CN108409854A - A kind of small-molecular-weight silkworm chrysalis collagen - Google Patents
A kind of small-molecular-weight silkworm chrysalis collagen Download PDFInfo
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Abstract
The invention discloses a kind of small-molecular-weight silkworm chrysalis collagen, molecular weight is 600 1000Da, and the preparation method of the collagen is：Degreasing agent degreasing is added into dried silkworm chrysalis meal, filters, filter residue drying；Phosphate buffer and neutral proteinase, enzymolysis are added into degreasing silkworm chrysalis, enzyme deactivation centrifuges after cooling, and supernatant is crude protein liquid；Chitosan solution is added into crude protein liquid, stirs, centrifuged supernatant is put into ice-water bath, ammonium sulfate is added, stirring is stood overnight to being completely dissolved, and is centrifuged, sediment is dissolved in phosphate buffer, dialysis, dialyzate is entered in hydroxyapatite cylinder, and buffer solution elution is added, freeze-drying is to get small-molecular-weight silkworm chrysalis collagen.It has the beneficial effect that：Small-molecular-weight silkworm chrysalis collagen purity of the present invention is high, activity is high, molecular weight is more concentrated, and preparation method simple possible, degreasing efficiency are high, yield is high, are easy to industrialized production, and safety higher improves silkworm chrysalis added value.
Description
Technical field
The present invention relates to silkworm chrysalis deep process technology fields, more particularly, to a kind of small-molecular-weight silkworm chrysalis collagen.
Background technology
Collagen or collagen are a kind of biological macromolecules synthesized by zooblast, are widely present in animal
In bone, tendon, cartilage, skin and other connective tissues, has the function of supporting organ, protection body, account for about animal total protein concentration
30%.Type of aquatic in ocean is various, and its form of different aquatic animals and body structure all have huge difference
It is different.As the cephalopodous squid of invertebrate, there is flourishing dermal tissue, internal collagen to be mainly distributed on
In dermal tissue.Collagen molecules structure is very stable, and immunogenicity is low, and biocompatibility is good, these properties determine
Its purposes, collagen purposes is very extensive, and product has various different shapes and purposes, multiple throughout food chemistry medicine etc.
Field, collagen are just becoming the new lover in food medicine and cosmetic industry.Developing and using collagen will be related to
Collagen isolates and purifies work.Purer collagen is only extracted from raw material could carry out follow-up study, extract work
Work is basis.So far, usually there are four types of methods for extraction collagen：That is acid system, salt method, enzyme process and Hot water extraction.
Since ancient times, Sericulture is just seized of critical role in the agricultural of China, and China is in the world earliest and maximum
Sericulture filature country.2014, China's silk cocoon annual output accounted for about the 80% of Gross World Product it is reported that there is 650,000 tons or so.
Silkworm chrysalis is that the by-product after silk cocoon filature is generally often produced the dry pupa of 1 ton of raw silk 0.8 ton of output of meeting, thus silkworm chrysalis yield ten
Divide huge.Modern scientific research finds that silkworm chrysalis is rich in protein, fat, chitin, carbohydrate and multiple protein hormone, dimension life
The bioactive ingredients such as element, trace element have very high nutrition, medical value and vast market prospect.Egg in silkworm chrysalis
Content highest of the white matter in all the components, for water content in the dry silkworm chrysalis of 4-6%, protein content is 50-65% or so, grease
Account for about 23-30%, chitin content is about 2-3%, and rich in carbohydrate, multivitamin, abundant trace element and antibacterial peptide etc.
Bioactive ingredients etc..Thus silkworm chrysalis has a wide range of applications in fields such as food, feed, chemical industry.And due to lacking deep processing
Technology, most of silkworm chrysalis are used for feed and fertilizer, and added value is relatively low always.Silkworm chrysalis passes through at the techniques such as degreasing, deodorization, decoloration
After reason, the pupa albumen of high-purity can be obtained.Pupa albumen amino acid containing there are many, receives extensive pass both domestic and external in recent years
Note, the exploitation of pupa albumen make silkworm chrysalis resource obtain further development and utilization.And prepare collagen also not with silkworm chrysalis
It appears in the newspapers.
Invention content
The purpose of the present invention is to provide a kind of purity, and high, active high, molecular weight is more concentrated, and preparation method simply may be used
Row, degreasing efficiency are high, yield is high, are easy to industrialized production, and safety higher improves a kind of small-molecular-weight silkworm of silkworm chrysalis added value
Pupa collagen.
The present invention in view of the above technology in the problem of mentioning, the technical solution taken is：
A kind of small-molecular-weight silkworm chrysalis collagen, molecular weight 600-1000Da, the collagen is full of nutrition, pupa albumen
Amino acid ratio is excellent, can improve lymphocyte activity, and body's immunity is improved by lymphatic system, enhances the anti-of body
Disease, disease resistance, moreover it is possible to remove the free radical in organism and promote body cell substitution, and with significant antitumor
Activity can be used as nourishing additive agent in bread, noodles, biscuit, flavouring or high protein beverage.
A kind of preparation method of small-molecular-weight silkworm chrysalis collagen, specifically includes following steps：
Degreasing：It is 1 by solid-liquid ratio:4-6（g/mL）Degreasing agent is added into dried silkworm chrysalis meal, then the degreasing 20- at 35-45 DEG C
30min is filtered after cooling, and filter residue is degreasing silkworm chrysalis, is then dried at 50-70 DEG C, spare, which has degreasing
Rate height, solvent noresidue, it is nontoxic nonflammable, protein is indeformable the advantages that, while degreasing agent be easy recycling, degreasing can be reduced
Cost；
Enzymolysis：It is 1 by solid-liquid ratio:20-30（g/mL）The phosphate buffer that pH is 7.5-8.5 is added into degreasing silkworm chrysalis, then adds
The neutral proteinase for entering degreasing silkworm chrysalis weight 2-4%, digests 1-2h at 45-55 DEG C, and temperature is then risen to 90-100 DEG C, protects
Warm 10-20min carries out enzyme deactivation, and 8- is centrifuged in the centrifuge that temperature is 1-5 DEG C, rotating speed is 8000-10000rpm after cooling
10min, supernatant are crude protein liquid, and enzymatic hydrolysis safety higher carries out positioning hydrolysis, hydrolytic process in a mild condition
Also it is easier to control, collagen is mainly enzyme dissolubility, is cut off the non-helical region of collagen molecules with protease
Afterwards, three spiral main regions of collagen are easy to be dissolved in extracting solution, and protease will not be to the triple-helix structure of collagen
It has an impact, extracted amount is very big, and its structure, as the collagen structure of acidity extraction, the collagen is loose and more
Hole, regular uniform cellular porous, this reticulated porous structures are suitable for doing pharmaceutical carrier, not only contribute to the equal of drug
Even distribution also helps the volatilization of liquid；
It saltouts：It is added the chitosan solution of a concentration of 1.8-2.2wt% into crude protein liquid, crude protein liquid and chitosan solution
W/v is 1:4-6, adjust pH to 6.5-7, stir 20-25min, temperature be 1-5 DEG C, rotating speed 8000-
8-10min is centrifuged in the centrifuge of 10000rpm, supernatant is put into ice-water bath, ammonium sulfate, which is added, makes its a concentration of 50-
55wt%, stirring be completely dissolved to ammonium sulfate, stand overnight, then temperature be 1-5 DEG C, rotating speed be 8000-10000rpm from
8-10min is centrifuged in scheming, sediment is salt precipitation object, and a large amount of ammonium sulfate is added in the step, so that protein is obtained molten
Xie Du is reduced and Precipitation, and this method advantage is that ammonium sulfate concentrations variation has continuity, and effect of saltouing is good, saves energy
Consumption；
Dialysis：Salt precipitation object is dissolved in phosphate buffer, is then charged into the bag filter that molecular cut off is 1000, row
Bubble removing, sealing, is placed in phosphate buffer and dialyses, and replaces buffer solution 4-6 times to get dialyzate, this method utilizes small point
Sub- substance in the solution can be by bag filter, and macromolecular substances it is miscellaneous cannot to reach separation small molecule by the property of bag filter
The purpose of matter, easy to operate, effect is good, saves energy consumption；
Purifying：The hydroxyapatite for impregnating 10-12h with buffer solution is fitted into the pillar containing pre-add buffer solution sum, is added saturating
Liquid is analysed, after cylinder, buffer solution elution is added, is freeze-dried to get small-molecular-weight silkworm chrysalis collagen, the collagen egg
It is white full of nutrition, lymphocyte activity can be improved, body's immunity is improved by lymphatic system, enhances the diseases prevention of body, resist
Sick ability, moreover it is possible to remove the free radical in organism and promote body cell substitution, and there is significant antitumor activity.
Preferably, degreasing agent is the mixture of n-hexane and limonene in defatting step, its ratio be 100:2.2-
2.7, the weight ratio of left-handed limonene and d-limonen is 1 in limonene:The degreasing effect of 0.3-0.4, the degreasing agent are good, energy
So that the grease in silkworm chrysalis, rapidly to external diffusion into the surface, within a short period of time in Fast Stripping to degreasing agent, degreasing efficiency is high,
Need not repeat degreasing can achieve the goal, and the addition of limonene can improve saturation degree of the silkworm chrysalis oil in degreasing solvent, into
And the dosage of degreasing agent is reduced, and the rational proportion of left-handed limonene and d-limonen can guarantee that the structure of collagen is not easy
Deformation, and then ensure that its activity is not destroyed.
Preferably, containing 0.03-0.05% sodium citrates and 0.01-0.02% benzene first in enzymolysis step in phosphate buffer
On the one hand the addition of sour sodium, sodium citrate and sodium benzoate can cooperate with the pH of phosphate buffer temperature enzymatic hydrolysis system, can make
Enzyme and silkworm chrysalis compound fast decoupled are obtained, quick release goes out collagen and enzyme, ensures related group on enzyme molecule active site
Stability, improve the reaction rate of reaction system, more enzymes can on the other hand be deep into inside pupa albumen and promotes egg
The hydrolysis of white matter, improves the degree of hydrolysis of pupa albumen, and the molecular weight of the collagen of acquisition is made more to concentrate, and is conducive to collagen egg
White is absorbed and utilized.
Preferably, chromatographic column specification is diameter 2-3cm in purification step, long 48-52cm, it is 6.5- that buffer solution, which selects pH,
7, the KCl buffer solutions of the phosphate buffer of a concentration of 0.008-0.012M and 0.1-0.12M, flow velocity 0.6-0.8mL/min.
Compared with the prior art, the advantages of the present invention are as follows：
1）The purity of small-molecular-weight silkworm chrysalis collagen of the present invention is high, activity is high, molecular weight is more concentrated, and preparation method is simple
It is feasible, degreasing efficiency is high, yield is high, is easy to industrialized production, safety higher greatly improves silkworm chrysalis added value；
2）The collagen that the preparation is amplified is loose and porous, regular uniform cellular porous, this mesh structural porous
Structure is suitable for doing pharmaceutical carrier, not only contributes to being uniformly distributed for drug, also helps the volatilization of liquid；
3）The degreasing effect of present invention degreasing agent is good, and degreasing efficiency is high, and need not repeat degreasing can achieve the goal, and degreasing
The dosage of agent is few, it is ensured that the structure of collagen is unlikely to deform, and then ensures that its activity is not destroyed；
4）The present invention enables to enzyme and silkworm chrysalis compound fast decoupled with phosphate buffer, quick release go out collagen and
Enzyme ensures the stability of related group on enzyme molecule active site, improves the reaction rate of reaction system, while can make more
Enzyme is deep into the hydrolysis for promoting protein inside pupa albumen, and the molecular weight of the collagen of acquisition is made more to concentrate.
Specific implementation mode
The present invention program is described further below by embodiment：
Embodiment 1：
A kind of small-molecular-weight silkworm chrysalis collagen, molecular weight 600-1000Da, the collagen is full of nutrition, pupa albumen
Amino acid ratio is excellent, can improve lymphocyte activity, and body's immunity is improved by lymphatic system, enhances the anti-of body
Disease, disease resistance, moreover it is possible to remove the free radical in organism and promote body cell substitution, and with significant antitumor
Activity can be used as nourishing additive agent in bread, noodles, biscuit, flavouring or high protein beverage.
A kind of preparation method of small-molecular-weight silkworm chrysalis collagen, specifically includes following steps：
1）Degreasing：It is 1 by solid-liquid ratio:6（g/mL）Degreasing agent is added into dried silkworm chrysalis meal, then the degreasing 30min at 35 DEG C, it is cooling
After filter, filter residue is degreasing silkworm chrysalis, is then dried at 50 DEG C, spare, which has that degreasing rate is high, solvent is without residual
The advantages that staying, be nontoxic nonflammable, protein is indeformable, while degreasing agent is easy recycling, can reduce degreasing cost；
2）Enzymolysis：It is 1 by solid-liquid ratio:30（g/mL）The phosphate buffer that pH is 7.5 is added into degreasing silkworm chrysalis, adds degreasing
The neutral proteinase of silkworm chrysalis weight 4% digests 2h at 45 DEG C, temperature is then risen to 90 DEG C, heat preservation 20min carries out enzyme deactivation, cold
But 8min is centrifuged after in the centrifuge that temperature is 1 DEG C, rotating speed is 10000rpm, supernatant is crude protein liquid, enzymatic hydrolysis
Safety higher carries out positioning hydrolysis in a mild condition, and hydrolytic process is also easier to control, and collagen is mainly that enzyme is molten
Property, after being cut off the non-helical region of collagen molecules with protease, three spiral main regions of collagen, which are easy to be dissolved in, to be carried
It takes in liquid, and protease will not have an impact the triple-helix structure of collagen, extracted amount is very big, and its structure and acid
The collagen structure of method extraction is the same, and the collagen is loose and porous, regular uniform cellular porous, this netted
Porous structure is suitable for doing pharmaceutical carrier, not only contributes to being uniformly distributed for drug, also helps the volatilization of liquid；
3）It saltouts：The chitosan solution of a concentration of 2.2wt%, the weight of crude protein liquid and chitosan solution are added into crude protein liquid
It is 1 to measure volume ratio:4, pH to 7 is adjusted, 20min is stirred, is centrifuged in the centrifuge that temperature is 1 DEG C, rotating speed is 10000rpm
Supernatant is put into ice-water bath by 8min, and ammonium sulfate, which is added, makes its a concentration of 55wt%, stirring to ammonium sulfate be completely dissolved, and stands
Overnight, 8min is then centrifuged in the centrifuge that temperature is 1 DEG C, rotating speed is 10000rpm, sediment is salt precipitation object, should
A large amount of ammonium sulfate is added in step, make protein obtain solubility reduce and Precipitation, this method advantage are that ammonium sulfate is dense
Degree variation has continuity, and effect of saltouing is good, saves energy consumption；
4）Dialysis：Salt precipitation object is dissolved in phosphate buffer, is then charged into bag filter, excludes bubble, sealing is placed in phosphorus
It dialyses in phthalate buffer, replaces buffer solution 4 times to get dialyzate, this method can pass through in the solution using small-molecule substance
Bag filter, and macromolecular substances cannot achieve the purpose that detach small molecular weight impurity by the property of bag filter, easy to operate, effect
Fruit is good, saves energy consumption；
5）Purifying：The hydroxyapatite for impregnating 12h with buffer solution is fitted into the pillar containing pre-add buffer solution sum, dialysis is added
Liquid is added buffer solution elution, is freeze-dried to get small-molecular-weight silkworm chrysalis collagen, the collagen after cylinder
It is full of nutrition, lymphocyte activity can be improved, body's immunity is improved by lymphatic system, enhances the diseases prevention, disease-resistant of body
Ability, moreover it is possible to remove the free radical in organism and promote body cell substitution, and there is significant antitumor activity.
Degreasing agent is the mixture of n-hexane and limonene in step 1, and its ratio be 100:2.2, left-handed lemon in limonene
The weight ratio of lemon alkene and d-limonen is 1:0.4, the degreasing effect of the degreasing agent is good, can so that silkworm chrysalis in grease rapidly to
Outer surface is spread, and within a short period of time in Fast Stripping to degreasing agent, degreasing efficiency is high, need not repeat degreasing and can reach
Purpose, and the addition of limonene can improve saturation degree of the silkworm chrysalis oil in degreasing solvent, and then the dosage of degreasing agent is reduced, and it is left
Rotation limonene and d-limonen rational proportion can guarantee collagen structure be unlikely to deform, and then ensure its activity not by
It destroys.
Contain 0.03% sodium citrate and 0.02% sodium benzoate, sodium citrate and benzoic acid in step 2 in phosphate buffer
On the one hand the addition of sodium can cooperate with the pH of phosphate buffer temperature enzymatic hydrolysis system, enzyme is enabled to quickly to divide with silkworm chrysalis compound
Solution, quick release go out collagen and enzyme, ensure the stability of related group on enzyme molecule active site, improve reaction system
On the other hand reaction rate can make more enzymes be deep into the hydrolysis for promoting protein inside pupa albumen, improve pupa albumen
Degree of hydrolysis, and the molecular weight of the collagen of acquisition is made more to concentrate, conducive to being absorbed and utilized for collagen.
Chromatographic column specification is diameter 2cm in step 5, and long 52cm, it is the phosphorus of 6.5, a concentration of 0.012M that buffer solution, which selects pH,
Phthalate buffer and 0.1 KCl buffer solutions, flow velocity 0.8mL/min.
Embodiment 2：
A kind of small-molecular-weight silkworm chrysalis collagen, molecular weight 600-1000Da.
A kind of preparation method of small-molecular-weight silkworm chrysalis collagen, specifically includes following steps：
1）Degreasing：It is 1 by solid-liquid ratio:4（g/mL）Degreasing agent is added into dried silkworm chrysalis meal, then the degreasing 20min at 45 DEG C, it is cooling
After filter, filter residue is degreasing silkworm chrysalis, is then dried at 70 DEG C, spare；
2）Enzymolysis：It is 1 by solid-liquid ratio:20（g/mL）The phosphate buffer that pH is 8.5 is added into degreasing silkworm chrysalis, adds degreasing
The neutral proteinase of silkworm chrysalis weight 2% digests 1h at 55 DEG C, and temperature is then risen to 100 DEG C, and heat preservation 10min carries out enzyme deactivation,
10min is centrifuged after cooling in the centrifuge that temperature is 5 DEG C, rotating speed is 8000rpm, supernatant is crude protein liquid；
3）It saltouts：The chitosan solution of a concentration of 1.8wt%, the weight of crude protein liquid and chitosan solution are added into crude protein liquid
It is 1 to measure volume ratio:6, pH to 6.5 is adjusted, 25min is stirred, is centrifuged in the centrifuge that temperature is 5 DEG C, rotating speed is 8000rpm
Supernatant is put into ice-water bath by 10min, and ammonium sulfate, which is added, makes its a concentration of 50wt%, and stirring to ammonium sulfate is completely dissolved, quiet
It sets overnight, 10min is then centrifuged in the centrifuge that temperature is 5 DEG C, rotating speed is 8000rpm, sediment is salt precipitation object；
4）Dialysis：Salt precipitation object is dissolved in phosphate buffer, is then charged into bag filter, excludes bubble, sealing is placed in phosphorus
It dialyses in phthalate buffer, replaces buffer solution 6 times to get dialyzate；
5）Purifying：The hydroxyapatite for impregnating 10h with buffer solution is fitted into the pillar containing pre-add buffer solution sum, dialysis is added
Liquid is added buffer solution elution, is freeze-dried to get small-molecular-weight silkworm chrysalis collagen after cylinder.
Degreasing agent is the mixture of n-hexane and limonene in step 1, and its ratio be 100:2.7, left-handed lemon in limonene
The weight ratio of lemon alkene and d-limonen is 1:0.3.
Contain 0.05% sodium citrate and 0.01% sodium benzoate in step 2 in phosphate buffer.
Chromatographic column specification is diameter 3cm in step 5, and long 48cm, it is the phosphoric acid of 7, a concentration of 0.008M that buffer solution, which selects pH,
The KCl buffer solutions of salt buffer and 0.12M, flow velocity 0.6mL/min.
Embodiment 3：
A kind of small-molecular-weight silkworm chrysalis collagen, molecular weight 600-1000Da.
A kind of preparation method of small-molecular-weight silkworm chrysalis collagen, specifically includes following steps：
1）Degreasing：It is 1 by solid-liquid ratio:5（g/mL）Degreasing agent is added into dried silkworm chrysalis meal, then the degreasing 25min at 40 DEG C, it is cooling
After filter, filter residue is degreasing silkworm chrysalis, is then dried at 60 DEG C, spare；
2）Enzymolysis：It is 1 by solid-liquid ratio:25（g/mL）The phosphate buffer that pH is 8.0 is added into degreasing silkworm chrysalis, adds degreasing
The neutral proteinase of silkworm chrysalis weight 3% digests 1.5h at 50 DEG C, and temperature is then risen to 95 DEG C, and heat preservation 15min carries out enzyme deactivation,
9min is centrifuged after cooling in the centrifuge that temperature is 2 DEG C, rotating speed is 9000rpm, supernatant is crude protein liquid；
3）It saltouts：The chitosan solution of a concentration of 2.0wt%, the weight of crude protein liquid and chitosan solution are added into crude protein liquid
It is 1 to measure volume ratio:5, adjust pH to 6.8, stir 20-25min, in the centrifuge that temperature is 2 DEG C, rotating speed is 9000rpm from
Heart 9min, supernatant is put into ice-water bath, and ammonium sulfate, which is added, makes its a concentration of 52wt%, and stirring to ammonium sulfate is completely dissolved, quiet
It sets overnight, 9min is then centrifuged in the centrifuge that temperature is 2 DEG C, rotating speed is 9000rpm, sediment is salt precipitation object；
4）Dialysis：Salt precipitation object is dissolved in phosphate buffer, is then charged into bag filter, excludes bubble, sealing is placed in phosphorus
It dialyses in phthalate buffer, replaces buffer solution 5 times to get dialyzate；
5）Purifying：The hydroxyapatite for impregnating 11h with buffer solution is fitted into the pillar containing pre-add buffer solution sum, dialysis is added
Liquid is added buffer solution elution, is freeze-dried to get small-molecular-weight silkworm chrysalis collagen after cylinder.
Degreasing agent is the mixture of n-hexane and limonene in step 1, and its ratio be 100:2.5, left-handed lemon in limonene
The weight ratio of lemon alkene and d-limonen is 1:0.35.
Contain 0.04% sodium citrate and 0.015% sodium benzoate in step 2 in phosphate buffer.
Chromatographic column specification is diameter 2.5cm in step 5, and long 50cm, it is the phosphorus of 6.8, a concentration of 0.01M that buffer solution, which selects pH,
The KCl buffer solutions of phthalate buffer and 0.11M, flow velocity 0.7mL/min.
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 (8)
1. a kind of small-molecular-weight silkworm chrysalis collagen, it is characterised in that：The molecular weight of the silkworm chrysalis collagen is 600-
1000Da。
2. a kind of small-molecular-weight silkworm chrysalis collagen according to claim 1, it is characterised in that：The system of the collagen
Preparation Method specifically includes following steps：
1）Degreasing：The degreasing agent of mixing n-hexane and limonene is added into dried silkworm chrysalis meal, degreasing filters after cooling, and filter residue is
Degreasing silkworm chrysalis, drying；
2）Enzymolysis：Phosphate buffer and neutral proteinase, enzymolysis are added into degreasing silkworm chrysalis, enzyme deactivation centrifuges, supernatant after cooling
As crude protein liquid；
3）It saltouts：Chitosan solution is added into crude protein liquid, adjusts pH to 6.5-7, stirs 20-25min, centrifugation, by supernatant
Liquid is put into ice-water bath, and ammonium sulfate is added, and stirring is completely dissolved to ammonium sulfate, stands overnight, and centrifuges, and it is heavy that sediment is as saltoutd
Starch；
4）Dialysis：Salt precipitation object is dissolved in phosphate buffer, is then charged into the bag filter that molecular cut off is 1000,
Bubble is excluded, sealing is placed in phosphate buffer and dialyses, and replaces buffer solution 4-6 times to get dialyzate；
5）Purifying：The hydroxyapatite for impregnating 10-12h with buffer solution is fitted into the pillar containing pre-add buffer solution sum, is added
Dialyzate is added buffer solution elution, is freeze-dried to get small-molecular-weight silkworm chrysalis collagen after cylinder.
3. a kind of preparation method of small-molecular-weight silkworm chrysalis collagen according to claim 2, it is characterised in that：The step
The solid-liquid ratio of dried silkworm chrysalis meal and degreasing agent is 1 in rapid 1:4-6（g/mL）, skimming temp is 35-45 DEG C, time 20-30min.
4. a kind of preparation method of small-molecular-weight silkworm chrysalis collagen according to claim 2, it is characterised in that：The step
The ratio of n-hexane and limonene is 100 in degreasing agent in rapid 1:2.2-2.7, left-handed limonene and dextrorotation lemon in the limonene
The weight ratio of lemon alkene is 1:0.3-0.4.
5. a kind of preparation method of small-molecular-weight silkworm chrysalis collagen according to claim 2, it is characterised in that：The step
The solid-liquid ratio of degreasing silkworm chrysalis and phosphate buffer is 1 in rapid 2:20-30（g/mL）, the pH of the phosphate buffer is 7.5-8.5,
Contain 0.03-0.05% sodium citrates and 0.01-0.02% sodium benzoates in phosphate buffer.
6. a kind of preparation method of small-molecular-weight silkworm chrysalis collagen according to claim 2, it is characterised in that：The step
The additive amount of neutral proteinase is the 2-4% of degreasing silkworm chrysalis weight in rapid 2, and hydrolysis temperature is 45-55 DEG C, time 1-2h.
7. a kind of preparation method of small-molecular-weight silkworm chrysalis collagen according to claim 2, it is characterised in that：The step
The w/v of crude protein liquid and chitosan solution is 1 in rapid 3:4-6, a concentration of 1.8-2.2wt% of chitosan solution,
It is 50-55wt% that the ammonium sulfate, which is added to solution concentration,.
8. a kind of preparation method of small-molecular-weight silkworm chrysalis collagen according to claim 2, it is characterised in that：The layer
It is diameter 2-3cm, long 48-52cm to analyse column specification, and it is the phosphorus of 6.5-7, a concentration of 0.008-0.012M that the buffer solution, which selects pH,
The KCl buffer solutions of phthalate buffer and 0.1-0.12M, flow velocity 0.6-0.8mL/min.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810138024.1A CN108409854A (en) | 2018-02-10 | 2018-02-10 | A kind of small-molecular-weight silkworm chrysalis collagen |
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| CN201810138024.1A CN108409854A (en) | 2018-02-10 | 2018-02-10 | A kind of small-molecular-weight silkworm chrysalis collagen |
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