糖基化改性对花生蛋白膜性能的影响及其作用机理研究

发布时间：2018-08-07 14:07

【摘要】：花生蛋白膜是一种绿色环保的新型材料,但是由于花生蛋白质基质间缺乏柔韧性,同时又含有大量亲水基团,导致蛋白膜具有脆而不耐水的缺点,限制了其在食品包装中的应用。针对以上问题,本研究对花生蛋白进行糖基化改性,并分析了改性对花生蛋白结构的影响,再以改性蛋白为原料制备具有良好性能的花生蛋白膜,同时分析糖基化改性过程中蛋白膜性能的变化规律,通过蛋白结构与蛋白膜性能之间的相关关系,明确糖基化改善花生蛋白膜性能的机理,最后将蛋白膜应用在方便面调料包装中,探讨其在食品包装应用中的可行性。确定了花生蛋白糖基化改性的最佳工艺条件。对比了木糖、甘露糖、乳糖、半乳糖、葡萄糖和蔗糖对花生蛋白膜各种性能的影响,木糖在提高蛋白膜机械性能的同时能改善花生蛋白膜的耐水性,因此筛选出木糖对花生蛋白进行糖基化改性。结合单因素和正交试验对改性条件进行优化,得到花生蛋白-木糖糖基化的最适改性条件为:木糖浓度10%、p H 9.5、温度91.5℃、时间95min。此条件下,利用改性蛋白制备的蛋白膜,其拉伸强度(TS)、断裂延伸率(E)和溶解性(TSM)分别为10.37 MPa、96.47%和35.94%,同时呈现致密、紧凑的网络结构,显著优于未改性花生蛋白膜。研究了花生蛋白结构在不同木糖浓度、p H、温度和时间的糖基化改性过程中的变化。SDS-PAGE结果表明,糖基化改性后,花生蛋白的条带组成并无变化,但在糖基化改性的作用下,花生蛋白内部发生交联,使花生球蛋白及伴花生球蛋白Ⅱ的条带变浅,同时生成分子量较大的(130 k Da)聚合物。接枝度方面,随着木糖浓度、p H、温度和时间的增加,花生蛋白接枝度呈上升趋势,在木糖浓度10%、p H 9、90℃反应90 min的条件下,接枝度为10.49%。红外光谱方面,改性蛋白光谱在1117 cm-l形成新的吸收峰,而糖基化使酰胺I和酰胺Ⅱ的吸收峰强度有所减弱。巯基含量方面,改性前花生蛋白中巯基含量为7.63μmol/g pro,随着蛋白和木糖之间交联程度的增加,蛋白结构得以伸展,内部巯基暴露使其含量增加,改性后巯基含量最高达9.72μmol/g pro。暴露在外的巯基在蛋白成膜过程中形成二硫键,从而使蛋白膜中巯基含量下降,最低降至1.65μmol/g pro。表面疏水指数方面,糖基化改性使蛋白表面疏水指数显著增加,增加木糖浓度、p H和温度,分别使表面疏水指数增加2.2-2.8倍、1.5-3.4倍和1.6-4.2倍。化学作用力方面,改性前花生蛋白中的化学作用力以氢键为主,糖基化使蛋白中离子键的作用消失,部分氢键被破坏,同时由于内部疏水基团暴露,使疏水相互作用得到加强。确定了利用木糖糖基化改性蛋白(PPI-X)制备蛋白膜的工艺。在上述最优改性条件下对花生蛋白进行改性,并通过喷雾干燥获得粉末状改性蛋白,通过分析改性蛋白的溶解温度及甘油添加量对蛋白膜性能的影响,最终确定蛋白膜制备工艺为PPI-X粉末溶解温度20℃、甘油添加量为25%,此条件下蛋白膜TS、E和TSM分别为10.37 MPa、90.11%和35.94%。研究了糖基化改性过程对花生蛋白膜性能的影响。溶解性方面,木糖浓度的增加使蛋白膜溶解性显著下降,木糖浓度达到10%时TSM比木糖含量为1%时下降17.4%;p H 9、温度90℃时,蛋白膜溶解性最低,仅为33.64%。膨胀性方面,不同改性条件对花生蛋白膜膨胀性均有影响,其中提高p H使膜的膨胀率显著增加,p H为11时蛋白膜膨胀率高达829.70,分别是p H 3、7和9时的7.7倍、3.5倍和2.0倍。表面疏水性方面,木糖浓度增加使花生蛋白膜的接触角(CA)呈现先上升后下降的趋势;改性p H和温度增加,蛋白膜表面疏水性逐渐增加,p H为11时或温度为90℃时,CA相对最高,分别为64.2°和68.91°;随着改性时间延长,蛋白膜表面疏水性整体呈上升趋势。微观结构形态方面,木糖浓度从1%增加至10%,蛋白膜从致密片状结构向网络结构转变;增加p H有利于网络结构的形成,p H为9时,蛋白膜网络结构清晰可见,但p H高达11时分子间产生的过度交联,使蛋白网络变得紧密;温度低于50℃时,蛋白变性程度低,蛋白膜呈现致密的片层结构,随着改性温度上升,蛋白分子结构伸展使蛋白膜呈现网络结构。其他性能方面,糖基化改性使蛋白膜颜色加深,同时能提高蛋白膜水蒸气阻隔性能,但对膜的氧气透过性和热稳定性影响不显著。初步探讨了糖基化改性改善花生蛋白膜性能的作用机理。对改性过程中花生蛋白结构变化与性能改变进行相关性分析,发现接枝度、蛋白巯基含量、表面疏水指数与蛋白膜的强度和延伸性均呈极显著的正相关,与TSM则呈极显著的负相关。上述结果表明,蛋白与木糖的接枝,使蛋白结构得以伸展,大量的疏水基团以及巯基暴露,在蛋白成膜过程中分别形成疏水相互作用和二硫键,使蛋白膜能承受更大程度的形变,故而强度和延伸性均有所增强。此外,疏水基团的暴露,使蛋白亲水性下降,疏水性增加,因此耐水性得到提高。研究了花生蛋白膜在方便面调料包装中应用的可行性。将花生蛋白膜包装植物油,植物油保质期可达180天以上,而无包装对照组和塑料包装的植物油保质期较短,均为40天左右。将花生蛋白膜包装调料粉,在贮藏60天后水分含量和菌落总数均符合标准要求。本研究通过糖基化改性显著改善了花生蛋白膜的机械性能和耐水性,同时明确了糖基化改性改善蛋白膜性能的机理,为花生蛋白膜的实际应用提供了理论依据,有利于实现花生蛋白膜在食品包装中的开发与利用。
[Abstract]:Peanut protein membrane is a new green material. However, due to the lack of flexibility between the peanut protein matrix and a large number of hydrophilic groups, the peanut protein membrane has the shortcoming of crisp and water resistant, which restricts its application in food packaging. The effect of modification on the structure of peanut protein was made, and the peanut protein membrane with good properties was prepared with modified protein as raw material, and the change law of the performance of the protein membrane during the process of glycosylation was analyzed. The mechanism of glycosylation to improve the performance of peanut protein membrane was determined by the relationship between the protein structure and the performance of the protein membrane. The feasibility of the application of white film in instant noodle dressing was discussed. The optimum technological conditions for the modification of peanut protein glycosylation were determined. The effects of xylose, mannose, lactose, galactose, glucose and sucrose on the performance of peanut protein membrane were compared. The xylose could improve the mechanical properties of the protein membrane while improving the mechanical properties of the protein membrane. The water resistance of peanut protein membrane was obtained. Therefore, the glycosylation of peanut protein was screened out by xylose. The modification conditions were optimized by single factor and orthogonal test. The optimum conditions for the modification of peanut protein xylose glycosylation were: the concentration of xylose concentration 10%, P H 9.5, temperature 91.5 C, time 95min., and the protein prepared by the modified protein The tensile strength (TS), fracture elongation (E) and solubility (TSM) of the membrane were 10.37 MPa, 96.47% and 35.94% respectively, showing a compact and compact network structure, which was significantly better than the unmodified peanut protein membrane. The changes of peanut protein structure in the glycosylated modification process of different xylose concentration, P H, temperature and time were studied. After glycosylation, the band composition of peanut protein has no change, but under the action of glycosylated modification, peanut protein and peanut globulin and peanut globulin II are shallower, and the molecular weight (130 K Da) polymer is produced. With the increase of the graft degree, the increase of the concentration of xylose, P H, temperature and time The graft degree of raw protein is on the rise. In the condition of the concentration of xylose 10%, P H 9,90 C reaction 90 min, the grafting degree is 10.49%. infrared spectrum, the modified protein spectrum forms a new absorption peak at 1117 cm-l, and the glycosylation makes the absorption peak intensity of amide I and amide II weaken. For 7.63 mol/g pro, with the increase of cross-linking degree between protein and xylose, the structure of protein is extended and the content of the internal sulfhydryl group is increased. The sulfhydryl group content of the modified sulfhydryl group is up to 9.72 mu mol/g and the sulfhydryl group is exposed to the protein film to form two sulfur bonds in the process of protein formation, thus the content of sulfhydryl group in the protein membrane is reduced, and the lowest to 1.65 mu mol/. G pro. surface hydrophobicity index, glycosylation modification makes the surface hydrophobicity index of protein increase significantly, increase the concentration of xylose, P H and temperature, make the surface hydrophobicity index increase 2.2-2.8 times, 1.5-3.4 times and 1.6-4.2 times respectively. In chemical force, the chemical force in the pre modified peanut protein is mainly hydrogen bond, and glycosylation makes the ionic bond in the protein. The effect was destroyed and some hydrogen bonds were destroyed, and the hydrophobic interaction was enhanced by the exposure of internal hydrophobic groups. The process of preparing protein membrane by xylose glycosylated protein (PPI-X) was determined. The peanut protein was modified under the above optimal modification conditions, and the powder modified protein was obtained by spray drying. The effect of the dissolved temperature of the modified protein and the amount of glycerol added on the performance of the protein membrane was determined. Finally, the preparation process of the protein membrane was PPI-X powder dissolution temperature 20, and glycerol 25%. The protein membrane TS, E and TSM were 10.37 MPa, 90.11% and 35.94%. respectively. The effect of glycosylated modification on the performance of peanut protein membrane was studied. When the concentration of xylose increased, the solubility of protein membrane decreased significantly. When the concentration of xylose reached 10%, TSM decreased by 17.4% when the content of xylose was 1%; P H 9, at the temperature 90, the solubility of the protein membrane was the lowest, only 33.64%. expansibility, and the different modification conditions had an effect on the expansibility of the peanut protein membrane, in which the increase of P H increased the expansion rate of the membrane significantly. When p H was 11, the swelling rate of protein membrane was up to 829.70, 7.7 times, 3.5 times and 2 times of P H 3,7 and 9, respectively. The increase of surface hydrophobicity made the contact angle (CA) of peanut protein film increase first and then decline; the modified P H and temperature increased, the surface hydrophobicity of protein membrane increased gradually, P H was 11 times or temperature 90 degrees, CA Relative highest, 64.2 and 68.91 degrees, respectively. With the prolongation of the modification time, the surface hydrophobicity of the protein film is increasing. In microstructure morphology, the concentration of xylose increases from 1% to 10%, the protein membrane is transformed from dense sheet structure to the network structure, and the increase of P H is beneficial to the formation of network structure. When p H is 9, the structure of the protein membrane network is clear. When p H is up to 11, the excessive cross-linking between molecules makes the protein network tight; when the temperature is lower than 50, the protein denaturation is low and the protein film presents a compact lamellar structure. With the increase of the temperature, the protein molecular structure extends to the network structure. The other performance, the glycosylation modification makes the protein film color deepened, At the same time, it can improve the water vapor barrier property of the protein membrane, but it has no significant influence on the oxygen permeability and thermal stability of the membrane. The mechanism of the effect of glycosylated modification on the performance of peanut protein membrane is preliminarily discussed. The correlation of the changes of the structure and performance of the peanut protein in the process of modification is analyzed, and the graft degree, the content of the protein sulfhydryl group and the surface of the protein are found. The hydrophobicity index has a very significant positive correlation with the strength and elongation of the protein membrane, which shows a very significant negative correlation with TSM. The results show that the graft of protein and xylose makes the protein structure extend, a large number of hydrophobic groups and sulfhydryl groups are exposed, and the hydrophobic interaction and two sulfur bonds are formed in the process of protein formation, and the protein membrane is made to make the protein membrane. It can bear a greater degree of deformation, so the strength and extensibility are enhanced. In addition, the hydrophobicity of the hydrophobic group decreases and the hydrophobicity increases, so the water resistance is increased. The feasibility of the application of peanut protein membrane in the instant noodle packing is studied. The vegetable oil is packaged with the peanut protein membrane, and the shelf life of the vegetable oil can reach 18. For more than 0 days, the shelf life of unpackaged control group and plastic package was shorter than 40 days. The water content of peanut protein membrane packing powder and the total number of colonies were in line with the standard requirements after 60 days of storage. The mechanical and water resistance of peanut egg white film were improved by glycosylation, and the sugar was clearly improved. The mechanism of improving the performance of the protein membrane by the modified modification provides a theoretical basis for the practical application of the peanut protein membrane, and is beneficial to the development and utilization of the peanut protein membrane in the food packaging.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：TS206.4

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