CN113150307B - Glycosylated sericin, preparation method and application thereof - Google Patents
Glycosylated sericin, preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
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- Materials Engineering (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
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Abstract
The invention discloses a glycosylated sericin protein, a preparation method and application thereof. Placing a silk raw material in a calcium hydroxide aqueous solution with the mass concentration of 0.01-0.1%, boiling/ultrasonically boiling in water to obtain a sericin alkaline solution, and adjusting the pH value to 9.0-11.0; mixing 1-20% sericin aqueous solution and reducing sugar aqueous solution according to the mass ratio of sericin to reducing sugar of 1.1-0.6, and reacting to obtain an ochre-colored glycosylated sericin product. The method for preparing the glycosylated sericin has the advantages of convenient operation, green and environment-friendly process, high reaction efficiency, effective improvement of the functions of water solubility, emulsibility, oxidation resistance and the like of the graft, change of the light odor of the original sericin, and sweet fragrance. The glycosylated sericin powder provided by the invention has industrial and large-scale application prospects in the fields of healthy food additives, auxiliary hypoglycemic foods, biological materials and the like.
Description
Technical Field
The invention relates to novel glycosylated sericin and a grafting method thereof, in particular to glycosylated sericin, a preparation method and application thereof, belonging to the technical field of sericin modification.
Background
The silk mulberry silk industry is always the traditional industry in China, and relates to conventional processing of raw silk, silk blank, silk wadding quilt, silk spinning and the like, or in the preparation process of silk fibroin in the processing process of modern silk biological materials, a large amount of silk fibroin can be discharged as industrial waste along with alkaline substances such as degumming agents, scouring agents and the like, so that the environment is seriously polluted, and a large amount of biological protein resources are wasted. Sericin is a mixture of a plurality of globular proteins with different molecular weights, and plays a role in adhesion in silk fibers. Sericin, which is not degraded seriously in general, is not easily soluble in cold water, and is easily soluble in hot water, particularly, very alkali hot water. Common sericin solvents comprise aqueous solutions of alkaline substances such as sodium carbonate and sodium bicarbonate, and strong bases such as sodium hydroxide which are widely applied in the processing of silk cotton quilt, and the recovery and utilization of sericin in the degumming waste liquid are difficult due to the existence of a large amount of metal ions. Another important problem is that the recovered sericin, especially sericin which is not easily dissolved in cold water and has a large molecular weight distribution range, has a light silkworm pupa odor and is not acceptable when being directly mixed into common foods or functional foods. Therefore, there are several difficulties in the recovery and utilization of sericin, which involve barriers to technology, cost, function, and market.
Of proteins
The covalent bonding of the amino groups to the reducing carbonyl end of the polysaccharide, the so-called Maillard reaction, is a major factor affecting the structure and quality of the protein and allows the obtainment of glycosylated grafts of proteins with superior functional properties. Not only retains the surface activity of protein, but also has the hydrophilic and water-holding properties of polysaccharide. Compared with the mixture formed by weak interaction of protein and polysaccharide, the glycosylated protein has higher adaptability to environmental conditions such as temperature, pH value, ionic strength and the like. Common proteins are easy to aggregate and denature when meeting higher temperature, so that the grafting reaction in a higher-temperature water phase system is difficult to realize. Taking the silk fibroin commonly used in the research and development of common nutritional food egg white and biological materials as an example, the egg white is easy to be gelatinized at the temperature higher than 50 ℃ to regenerate the silk fibroinIt is easy to be denatured by denaturation due to the formation of "b", and is not favorable for the maillard reaction between protein and reducing sugar. Thus, to date, it has not been possible to effectively carry out the Maillard reaction of polysaccharides and proteins in aqueous solutions, particularly at high temperatures.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a novel sericin glycosylation and preparation method and application thereof, and the technical scheme of the invention has the advantages of green and environment-friendly processing technology, convenient operation, stable performance, no peculiar smell of the obtained product, easy dissolution in cold water, antibacterial, antioxidant, in vitro and in vivo blood sugar reducing and other functions, overcomes the defect that the industrial utilization of the conventional recovered sericin is difficult to realize, and has good industrial, large-scale, functional and commercialized application prospects.
The technical scheme for realizing the aim of the invention is to provide a preparation method of glycosylated sericin, which comprises the following steps:
(1) Placing a silk raw material in a calcium hydroxide aqueous solution with the mass concentration of 0.01-0.1%, boiling/ultrasonically boiling to hydrolyze sericin, filtering or centrifuging the obtained sericin dissolved solution, and removing impurities to obtain a sericin alkaline solution; adjusting the pH value to 9.0-11.0, and then filtering or centrifuging to obtain a sericin aqueous solution with the mass concentration of 1-20%;
(2) Mixing a reducing sugar aqueous solution with the mass concentration of 1-10% and a sericin aqueous solution according to the mass ratio of sericin to reducing sugar of 1: 0.1-0.6, and stirring or shaking for reaction for 3-10 h at the temperature of 90-100 ℃ to obtain a ochre-colored glycosylated sericin protein product.
The silk raw materials comprise silk fibers, silkworm cocoon shells, raw silk, silk floss pieces, spun silk and blank silk.
The invention provides a preparation method of glycosylated sericin, which has the preferable technical scheme that:
in the step (1), the treatment time of boiling/ultrasonic boiling hydrolysis sericin is 0.5-2.0 h; the pH is adjusted to 10.0 with an acid or base.
In the step (2), the mass ratio of sericin to reducing sugar is 1:0.3, and the mass concentration of a reducing sugar aqueous solution is 2.4%; the reaction temperature is 95 ℃; the reaction time was 6h.
And (3) performing spray drying or freeze drying on the product obtained in the step (2) to prepare glycosylated sericin powder.
The technical scheme of the invention comprises the glycosylated sericin obtained by the preparation method.
The technical scheme of the invention also comprises the application of the glycosylated sericin protein in the surface modification of food additives, medical dressings, daily chemical products, cell culture substrates and materials.
The specific application comprises the following steps: the food additive is an auxiliary hypoglycemic additive; the medical dressing is a wound healing dressing; the daily chemical product is a cosmetic.
The invention uses the water solution of calcium hydroxide as an environment-friendly sericin alkaline dissolving agent, and is applied to the dissolution of sericin in the degumming or refining of a series of silks such as the refining of cocoon shells, raw silks and silk floss sheets, the refining of silk spinning, the refining of silk blank materials and the like. The sericin alkaline solution provided by the invention can neutralize redundant calcium ions and precipitate in a salt form by adjusting the pH to about 10, thereby achieving the purpose of separating sericin. The process not only recovers sericin, but also recovers precipitate calcium salt, thereby avoiding environmental pollution caused by a large amount of silk degumming alkaline waste liquid.
Compared with the prior art, the invention has the beneficial effects that:
1. the technical scheme provided by the invention solves the problem that the outer layer water-soluble sericin extracted by water boiling and the sericin prepared by a high-temperature high-pressure water boiling method in the prior art are difficult to carry out glycosylation reaction, and the obtained glycosylated sericin product overcomes the defects that protofilament sericin needs to be dissolved by hot water and the original silkworm pupae has light odor; meanwhile, the antibacterial property, the antioxidant capacity, the whitening capacity and the in-vitro glycosidase inhibiting capacity of the composition are obviously improved, and the composition has a remarkable blood sugar reducing function when being orally taken by diabetic rats.
2. The glycosylated sericin powder provided by the invention has a simple preparation process, is green and environment-friendly, and has wide application potential in the aspects of industrial product surface modification materials, cell culture matrixes, high-grade cosmetics or daily chemical products, health foods, auxiliary blood sugar lowering functional foods and the like.
Drawings
FIG. 1 shows UV-VIS absorption spectra of reaction products of sericin and seven reducing sugars, respectively, according to an embodiment of the present invention;
FIG. 2 is a histogram of absorbance values for seven reducing sugars provided by the examples of the present invention;
FIG. 3 is a bar graph of absorbance versus Maillard reaction of sericin with arabinose (Ara), glucose Glc) and xylose (Xyl) using different reaction temperatures in examples of the present invention;
FIG. 4 is a bar graph of absorbance versus Maillard reaction using different mass ratios of sericin and reducing sugar in the examples of the present invention;
FIG. 5 is a graph showing the comparison of absorbance of products obtained by using different reaction times of sericin and reducing sugar according to an embodiment of the present invention;
FIG. 6 is a graph showing the comparison of absorbance of products obtained by using different concentrations of sericin and reducing sugar according to an example of the present invention;
FIG. 7 is a bar graph of the absorbance comparison of Maillard reaction products of three sericin proteins and seven reducing sugars, as provided in the example of the invention.
Detailed Description
Example 1
The embodiment provides a method for synthesizing glycosylated sericin, which comprises the following steps:
(1) Soaking clean silkworm cocoon shell or other silk material in 0.05% Ca (OH) 2 Ultrasonically boiling for 20 min' 2 in the aqueous solution;
(2) Taking out the degummed silk fibroin for 2 times, combining the sericin alkaline solutions for 2 times, and filtering or centrifuging to remove calcium salt precipitates;
(3) Adding reducing sugar into the sericin alkaline filtrate or supernatant, and stirring at 95 ℃ for reaction for 6h to obtain a brown reaction product, namely a glycosylated sericin protein solution;
(4) Spray drying or freeze drying to obtain glycosylated silk protein powder.
The glycosylated sericin protein product prepared by the method can be applied to preparation of modified protein or food additives, preparation of food products for assisting blood sugar reduction, preparation of wound healing dressing products and the like, and can also be widely applied to the aspects of industrial product surface modification materials, cell culture substrates, cosmetics, daily chemical products and the like.
Example 2
Calcium hydroxide is prepared into water solution with the concentration of 0.01-0.10%, and 10.0 g of silkworm cocoon shell is prepared by mixing the following components in a ratio of 1:60 (g/mL) bath ratio is respectively immersed in calcium hydroxide aqueous solution, boiling or ultrasonic treatment is respectively carried out for 10-30 min, the calcium hydroxide aqueous solution with the same concentration is boiled repeatedly or once by ultrasonic boiling, silk with sericin dissolved away is taken out, namely fibroin fiber, then sericin alkaline aqueous solution is combined for 2 times, the pH is adjusted to about 10, after standing for several hours or overnight, precipitate is removed by filtration or centrifugation, concentration or rotary concentration is carried out until 1-20% sericin aqueous solution is placed at 4 ℃ for standby, or sericin powder is prepared by direct spray drying or freeze drying and placed at 4 ℃ for standby. The sericin sample provided in this example was whole sericin (denoted as SS) degraded by an aqueous calcium hydroxide solution.
Adding reducing sugar such as glucose or xylose with the mass ratio of 1.1-0.6 into a sericin alkaline aqueous solution with the concentration of 1-20% (pH = 10) and most suitable for 8% sericin alkaline aqueous solution, and placing the solution in a shaking table at the temperature of 90-100 ℃ for reaction or in an environment at the temperature of 90-100 ℃ for stirring and reacting for 3-10 h; after reaction for 6h, the mixture is taken out and filtered or centrifuged to remove impurities, and then the glycosylated sericin aqueous solution is obtained and is placed at 4 ℃ for standby, or sericin powder is prepared by direct spray drying or freeze drying and is placed at 4 ℃ for standby.
Whole sericin (SS) prepared in this example was subjected to Maillard reaction with 7 reducing sugars, arabinose (Ara), fructose (Fru), galactose (Gal), glucose (Glc), mannose (Man), rhamnose (Rha) and xylose (Xyl), respectively.
According to the technical scheme of the embodiment, 2.0 percent sericin water solution and 0.6 percent reducing sugar water are mixedPreparing 5 mL of reaction solution by weight ratio of 1:0.3, placing the reaction solution at 90 ℃ for continuous reaction for 6h, cooling, centrifuging the reaction solution, taking supernatant fluid to dilute by 10 times, placing the supernatant fluid in a Hitachi 200-20 type ultraviolet-visible spectrophotometer to perform spectral scanning and measuring absorbance value at 320 nm on an MD-5 microplate reader,n = 3。referring to FIG. 1, there are provided UV-VIS absorption spectra of reaction products of sericin and 7 reducing sugars, respectively, in this example; the absorbance values at 320 nm for 7 sericin-reducing sugar reaction products (the absorbance value at 320 nm for blank samples sericin has been subtracted).
Referring to fig. 2, a bar graph of absorbance values of 7 reducing sugar reaction products of sericin (SS) with arabinose (Ara), fructose (Fru), galactose (Gal), glucose (Glc), mannose (Man), rhamnose (Rha) and xylose (Xyl) is provided in this example.
Example 3
In this example, different reaction temperatures were used for Maillard reaction of sericin with arabinose (Ara), glucose Glc) and xylose (Xyl) under the following conditions: example 2 the sericin aqueous solution with a concentration of 2.0% and the reducing sugar aqueous solution with a concentration of 0.6% were prepared into 5 mL of a reaction solution at a weight ratio of 1.3, placed at different temperatures (see the abscissa "reaction temperature" in FIG. 3, 80 ℃, 85 ℃, 90 ℃, 95 ℃ and 100 ℃ respectively) and shaken for continuous reaction for 6 hours, the reaction solution was centrifuged after cooling, the supernatant was diluted 10 times and the absorbance value at 320 nm was measured,n = 3。
referring to FIG. 3, a bar graph of absorbance versus Maillard reaction of sericin with arabinose (Ara), glucose Glc) and xylose (Xyl) using different reaction temperatures is shown for this example.
Example 4
In this example, different mass ratios of sericin, arabinose (Ara), glucose (Glc) and xylose (Xyl) were used for Maillard reaction under the following conditions: example 2 provides a 2.0% aqueous sericin solution and a 0.6% aqueous reducing sugar solution, each sericin as represented by the abscissa of the accompanying figure 4: mixing reducing sugar (W: W) at different ratio, placing at 90 deg.C, shaking for continuous reaction for 6 hr, cooling, centrifuging, collecting supernatantMeasuring the absorbance value at 320 nm after diluting by 10 times,n = 3。
referring to FIG. 4, a bar graph of absorbance versus reducing sugar ratio for the Maillard reaction using different mass ratios of sericin and reducing sugar is shown in this example.
Example 5
In this example, different reaction times were used for the maillard reaction between sericin and reducing sugar, and the reaction conditions were as follows: the sericin aqueous solution with the concentration of 2.0% provided in example 2 is respectively prepared into 5 mL of reaction solution with 0.6% reducing sugar arabinose (Ara), glucose (Glc) and xylose (Xyl) aqueous solution according to the weight ratio of 1:0.3, the reaction solution is placed at 90 ℃ for shaking and continuous reaction for 0-48 h, the reaction solution is centrifuged after cooling, the supernatant is diluted by 10 times, and the absorbance value at 320 nm is measured,n = 3.
referring to FIG. 5, a graph comparing the absorbance of products obtained using different reaction times of sericin and reducing sugar is provided for the example.
Example 6
In this example, aqueous solutions of sericin and reducing sugar (mass ratio 1.
Referring to FIG. 6, the absorbance contrast plots for the products obtained using different concentrations of sericin and reducing sugar provided for this example; preparing reaction solution with a total volume of 5 mL according to the concentration ratio of sericin to three reducing sugars in the abscissa of the graph, placing the reaction solution at 90 ℃ for shaking continuous reaction for 6 hours, centrifuging the reaction solution after cooling, taking supernatant, diluting the supernatant by 10 times, measuring the absorbance value at 320 nm,n = 3。
example 7
Soaking clean silkworm cocoon shells in hot water with a volume ratio of 1: 50 overnight to swell cocoon shells, then boiling for 60 min, taking out degumming silks, changing to the same volume of pure water, repeatedly boiling for 60 min, combining silk water boiling solution for 2 times, concentrating filtered or centrifuged sericin aqueous solution to a concentration range of 1-10%, namely outer-layer sericin (marked as OLS), and placing at 4 ℃ for later use, or directly preparing outer-layer sericin powder by spray drying or freeze drying and placing at 4 ℃ for later use.
The method provided by the embodiment comprises the steps of boiling off silk fibers of the outer sericin layer by 2 times of water, immersing the silk fibers in purified water with the volume ratio of 1: 50, placing the silk fibers in a high-temperature high-pressure cooker at 120 ℃ for heat treatment for 1 to 2 hours, filtering or centrifuging the sericin solution except the silk fibers, concentrating the sericin aqueous solution to the concentration range of 1 to 10 percent to obtain inner sericin (marked as ILS), placing the inner sericin in 4 ℃ for later use, or directly carrying out spray drying or freeze drying to prepare inner sericin powder and placing the inner sericin powder in 4 ℃ for later use.
The outer sericin (OLS) provided in this example, the inner sericin (ILS) and the whole sericin (SS) provided in example 2 were subjected to maillard reaction with 7 reducing sugars, arabinose (Ara), fructose (Fru), galactose (Gal), glucose (Glc), mannose (Man), rhamnose (Rha) and xylose (Xyl), respectively. The specific method comprises the following steps: after mixing OLS, ILS and SS sericin aqueous solutions with the concentration of 2.0% and seven reducing sugars respectively according to the mass ratio of 1.
Referring to fig. 7, a bar graph comparing the absorbance of Maillard reaction products of three sericin proteins and seven reducing sugars; in the figure, OLS is outer sericin recovered from silk fiber after water boiling; ILS is inner-layer sericin obtained by boiling silk fiber subjected to degumming in water and removal of outer-layer sericin for 2 hours in high-temperature high-pressure water; SS is the full sericin which is dissolved and degraded by the calcium hydroxide aqueous solution by adopting the technical scheme of the invention,n = 3。
the result of this example shows that the conventional method is used to carry out Maillard grafting reaction on the water-soluble outer-layer sericin obtained by boiling silk in water and the inner-layer sericin dissolved at high temperature and high pressure water (120 ℃), and the glycosylation efficiency is very low. By adopting the technical scheme provided by the invention, the glycosylation efficiency of the sericin is effectively improved, the obtained glycosylated sericin overcomes the defects that protocollagen needs to be dissolved in hot water and silkworm pupae has light odor, can be widely applied to the fields of food additives, medical dressings, daily chemical products, cell culture matrixes, surface modification of materials and the like, and particularly has wide application in the aspects of assisting blood sugar reduction, wound healing dressings, cosmetics and the like.
Claims (8)
1. A preparation method of glycosylated sericin is characterized by comprising the following steps:
(1) Placing a silk raw material in a calcium hydroxide aqueous solution with the mass concentration of 0.01-0.1%, and ultrasonically boiling to hydrolyze sericin for 0.5-2.0 h; filtering or centrifuging the obtained sericin dissolving solution to remove impurities to obtain a sericin alkaline solution; adjusting the pH value to 9.0-11.0, and then filtering or centrifuging to obtain a sericin aqueous solution with the mass concentration of 1-20%;
(2) Mixing a reducing sugar aqueous solution with the mass concentration of 1-10% with a sericin aqueous solution according to the mass ratio of sericin to reducing sugar of 1.1-0.6 and the reducing sugar being xylose, and stirring or shaking for reaction for 3-10 h at the temperature of 90-100 ℃ to obtain a ochre-colored glycosylated sericin protein product.
2. The method according to claim 1, wherein the glycosylated sericin is prepared by the method comprising the steps of: the silk raw material comprises silk fiber, silkworm cocoon shell, raw silk, silk cotton sheet, silk fabric and blank silk.
3. The method according to claim 1, wherein the glycosylated sericin is prepared by the method comprising the steps of: in the step (1), the pH is adjusted to 10.0 with an acid or a base.
4. The method according to claim 1, wherein the glycosylated sericin is prepared by the method comprising the steps of: in the step (2), the mass ratio of sericin to reducing sugar is 1.3, and the mass concentration of the reducing sugar aqueous solution is 2.4%; the reaction temperature is 95 ℃; the reaction time was 6h.
5. The method for producing glycosylated sericin according to claim 1, wherein: and (3) performing spray drying or freeze drying on the product obtained in the step (2) to prepare glycosylated sericin powder.
6. A glycosylated sericin obtained by the production method according to claim 1.
7. The use of a glycosylated sericin according to claim 6, wherein: it can be used for surface modification of food additive, medical dressing, daily chemical product, cell culture matrix and material.
8. The use of a glycosylated sericin according to claim 7, wherein: the food additive is an auxiliary hypoglycemic additive; the medical dressing is a wound healing dressing; the daily chemical product is a cosmetic.
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