pH调节法优化鸢乌贼分离蛋白制备的工艺研究
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摘要
本文采用p H调节法对鸢乌贼分离蛋白的制备工艺进行优化,以溶解得率与蛋白浓度作为评价因子,分析p H、料液比、溶解时间与溶解温度等工艺条件对可溶性蛋白提取率的影响,从而优化其提取条件,并对不同溶解和沉淀p H条件下的蛋白进行聚丙烯酰胺凝胶电泳分析。结果表明,在p H12.0、料液比1∶9(g/m L)、溶解温度4℃与溶解时间10 min的条件下,制备得到的分离蛋白溶解得率最高(80%);在最佳酸、碱溶解p H前提下沉淀蛋白,p H5.5沉淀条件下回收蛋白的得率最高,且酸沉得到的分离蛋白沉淀得率达60%,大于碱沉;p H对鸢乌贼分离蛋白各组分的影响存在差异。其中,肌球蛋白重链(Myosin heavy chain,MHC)、副肌球蛋白(Paramyosin,PM)在各p H条件下降解速率较快,酸性条件对原肌球蛋白(Tropomyosin,TM)降解效果影响较大,而肌动蛋白(Actin,A)在酸、碱性环境下变化较不明显。该工艺可用于工业化大规模制备鸢乌贼分离蛋白,为分离蛋白的制备及来源提供了新途径。
Regarded yield and concentration of soluble protein as eveluation factors,the optimum preparation process of isolate protein from Sthenoteuthis oualaniensis by p H-regulation method was researched. The effects of p H value,solid-liquid ratio,dissolution time and dissolving temperature on the extraction rate of soluble protein were analyzed,and the extraction conditions were optimized.The soluble protein under different soluble and precipitated p H value were measured by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis( SDS-PAGE). The results of optimum preparation conditions were as follows,p H12.0,solid-liquid ratio 1∶ 9( g/m L),dissolving temperature 4 ℃ and dissolution time 10 min.The result showed that dissolution rate of isolated protein was the highest and reached 80% under this condition.The yield of protein recovered was the highest when p H = 5.5 with the optimum condition of acid or alkaline dissolution p H value and the precipitation rate of isolated protein precipitated by acid precipitation reached 60%,which was higher than that of alkaline precipitation.The effects of p H values on the components of protein isolates from Sthenoteuthis oualaniensis were different. Myosin heavy chain( MHC) and paramyosin( PM) were affected by p H values and degradated quickly,actin( A) was less affected by acid and alkaline conditions,tropomyosin( TM) was affected by acidic conditions and the degradation effect was great.The preparation process can be used to large-scale produce isolate protein from Sthenoteuthis oualaniensis and it provides a new isolated protein preparation method.
Regarded yield and concentration of soluble protein as eveluation factors,the optimum preparation process of isolate protein from Sthenoteuthis oualaniensis by p H-regulation method was researched. The effects of p H value,solid-liquid ratio,dissolution time and dissolving temperature on the extraction rate of soluble protein were analyzed,and the extraction conditions were optimized.The soluble protein under different soluble and precipitated p H value were measured by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis( SDS-PAGE). The results of optimum preparation conditions were as follows,p H12.0,solid-liquid ratio 1∶ 9( g/m L),dissolving temperature 4 ℃ and dissolution time 10 min.The result showed that dissolution rate of isolated protein was the highest and reached 80% under this condition.The yield of protein recovered was the highest when p H = 5.5 with the optimum condition of acid or alkaline dissolution p H value and the precipitation rate of isolated protein precipitated by acid precipitation reached 60%,which was higher than that of alkaline precipitation.The effects of p H values on the components of protein isolates from Sthenoteuthis oualaniensis were different. Myosin heavy chain( MHC) and paramyosin( PM) were affected by p H values and degradated quickly,actin( A) was less affected by acid and alkaline conditions,tropomyosin( TM) was affected by acidic conditions and the degradation effect was great.The preparation process can be used to large-scale produce isolate protein from Sthenoteuthis oualaniensis and it provides a new isolated protein preparation method.
引文
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[11]唐小丹.罗非鱼分离蛋白的制备及其性质研究[D].湛江:广东海洋大学,2011.
[12]Choi Y J,Park J W.Acid-aided protein recovery from enzyme-rich Pacific whiting[J].Journal of Food Science,2002,67(8):2962-2967.
[13]Yongsawatdigul J,Park J W.Effects of alkali and acid solubilization on gelation characteristics of rockfish muscle proteins[J].Journal of Food Science,2004,69(7):499-505.
[14]Cortes-Ruiz J A,Pacheco-Aguilar R,Garcia-Sanchez G,et al.Functional characterization of a protein concentrate from bristly sardine made under acidic conditions[J].Journal of Aquatic Food Product Technology,2001,10:5-23.
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[16]陈申如,张其标,倪辉.酸法提取鲢鱼鱼肉蛋白质技术的研究[J].海洋水产研究,2004,25(5):61-64.
[17]刘诗长,周春霞,洪鹏志,等.罗非鱼下脚料分离蛋白的制备及其性质研究[J].食品研究与开发,2011,32(6):38-42.
[18]邱月,曾少葵,章超桦,等.鸢乌贼和杜氏枪乌贼蛋白分离及其性质初探[J].食品与发酵工业,2016,42(9):81-86.
[19]Halldórsdóttir S M,Hamaguchi P Y,Sveinsdóttir H,et al.Properties of hydrolysed saithe protein isolates prepared via p H shift process with and without dewatering[J].LWT-Food Science and Technology,2011,44:1999-2004.
[20]Blanco-Pascual N,Fernández-Martín F,Montero P.Jumbo squid(Dosidicus gigas)myofibrillar protein concentrate for edible packaging films and storage stability[J].LWT-Food Science and Technology,2014,55(2):543-550.
[21]Abdelmalek B E,Gómez-Estaca J,Sila A,et al.Characteristics and functional properties of gelatin extracted from squid(Loligo vulgaris)skin[J].LWT-Food Science and Technology,2016,65:924-931.
[22]Asaduzzaman A K M,Chun B.Recovery of functional materials with thermally stable antioxidative properties in squid muscle hydrolyzates by subcritical water[J].Journal of Food Science and Technology,2015,52(2):793-802.
[2]邹建伟,陈立峰,林蒋进,等.南海外海灯光罩网主要渔场分布及变动研究—基于广西渔船的生产监测统计[J].南方水产,2014,10(4):78-84.
[3]张俊,陈作志,陈国宝,等.南海鸢乌贼水声学测量和评估相关技术研究[J].南方水产,2014(6):1-11.
[4]范江涛,冯雪,邱永松,等.南海鸢乌贼生物学研究进展[J].广东农业科学,2013(23):122-128.
[5]江艳娥,陈作志,林昭进,等.南海中部海域鸢乌贼耳石形态特征分析[J].南方水产,2014,10(4):85-90.
[6]于刚,张洪杰,杨少玲,等.南海鸢乌贼营养成分分析与评价[J].食品工业科技,2014,35(18):358-372.
[7]Hultin H O,Kelleher S D.Process for isolating a protein composition from a muscle source and protein composition:U.S.Patent Ser 0848911[P].1997-2-12.
[8]Palafox H,Córdova-Murueta J H,Navarrete del Toro M A,et al.Protein isolates from jumbo squid by p H-shift processing[J].Process Biochemistry,2009,44:584-587.
[9]Cortés-Ruiz J A,Pacheco-Aguilar R,Lugo-Sánchez M E,et al.Production and functional evaluation of a protein concentrate from giant squid(Dosidicus gigas)by acid dissolution and isoelectric precipitation[J].Food Chemistry,2008,110:485-492.
[10]Laemmli U K.Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J].Nature,1970,227:680-685.
[11]唐小丹.罗非鱼分离蛋白的制备及其性质研究[D].湛江:广东海洋大学,2011.
[12]Choi Y J,Park J W.Acid-aided protein recovery from enzyme-rich Pacific whiting[J].Journal of Food Science,2002,67(8):2962-2967.
[13]Yongsawatdigul J,Park J W.Effects of alkali and acid solubilization on gelation characteristics of rockfish muscle proteins[J].Journal of Food Science,2004,69(7):499-505.
[14]Cortes-Ruiz J A,Pacheco-Aguilar R,Garcia-Sanchez G,et al.Functional characterization of a protein concentrate from bristly sardine made under acidic conditions[J].Journal of Aquatic Food Product Technology,2001,10:5-23.
[15]Undeland I,Kelleher S D,Hultin H O.Recovery of functional proteins from herring(Clupea harengus)light muscle by an acid or alkaline solubilization process[J].Journal of Agricultural and Food Chemistry,2002,5025:7371-7379.
[16]陈申如,张其标,倪辉.酸法提取鲢鱼鱼肉蛋白质技术的研究[J].海洋水产研究,2004,25(5):61-64.
[17]刘诗长,周春霞,洪鹏志,等.罗非鱼下脚料分离蛋白的制备及其性质研究[J].食品研究与开发,2011,32(6):38-42.
[18]邱月,曾少葵,章超桦,等.鸢乌贼和杜氏枪乌贼蛋白分离及其性质初探[J].食品与发酵工业,2016,42(9):81-86.
[19]Halldórsdóttir S M,Hamaguchi P Y,Sveinsdóttir H,et al.Properties of hydrolysed saithe protein isolates prepared via p H shift process with and without dewatering[J].LWT-Food Science and Technology,2011,44:1999-2004.
[20]Blanco-Pascual N,Fernández-Martín F,Montero P.Jumbo squid(Dosidicus gigas)myofibrillar protein concentrate for edible packaging films and storage stability[J].LWT-Food Science and Technology,2014,55(2):543-550.
[21]Abdelmalek B E,Gómez-Estaca J,Sila A,et al.Characteristics and functional properties of gelatin extracted from squid(Loligo vulgaris)skin[J].LWT-Food Science and Technology,2016,65:924-931.
[22]Asaduzzaman A K M,Chun B.Recovery of functional materials with thermally stable antioxidative properties in squid muscle hydrolyzates by subcritical water[J].Journal of Food Science and Technology,2015,52(2):793-802.