【作者】 刘诗长；

【导师】 洪鹏志；

【作者基本信息】 广东海洋大学 ， 水产品加工及贮藏工程， 2011， 硕士

【摘要】 针对水产蛋白加工利用过程中存在的提取率低、产品杂质含量高、功能特性和风味差等问题,本研究拟以罗非鱼肉及其加工下脚料为原料,采用pH值调节法(pH-shifting)提取鱼蛋白。在低温偏离蛋白质等电点的酸性(pH<3.5)或碱性(pH>10.5)条件下使其溶解,高速离心除去脂肪和不溶性杂质,然后在等电点条件下使蛋白沉淀,制备罗非鱼肉及其下脚料分离蛋白。主要探讨pH值调节法提取罗非鱼蛋白的最佳工艺条件,分离蛋白的营养特性、功能特性和结构特征以及提取过程中蛋白质分子结构和性质的变化。其主要目的是制备功能特性和营养特性优良的鱼分离蛋白,为低值水产蛋白资源的高值化利用以及新型蛋白资源的开发提供理论依据。主要结果如下:1、以罗非鱼肉为原料,探讨了广泛pH值、料液比、溶解时间等对可溶性蛋白得率以及pH值对可溶性蛋白沉淀得率的影响,确定pH值调节法提取罗非鱼肉分离蛋白的溶解pH值为pH 2.0、3.0、11.0和12.0,料液比为1:9(w/v),溶解时间10min;酸/碱可溶性蛋白的最佳沉淀条件为pH 5.5。SDS-PAGE电泳分析显示,在极端酸性(pH≤3)和碱性(pH≥11)条件下,可溶性蛋白包括肌球蛋白重链、肌动蛋白、肌球蛋白轻链、肌浆蛋白和小分子水溶性蛋白,符合典型的鱼蛋白电泳图谱;酸/碱可溶性蛋白经pH 5.5条件沉淀处理后,上清液在电泳图谱中基本不显示蛋白条带,沉淀过程的蛋白回收率较高;比较而言,碱溶性蛋白沉淀得率较低。2、在极端酸性(pH 2.0和3.0)和碱性(pH 11.0和12.0)条件下溶解,在pH 5.5条件下沉淀,制备罗非鱼鱼肉分离蛋白,其提取得率为56.06%~64.95%。冷冻干燥得到鱼分离蛋白分,色泽洁白,无腥味,其干基蛋白含量在95%以上,脂肪含量在1%左右,灰分含量低于2.03%,且碱提蛋白的脂肪和灰分含量较酸提蛋白低,表明碱提法较酸提法更能有效地去除鱼肉中的脂肪和灰分等杂质。分离蛋白的氨基酸种类齐全,必须氨基酸含量占氨基酸总量的49%左右,赖氨酸含量高,符合FAO/WHO的推荐模式,产品可用作蛋白食品添加剂。3、对四种分离蛋白溶解性的分析显示,pH值对罗非鱼肉分离蛋白溶解性的影响比较明显,总的趋势与鱼肉蛋白的pH溶解曲线相似,比较而言,碱提蛋白的溶解性较好,其水溶性蛋白和盐溶性蛋白的含量均较高;SDS-PAGE分析显示,分离蛋白中盐溶性蛋白含量高,显示了肌球蛋白重链、肌动蛋白、肌球蛋白轻链和小分子的水溶性蛋白条带;但比较而言,碱提蛋白的组成相对比较齐全,总巯基含量较高,其产品凝胶强度及凝胶持水性较好,而酸提蛋白中有少量肌浆蛋白损失,其表面疏水性较高,色氨酸荧光发射峰相对红移,表明鱼肉分离蛋白在酸法提取过程中变性程度较大;与鱼肉蛋白相比,四种分离蛋白中水溶性蛋白的含量均降低,可能与酸提过程中蛋白质分子的降解及沉淀过程中的损失有关。因此,碱提法更适合用于提取鱼肉分离蛋白。4、进一步对pH调节处理中酸/碱溶解过程对分离蛋白结构和性质的影响研究显示,在极端酸性(pH 2.0、3.0)和碱性(pH 11.0、12.0)条件下,鱼肉蛋白溶解性较好,可溶性蛋白总巯基含量较低,相对表面疏水性降低,色氨酸荧光发射峰红移,表明鱼蛋白在极端pH条件下分子部分展开和变性,并且酸性条件下蛋白质的变性更为明显,蛋白质部分降解,可溶性蛋白的SDS-PAGE电泳条带部分消失。5、等电点沉淀过程的研究显示,酸/碱可溶性蛋白经pH 5.5沉淀回收后,溶解性明显降低,SDS-PAGE电泳图谱中部分肌浆蛋白条带消失,活性巯基占总巯基的比例下降;比较而言,等电点沉淀对水溶性蛋白的影响比较明显,pH 2.0、3.0和12.0条件下提取的可溶性蛋白,经过等电点沉淀处理后水溶性组分表面疏水性明显升高,色氨酸荧光发射峰发生明显偏移;而pH 11.0条件下提取的可溶性蛋白,经过等电点沉淀处理后水溶性组分的表面疏水性降低,但对盐溶性蛋白的影响不明显。总体分析,在实验范围内,pH 11.0条件下提取的碱溶蛋白的分子结构和性质的保持较好。6、以罗非鱼下脚料为原料,采用pH值调节法,分别在pH 2.0、3.0和pH 12.0、13.0溶解,pH 5.5沉淀回收下脚料蛋白,其提取得率均达52%以上。冷冻干燥得到蛋白粉,其干基蛋白含量达84%以上,灰分含量小于4%,其中酸提蛋白中Ca、Mg等矿质元素的含量较高。下脚料分离蛋白的氨基酸种类齐全且组成均衡合理,符合FAO/WHO的推荐量,是一种优质蛋白。功能特性研究显示,pH 12.0条件下提取的蛋白溶解性和乳化性均最好;SDS-PAGE电泳分析显示,四种下脚料分离蛋白的分子量分布相差不大,含有典型的鱼蛋白条带。另外,酸提蛋白在66.4~44.3kDa处有蛋白条带缺失,可能在酸法提取蛋白的过程中,部分下脚料蛋白降解,与罗非鱼肉分离蛋白的研究结果类似。更多还原

【Abstract】 For the existence of low protein extraction yield, high impurity content, poor functional properties and flavor in processing and utilization of aquatic protein, fish protein isolates (FPI) was prepared from Tilapia muscle and by-products by pH-shifting in this study. Fish muscle proteins were extracted at either acidic (pH<3.5) or alkaline (pH>10.5) at low temperature to obtain the maximum solubility and remove fat and insoluble impurities via high-speed centrifugation followed by the recovery of precipitated proteins at their isoelectric point. The optimum processing conditions of protein isolates from Tilapia muscle via pH-shifting was determined. And the nutritional properties, functional characteristics, structural features, as well as the changes of molecular structure and properties in protein extraction processing were investigated. The main purpose of this study was preparing fish protein isolates with excellent functional and nutritional properties, and providing theoretical basis for high-efficient utilization of low-valuable aquatic protein and new protein resources development. The main results are as follows:1. Effect of the wide range of pH, solid to liquid ratio and dissolution time on the yield of soluble protein from Tilapia muscle was determined. And then effect of pH value on precipitated protein yield from acid-soluble and alkali-soluble was determined as well. Results of soluble protein yield showed that the optimum conditions of protein extracting from Tilapia muscle using pH-shifting were as follows: dissolve conditions was pH 2.0, 3.0, 11.0, and 12.0, ratio of solid to liquid of 1:9 (w/v), dissolving time of 10 min, and the temperature of 4℃. Studies of protein yield of precipitation from soluble protein showed that the optimum condition was pH 5.5. SDS-PAGE electrophoresis analysis showed that, under the extreme acidic (pH≤3) and alkaline (pH≥11) conditions, the soluble proteins strips included myosin heavy chain, actin, myosin light chain, sarcoplasmic proteins and small water-soluble protein band, which was in line with the typical fish protein electrophoresis pattern. When the acid-soluble and alkali-soluble was precipitated at pH 5.5, no protein strips of resulting supernatant displayed at SDS-PAGE profile, and so a higher protein recovery from precipitation process was obtained. Comparatively, the precipitation yield of alkali-soluble protein was relatively lower.2. Fish protein isolates was prepared from Tilapia muscle by solving at pH 2.0, 3.0, 11.0 and 12.0 and then precipitated at pH 5.5, in which protein yield was from 56.06% to 64.95%. The protein powder with white color and no smell was obtained by freeze drying, in which the content of crude protein in dry basis was more than 95%,fat content was 1% approximately, ash content was below 2.03%. And fat and ash content of alkali-made protein were lower than that of acid-made protein. These results indicated that for removing fat and ash impurities, alkali-soluble processing was more effective than acid alkali-soluble processing. Protein isolates contained a full range of amino acid with balanced composition, especially with high lysine content, and the total essential amino acids account for about 49 percent of total amino acids, which was in line with FAO/WHO recommended model. Therefore, these fish protein isolates could be used as protein food additive.3. Studies of solubility of four kinds of protein isolates showed that effect of pH value on solubility of protein isolate from Tilapia muscle was obvious, and the general trend was similar to fish protein pH solving curve. Solubility of alkali-made protein was higher, and its water-soluble and salt-soluble protein content were higher. Studies of SDS-PAGE showed that the salt-soluble content of protein isolate was high, and myosin heavy chain, actin, myosin light chain and small molecule water-soluble protein bands were displayed. Comparatively, the more composition and higher total sulfhydryl content of alkali-made protein were observed, and so did better gel strength and water holding capacity However, and a loss of small amount of sarcoplasmic protein was observed in acid-made protein, and the higher relative surface hydrophobicity and red shifting of tryptophan fluorescence emission peaks was detected. These results indicated a greater degree of protein denaturation in acid-extracted process of fish protein isolate. Compared with Tilapia muscle protein, water-soluble protein content of four protein isolates decreased, this may be related to the degradation in acid solving process and the protein loss during precipitation process. Therefore, alkali solubilization and isoelectric precipitation was more suitable for extracting fish protein isolates.4. Effect of acid and alkali dissolution process on structure and properties of protein isolates was further studied. Results showed that, in extreme acidic (pH 2.0 and 3.0) and alkaline (pH 11.0 and 12.0) conditions, the solubility of fish protein and total thiol content of soluble protein was better, relative surface hydrophobicity was lower, and red shifting of tryptophan fluorescence emission peak was observed, indicating occurrence of protein unfolding and denaturation in extreme pH conditions. Especially in acidic conditions, part of degradation of protein molecules resulted to disappearance of some soluble protein bands on SDS-PAGE profile. 5. Studies on isoelectric precipitation process showed that the solubility of acid-soluble and alkali-soluble protein decreased by precipitation at pH 5.5, and some sarcoplasmic protein bands on SDS-PAGE profile disappeared and the active thiol reduction in the proportion of total sulfhydryl decreased. Comparatively, effect of isoelectric precipitation on soluble protein was more obvious. Soluble proteins solved at pH 2.0, 3.0 and 12.0 followed by the recovery of precipitated proteins at their isoelectric point resulted in significantly increasing surface hydrophobicity and obviously shifting of tryptophan emission peak of water-soluble fractions. And soluble proteins solved at pH 11.0 followed by the recovery of precipitated proteins at their isoelectric point resulted to significantly decreasing surface hydrophobicity, but no significant effect on salt-soluble proteins was observed. The overall analysis showed that, in the range of experiment, molecular structure and properties for alkali-soluble protein extracted at pH 11.0 were well maintained.6. Fish protein isolates was prepared by pH-shifting from Tilapia by-products. Fish by-products was extracted at pH 2.0, 3.0 and pH 12.0, 13.0, and then was precipitated at pH 5.5, in which protein yield was more than 52%. Protein isolates powder was obtained by freeze-drying, and the content of crude protein in dry basis was more than 85% and ash content was below 4%. Comparatively, mineral elements such as Ca and Mg of acid-made protein isolates were higher. Besides, fish protein isolates from Tilapia by-products contained a full range of amino acid with balanced composition and high lysine content, which accorded with FAO/WHO recommended model, and was a kind of high quality protein. Studies of functional properties showed that protein isolates extracted at pH 12.0 had the best solubility and emulsifying activity. Studies of SDS-PAGE showed that several protein isolates from different extracting conditions had similar and complex molecular weight distribution, and typical fish protein strips was displayed on SDS-PAGE profile. In addition, no protein strips displayed at 66.4~44.3kDa for acid-made protein on SDS-PAGE profile, this may be connected with some degradation of protein during acid-extracted process, which was similar to the result of protein isolates from Tilapia muscle.更多还原