CN113150307B - Glycosylated sericin, preparation method and application thereof - Google Patents
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Abstract
本发明公开了一种糖基化丝胶蛋白、制备方法及其应用。将蚕丝原材料置于质量浓度为0.01~0.1%的氢氧化钙水溶液中,煮沸/超声煮沸水处理得到丝胶碱性溶液,调节pH至9.0~11.0;按丝胶与还原糖的质量比为1:0.1~0.6,将质量浓度为1~20%的丝胶水溶液与还原糖水溶液混合,反应后得到一种赭石色的糖基化丝胶蛋白产物。本发明制备糖基化丝胶蛋白的方法操作方便,工艺绿色环保,反应效率高,接枝物的水溶性、乳化性、抗氧化性等功能得到有效提高,且改变了原有丝胶蛋白的淡臭味,具有甜香味。本发明提供的糖基化丝胶粉末在健康食品添加剂、辅助降血糖食品以及生物材料等领域,具有工业化、规模化的应用前景。
The invention discloses a glycosylated sericin, a preparation method and an application thereof. Put the silk raw material in an aqueous calcium hydroxide solution with a mass concentration of 0.01-0.1%, boil/ultrasonic boil water to obtain a sericin alkaline solution, and adjust the pH to 9.0-11.0; the mass ratio of sericin to reducing sugar is 1 : 0.1 to 0.6, mix the sericin aqueous solution with a mass concentration of 1 to 20% and the reducing sugar aqueous solution to obtain an ocher-colored glycosylated sericin product after the reaction. The method for preparing glycosylated sericin in the present invention is easy to operate, the process is green and environmentally friendly, and the reaction efficiency is high. Light odor, with a sweet fragrance. The glycosylated sericin powder provided by the invention has industrialized and large-scale application prospects in the fields of health food additives, auxiliary hypoglycemic foods, biological materials and the like.
Description
技术领域technical field
本发明涉及一种新型的糖基化丝胶蛋白及其接枝方法,特别涉及一种糖基化丝胶蛋白、制备方法及其应用,属于丝胶蛋白改性技术领域。The invention relates to a novel glycosylated sericin protein and a grafting method thereof, in particular to a glycosylated sericin protein, a preparation method and an application thereof, and belongs to the technical field of sericin protein modification.
背景技术Background technique
蚕桑丝绸产业一直是我国的传统产业,有关生丝、丝绸坯料、丝绵被、绢纺等常规加工,或者是现代蚕丝生物材料的加工过程中丝蛋白的制备过程中,大量的丝胶蛋白会随着脱胶剂、精练剂等碱性物质作为工业废弃物一起排放,对环境产生严重污染,也造成大量生物蛋白资源的浪费。丝胶是一类分子量大小不一的多种球状蛋白的混合物,在蚕丝纤维中起到的胶粘作用。一般情况下降解不严重的丝胶蛋白不易溶于冷水,易溶于热水特别是非常易溶于碱性热水中。常见的丝胶溶剂有碳酸钠、碳酸氢钠等碱性物质的水溶液,丝棉被加工中大量应用的氢氧化钠等强碱,这些脱胶废液中丝胶蛋白的回收和利用因大量金属离子的存在而变得困难重重。另一个重要问题是回收的丝胶蛋白,特别是冷水中不易溶解的分子量分布范围较大的丝胶蛋白有一股淡淡的蚕蛹臭味,直接混合到常见食品或功能食品中比较难以接受。所以,丝胶蛋白的回收与利用面临多个方面的难题,它涉及到技术、成本、功能和市场等多个方面的壁垒。The sericulture and silk industry has always been a traditional industry in my country. During the conventional processing of raw silk, silk blanks, silk quilts, and silk spinning, or the preparation of silk proteins in the processing of modern silk biomaterials, a large amount of sericin will be produced along with the Alkaline substances such as degumming agent and scouring agent are discharged together as industrial waste, causing serious pollution to the environment and also causing a waste of a large amount of biological protein resources. Sericin is a mixture of various globular proteins with different molecular weights, which plays an adhesive role in silk fibers. In general, sericin, which is not seriously degraded, is not easily soluble in cold water, but easily soluble in hot water, especially in alkaline hot water. Common sericin solvents include aqueous solutions of alkaline substances such as sodium carbonate and sodium bicarbonate, and strong alkalis such as sodium hydroxide that are widely used in silk quilt processing. The recovery and utilization of sericin in these degumming waste liquids is due to the large amount of metal ions existence makes it difficult. Another important problem is that recovered sericin, especially sericin which is not easily soluble in cold water and has a large molecular weight distribution range, has a faint smell of silkworm chrysalis, and it is unacceptable to mix it directly into common foods or functional foods. Therefore, the recovery and utilization of sericin face many difficulties, which involve barriers in technology, cost, function and market.
蛋白质的-氨基与多糖的还原性羰基末端共价结合即所谓的Maillard反应,是影响蛋白质结构和品质的主要因素,可以得到具有优越功能性质的蛋白质糖基化接枝物。既保留了蛋白质的表面活性又具有多糖亲水和持水的性能。与蛋白质多糖弱相互作用形成的混合物对比,糖基化蛋白对环境条件如温度、pH值、离子强度等具有较高的适应性。常见蛋白质遇较高的温度就易聚集变性,所以在较高温度的水相体系中进行接枝反应难以实现。以普通营养食品蛋清和生物材料研发中常用的丝素蛋白为例,高于50℃蛋清易发生胶凝化而再生丝素蛋白易发生b化而变性,不利于蛋白质与还原糖的美德拉反应。因此,到目前为至,始终无法使多糖与蛋白的Maillard 反应在水溶液中特别是高温水溶液中有效进行。protein - The covalent combination of the amino group and the reducing carbonyl end of the polysaccharide is the so-called Maillard reaction, which is the main factor affecting the structure and quality of the protein, and the protein glycosylation graft with superior functional properties can be obtained. It not only retains the surface activity of the protein but also has the hydrophilic and water-holding properties of polysaccharides. Compared with the mixture formed by the weak interaction of proteoglycans, glycosylated proteins have higher adaptability to environmental conditions such as temperature, pH value, and ionic strength. Common proteins tend to aggregate and denature at higher temperatures, so it is difficult to carry out grafting reactions in higher temperature aqueous phase systems. Take the egg white of ordinary nutritional food and the silk fibroin commonly used in the research and development of biomaterials as an example. The egg white tends to gelatinize and the regenerated silk fibroin tends to be b-formed and denatured when the temperature is higher than 50°C, which is not conducive to the Medela reaction between protein and reducing sugar. . Therefore, until now, the Maillard reaction between polysaccharides and proteins cannot be effectively carried out in aqueous solution, especially in high-temperature aqueous solution.
发明内容Contents of the invention
本发明针对现有技术存在的不足,提供一种新型的丝胶蛋白糖基化、制备方法及其应用,本发明技术方案具有加工工艺绿色环保、操作方便、性能稳定、所得产物无异味、冷水易溶,具有抗菌、抗氧化、体外和体内降血糖等功能,克服了现有回收的丝胶蛋白难以实现工业化利用的缺陷,产品具有良好的工业化、规模化、功能化和商品化的应用前景。The present invention aims at the deficiencies in the prior art, and provides a novel sericin protein glycosylation, preparation method and application thereof. The technical scheme of the present invention has the advantages of green and environmental protection processing technology, convenient operation, stable performance, no peculiar smell of the obtained product, cold water It is easy to dissolve and has the functions of antibacterial, anti-oxidation, hypoglycemic in vitro and in vivo, etc. It overcomes the defect that the existing recovered sericin protein is difficult to realize industrial utilization, and the product has good application prospects of industrialization, scale, function and commercialization .
实现本发明目的的技术方案是提供一种糖基化丝胶蛋白的制备方法,包括如下步骤:The technical solution for realizing the purpose of the present invention is to provide a method for preparing glycosylated sericin, comprising the following steps:
(1)将蚕丝原材料置于质量浓度为0.01~0.1%的氢氧化钙水溶液中,煮沸/超声煮沸水解丝胶蛋白,得到的丝胶溶解液经过滤或离心处理,去除杂质,获得丝胶碱性溶液;调节pH至9.0 ~11.0,再经过滤或离心处理,得到质量浓度为1~20%的丝胶水溶液;(1) Put silk raw materials in calcium hydroxide aqueous solution with a mass concentration of 0.01-0.1%, boil/ultrasonic boil to hydrolyze sericin protein, and filter or centrifuge the obtained sericin solution to remove impurities and obtain sericin base neutral solution; adjust the pH to 9.0-11.0, and then filter or centrifuge to obtain a sericin aqueous solution with a mass concentration of 1-20%;
(2)按丝胶与还原糖的质量比为1: 0.1~0.6,将质量浓度为1~10%的还原糖水溶液与丝胶水溶液混合,在温度为 90~100℃的条件下搅动或摇动反应3~10 h,得到一种赭石色的糖基化丝胶蛋白产物。(2) According to the mass ratio of sericin and reducing sugar as 1: 0.1 to 0.6, mix the aqueous solution of reducing sugar with a mass concentration of 1 to 10% and the aqueous solution of sericin, and stir or shake at a temperature of 90 to 100°C After reacting for 3-10 h, an ocher-colored glycosylated sericin product was obtained.
本发明所述的蚕丝原材料包括蚕丝纤维、蚕茧壳、生丝、丝绵片、绢纺及坯绸。The silk raw materials described in the present invention include silk fibers, cocoon shells, raw silk, silk floss sheets, spun silk and gray silk.
本发明提供的一种糖基化丝胶蛋白的制备方法,优选的技术方案是:A kind of preparation method of glycosylated sericin provided by the present invention, the preferred technical scheme is:
步骤(1)中,煮沸/超声煮沸水解丝胶蛋白的处理时间为0.5~2.0 h;用酸或碱调节pH为10.0。In step (1), the treatment time of boiling/ultrasonic boiling hydrolyzed sericin is 0.5-2.0 h; the pH is adjusted to 10.0 with acid or alkali.
步骤(2)中,丝胶与还原糖的质量比为1: 0.3,还原糖水溶液的质量浓度为2.4%;反应温度为95℃;反应时间为6 h。In step (2), the mass ratio of sericin to reducing sugar was 1:0.3, the mass concentration of reducing sugar aqueous solution was 2.4%; the reaction temperature was 95°C; the reaction time was 6 h.
对步骤(2)得到的产物采用喷雾干燥或冷冻干燥,制成糖基化丝胶蛋白粉末。The product obtained in step (2) is spray-dried or freeze-dried to prepare glycosylated sericin powder.
本发明技术方案包括按上述制备方法得到的一种糖基化丝胶蛋白。The technical solution of the present invention includes a kind of glycosylated sericin obtained by the above preparation method.
本发明技术方案还包括所述的一种糖基化丝胶蛋白的应用,将其用于食品添加剂、医用敷料、日化用品、细胞培养基质及材料的表面改性。The technical solution of the present invention also includes the application of the glycosylated sericin, which is used for surface modification of food additives, medical dressings, daily chemical products, cell culture substrates and materials.
具体的应用包括:所述的食品添加剂为辅助降血糖添加剂;所述的医用敷料为伤口愈合敷料;所述的日化用品为化妆品。Specific applications include: the food additive is an auxiliary hypoglycemic additive; the medical dressing is a wound healing dressing; the daily chemical product is a cosmetic.
本发明利用氢氧化钙的水溶液作为一种环保的丝胶碱性溶解剂,应用于一系列蚕丝的脱胶或精练如蚕茧壳、生丝和丝绵片精练、绢纺精练、真丝绸坯料精练等加工过程中丝胶蛋白的溶解。本发明提供的丝胶碱性溶解液可以通过调节pH至10左右,将多余的钙离子中和后以盐的形式沉淀,达到与丝胶蛋白分离的目的。这一过程不仅回收了丝胶蛋白,同时也回收了沉淀物钙盐,从而免除了大量蚕丝脱胶碱性废液的环境污染。The present invention uses the aqueous solution of calcium hydroxide as an environmentally friendly sericin alkaline dissolving agent, which is applied to a series of degumming or scouring of silk such as silkworm cocoon shells, scouring of raw silk and silk floss, scouring of silk spinning, scouring of real silk blanks, etc. Solubilization of sericin. The alkaline solution of sericin provided by the present invention can be separated from sericin by adjusting the pH to about 10, neutralizing excess calcium ions and then precipitating in the form of salt. This process not only recovers the sericin protein, but also recovers the calcium salt of the precipitate, thereby avoiding the environmental pollution of a large amount of silk degumming alkaline waste liquid.
与现有技术相比,本发明的有益效果在于:Compared with prior art, the beneficial effect of the present invention is:
1.本发明提供的技术方案解决了现有技术中水煮提取的外层水溶性丝胶蛋白和高温高压水煮法制备的丝胶蛋白难以进行糖基化反应的问题,获得的糖基化丝胶蛋白产物克服了原丝胶蛋白需要热水溶解和原有蚕蛹具有淡臭味的不足,本发明所制备的糖基化丝胶粉末能溶于冷水,味甘甜醇香;同时,其抗菌性、抗氧化能力、美白能力、体外抑制糖苷酶能力有明显增加,糖尿病大鼠口服有显著的降血糖功能。1. The technical solution provided by the present invention solves the problem that the outer water-soluble sericin protein extracted by boiling water and the sericin protein prepared by high-temperature and high-pressure boiling method are difficult to undergo glycosylation reaction in the prior art, and the obtained glycosylation The sericin product overcomes the disadvantages that the original sericin needs to be dissolved in hot water and the original silkworm chrysalis has a light odor. The glycosylated sericin powder prepared by the present invention can be dissolved in cold water, and tastes sweet and mellow; at the same time, its antibacterial property , anti-oxidation ability, whitening ability, and ability to inhibit glycosidase in vitro are significantly increased, and oral administration of diabetic rats has a significant hypoglycemic function.
2.本发明提供的糖基化丝胶蛋白粉末制备工艺简单,绿色环保,在工业产品表面改性材料、细胞培养基质、高级化妆品或日化用品、健康食品、辅助降血糖功能食品等方面具有广泛应用的潜能。2. The preparation process of the glycosylated sericin powder provided by the present invention is simple, green and environmentally friendly, and has advantages in surface modification materials for industrial products, cell culture substrates, high-grade cosmetics or daily chemical products, health foods, and auxiliary hypoglycemic functional foods. Potential for wide application.
附图说明Description of drawings
图1为本发明实施例提供的丝胶分别与七种还原糖反应产物的紫外-可见吸收光谱;Fig. 1 is the ultraviolet-visible absorption spectrum of sericin provided by the embodiment of the present invention and seven kinds of reducing sugar reaction products respectively;
图2为本发明实施例提供的七种还原糖的吸光度值柱状图;Fig. 2 is a histogram of the absorbance values of seven reducing sugars provided by the embodiments of the present invention;
图3为本发明实施例采用不同的反应温度对丝胶与阿拉伯糖(Ara)、葡萄糖Glc)和木糖(Xyl)进行美拉德反应的吸光度对比柱状图;Fig. 3 is a histogram of absorbance comparison of maillard reaction of sericin with arabinose (Ara), glucose (Glc) and xylose (Xyl) at different reaction temperatures in an embodiment of the present invention;
图4为本发明实施例采用丝胶与还原糖不同的质量比进行美拉德反应的吸光度对比柱状图;Fig. 4 is the histogram of the absorbance comparison of Maillard reaction using different mass ratios of sericin and reducing sugar in the embodiment of the present invention;
图5为本发明实施例提供的采用不同的丝胶与还原糖反应时间得到的产物的吸光度对比曲线图;Fig. 5 is the absorbance contrast curve of the products obtained by adopting different reaction times of sericin and reducing sugar provided by the embodiment of the present invention;
图6为本发明实施例提供的采用不同的丝胶与还原糖的浓度得到的产物的吸光度对比曲线图;Fig. 6 is a curve chart of absorbance comparison of products obtained by adopting different concentrations of sericin and reducing sugar provided by the embodiment of the present invention;
图7为本发明实施例提供的三种丝胶蛋白与七种还原糖的美拉德反应产物的吸光度对比柱状图。Fig. 7 is a histogram of absorbance comparison of Maillard reaction products of three kinds of sericin proteins and seven kinds of reducing sugars provided by the embodiment of the present invention.
具体实施方式Detailed ways
实施例1Example 1
本实施例提供一种糖基化丝胶蛋白的合成方法,包括如下步骤:This embodiment provides a method for synthesizing glycosylated sericin, comprising the following steps:
(1)洁净的蚕茧壳或其它蚕丝材料浸入0.05% Ca(OH)2水溶液中,超声煮沸20 min´ 2;(1) Clean cocoon shells or other silk materials are immersed in 0.05% Ca(OH) 2 aqueous solution, and ultrasonically boiled for 20 min´ 2;
(2)取出脱胶2次的丝素纤维,将2次丝胶碱性液合并后,过滤或离心去除钙盐沉淀物;(2) Take out the silk fiber that has been degummed twice, combine the two times of sericin alkaline solution, filter or centrifuge to remove the calcium salt precipitate;
(3)丝胶碱性过滤液或上清液加入还原糖后置于95℃搅拌反应6 h,获得的棕色反应产物—糖基化丝胶蛋白溶液;(3) Sericin alkaline filtrate or supernatant was added with reducing sugar, then placed at 95°C and stirred for 6 hours to obtain a brown reaction product—glycosylated sericin solution;
(4)喷雾干燥或冷冻干燥制成糖基化丝蛋白粉末。(4) Spray drying or freeze drying to make glycosylated silk protein powder.
本发明制备的糖基化丝胶蛋白产物可应用于制备改性蛋白或食品添加剂、制备辅助降血糖食品产品、制备伤口愈合敷料产品等,也可在工业品表面改性材料、细胞培养基质、化妆品、日化用品等方面得到广泛应用。The glycosylated sericin product prepared by the present invention can be applied to the preparation of modified proteins or food additives, the preparation of auxiliary hypoglycemic food products, the preparation of wound healing dressing products, etc., and can also be used in surface modification materials of industrial products, cell culture substrates, It is widely used in cosmetics and daily chemical products.
实施例2Example 2
将氢氧化钙配制成浓度0.01~0.10%水溶液,10.0 g蚕茧壳以1:60(g/mL)浴比分别浸入氢氧化钙水溶液中,分别煮沸或超声处理10~30 min,以同样浓度的氢氧化钙水溶液再重复煮沸或超声煮沸一次,取出已经溶掉丝胶的蚕丝即丝素纤维后合并2次丝胶碱性水溶液,调节pH至10左右,静止数小时或过夜后,过滤或离心去除沉淀物,浓缩或旋转浓缩至1~20%丝胶水溶液置于4℃备用,或者直接喷雾干燥或冷冻干燥制成丝胶粉末置于4℃备用。本实施例提供的丝胶样品即为氢氧化钙水溶液降解的全丝胶蛋白(记作SS)。Calcium hydroxide was prepared as an aqueous solution with a concentration of 0.01-0.10%, and 10.0 g of silkworm cocoon shells were respectively immersed in the aqueous calcium hydroxide solution at a bath ratio of 1:60 (g/mL). Calcium hydroxide aqueous solution is boiled again or ultrasonically boiled again, take out the silk that has dissolved the sericin, i.e. silk fiber, and combine the sericin alkaline aqueous solution twice, adjust the pH to about 10, stand for several hours or overnight, filter or centrifuge Remove the precipitate, concentrate or spin concentrate to 1-20% sericin aqueous solution and store at 4°C for later use, or directly spray-dry or freeze-dry to make sericin powder and store at 4°C for later use. The sericin sample provided in this example is the whole sericin protein (referred to as SS) degraded by calcium hydroxide aqueous solution.
在浓度为1~20%丝胶碱性水溶液(pH=10),最适在8%丝胶碱性水溶液中加入质量比为1:0.1~0.6还原糖如葡萄糖或木糖,置于90~100℃摇床中反应或置于90~100℃环境中搅拌反应3~10 h;一般反应6 h后取出过滤或离心去除杂质后即获得糖基化丝胶蛋白水溶液置于4℃备用,或者直接喷雾干燥或冷冻干燥制成丝胶粉末置于4℃备用。In the concentration of 1-20% sericin alkaline aqueous solution (pH=10), it is most suitable to add reducing sugars such as glucose or xylose with a mass ratio of 1:0.1-0.6 to 8% sericin alkaline aqueous solution, and place it at 90- React in a shaker at 100°C or place it in an environment of 90-100°C and stir for 3-10 hours; after 6 hours of reaction, take out and filter or centrifuge to remove impurities to obtain an aqueous solution of glycosylated sericin protein and place it at 4°C for use, or Spray-dry or freeze-dry directly to make sericin powder and store it at 4°C for later use.
用本实施例制备的全丝胶蛋白(SS)分别与7种还原糖阿拉伯糖(Ara)、果糖(Fru)、半乳糖(Gal)、葡萄糖(Glc)、甘露糖(Man)、鼠李糖(Rha)和木糖(Xyl)进行美拉德反应。The whole sericin (SS) prepared in this example was mixed with seven reducing sugars, arabinose (Ara), fructose (Fru), galactose (Gal), glucose (Glc), mannose (Man), rhamnose (Rha) and xylose (Xyl) undergo a Maillard reaction.
按本实施例技术方案,将2.0%丝胶水溶液与0.6%还原糖水溶液以重量比1:0.3配制成5 mL反应液,置于90℃摇动连续反应6 h,冷却后反应液离心,取上清液稀释10倍后,置于日立200-20型紫外可见分光光度计进行光谱扫描和MD-5酶标仪上320 nm测定吸光度值,n = 3。参见附图1,为本实施例提供的丝胶分别与7种还原糖反应产物的紫外-可见吸收光谱;7种丝胶-还原糖反应产物在320 nm处的吸光度值(已扣除空白样品丝胶320 nm的吸光度值)。According to the technical scheme of this example, 2.0% sericin aqueous solution and 0.6% reducing sugar aqueous solution were prepared into 5 mL reaction solution at a weight ratio of 1:0.3, placed at 90°C and shaken for continuous reaction for 6 h, after cooling, the reaction solution was centrifuged, and the upper After the serum was diluted 10 times, it was placed on a Hitachi 200-20 UV-Vis spectrophotometer for spectral scanning and the absorbance value was measured at 320 nm on an MD-5 microplate reader, n=3. Referring to accompanying drawing 1, the ultraviolet-visible absorption spectrum of sericin and 7 kinds of reducing sugar reaction products provided for this embodiment respectively; Absorbance value at 320 nm of 7 kinds of sericin-reducing sugar reaction products absorbance at 320 nm).
参见附图2,为本实施例提供的丝胶(SS)与阿拉伯糖(Ara)、果糖(Fru)、半乳糖(Gal)、葡萄糖(Glc)、甘露糖(Man)、鼠李糖(Rha)和木糖(Xyl)7种还原糖反应产物的吸光度值柱状图。Referring to accompanying drawing 2, sericin (SS) and arabinose (Ara), fructose (Fru), galactose (Gal), glucose (Glc), mannose (Man), rhamnose (Rha) provided for this example ) and xylose (Xyl) seven kinds of reducing sugar reaction products of the absorbance histogram.
实施例3Example 3
本实施例采用不同的反应温度对丝胶与阿拉伯糖(Ara)、葡萄糖Glc)和木糖(Xyl)进行美拉德反应,反应条件如下:实施例2提供的浓度为2.0%的丝胶水溶液与0.6%还原糖水溶液以重量比1:0.3配制成5 mL反应液,置于不同温度(参见附图3横坐标“反应温度”,分别为80℃、85℃、90℃、95℃和100℃)下摇动连续反应6 h,冷却后反应液离心,取上清液稀释10倍后测定320 nm吸光度值,n = 3。 In this example, different reaction temperatures are used to carry out Maillard reaction of sericin with arabinose (Ara), glucose (Glc) and xylose (Xyl). Prepare 5 mL of reaction solution with 0.6% reducing sugar aqueous solution at a weight ratio of 1:0.3, and place them at different temperatures (see the abscissa "reaction temperature" in Figure 3, which are 80°C, 85°C, 90°C, 95°C and 100°C, respectively. ℃) and shake continuously for 6 h, after cooling, the reaction solution was centrifuged, and the supernatant was diluted 10 times, and the absorbance value at 320 nm was measured, n = 3.
参见附图3,为本实施例采用不同的反应温度对丝胶与阿拉伯糖(Ara)、葡萄糖Glc)和木糖(Xyl)进行美拉德反应的吸光度对比柱状图。Refer to Fig. 3, which is a histogram of absorbance comparison of maillard reaction of sericin with arabinose (Ara), glucose (Glc) and xylose (Xyl) at different reaction temperatures in this embodiment.
实施例4Example 4
本实施例采用丝胶与阿拉伯糖(Ara)、葡萄糖Glc)和木糖(Xyl)不同的质量比进行美拉德反应,反应条件如下:实施例2提供的浓度为2.0%的丝胶水溶液与0.6%还原糖水溶液,按附图4横坐标所示的各丝胶:还原糖(W:W)的不同比例混合后,置于90℃摇动连续反应6 h,冷却后反应液离心,取上清液稀释10倍后测定320 nm吸光度值,n = 3。 In this example, different mass ratios of sericin to arabinose (Ara), glucose (Glc) and xylose (Xyl) were used to carry out the Maillard reaction. 0.6% reducing sugar aqueous solution, mixed with different ratios of sericin: reducing sugar (W:W) shown in the abscissa of attached drawing 4, placed at 90°C and shaken for continuous reaction for 6 h, after cooling, the reaction solution was centrifuged, and the upper After the supernatant was diluted 10 times, the absorbance at 320 nm was measured, n = 3.
参见附图4,为本实施例采用丝胶与还原糖不同的质量比进行美拉德反应的吸光度对比柱状图。Referring to accompanying drawing 4, it is a histogram of absorbance comparison of maillard reaction with different mass ratios of sericin and reducing sugar in this embodiment.
实施例5Example 5
本实施例以丝胶与还原糖采用不同的反应时间进行美拉德反应,反应条件如下:实施例2提供的浓度为2.0%丝胶水溶液分别与0.6%还原糖阿拉伯糖(Ara)、葡萄糖(Glc)和木糖(Xyl)水溶液以重量比1:0.3配制成5 mL反应液,置于90℃温度下摇动连续反应0 ~ 48h,冷却后反应液离心,取上清液稀释10倍后测定320 nm吸光度值,n = 3.In this example, sericin and reducing sugars were used to carry out Maillard reaction with different reaction times, and the reaction conditions were as follows: the concentration of 2.0% sericin aqueous solution provided in Example 2 was mixed with 0.6% reducing sugar arabinose (Ara), glucose ( Glc) and xylose (Xyl) aqueous solution were prepared at a weight ratio of 1:0.3 to prepare 5 mL of reaction solution, placed at 90°C and shaken for continuous reaction for 0 ~ 48h, after cooling, the reaction solution was centrifuged, and the supernatant was diluted 10 times before
参见附图5,为本实施例提供的采用不同的丝胶与还原糖反应时间得到的产物的吸光度对比曲线图。Referring to Fig. 5 , it is a curve chart of absorbance comparison of products obtained by adopting different reaction times of sericin and reducing sugar provided in this example.
实施例6Example 6
本实施例采用不同浓度的丝胶与还原糖(质量比1:0.3)的水溶液进行糖基化反应。In this example, aqueous solutions of sericin and reducing sugar (mass ratio 1:0.3) with different concentrations were used for glycosylation reaction.
参见附图6,为本实施例提供的采用不同的丝胶与还原糖的浓度得到的产物的吸光度对比曲线图;按图横坐标中丝胶与三种还原糖的浓度比配制成总体积5 mL的反应液,置于90℃温度下摇动连续反应6 h,冷却后反应液离心,取上清液稀释10倍后测定320 nm吸光度值,n = 3。Referring to accompanying drawing 6, the absorbance contrast graph of the product that adopts the concentration of different sericin and reducing sugar that the present embodiment provides; mL of the reaction solution was shaken at 90°C for 6 h. After cooling, the reaction solution was centrifuged. The supernatant was diluted 10 times and the absorbance at 320 nm was measured, n = 3 .
实施例7Example 7
洁净的蚕茧壳浸入1: 50体积比的热水中过夜,使茧层溶胀,然后煮沸60 min,取出脱胶丝换同体积净水重复煮沸60 min,合并2次蚕丝水煮溶解液,过滤或离心的丝胶水溶液浓缩至1~10%浓度范围,即为外层丝胶蛋白(记作OLS),置于4℃备用,或者直接喷雾干燥或冷冻干燥制成外层丝胶蛋白粉末置于4℃备用。Soak the clean cocoon shells in hot water with a volume ratio of 1:50 overnight to swell the cocoon layer, then boil for 60 minutes, take out the degummed silk and replace it with the same volume of clean water and repeat the boiling for 60 minutes, combine the silk boiling solution twice, filter or The centrifuged sericin aqueous solution is concentrated to a concentration range of 1-10%, which is the outer layer sericin protein (referred to as OLS), which is stored at 4°C for later use, or directly spray-dried or freeze-dried to make the outer layer sericin protein powder and placed in Store at 4°C for later use.
按本实施例提供的上述方法经过2次水煮去掉外层丝胶蛋白的丝纤维,浸入以1:50体积比的净水中置于120℃高温高压锅中热处理1~2 h,去除蚕丝纤维外的丝胶溶液过滤或离心后,其丝胶水溶液浓缩至1~10%浓度范围,即为内层丝胶蛋白(记作ILS),置于4℃备用,或者直接喷雾干燥或冷冻干燥制成内层丝胶蛋白粉末置于4℃备用。According to the above method provided in this example, the silk fibers of the outer sericin protein were boiled twice, immersed in clean water with a volume ratio of 1:50 and placed in a high-temperature and high-pressure cooker at 120°C for 1-2 hours to remove the silk fibers. After the outer sericin solution is filtered or centrifuged, the sericin aqueous solution is concentrated to a concentration range of 1 to 10%, which is the inner sericin protein (referred to as ILS), which is stored at 4°C for later use, or directly spray-dried or freeze-dried to produce The inner sericin powder was placed at 4°C for later use.
将本实施例提供的外层丝胶(OLS)、内层丝胶(ILS)和实施例2提供全丝胶蛋白(SS)分别与阿拉伯糖(Ara)、果糖(Fru)、半乳糖(Gal)、葡萄糖(Glc)、甘露糖(Man)、鼠李糖(Rha)和木糖(Xyl)7种还原糖进行美拉德反应。具体方法:分别将浓度为2.0% 的OLS、 ILS和SS丝胶水溶液以质量比为1:0.3与七种还原糖分别混合后,配制成反应总体积为5 mL(pH=10.0)置于90℃摇动反应6 h, 冷却后反应液离心,取上清液稀释10倍后测定320 nm吸光度值。The outer layer sericin (OLS), the inner layer sericin (ILS) provided in this example and the whole sericin (SS) provided in Example 2 were mixed with arabinose (Ara), fructose (Fru), galactose (Gal ), glucose (Glc), mannose (Man), rhamnose (Rha) and xylose (Xyl) seven reducing sugars undergo Maillard reaction. Specific method: Mix 2.0% OLS, ILS and SS sericin aqueous solutions with a mass ratio of 1:0.3 and seven kinds of reducing sugars respectively, and prepare a total reaction volume of 5 mL (pH=10.0) at 90 The reaction solution was shaken at ℃ for 6 h, and after cooling, the reaction solution was centrifuged, and the supernatant was diluted 10 times and the absorbance at 320 nm was measured.
参见附图7,为三种丝胶蛋白与七种还原糖的美拉德反应产物的吸光度对比柱状图;图中,OLS为蚕丝纤维水煮后回收的外层丝胶蛋白;ILS为水煮脱胶去除外层丝胶后的蚕丝纤维用高温高压水煮2 h获得的内层丝胶蛋白;SS为采用本发明技术方案氢氧化钙水溶液溶解和降解的全丝胶蛋白,n = 3。See Figure 7, which is a histogram of the absorbance comparison of the Maillard reaction products of three sericin proteins and seven reducing sugars; in the figure, OLS is the outer sericin protein recovered after boiling silk fibers; ILS is the boiled silk fiber The inner layer sericin protein obtained by degumming and removing the outer layer sericin from the silk fiber is boiled in high temperature and high pressure water for 2 hours; SS is the whole sericin protein dissolved and degraded by the calcium hydroxide aqueous solution of the technical solution of the present invention, n = 3 .
由于蚕丝的丝胶蛋白是热水中易溶的球状胶体蛋白,水煮后易溶解,冷却后又会凝聚胶凝化,本实施例结果表明,采用常规的方法对水煮蚕丝获得的水溶性外层丝胶蛋白和随后高温高压水(120℃)溶解的内层丝胶蛋白在高温下进行美拉德接枝反应,糖基化效率非常低下。采用本发明提供的技术方案,有效提高了丝胶蛋白的糖基化效率,且获得的糖基化丝胶蛋白克服了原丝胶蛋白需热水溶解和蚕蛹具有淡臭味的不足,可广泛应用于食品添加剂、医用敷料、日化用品、细胞培养基质及材料的表面改性等领域,尤其是在辅助降血糖、伤口愈合敷料和化妆品等方面具有广泛的应用。Since silk sericin is a globular colloid protein that is easily soluble in hot water, it is easy to dissolve after boiling, and will coagulate and gel after cooling. The results of this example show that the water-soluble The outer sericin protein and the inner sericin protein dissolved in high-temperature and high-pressure water (120°C) undergo Maillard grafting reaction at high temperature, and the glycosylation efficiency is very low. By adopting the technical scheme provided by the present invention, the glycosylation efficiency of sericin is effectively improved, and the obtained glycosylated sericin overcomes the disadvantages that original sericin needs to be dissolved in hot water and silkworm chrysalis has light odor, and can be widely used It is widely used in food additives, medical dressings, daily chemical products, cell culture substrates and surface modification of materials, especially in auxiliary hypoglycemic, wound healing dressings and cosmetics.
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