates and purifies effect；
4）Separation：It is that chitosan oligosaccharide is added into zymotic fluid by 0.1mg/mL by additive amount, absorption is vibrated under conditions of 40 DEG C
25min is subsequently placed in the centrifuge that rotating speed is 6000r/min and centrifuges 8min, takes 15 times of the supernatant concentration, freeze-drying to be
Drone pupae polypeptide, the step occur mainly in the absorption of peptide the outer surface of microballoon, without interior diffusion phenomena, therefore reaction rate
Constant is big, and time of equilibrium adsorption is short, improves the separative efficiency of polypeptide, and obtained drone pupae polypeptide is without bitter taste, of light color, safe nothing
Poison has effects that enhance immunological regulation, antitumor, anti-oxidant, antifatigue and somatotrophic, can be applied to food, medicine, guarantor
Strong product field.
Chitosan oligosaccharide is chitosan oligosaccharide magnetic particle in above-mentioned separating step, and preparation method is：
Step 1：It is 1 by solid-liquid ratio:16（g/L）Chitosan is added to the water, its pH to 4.5 is adjusted, it is 7% then to press enzyme concentration
Complex enzyme is added, the proportioning of papain and cellulase is 1 in complex enzyme:0.4, it is digested under conditions of temperature is 50 DEG C
4h is aided with hydrogen peroxide degradation in enzymolysis process, then adjusts the pH value of enzymolysis liquid to neutrality, precipitates undegradable chitosan,
Supernatant concentration, vacuum drying obtain water-soluble chitosan oligosaccharide, which was combined using chitosan as raw material using double enzymolysis method
Hydrogen oxide method degradation chitosan prepares chitosan oligosaccharide, can cut off the glycosidic bond between GlcN and GlcNAC, preferential degradation is compared with long molecule
Chain and the higher part of the degree of polymerization, relative molecular weight is low and the chitosan oligosaccharide of narrowly distributing for production, the preparation method reaction condition temperature
With easily controllable, free from environmental pollution, speed is fast；
Step 2：0.6 parts by weight chitosan oligosaccharide is dissolved in 18 parts by weight of deionized water, 0.3 parts by weight Fe3O4 magnetic particles are added,
Be in ultrasonic power, under conditions of be ultrasonically treated 1min, be then added slowly with stirring 90 part by weight of liquid paraffin and 3 weights
Part Tween 80 is measured, 20min is stirred under the conditions of 1000rpm, adds 6 parts by weight glutaraldehydes, continues to stir 2h, then be placed in 70 DEG C
Lower heat preservation 1.5h, product petroleum ether, acetone, deionized water are washed repeatedly, are finally dried in vacuo to get chitosan oligosaccharide magnetic at 45 DEG C
Property particle, in chitosan oligosaccharide structure on C2 there are one-NH2, on the positions C3 there are one-OH, be all equatorial bond, be conducive to metal from
Son forms complex, which is prepared into magnetic-particle, it is made to have magnetic responsiveness, can be under external magnetic field from solution
Middle quick separating can be recycled.
The above-mentioned hydrogenperoxide steam generator addition time is to digest to terminate preceding 50min, a concentration of the 12% of hydrogenperoxide steam generator,
The volume ratio of H2O2 and enzyme solution is 1:6.The effect of the assistant degradation of hydrogen peroxide is good, and high concentration H2O2 solution meetings are added too early
The vigor for influencing enzyme, hinders the release of reduced sugar, and the condition can make hydrolysis result and degradation effect reach best, while weak acid
Chitosan dissolves just in solution, and is not easy to form ammonium salt, and oxidation site is more, and degradation speed is fast so that and molecular weight of product is low, and
Hydrogen peroxide degradation method is at low cost, easy to operate, while being influenced on the subsequent processing of product small.
Catechol containing 0.3mM and 0.01mM ellagic acids in above-mentioned hydrogenperoxide steam generator, catechol and ellagic acid can
The functional group that chitosan oligosaccharide is enriched by chemical modification improves its physico-chemical property, improves the associativity of itself and Fe3O4 magnetic particles
It can so that chitosan oligosaccharide is successfully modified on Fe3O4 magnetic particles surface, while can protect amino in chitosan, hydroxyl isoreactivity group
The stability of amino avoids that agglomeration occurs between particle, improves the leakproofness of chitosan oligosaccharide package Fe3O4 magnetic particles, finally
The absorption property for improving chitosan oligosaccharide magnetic particle, improves the separative efficiency of polypeptide, and be avoided that the generation of monosaccharide, improves chitosan oligosaccharide
Yield, reduce extraction cost.
Embodiment 2：
A kind of drone pupae polypeptide extract, the preparation method of the drone pupae polypeptide extract, including pretreatment, drone pupae protease
Solution, ultrafiltration, separation, the specific steps are：
1）Pretreatment：Drone pupae is crushed, sieves with 100 mesh sieve to obtain drone pupae powder, it is 1 then to press solid-liquid ratio:8（g/mL）To drone
Petroleum ether is added in pupa powder, is heated to reflux 5h to get drone pupae albumen, it is spare；
2）Drone pupae proteolysis：It is 1 by solid-liquid ratio:25（g/mL）Distilled water is added into drone pupae albumen powder, is uniformly mixed
After sterilize, be cooled to room temperature, then adjust solution pH to 6.5, by inoculum concentration be 1.2% be inoculated with composite bacteria, add compound
The arabinose of strain total amount 0.02%, then fermented and cultured 25h obtains zymotic fluid under conditions of 35 DEG C, spare, above-mentioned compound bacteria
It is 1 that kind, which is weight ratio,:0.4:0.2 bacillus subtilis, aspergillus niger and saccharomycete, the D- that arabinose contains 1.5% are Arabic
Sugar；
3）Ultrafiltration：Enzymolysis liquid is subjected to ultrafiltration through the ultrafiltration membrane that molecular cut off is 6kDa and obtains ultrafiltration enzymolysis liquid, it is spare；
4）Separation：It is that chitosan oligosaccharide is added into zymotic fluid by 0.2mg/mL by additive amount, absorption is vibrated under conditions of 30 DEG C
35min is subsequently placed in the centrifuge that rotating speed is 4000r/min and centrifuges 15min, takes 8 times of the supernatant concentration, freeze-drying to be
Drone pupae polypeptide.
Chitosan oligosaccharide is chitosan oligosaccharide magnetic particle in above-mentioned separating step, and preparation method is：
Step 1：It is 1 by solid-liquid ratio:12（g/L）Chitosan is added to the water, its pH to 5.5 is adjusted, it is 5% then to press enzyme concentration
Complex enzyme is added, the proportioning of papain and cellulase is 1 in complex enzyme:0.6, it is digested under conditions of temperature is 40 DEG C
6h is aided with hydrogen peroxide degradation in enzymolysis process, then adjusts the pH value of enzymolysis liquid to neutrality, precipitates undegradable chitosan,
Supernatant concentration, vacuum drying obtain water-soluble chitosan oligosaccharide；
Step 2：0.4 parts by weight chitosan oligosaccharide is dissolved in 22 parts by weight of deionized water, 0.2 parts by weight Fe3O4 magnetic particles are added,
Be in ultrasonic power, under conditions of be ultrasonically treated 2min, be then added slowly with stirring 70 part by weight of liquid paraffin and 5 weights
Part Tween 80 is measured, 40min is stirred under the conditions of 700rpm, adds 4 parts by weight glutaraldehydes, continues to stir 3h, then be placed in 50 DEG C
Lower heat preservation 2.5h, product petroleum ether, acetone, deionized water are washed repeatedly, are finally dried in vacuo to get chitosan oligosaccharide magnetic at 35 DEG C
Property particle.The above-mentioned hydrogenperoxide steam generator addition time is that enzymolysis terminates preceding 70min, a concentration of 8%, the H2O2 of hydrogenperoxide steam generator
Volume ratio with enzyme solution is 1:10.Catechol containing 0.2mM and 0.1mM ellagic acids in above-mentioned hydrogenperoxide steam generator.
Embodiment 3：
A kind of drone pupae polypeptide extract, the preparation method of the drone pupae polypeptide extract, including pretreatment, drone pupae protease
Solution, ultrafiltration, separation, the specific steps are：
1）Pretreatment：Drone pupae is crushed, 150 mesh is crossed and sieves to obtain drone pupae powder, it is 1 then to press solid-liquid ratio:6（g/mL）To drone
Petroleum ether is added in pupa powder, is heated to reflux 5.5h to get drone pupae albumen, it is spare；
2）Drone pupae proteolysis：It is 1 by solid-liquid ratio:22（g/mL）Distilled water is added into drone pupae albumen powder, is uniformly mixed
After sterilize, be cooled to room temperature, then adjust solution pH to 7.2, by inoculum concentration be 1.0% be inoculated with composite bacteria, add compound
The arabinose of strain total amount 0.2%, then fermented and cultured 28h obtains zymotic fluid under conditions of 30 DEG C, spare, above-mentioned compound bacteria
It is 1 that kind, which is weight ratio,:0.35:0.25 bacillus subtilis, aspergillus niger and saccharomycete, arabinose contain 1.0% D- I
Uncle's sugar；
3）Ultrafiltration：Enzymolysis liquid is subjected to ultrafiltration through the ultrafiltration membrane that molecular cut off is 8kDa and obtains ultrafiltration enzymolysis liquid, it is spare；
4）Separation：It is that chitosan oligosaccharide is added into zymotic fluid by 0.15mg/mL by additive amount, absorption is vibrated under conditions of 35 DEG C
30min is subsequently placed in the centrifuge that rotating speed is 5000r/min and centrifuges 12min, takes 12 times of the supernatant concentration, freeze-drying to be
For drone pupae polypeptide.
Chitosan oligosaccharide is chitosan oligosaccharide magnetic particle in above-mentioned antibacterial peptide separating step, and preparation method is：
Step 1：It is 1 by solid-liquid ratio:14（g/L）Chitosan is added to the water, its pH to 5.0 is adjusted, it is 6% then to press enzyme concentration
Complex enzyme is added, the proportioning of papain and cellulase is 1 in complex enzyme:0.5, it is digested under conditions of temperature is 45 DEG C
5h is aided with hydrogen peroxide degradation in enzymolysis process, then adjusts the pH value of enzymolysis liquid to neutrality, precipitates undegradable chitosan,
Supernatant concentration, vacuum drying obtain water-soluble chitosan oligosaccharide；
Step 2：0.5 parts by weight chitosan oligosaccharide is dissolved in 20 parts by weight of deionized water, 0.25 parts by weight Fe3O4 magnetic grains are added
Son, be in ultrasonic power, under conditions of be ultrasonically treated 1min, be then added slowly with stirring 80 part by weight of liquid paraffin and
4 parts by weight Tween 80s, 30min is stirred under the conditions of 800rpm, adds 5 parts by weight glutaraldehydes, continues to stir 2.5h, then be placed in
2.0h is kept the temperature at 60 DEG C, product petroleum ether, acetone, deionized water are washed repeatedly, are finally dried in vacuo at 40 DEG C to get shell widow
Sugared magnetic particle.The above-mentioned hydrogenperoxide steam generator addition time is that enzymolysis terminates preceding 50-70min, and the time is added in hydrogenperoxide steam generator
Terminate preceding 60min for enzymolysis, a concentration of 10%, the H2O2 of hydrogenperoxide steam generator and the volume ratio of enzyme solution are 1:8；Hydrogen peroxide is molten
Contain 0.25mM catechols and 0.05mM ellagic acids in liquid.
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme of the present invention is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of drone pupae polypeptide extract, it is characterised in that：The preparation method of the extract includes the following steps：
1）Pretreatment：Drone pupae is crushed, sieving obtains drone pupae powder, and petroleum ether is then added, is heated to reflux to get drone pupae
Albumen；
2）Drone pupae proteolysis：Distilled water is added into drone pupae albumen powder, sterilizes after mixing, is cooled to room temperature, so
The pH for adjusting solution afterwards, is inoculated with composite bacteria, adds arabinose, fermented and cultured obtains zymotic fluid, spare；
3）Ultrafiltration：Enzymolysis liquid is subjected to ultrafiltration through ultrafiltration membrane and obtains ultrafiltration enzymolysis liquid, it is spare；
4）Separation：Chitosan oligosaccharide is added into zymotic fluid, oscillation absorption, centrifugation, supernatant concentration, freeze-drying is that drone pupae is more
Peptide.
2. a kind of drone pupae polypeptide extract according to claim 1, it is characterised in that：Drone pupae powder in the step 1
Solid-liquid ratio with petroleum ether is 1:5-8（g/mL）, it is heated to reflux 5-6h.
3. a kind of drone pupae polypeptide extract according to claim 1, it is characterised in that：Composite bacteria in the step 2
It is 1 for weight ratio:0.3-0.4:Bacillus subtilis, aspergillus niger and the saccharomycete of 0.2-0.3, inoculum concentration 0.8-1.2%, hair
Ferment cultivation temperature is 25-35 DEG C, time 25-30h.
4. a kind of drone pupae polypeptide extract according to claim 1, it is characterised in that：Arabinose in the step 2
Addition be composite bacteria total amount 0.02-0.5%, arabinose contains the D-arabinose of 0.1-1.5%.
5. a kind of drone pupae polypeptide extract according to claim 4, it is characterised in that：Chitosan oligosaccharide in the step 4
Additive amount is 0.1-0.2mg/mL, oscillation adsorption temp be 30-40 DEG C, time 25-35min.
6. a kind of drone pupae polypeptide extract according to claim 1, it is characterised in that：Chitosan oligosaccharide is in the step 4
Chitosan oligosaccharide magnetic particle, preparation method are：
Step 1：It is 1 by solid-liquid ratio:12-16（g/L）Chitosan is added to the water, its pH to 4.5-5.5 is adjusted, is then pressed enzyme
Amount is that complex enzyme is added in 5-7%, and the proportioning of papain and cellulase is 1 in complex enzyme:0.4-0.6 is 40- in temperature
4-6h is digested under conditions of 50 DEG C, is aided with hydrogen peroxide degradation in enzymolysis process, is then adjusted the pH value of enzymolysis liquid to neutrality, is sunk
Form sediment undegradable chitosan, supernatant concentration, and vacuum drying obtains water-soluble chitosan oligosaccharide；
Step 2：0.4-0.6 parts by weight chitosan oligosaccharides are dissolved in 18-22 parts by weight of deionized water, 0.2-0.3 parts by weight are added
Fe3O4 magnetic particles, be in ultrasonic power, under conditions of be ultrasonically treated 1-2min, be then added slowly with stirring 70-90 weights
Part atoleine and 3-5 parts by weight Tween 80s are measured, 20-40min is stirred under the conditions of 700-1000rpm, adds 4-6 parts by weight
Glutaraldehyde continues to stir 2-3h, then is placed at 50-70 DEG C and keeps the temperature 1.5-2.5h, and product petroleum ether, acetone, deionized water are anti-
After backwashing is washed, and is finally dried in vacuo to get chitosan oligosaccharide magnetic particle at 35-45 DEG C.
7. a kind of drone pupae polypeptide extract according to claim 6, it is characterised in that：The hydrogenperoxide steam generator is added
Time is to digest to terminate preceding 50-70min, and the volume ratio of a concentration of 8-12% of hydrogenperoxide steam generator, H2O2 and enzyme solution is 1:6-
10。
8. a kind of drone pupae polypeptide extract according to claim 6, it is characterised in that：Contain in the hydrogenperoxide steam generator
There are the catechol and 0.01-0.1mM ellagic acids of 0.2-0.3mM.
9. can be applied to food, doctor as product or raw material according to any one of claim 1 to 7 drone pupae polypeptide extract
Medicine, field of health care products.
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|---|---|---|---|
| CN201810130220.4A CN108504715A (en) | 2018-02-08 | 2018-02-08 | A kind of drone pupae polypeptide extract |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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ID=63375637
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117837704A (en) * | 2024-03-08 | 2024-04-09 | 北京衡美金叶营养健康科技有限公司 | Liquid beverage for increasing skin collagen content and preparation method and application thereof |
| CN117837704B (en) * | 2024-03-08 | 2024-06-21 | 北京衡美金叶营养健康科技有限公司 | Liquid beverage containing collagen peptide and preparation method and application thereof |
-
2018
- 2018-02-08 CN CN201810130220.4A patent/CN108504715A/en not_active Withdrawn
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117837704A (en) * | 2024-03-08 | 2024-04-09 | 北京衡美金叶营养健康科技有限公司 | Liquid beverage for increasing skin collagen content and preparation method and application thereof |
| CN117837704B (en) * | 2024-03-08 | 2024-06-21 | 北京衡美金叶营养健康科技有限公司 | Liquid beverage containing collagen peptide and preparation method and application thereof |
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Application publication date: 20180907 |