蓝圆鲹酸/碱等电点沉淀法分离蛋白凝胶特性与消化特性
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摘要
采用酸/碱等电点沉淀(isoelectric solubilization/precipitation,ISP)法制备蓝圆鲹肌肉分离蛋白(acid/alkaline aided protein isolate,API/KPI),并对全蛋白(total protein,TP)、肌原纤维蛋白(myofibrillar protein,MP)与分离蛋白的理化特性、凝胶特性以及消化稳定性进行比较研究。结果表明,蓝圆鲹肌肉蛋白在碱性条件下溶解性显著高于酸性条件,经优化后的API与KPI的回收率分别为65.0%与84.6%,显著高于MP(54.0%)。KPI、API与MP的脂肪与灰分含量明显低于TP,其中KPI的粗蛋白含量最高。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示KPI与API的蛋白组成与TP相近,但KPI与API的氨基酸组成中甘氨酸与脯氨酸含量显著低于TP。质构与流变学分析结果显示,KPI与API的凝胶强度与储能模量(G’)均明显低于TP与MP,其中TP、MP与KPI的储能模量在50～55℃会出现明显的下降趋势,表明发生凝胶劣化现象,而API组无明显变化。扫描电子显微镜结果表明,与90℃相比,经55℃加热处理的KPI、TP与MP组凝胶结构更加疏松、多孔,这与流变学分析的结果一致。体外模拟胃肠液消化实验表明,MP具有最佳的消化性,API与KPI的消化性相当,且显著高于TP。综上所述,利用ISP制备分离蛋白可显著提高蛋白回收率,且分离蛋白的消化性明显优于TP。由于分离蛋白失去凝胶化能力,故可考虑将其应用于食品蛋白配料领域。
In the present study, protein isolates were recovered from the muscle of blue round scads(Decapterus maruadsi)using acidic-or alkaline-aided isoelectric solubilization/precipitation. The physiochemical, gelation, and digestive properties of acid(API)-and alkali(KPI)-recovered proteins were investigated, as compared to total proteins(TP) and myofibrillar proteins(MP). D. maruadsi muscle proteins were even more soluble in alkali than in acid. The recovery yield(65.0% and84.6%, respectively) of API and KPI were much higher as compared with MP(54.0%). API, KPI and MP contained far smaller amounts of lipid and ash, whereas KPI contained the highest protein content. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis showed that there were no significant differences in the protein composition of TP, KPI and API. However, compared with TP, API and KPI had lower contents of Gly and Pro. Textural and rheological analysis revealed that API and KPI possessed lower gel strength and storage module G' than TP and MP. Meanwhile,although no significant changes occurred in API, remarkable decreases in the G' values of TP, MP and KPI were observed in the temperature range from 50 to 55 ℃, indicating the occurrence of modori phenomenon. This finding was well consistent with the microstructure observed by scanning electron microscopy(SEM), whereas KPI, TP and MP showed looser, porous and disorganized network when incubated at 55 ℃ for 30 min than at 90 ℃. The results of simulated gastrointestinal digestion showed that MP had the best digestibility among four proteins, while KPI and API possessed better digestibility than TP. In conclusion, ISP can greatly improve the recovery yield of protein from blue round scads. Due to their higher digestibility and poorer gelation ability, the use of the protein isolates as protein ingredients in food products is proposed.
In the present study, protein isolates were recovered from the muscle of blue round scads(Decapterus maruadsi)using acidic-or alkaline-aided isoelectric solubilization/precipitation. The physiochemical, gelation, and digestive properties of acid(API)-and alkali(KPI)-recovered proteins were investigated, as compared to total proteins(TP) and myofibrillar proteins(MP). D. maruadsi muscle proteins were even more soluble in alkali than in acid. The recovery yield(65.0% and84.6%, respectively) of API and KPI were much higher as compared with MP(54.0%). API, KPI and MP contained far smaller amounts of lipid and ash, whereas KPI contained the highest protein content. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis showed that there were no significant differences in the protein composition of TP, KPI and API. However, compared with TP, API and KPI had lower contents of Gly and Pro. Textural and rheological analysis revealed that API and KPI possessed lower gel strength and storage module G' than TP and MP. Meanwhile,although no significant changes occurred in API, remarkable decreases in the G' values of TP, MP and KPI were observed in the temperature range from 50 to 55 ℃, indicating the occurrence of modori phenomenon. This finding was well consistent with the microstructure observed by scanning electron microscopy(SEM), whereas KPI, TP and MP showed looser, porous and disorganized network when incubated at 55 ℃ for 30 min than at 90 ℃. The results of simulated gastrointestinal digestion showed that MP had the best digestibility among four proteins, while KPI and API possessed better digestibility than TP. In conclusion, ISP can greatly improve the recovery yield of protein from blue round scads. Due to their higher digestibility and poorer gelation ability, the use of the protein isolates as protein ingredients in food products is proposed.
引文
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[12]KRISTINSSON H G,HULTIN H O.Changes in conformation and subunit assembly of cod myosin at low and high pH and after subsequent refolding[J].Journal of Agricultural and Food Chemistry,2003,51(24):7187-7196.DOI:10.1021/jf026193m.
[13]UNDELAND I,KELLEHER S D,HULTIN H O,et al.Consistency and solubility changes in herring(Clupea harengus)light muscle homogenates as a function of pH[J].Journal of Agricultural and Food Chemistry,2003,51(14):3992-3998.DOI:10.1021/jf0210932.
[14]CHANG T,WANG C J,YANG H,et al.Comparison of conventional washing processing and pH shift processing on gelation characteristics of bighead carp(Aristichthys nobilis)muscle proteins[J].Journal of Aquatic Food Product Technology,2017,26(1):103-114.DOI:10.1080/10498850.2015.1099067.
[15]RAWDKUEN S,SAI-UT S,KHAMSORN S,et al.Biochemical and gelling properties of tilapia surimi and protein recovered using an acid-alkaline process[J].Food Chemistry,2009,112(1):112-119.DOI:10.1016/j.foodchem.2008.05.047.
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[17]孙莎.皱纹盘鲍肌肉氨肽酶的酶学特性及呈味作用研究[D].厦门:集美大学,2016:15.
[18]LAEMMLI U K.Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J].Nature,1970,227:680-685.DOI:10.1038/227680a0.
[19]United States Pharmacopoeia.The United States Pharmacopeia 23,the national formulary 18[M].Rockville,MD:The United States Pharmacopeial Convention,Inc.,1995:2053.
[20]万楚君,游银川,翁凌,等.四种加工方式对皱纹盘鲍制品消化特性的影响[J].水产学报,2017,41(6):928-936.DOI:10.11964/jfc.20161010574.
[21]LOWRY O H,ROSEBROUGH N J,FARR A L.Protein measurement with the Folin phenol reagent[J].Journal of Biological Chemistry,1951,193(1):265-275.
[22]KLOMKLAO S,BENJAKUL S,KISHIMURA H,et al.Trypsin inhibitor from yellowfin tuna(Thunnus albacores)roe:effects on gel properties of surimi from bigeye snapper(Priacanthus macracanthus)[J].LWT-Food Science and Technology,2016,65:122-127.DOI:10.1016/j.lwt.2015.07.074.
[23]ABDOLLAHI M,REZAEI M,JAFARPOUR A,et al.Dynamic rheological,microstructural and physicochemical properties of blend fish protein recovered from kilka(Clupeonella cultriventris)and silver carp(Hypophthalmichthys molitrix)by the pH-shift process or washing-based technology[J].Food Chemistry,2017,229:695-709.DOI:10.1016/j.foodchem.2017.02.133.
[24]WENG W Y,ZHENG W X.Silver carp(Hypophthalmichthys molitrix)surimi acid-induced gel extract characteristics:a comparison with heat-induced gel[J].International Journal of Food Properties,2015,18(4):821-832.DOI:10.1080/10942912.2013.864675.
[25]TIAN Y Y,WANG W,YUAN C H,et al.Nutritional and digestive properties of protein isolates extracted from the muscle of the common carp using pH-shift processing[J].Journal of Food Processing and Preservation,2017,41(1):1-9.DOI:10.1111/jfpp.12847.
[26]TAKTAK W,NASRI R,HAMDI M,et al.Physicochemical,textural,rheological and microstructural properties of protein isolate gels produced from European eel(Anguilla anguilla)by heatinduced gelation process[J].Food Hydrocolloids,2018,82:278-287.DOI:10.1016/j.foodhyd.2018.04.008.
[27]CHEN Y C,JACZYNSKI J.Protein recovery from rainbow trout(Oncorhynchus mykiss)processing byproducts via isoelectric solubilization/precipitation and its gelation properties as affected by functional additives[J].Journal of Agricultural and Food Chemistry,2007,55(22):9079-9088.DOI:10.1021/jf071992w.
[28]渡部终五,林华娟,毛伟杰.水产利用化学基础[M].北京:化学工业出版社,2017:14.
[29]杨伊然,胡晓,杨贤庆,等.蓝圆鲹蛋白酶解物的螯合矿物离子活性研究[J].食品科学,2017,38(3):88-93.DOI:10.7506/spkx1002-6630-201703015.
[30]SUN L C,YOSHIDA A,CAI Q F,et al.Mung bean trypsin inhibitor is effective in suppressing the degradation of myofibrillar proteins in the skeletal muscle of blue scad(Decapterus maruadsi)[J].Journal of Agricultural and Food Chemistry,2010,58(24):12986-12992.DOI:10.1021/jf103526e.
[31]ZHONG C,CAI Q F,LIU G M,et al.Purification and characterisation of cathepsin L from the skeletal muscle of blue scad(Decapterus maruadsi)and comparison of its role with myofibril-bound serine proteinase in the degradation of myofibrillar proteins[J].Food Chemistry,2012,133(4):1560-1568.DOI:10.1016/j.foodchem.2012.02.050.
[32]王梦想,钟婵,蔡秋凤,等.蓝圆鲹肌肉中丝氨酸蛋白酶的分离纯化及性质研究[J].食品工业科技,2012,33(19):58-62.DOI:10.13386/j.issn1002-0306.2012.19.029.
[33]VISESSANGUAN W,OGAWA M,NAKAI S,et al.Physicochemical changes and mechanism of heat-induced gelation of arrowtooth flounder myosin[J].Journal of Agricultural and Food Chemistry,2000,48(4):1016-1023.DOI:10.1021/jf9900332.
[34]EGELANDSDAL B,MARTINSEN B,AUTIO K.Rheological parameters as predictors of protein functionality:a model study using myofibrils of different fibre-type composition[J].Meat Science,1995,39(1):97-111.DOI:10.1016/0309-1740(95)80011-5.
[35]刘海梅.鲢鱼糜凝胶及形成机理的研究[D].武汉:华中农业大学,2007:88.
[36]BUAMARD N,BENJAKUL S.Improvement of gel properties of sardine(Sardinella albella)surimi using coconut husk extracts[J].Food Hydrocolloids,2015,51:146-155.DOI:10.1016/j.foodhyd.2015.05.011.
[2]农业部渔业渔政管理局.中国渔业统计年鉴[M].北京:中国农业出版社,2017:41.
[3]陈晓婷,吴靖娜,路海霞,等.蓝圆鲹肌肉中营养成分分析与评价[J].渔业现代化,2016,43(1):56-61.DOI:10.3969/j.issn.1007-9580.2016.01.011.
[4]张浩,李丹辰,洪佳敏,等.漂洗条件对蓝圆鯵鱼糜特性的影响[J].食品工业,2014,35(3):18-21.
[5]FRIEDMAN M.Nutritional and toxicological significance of enzyme inhibitors in foods[M].New York:Springer,1986:349-355.
[6]王伟,刘俊荣,马永生,等.等电点沉淀法鱼分离蛋白结构与功能变化研究进展[J].食品科学,2015,36(1):250-255.DOI:10.7506/spkx1002-6630-201501048.
[7]K R I S T I N S S O N H G,T H E O D O R E A E,D E M I R N,e t a l.A comparative study between acid-and alkali-aided processing and surimi processing for the recovery of proteins from channel catfish muscle[J].Journal of Food Science,2005,70(4):C298-C306.DOI:10.1111/j.1365-2621.2005.tb07177.x.
[8]KRISTINSSON H G,LIANG Y.Effect of pH-shift processing and surimi processing on atlantic croaker(Micropogonias undulates)muscle proteins[J].Journal of Food Science,2006,71(5):C304-C312.DOI:10.1111/j.1750-3841.2006.00046.x.
[9]CHOMNAWANG C,YONGSAWATDIGUL J.Protein recovery of tilapia frame by-products by pH-shift method[J].Journal of Aquatic Food Product Technology,2013,22(2):112-120.DOI:10.1080/10498850.2011.629077.
[10]ABDOLLAHI M,REZAEI M,JAFARPOUR A,et al.Dynamic rheological,microstructural and physicochemical properties of blend fish protein recovered from kilka(Clupeonella cultriventris)and silver carp(Hypophthalmichthys molitrix)by the pH-shift process or washing-based technology[J].Food Chemistry,2017,229:695-709.DOI:10.1016/j.foodchem.2017.02.133.
[11]PARK J D,YONGSAWATDIGUL J,CHOI Y J,et al.Biochemical and conformational changes of myosin purified from pacific sardine at various pHs[J].Journal of Food Science,2008,73(3):C191-C197.DOI:10.1111/j.1750-3841.2008.00691.x.
[12]KRISTINSSON H G,HULTIN H O.Changes in conformation and subunit assembly of cod myosin at low and high pH and after subsequent refolding[J].Journal of Agricultural and Food Chemistry,2003,51(24):7187-7196.DOI:10.1021/jf026193m.
[13]UNDELAND I,KELLEHER S D,HULTIN H O,et al.Consistency and solubility changes in herring(Clupea harengus)light muscle homogenates as a function of pH[J].Journal of Agricultural and Food Chemistry,2003,51(14):3992-3998.DOI:10.1021/jf0210932.
[14]CHANG T,WANG C J,YANG H,et al.Comparison of conventional washing processing and pH shift processing on gelation characteristics of bighead carp(Aristichthys nobilis)muscle proteins[J].Journal of Aquatic Food Product Technology,2017,26(1):103-114.DOI:10.1080/10498850.2015.1099067.
[15]RAWDKUEN S,SAI-UT S,KHAMSORN S,et al.Biochemical and gelling properties of tilapia surimi and protein recovered using an acid-alkaline process[J].Food Chemistry,2009,112(1):112-119.DOI:10.1016/j.foodchem.2008.05.047.
[16]SUN L C,KANEKO K,OKAZAKI E,et al.Comparative study of proteins recovered from whole north pacific krill Euphausia pacifica by acidic and alkaline treatment during isoelectric solubilization/precipitation[J].Fisheries Science,2013,79(3):537-546.DOI:10.1007/s12562-013-0625-6.
[17]孙莎.皱纹盘鲍肌肉氨肽酶的酶学特性及呈味作用研究[D].厦门:集美大学,2016:15.
[18]LAEMMLI U K.Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J].Nature,1970,227:680-685.DOI:10.1038/227680a0.
[19]United States Pharmacopoeia.The United States Pharmacopeia 23,the national formulary 18[M].Rockville,MD:The United States Pharmacopeial Convention,Inc.,1995:2053.
[20]万楚君,游银川,翁凌,等.四种加工方式对皱纹盘鲍制品消化特性的影响[J].水产学报,2017,41(6):928-936.DOI:10.11964/jfc.20161010574.
[21]LOWRY O H,ROSEBROUGH N J,FARR A L.Protein measurement with the Folin phenol reagent[J].Journal of Biological Chemistry,1951,193(1):265-275.
[22]KLOMKLAO S,BENJAKUL S,KISHIMURA H,et al.Trypsin inhibitor from yellowfin tuna(Thunnus albacores)roe:effects on gel properties of surimi from bigeye snapper(Priacanthus macracanthus)[J].LWT-Food Science and Technology,2016,65:122-127.DOI:10.1016/j.lwt.2015.07.074.
[23]ABDOLLAHI M,REZAEI M,JAFARPOUR A,et al.Dynamic rheological,microstructural and physicochemical properties of blend fish protein recovered from kilka(Clupeonella cultriventris)and silver carp(Hypophthalmichthys molitrix)by the pH-shift process or washing-based technology[J].Food Chemistry,2017,229:695-709.DOI:10.1016/j.foodchem.2017.02.133.
[24]WENG W Y,ZHENG W X.Silver carp(Hypophthalmichthys molitrix)surimi acid-induced gel extract characteristics:a comparison with heat-induced gel[J].International Journal of Food Properties,2015,18(4):821-832.DOI:10.1080/10942912.2013.864675.
[25]TIAN Y Y,WANG W,YUAN C H,et al.Nutritional and digestive properties of protein isolates extracted from the muscle of the common carp using pH-shift processing[J].Journal of Food Processing and Preservation,2017,41(1):1-9.DOI:10.1111/jfpp.12847.
[26]TAKTAK W,NASRI R,HAMDI M,et al.Physicochemical,textural,rheological and microstructural properties of protein isolate gels produced from European eel(Anguilla anguilla)by heatinduced gelation process[J].Food Hydrocolloids,2018,82:278-287.DOI:10.1016/j.foodhyd.2018.04.008.
[27]CHEN Y C,JACZYNSKI J.Protein recovery from rainbow trout(Oncorhynchus mykiss)processing byproducts via isoelectric solubilization/precipitation and its gelation properties as affected by functional additives[J].Journal of Agricultural and Food Chemistry,2007,55(22):9079-9088.DOI:10.1021/jf071992w.
[28]渡部终五,林华娟,毛伟杰.水产利用化学基础[M].北京:化学工业出版社,2017:14.
[29]杨伊然,胡晓,杨贤庆,等.蓝圆鲹蛋白酶解物的螯合矿物离子活性研究[J].食品科学,2017,38(3):88-93.DOI:10.7506/spkx1002-6630-201703015.
[30]SUN L C,YOSHIDA A,CAI Q F,et al.Mung bean trypsin inhibitor is effective in suppressing the degradation of myofibrillar proteins in the skeletal muscle of blue scad(Decapterus maruadsi)[J].Journal of Agricultural and Food Chemistry,2010,58(24):12986-12992.DOI:10.1021/jf103526e.
[31]ZHONG C,CAI Q F,LIU G M,et al.Purification and characterisation of cathepsin L from the skeletal muscle of blue scad(Decapterus maruadsi)and comparison of its role with myofibril-bound serine proteinase in the degradation of myofibrillar proteins[J].Food Chemistry,2012,133(4):1560-1568.DOI:10.1016/j.foodchem.2012.02.050.
[32]王梦想,钟婵,蔡秋凤,等.蓝圆鲹肌肉中丝氨酸蛋白酶的分离纯化及性质研究[J].食品工业科技,2012,33(19):58-62.DOI:10.13386/j.issn1002-0306.2012.19.029.
[33]VISESSANGUAN W,OGAWA M,NAKAI S,et al.Physicochemical changes and mechanism of heat-induced gelation of arrowtooth flounder myosin[J].Journal of Agricultural and Food Chemistry,2000,48(4):1016-1023.DOI:10.1021/jf9900332.
[34]EGELANDSDAL B,MARTINSEN B,AUTIO K.Rheological parameters as predictors of protein functionality:a model study using myofibrils of different fibre-type composition[J].Meat Science,1995,39(1):97-111.DOI:10.1016/0309-1740(95)80011-5.
[35]刘海梅.鲢鱼糜凝胶及形成机理的研究[D].武汉:华中农业大学,2007:88.
[36]BUAMARD N,BENJAKUL S.Improvement of gel properties of sardine(Sardinella albella)surimi using coconut husk extracts[J].Food Hydrocolloids,2015,51:146-155.DOI:10.1016/j.foodhyd.2015.05.011.