中国水牛奶酸凝胶特性及其形成机制研究
摘要
凝胶在食品蛋白加工过程中扮演着重要角色,备受国内外学者关注。酸乳作为一种营养、风味俱佳的食品,是世界性流行乳制品。目前,绝大部分酸乳凝胶是由荷斯坦牛奶经乳酸菌发酵形成的,而水牛奶作为一种优质奶源,营养价值较高,利用水牛奶制作的酸乳凝胶在乳制品中具有巨大的发展潜力。酸乳凝胶特性对酸乳质量具有重要影响,了解酸乳凝胶中生物大分子间的相互作用和形成机制是控制和提高酸乳质量的理论基础,也是乳制品工艺理论的关键内容。本文对3种水牛奶的基本乳成分及其理化特性进行了分析,获取水牛奶加工工艺参数,并对水牛奶酸凝胶的质构特性及其受胞外多糖的影响进行了研究,另外对酸凝胶的主要组分酪蛋白(CN)在酸化凝胶过程中结构变化及其与乳清蛋白、胞外多糖等生物大分子的相互作用进行了研究,主要得到以下几方面实验结果：
通过对3种水牛奶的工艺学特性研究,发现水牛杂交后代牛奶乳蛋白、脂肪、总固形物含量等多个指标高于摩拉水牛奶和荷斯坦牛奶,且前者的稳定性也较高。另外不同品系水牛奶的缓冲容量不同。同时,发现牛奶浓度对其粘度、表面张力及电导率影响显著,加热温度对其粘度和表面张力有不同程度的影响。4种牛奶的粘度均随温度的上升、稀释倍数的增大而逐渐变小。pH值对其zeta电位、电导率和稳定系数影响显著。当温度为45℃、pH6.6、稀释倍数为6时,牛奶的表面张力较小。聚丙烯酰胺凝胶电泳(SDS-PAGE)和质谱结果表明水牛奶与荷斯坦牛奶酪蛋白不同,水牛奶β-酪蛋白的分子量稍高于荷斯坦牛奶,且水牛奶缺少αs2-酪蛋白。
采用3种水牛奶为原料,利用乳酸菌发酵制作酸凝胶,对其凝胶理化特性及质构特性进行分析,发现不同种类水牛奶酸凝胶的流变学、质构特性存在一定差异。酸凝胶的表观黏度与牛奶的总固形物含量成正比,杂交后代水牛奶酸凝胶的硬度比摩拉水牛奶低,但胶体脱水收缩作用敏感性(STS)较摩拉水牛奶高。同时,对不同种类水牛奶酸凝胶的差异性与相关性进行了研究,并对数据进行简单相关和典型相关分析,得出酸乳质构特性中的硬度和胶粘性与乳成分中的蛋白质含量显著相关。
将发酵酸凝胶中提取的胞外多糖(EPS)添加到水牛奶和荷斯坦牛奶中,利用乳酸菌发酵制作酸乳凝胶,研究EPS添加量对不同种类牛奶形成酸凝胶的质构特性的影响及其差异性,发现EPS对不同种类牛奶酸凝胶影响不同,其中以杂交一代(F1)最显著。EPS对水牛奶酸凝胶的形成具有一定的促进作用,凝胶质构特性更致密,添加少量EPS使酸凝胶粘度下降。利用激光共聚焦扫描显微镜(CLSM)和扫描电子显微镜(SEM)观察添加EPS水牛奶酸凝胶的微观结构,发现随着EPS添加量增加,蛋白与EPS间静电相互作用及EPS的空间位垒作用破坏了胶束的原有结构,酪蛋白胶束聚集方式发生改变,促使发酵酸凝胶体系形成新的网状立体结构。
采用圆二色谱(CD)、荧光光谱和纳米粒度分析对不同浓度、酸度和离子强度下的水牛奶CN各级结构进行了研究,结果表明蛋白浓度、环境的酸度和离子强度对水牛奶CN结构影响显著。水牛奶CN浓度明显影响CN二级结构。蛋白浓度较低时,其三级结构变化不明显；蛋白浓度较高时,荧光强度随蛋白浓度呈线性增强趋势,并使酪蛋白分子发生重排,疏水基团暴露,CN疏水性增强,CN颗粒聚集增大。在本研究范围内,当pH6.0时,水牛奶CN二级结构最稳定。当离子强度较大、pH较低时,对水牛奶CN三级结构影响较大,使CN的疏水基团暴露。
采用CD、色氨酸(Trp)和ANS荧光光谱分析CN/WP体系中分子的相互作用,发现蛋白浓度、环境中离子强度、pH均对CN/WP体系有不同影响。当二者浓度比为5：3时,CN/WP体系中分子二、三级结构发生显著变化,a-螺旋、p-折叠含量增多。随着离子强度增加,引起CN/WP分子结构改变,且在离子强度由0.01M增至0.15M过程中较为显著,导致Trp分子内猝灭作用增加。环境pH值由pH6.0降至pH5.0的过程中,体系分子中p-折叠含量增加,导致Trp分子暴露在疏水环境中,Trp分子内淬灭作用减弱,疏水基团暴露。
采CD、Trp和ANS荧光光谱分析CN与EPS的相互作用,发现EPS浓度、环境中离子强度、pH均影响CN与EPS的相互作用。随着pH降低,EPS影响CN Trp分子猝灭,低浓度EPS对CN分子二、三级结构影响显著,主要表现为CN分子α-螺旋结构增多,Trp发生分子内猝灭,疏水基团被遮蔽,而高浓度EPS则导致CN分子p-折叠增多。环境中离子强度较高(0.15M-0.30M)时,EPS浓度对CN的结构影响较小。
采用CD、Trp和ANS荧光光谱分析CN与油酸相互作用,发现蛋白浓度、环境中离子强度、pH对CN与油酸的相互作用影响不同。在中性条件下,低浓度油酸对CN分子二级结构影响较小,而高浓度油酸对CN分子二、三级结构影响显著,表现为α-螺旋结构增多,Trp残基暴露在疏水环境,疏水性基团被遮蔽,CN分子自身聚集程度增加。在低pH条件下,油酸使CN分子结构发生了明显变化,表现为p-折叠结构增多。环境中离子强度较低时,油酸对CN分子结构影响显著,环境中离子强度较高时,油酸对CN分子二级结构影响较小,但对其三级结构影响较大,CN疏水基团被遮蔽。
The gel plays an important role in food protein making process. Yogurt gel as one of the nutritional and delicious dairy products is popular all over the world. Currently, most of the yogurt gel was fermented using Holstein milk by lactic acid bacteria fermentation. Buffalo milk as a high quality and nutritional value of milk resource, fermented to buffalo yogurt gel occupies an extremely important status and a huge potential development. Yogurt gel property is essential for yogurt quality; thus, it is necessary to understand the macromolecules interaction between the yogurt gel and the formation mechanism to improve the theoretical basis. Therefore, in our work, the physical and chemical characteristics of the three kinds of buffalo milk during milk making process was studied; the textural properties of buffalo milk acid gel with or without exopolysaccharides (EPS) were analyzed; meanwhile the structure changes of casein (CN) and the interaction between CN and whey protein (WP), EPS, oleic acid were also explored. The following are some main results from our work.
Comparing the components among different types of buffalo milk, the protein, fat and total solids contents in hybrid buffalo milk were higher than those of Murrah and Holstein milks, so did the stability. The buffer capacity of buffalo milk was higher than that of Holstein milk, while Murrah milk was higher than hybrid offspring, but there were no differences between hybrid offspring milks. Viscosities of four kinds of milk decreased gradually with the increasing temperature and dilution factors. The surface tension of milk dispersion system was smaller under45℃, pH6.6, or6times dilution. Zeta potential, conductivity and stability of the milk were significantly affected by pH value. SDS-PAGE displayed slightly smaller molecular weight of αs-casein in the buffalo milk compared to the cow milk. On the contrast, the molecular weight of P-casein of the former was slightly larger than that of the latter. Furthermore, Guangxi buffalo milk casein was lack of one kind of as-casein.
Three buffalo milk was used to ferment yogurt gel by lactic acid bacteria. It is found that the rheological and textural properties of different kinds of buffalo acid gel were different. The apparent viscosity of the acid gel was proportional to the total solids content of the milk. The gel hardness made from hybrids buffalo milk was lower than that made from the male parent (Murrah buffalo milk); however, the syneresis (STS) was higher than Murrah buffalo milk high. The simple correlation and canonical correlation analysis showed the significantly related between hardness, gumminess and milk protein content.
The isolated exopolysaccharides (EPS) were added to the different types of buffalo milk and Holstein milk, fermented by lactic acid bacteria. The effect of EPS addition on textural properties of different types of milk acid gel was different. The impact of EPS on the properties of (hybrids) F1was remarkable. EPS had a certain role in promoting the formation of milk acid gel and the denser texture characteristics, its viscoelastic and water holding capacity (WHC) were proportional to the amount of EPS addition. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) were used to observe the microstructure of buffalo milk acid gel with or without EPS addition. The original structure of the micelles was damaged by the electrostatic interactions and steric barrier of EPS and protein with the increasing EPS addition. The dimensional network structure of casein micelles gathered and rearranged, which resulted in forming the new three-dimensional structure of acid gel system, showing the hexagonal porous state.
Circular dichroism (CD), fluorescence spectroscopy and particle size analysis were used to explore the secondary, tertiary and quaternary structure of CN as functions of protein concentrations, pH and ionic strength. The results showed that the CN structure was significantly influenced by protein concentration, pH and ionic strength. Buffalo milk concentration significantly affected the secondary structure of the CN. When the protein concentration was low, the effect on tertiary structure was not obvious. However, the fluorescence intensity linear increased with the protein concentration, resulting casein rearrangement, hydrophobic groups exposed, and the particle size increased. Within the scope of this study, the secondary structure of buffalo milk CN was the most stable at pH6.0. In the higher ionic strength, lower pH value environment, the tertiary structure of buffalo CN was influenced evidently.
CD, Trp and ANS fluorescence spectroscopy were used to characterize the interaction between CN and whey protein (WP). It is found that the protein concentration, ionic strength and pH affected the interaction between CN and WP. The secondary structure of CN/WP system was influenced by both the protein concentration ratio and the ionic strength, and strongly affected by the environmental pH value. When the ratio of CN/WP was5:3, the secondary and tertiary structure of CN/WP system changed visibly. The structure of the CN/WP system changed with the gradually increasing ionic strength, showing Trp intermolecular quenching increased, especially during0.01M-0.15M. A certain degree of interaction between NC and WP occurred in pH6.0-pH5.0, and significantly in pH6.0, the secondary structure of the CN/WP system expressed as the increase of the β-sheet; meanwhile, it affected the tertiary structure of the CN/WP system, resulting in Trp exposed in a hydrophobic environment, the hydrophobic groups exposed.
CD, Trp and ANS fluorescence spectroscopy were used to characterize the interaction between CN and EPS. It is found that the protein concentration, ionic strength and pH affected the interaction between CN and EPS. The CN structure changed significantly accompanied by the emergence of the EPS, under different pH value. The lower concentration of EPS expressed the stronger impact on the secondary and tertiary structure of CN; furthermore, the marked impact on CN structure under the lower pH value, the low concentration of EPS increased the a-helix content of CN, while the high concentration of EPS resulted in the secondary structure of CN mainly in β-sheet. The secondary structure of CN was changed by the emergence of the EPS as a function of ionic strength, showing the a-helix was destroyed. However, compared with the pH, the higher ionic strength slightly affected the CN structure. EPS concentration was less impact on the tertiary structure of CN when the ionic strength was in0.15M-0.30M.
CD, Trp and ANS fluorescence spectroscopy were used to characterize the interaction between CN and oleic acid. The interaction between CN and oleic acid was affected the protein concentration, ionic strength and pH. CN structure changed significantly with oleic acid added varied pH, and the impact was the greatest under the neutral condition. Nevertheless, high concentration of oleic acid influenced the secondary and tertiary structure of CN, showing the increased a-helix content of CN, the increased aggregation, and a large number of hydrophobic groups were masked and Trp was exposed to the hydrophobic environment. While the low concentration of oleic acid slightly impacted on the secondary structure of CN. The (3-sheet of CN increased with the emergence of the oleic acid under pH5.0. The secondary structure CN changed obviously with the amount of oleic acid added range from0.5mM to1.0mM; however,0.5mM oleic acid had a marked impact on the tertiary structure of CN. Oleic acid had notable effect on CN structure under the lower ionic strength, but slight effect on the secondary structure of CN and a marked impact on the tertiary structure of CN and under the higher ionic strength, the hydrophobic groups were hided.
通过对3种水牛奶的工艺学特性研究,发现水牛杂交后代牛奶乳蛋白、脂肪、总固形物含量等多个指标高于摩拉水牛奶和荷斯坦牛奶,且前者的稳定性也较高。另外不同品系水牛奶的缓冲容量不同。同时,发现牛奶浓度对其粘度、表面张力及电导率影响显著,加热温度对其粘度和表面张力有不同程度的影响。4种牛奶的粘度均随温度的上升、稀释倍数的增大而逐渐变小。pH值对其zeta电位、电导率和稳定系数影响显著。当温度为45℃、pH6.6、稀释倍数为6时,牛奶的表面张力较小。聚丙烯酰胺凝胶电泳(SDS-PAGE)和质谱结果表明水牛奶与荷斯坦牛奶酪蛋白不同,水牛奶β-酪蛋白的分子量稍高于荷斯坦牛奶,且水牛奶缺少αs2-酪蛋白。
采用3种水牛奶为原料,利用乳酸菌发酵制作酸凝胶,对其凝胶理化特性及质构特性进行分析,发现不同种类水牛奶酸凝胶的流变学、质构特性存在一定差异。酸凝胶的表观黏度与牛奶的总固形物含量成正比,杂交后代水牛奶酸凝胶的硬度比摩拉水牛奶低,但胶体脱水收缩作用敏感性(STS)较摩拉水牛奶高。同时,对不同种类水牛奶酸凝胶的差异性与相关性进行了研究,并对数据进行简单相关和典型相关分析,得出酸乳质构特性中的硬度和胶粘性与乳成分中的蛋白质含量显著相关。
将发酵酸凝胶中提取的胞外多糖(EPS)添加到水牛奶和荷斯坦牛奶中,利用乳酸菌发酵制作酸乳凝胶,研究EPS添加量对不同种类牛奶形成酸凝胶的质构特性的影响及其差异性,发现EPS对不同种类牛奶酸凝胶影响不同,其中以杂交一代(F1)最显著。EPS对水牛奶酸凝胶的形成具有一定的促进作用,凝胶质构特性更致密,添加少量EPS使酸凝胶粘度下降。利用激光共聚焦扫描显微镜(CLSM)和扫描电子显微镜(SEM)观察添加EPS水牛奶酸凝胶的微观结构,发现随着EPS添加量增加,蛋白与EPS间静电相互作用及EPS的空间位垒作用破坏了胶束的原有结构,酪蛋白胶束聚集方式发生改变,促使发酵酸凝胶体系形成新的网状立体结构。
采用圆二色谱(CD)、荧光光谱和纳米粒度分析对不同浓度、酸度和离子强度下的水牛奶CN各级结构进行了研究,结果表明蛋白浓度、环境的酸度和离子强度对水牛奶CN结构影响显著。水牛奶CN浓度明显影响CN二级结构。蛋白浓度较低时,其三级结构变化不明显；蛋白浓度较高时,荧光强度随蛋白浓度呈线性增强趋势,并使酪蛋白分子发生重排,疏水基团暴露,CN疏水性增强,CN颗粒聚集增大。在本研究范围内,当pH6.0时,水牛奶CN二级结构最稳定。当离子强度较大、pH较低时,对水牛奶CN三级结构影响较大,使CN的疏水基团暴露。
采用CD、色氨酸(Trp)和ANS荧光光谱分析CN/WP体系中分子的相互作用,发现蛋白浓度、环境中离子强度、pH均对CN/WP体系有不同影响。当二者浓度比为5：3时,CN/WP体系中分子二、三级结构发生显著变化,a-螺旋、p-折叠含量增多。随着离子强度增加,引起CN/WP分子结构改变,且在离子强度由0.01M增至0.15M过程中较为显著,导致Trp分子内猝灭作用增加。环境pH值由pH6.0降至pH5.0的过程中,体系分子中p-折叠含量增加,导致Trp分子暴露在疏水环境中,Trp分子内淬灭作用减弱,疏水基团暴露。
采CD、Trp和ANS荧光光谱分析CN与EPS的相互作用,发现EPS浓度、环境中离子强度、pH均影响CN与EPS的相互作用。随着pH降低,EPS影响CN Trp分子猝灭,低浓度EPS对CN分子二、三级结构影响显著,主要表现为CN分子α-螺旋结构增多,Trp发生分子内猝灭,疏水基团被遮蔽,而高浓度EPS则导致CN分子p-折叠增多。环境中离子强度较高(0.15M-0.30M)时,EPS浓度对CN的结构影响较小。
采用CD、Trp和ANS荧光光谱分析CN与油酸相互作用,发现蛋白浓度、环境中离子强度、pH对CN与油酸的相互作用影响不同。在中性条件下,低浓度油酸对CN分子二级结构影响较小,而高浓度油酸对CN分子二、三级结构影响显著,表现为α-螺旋结构增多,Trp残基暴露在疏水环境,疏水性基团被遮蔽,CN分子自身聚集程度增加。在低pH条件下,油酸使CN分子结构发生了明显变化,表现为p-折叠结构增多。环境中离子强度较低时,油酸对CN分子结构影响显著,环境中离子强度较高时,油酸对CN分子二级结构影响较小,但对其三级结构影响较大,CN疏水基团被遮蔽。
The gel plays an important role in food protein making process. Yogurt gel as one of the nutritional and delicious dairy products is popular all over the world. Currently, most of the yogurt gel was fermented using Holstein milk by lactic acid bacteria fermentation. Buffalo milk as a high quality and nutritional value of milk resource, fermented to buffalo yogurt gel occupies an extremely important status and a huge potential development. Yogurt gel property is essential for yogurt quality; thus, it is necessary to understand the macromolecules interaction between the yogurt gel and the formation mechanism to improve the theoretical basis. Therefore, in our work, the physical and chemical characteristics of the three kinds of buffalo milk during milk making process was studied; the textural properties of buffalo milk acid gel with or without exopolysaccharides (EPS) were analyzed; meanwhile the structure changes of casein (CN) and the interaction between CN and whey protein (WP), EPS, oleic acid were also explored. The following are some main results from our work.
Comparing the components among different types of buffalo milk, the protein, fat and total solids contents in hybrid buffalo milk were higher than those of Murrah and Holstein milks, so did the stability. The buffer capacity of buffalo milk was higher than that of Holstein milk, while Murrah milk was higher than hybrid offspring, but there were no differences between hybrid offspring milks. Viscosities of four kinds of milk decreased gradually with the increasing temperature and dilution factors. The surface tension of milk dispersion system was smaller under45℃, pH6.6, or6times dilution. Zeta potential, conductivity and stability of the milk were significantly affected by pH value. SDS-PAGE displayed slightly smaller molecular weight of αs-casein in the buffalo milk compared to the cow milk. On the contrast, the molecular weight of P-casein of the former was slightly larger than that of the latter. Furthermore, Guangxi buffalo milk casein was lack of one kind of as-casein.
Three buffalo milk was used to ferment yogurt gel by lactic acid bacteria. It is found that the rheological and textural properties of different kinds of buffalo acid gel were different. The apparent viscosity of the acid gel was proportional to the total solids content of the milk. The gel hardness made from hybrids buffalo milk was lower than that made from the male parent (Murrah buffalo milk); however, the syneresis (STS) was higher than Murrah buffalo milk high. The simple correlation and canonical correlation analysis showed the significantly related between hardness, gumminess and milk protein content.
The isolated exopolysaccharides (EPS) were added to the different types of buffalo milk and Holstein milk, fermented by lactic acid bacteria. The effect of EPS addition on textural properties of different types of milk acid gel was different. The impact of EPS on the properties of (hybrids) F1was remarkable. EPS had a certain role in promoting the formation of milk acid gel and the denser texture characteristics, its viscoelastic and water holding capacity (WHC) were proportional to the amount of EPS addition. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) were used to observe the microstructure of buffalo milk acid gel with or without EPS addition. The original structure of the micelles was damaged by the electrostatic interactions and steric barrier of EPS and protein with the increasing EPS addition. The dimensional network structure of casein micelles gathered and rearranged, which resulted in forming the new three-dimensional structure of acid gel system, showing the hexagonal porous state.
Circular dichroism (CD), fluorescence spectroscopy and particle size analysis were used to explore the secondary, tertiary and quaternary structure of CN as functions of protein concentrations, pH and ionic strength. The results showed that the CN structure was significantly influenced by protein concentration, pH and ionic strength. Buffalo milk concentration significantly affected the secondary structure of the CN. When the protein concentration was low, the effect on tertiary structure was not obvious. However, the fluorescence intensity linear increased with the protein concentration, resulting casein rearrangement, hydrophobic groups exposed, and the particle size increased. Within the scope of this study, the secondary structure of buffalo milk CN was the most stable at pH6.0. In the higher ionic strength, lower pH value environment, the tertiary structure of buffalo CN was influenced evidently.
CD, Trp and ANS fluorescence spectroscopy were used to characterize the interaction between CN and whey protein (WP). It is found that the protein concentration, ionic strength and pH affected the interaction between CN and WP. The secondary structure of CN/WP system was influenced by both the protein concentration ratio and the ionic strength, and strongly affected by the environmental pH value. When the ratio of CN/WP was5:3, the secondary and tertiary structure of CN/WP system changed visibly. The structure of the CN/WP system changed with the gradually increasing ionic strength, showing Trp intermolecular quenching increased, especially during0.01M-0.15M. A certain degree of interaction between NC and WP occurred in pH6.0-pH5.0, and significantly in pH6.0, the secondary structure of the CN/WP system expressed as the increase of the β-sheet; meanwhile, it affected the tertiary structure of the CN/WP system, resulting in Trp exposed in a hydrophobic environment, the hydrophobic groups exposed.
CD, Trp and ANS fluorescence spectroscopy were used to characterize the interaction between CN and EPS. It is found that the protein concentration, ionic strength and pH affected the interaction between CN and EPS. The CN structure changed significantly accompanied by the emergence of the EPS, under different pH value. The lower concentration of EPS expressed the stronger impact on the secondary and tertiary structure of CN; furthermore, the marked impact on CN structure under the lower pH value, the low concentration of EPS increased the a-helix content of CN, while the high concentration of EPS resulted in the secondary structure of CN mainly in β-sheet. The secondary structure of CN was changed by the emergence of the EPS as a function of ionic strength, showing the a-helix was destroyed. However, compared with the pH, the higher ionic strength slightly affected the CN structure. EPS concentration was less impact on the tertiary structure of CN when the ionic strength was in0.15M-0.30M.
CD, Trp and ANS fluorescence spectroscopy were used to characterize the interaction between CN and oleic acid. The interaction between CN and oleic acid was affected the protein concentration, ionic strength and pH. CN structure changed significantly with oleic acid added varied pH, and the impact was the greatest under the neutral condition. Nevertheless, high concentration of oleic acid influenced the secondary and tertiary structure of CN, showing the increased a-helix content of CN, the increased aggregation, and a large number of hydrophobic groups were masked and Trp was exposed to the hydrophobic environment. While the low concentration of oleic acid slightly impacted on the secondary structure of CN. The (3-sheet of CN increased with the emergence of the oleic acid under pH5.0. The secondary structure CN changed obviously with the amount of oleic acid added range from0.5mM to1.0mM; however,0.5mM oleic acid had a marked impact on the tertiary structure of CN. Oleic acid had notable effect on CN structure under the lower ionic strength, but slight effect on the secondary structure of CN and a marked impact on the tertiary structure of CN and under the higher ionic strength, the hydrophobic groups were hided.
引文
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