王喜波 王 健 張澤宇 陳 爽 高婷婷 江連洲
(東北農(nóng)業(yè)大學(xué)食品學(xué)院,哈爾濱150030)
物理改性對(duì)大豆蛋白柔性與乳化性的影響及其相關(guān)性分析
王喜波 王 健 張澤宇 陳 爽 高婷婷 江連洲
(東北農(nóng)業(yè)大學(xué)食品學(xué)院,哈爾濱150030)
通過不同物理改性方法(熱處理、超聲處理、高壓均質(zhì)處理、微波處理)分別得到不同柔性的大豆分離蛋白(SPI),并利用SPI對(duì)胰蛋白酶的敏感性表征柔性,研究物理改性對(duì)SPI柔性與乳化性的影響并分析兩者之間的相關(guān)性。結(jié)果表明,各改性方式對(duì)SPI柔性和乳化性產(chǎn)生不同的影響且乳化性隨柔性的上升而上升。與其他處理?xiàng)l件相比,121℃熱處理10min得到最高的柔性和乳化活性,高壓均質(zhì)處理對(duì)SPI柔性影響小但對(duì)乳化活性影響大。相關(guān)性分析結(jié)果表明:熱處理、超聲處理?xiàng)l件下SPI柔性與乳化活性、乳化穩(wěn)定性呈極顯著正相關(guān),相關(guān)系數(shù)分別為0.969、0.950和0.942、0.954。超高壓均質(zhì)處理?xiàng)l件下SPI柔性與乳化活性、乳化穩(wěn)定性呈正相關(guān),相關(guān)系數(shù)分別為0.771、0.720。微波處理?xiàng)l件下SPI柔性與乳化活性呈極顯著正相關(guān),與乳化穩(wěn)定性呈顯著正相關(guān),相關(guān)系數(shù)分別為0.976、0.862。
大豆分離蛋白;柔性;乳化性;物理改性;相關(guān)性分析
大豆分離蛋白(SPI)由于其良好的功能性質(zhì)和營(yíng)養(yǎng)價(jià)值而一直成為國(guó)內(nèi)外的研究熱點(diǎn)并被廣泛應(yīng)用于食品的多個(gè)領(lǐng)域[1-3]。大豆分離蛋白的功能性質(zhì)包括起泡性、乳化性、溶解性、凝膠性等,普遍認(rèn)為與蛋白的濃度、溶解度[4]、表面疏水性[5]、分子量[6]等相關(guān)。蛋白柔性定義為蛋白中各個(gè)結(jié)構(gòu)域的相對(duì)運(yùn)動(dòng)或者其多肽鏈中氨基酸殘基的重新取向松弛率[7]。因其在決定蛋白功能性質(zhì)尤其是界面功能性質(zhì)中的關(guān)鍵作用而受到越來(lái)越多學(xué)者的關(guān)注。
TANG等[8]研究牛血清蛋白結(jié)構(gòu)與功能性質(zhì)關(guān)系時(shí)發(fā)現(xiàn),柔性越高的蛋白越易在界面形成更好的粘彈性蛋白膜,從而表現(xiàn)出更好的乳化性質(zhì)。KATO等[9]利用不同蛋白對(duì)胰蛋白的敏感性來(lái)表征柔性,結(jié)果發(fā)現(xiàn)起泡性、乳化性與蛋白柔性具有較好的相關(guān)性,因此認(rèn)為蛋白柔性是影響乳化性和起泡性的重要因素。POON等[10]通過破壞S-S和非共價(jià)鍵得到不同柔性的蛋白,探究蛋白柔性與乳化能力的關(guān)系,并發(fā)現(xiàn)蛋白柔性的增加提高了蛋白乳化能力。這些研究主要從化學(xué)改性的角度研究蛋白柔性與功能性質(zhì)的關(guān)系,而柔性與功能性質(zhì)在物理改性條件下的關(guān)系尚未見報(bào)道研究。
本文采用加熱、超聲、高壓均質(zhì)、微波等不同方式處理SPI,研究不同處理方式對(duì)SPI柔性的影響,并分析和建立SPI柔性與乳化性相關(guān)性關(guān)系,為進(jìn)一步研究 SPI結(jié)構(gòu)與功能性質(zhì)的關(guān)系提供理論依據(jù)。
1.1 材料與試劑
大豆由東北農(nóng)業(yè)大學(xué)大豆研究所提供;大豆油(九三集團(tuán)哈爾濱惠康食品有限公司);三氯乙酸(永華精細(xì)化學(xué)品有限公司);SDS、Tris、胰蛋白酶(Sigma公司);其他試劑均為分析純。
1.2 儀器與設(shè)備
T18 Basic型高速分散機(jī)/勻漿機(jī)(德國(guó)IKA公司);LD4-2A型低速離心機(jī)(北京醫(yī)用離心機(jī)廠); TU-1800型紫外可見分光光度計(jì)(北京普析通用儀器有限責(zé)任公司);ALPHA 1-4 LSC型冷凍干燥機(jī)(德國(guó)Christ公司);ALC-310.3型分析天平(德國(guó)艾科勒ACCULAB公司);恒溫?cái)?shù)顯水浴鍋(賽普實(shí)驗(yàn)儀器廠);實(shí)驗(yàn)型高壓均質(zhì)機(jī)(英國(guó) Stansted Fluid Power公司);超聲波細(xì)胞破碎儀(寧波新芝生物科技股份有限公司);KD238-DA型微波爐(廣東美的微波爐制造有限公司)。
1.3 方法
1.3.1 SPI制備
參照SORGENTINI等[11]方法并略作改動(dòng)。大豆去皮、粉碎后過60目篩,乙醚脫脂,脫脂后豆粉與蒸餾水以0.1 g/mL的料液比混合,調(diào)pH值至8.5,室溫(25℃)低速攪拌2 h溶解。離心(4 000 g) 20min,取上清液,用2 mol/L HCl調(diào)pH值至4.5,4℃靜置12 h,去上清液,離心(4 000 g)5 min,水洗沉淀,離心2次,沉淀復(fù)溶調(diào)其pH值至7.0后冷凍干燥。
1.3.2 SPI組分測(cè)定
蛋白含量測(cè)定參照GB 5009.5—2010;含水率測(cè)定參照GB 5009.3—2010;灰分含量的測(cè)定參照GB 5009.4—2010;粗脂肪含量的測(cè)定參照 GB/T 14772—2008。
1.3.3 樣品制備
將SPI溶于緩沖液(0.2mol/L、pH值7.0磷酸鹽緩沖液),質(zhì)量濃度為20mg/mL,室溫?cái)嚢? h,再用分散機(jī)(10 000 r/min)處理1min,靜置12 h。
1.3.4 熱處理
將制備好的樣品在不同溫度60、70、80、90、100℃(水浴)及121℃(滅菌鍋)處理10min,迅速冰水冷卻5min備用。
1.3.5 超聲處理
選擇超聲功率100~800W、處理時(shí)間10 min,將超聲儀探頭浸入樣品液面約2 cm進(jìn)行超聲處理,并將樣品置于冰水浴中。
1.3.6 高壓均質(zhì)處理
選擇均質(zhì)壓力為100~450 MPa,對(duì)樣品進(jìn)行高壓均質(zhì)處理,均質(zhì)次數(shù)為1次。
1.3.7 微波處理
選擇微波處理功率為800W,處理時(shí)間為20~60 s,單次處理量為100mL。
1.3.8 柔性測(cè)定
參照KATO等[12]的方法略作改動(dòng)。利用SPI對(duì)胰蛋白酶的敏感性來(lái)表征柔性。取250μL質(zhì)量濃度1mg/mL的胰蛋白酶溶液(0.05mol/L、pH值8.0的Tris-HCl緩沖液)加入到4mL 1mg/mL處理后蛋白溶液中(蛋白溶液與胰蛋白酶溶液體積比16∶1),38℃保溫酶解5 min,酶解反應(yīng)結(jié)束后,加4mL 5%TCA終止酶解反應(yīng),離心,取上清液在280 nm測(cè)定其吸光度。用吸光度A表示柔性。
1.3.9 乳化性測(cè)定
參照TANG等[13]的方法,將改性后的蛋白樣品稀釋到2mg/mL,處理后蛋白樣品與大豆油以體積比3∶1的比例混合,分散機(jī)(10 000 r/min)處理1min,迅速吸取底部乳液50μL加入到5 mL 0.1% SDS溶液中,混合均勻。分別測(cè)定0 min和10 min吸光度,乳化活性(EAI)用吸光度A表示,乳化穩(wěn)定性計(jì)算公式為
式中 W——乳化穩(wěn)定性,min
A0——0min時(shí)測(cè)得的吸光度
A10——10min時(shí)測(cè)得的吸光度
1.3.10 數(shù)據(jù)統(tǒng)計(jì)分析
每次試驗(yàn)做3次平行,結(jié)果用平均值±標(biāo)準(zhǔn)差表示,組間差異顯著性采用t檢驗(yàn)分析(p<0.05)。數(shù)據(jù)統(tǒng)計(jì)分析采用 SPSS 17.0軟件,試驗(yàn)數(shù)據(jù)用Origin 9.0軟件繪制趨勢(shì)曲線圖。
2.1 大豆分離蛋白的組成
試驗(yàn)所制得的SPI蛋白質(zhì)、水分、灰分和粗脂肪質(zhì)量分?jǐn)?shù)分別為(87.22±0.44)%、(3.24± 0.67)%、(3.39±0.53)%和(0.47±0.06)%,符合試驗(yàn)要求。
2.2 不同物理改性對(duì)大豆分離蛋白柔性和乳化性的影響
由圖1(圖中不同小寫字母表示柔性差異顯著,下同)和圖2(圖中不同小寫字母表示乳化活性差異顯著,不同大寫字母表示乳化穩(wěn)定性差異顯著,下同)可以看出,當(dāng)處理溫度低于80℃時(shí),熱處理對(duì)SPI柔性的影響不大,當(dāng)熱處理溫度大于80℃時(shí),隨著熱處理溫度的提高,SPI的柔性不斷上升,并在121℃時(shí)柔性達(dá)到最高。伴隨著柔性上升,SPI乳化活性和乳化穩(wěn)定性也不斷上升,這與PALAZOLO等[14]的結(jié)果相似,該研究也認(rèn)為適當(dāng)?shù)臒崽幚砜梢杂行У靥岣叽蠖沟鞍椎娜榛再|(zhì)。而KATO等[15]研究熱處理對(duì)大豆伴球蛋白功能性質(zhì)影響發(fā)現(xiàn),乳化性(乳化活性和乳化穩(wěn)定性)與表面疏水性指數(shù)呈線性相關(guān)。
圖1 不同熱處理?xiàng)l件對(duì)SPI柔性的影響Fig.1 Effect of different heat treatment conditions on flexibility of soy protein isolate
圖2 不同熱處理?xiàng)l件對(duì)SPI乳化性的影響Fig.2 Effect of different heat treatment conditions on emulsion property of soy protein isolate
蛋白的柔性受氫鍵、范德華力、靜電引力和疏水相互作用等非共價(jià)鍵影響,高于80℃的熱處理可以破壞這些鍵從而使SPI緊密的結(jié)構(gòu)伸展開[16],柔性增大。而處理溫度為121℃時(shí),蛋白柔性顯著增大,可能是由于高溫處理使蛋白分子內(nèi)二硫鍵斷裂[17],造成柔性增加。柔性蛋白能快速吸附到界面,使SPI更容易形成界面膜,從而改善大豆分離蛋白的乳化性能[8,18-19]。
從圖3、4可以看出,當(dāng)處理功率小于300W時(shí),隨著功率的增加,SPI乳化活性和乳化穩(wěn)定性都隨柔性增加而增加,當(dāng)處理功率大于300W時(shí),三者變化都不大。有研究表明,超聲產(chǎn)生的空化效應(yīng)和機(jī)械效應(yīng)會(huì)破壞蛋白質(zhì)的四級(jí)結(jié)構(gòu),同時(shí)釋放出小分子的亞基和肽[20],使蛋白柔性增加而導(dǎo)致乳化性的上升。JAMBRAK等[21]研究表明,超聲處理可降低蛋白粒徑,使蛋白質(zhì)分子質(zhì)量顯著降低,還可改變蛋白中柔性區(qū)域與剛性區(qū)域的組成,從而降低了蛋白柔性。
圖3 不同超聲處理?xiàng)l件對(duì)SPI柔性的影響Fig.3 Effect of different ultrasonic treatment conditions on flexibility of soy protein isolate
由圖5、6可以看出,隨著均質(zhì)壓力的升高,SPI柔性升高,在均質(zhì)處理壓力為350MPa時(shí)達(dá)到最高,隨后下降。而SPI乳化活性在300 MPa時(shí)達(dá)到最高,乳化穩(wěn)定性則在不斷地上升。這歸因于高壓均質(zhì)產(chǎn)生的壓力作用、高頻振蕩和對(duì)流撞擊等機(jī)械力使SPI結(jié)構(gòu)發(fā)生改變,增加了柔性,從而造成乳化性的提升[22-23]。
圖4 不同超聲處理?xiàng)l件對(duì)SPI乳化性的影響Fig.4 Effect of different ultrasonic treatment conditionson emulsion property of soy protein isolate
圖5 不同高壓均質(zhì)處理?xiàng)l件對(duì)SPI柔性的影響Fig.5 Effect of different high pressure homogenization conditions on flexibility of soy protein isolate
圖6 不同高壓均質(zhì)處理?xiàng)l件對(duì)SPI乳化性的影響Fig.6 Effect of different high pressure homogenization conditions on emulsion property of soy protein isolate
由圖7、8可以看出,隨著微波處理時(shí)間的延長(zhǎng),SPI乳化活性和乳化穩(wěn)定性隨柔性上升都呈上升趨勢(shì),可能是由于微波場(chǎng)誘導(dǎo)SPI分子體系產(chǎn)生極化現(xiàn)象破壞了SPI的剛性結(jié)構(gòu),使柔性增加,內(nèi)部的疏水殘基暴露在蛋白表面,更容易在界面上展開和重排,促進(jìn)水-油界面的形成[18-19],使大豆分離蛋白乳化性質(zhì)提高。BOHR等[24]研究微波處理對(duì)球狀蛋白構(gòu)象的影響,發(fā)現(xiàn)微波處理可促進(jìn)蛋白質(zhì)結(jié)構(gòu)的柔性展開,使蛋白質(zhì)柔性增加,進(jìn)而改善蛋白質(zhì)的功能特性。
圖7 不同微波處理?xiàng)l件對(duì)SPI柔性的影響Fig.7 Effect of differentmicrowave treatment conditions on flexibility and emulsion stability of soy protein isolate
圖8 不同微波處理?xiàng)l件對(duì)SPI乳化性的影響Fig.8 Effect of differentmicrowave treatmentconditions on emulsion property of soy protein isolate
2.3 SPI柔性與乳化性相關(guān)性分析
為了進(jìn)一步分析柔性與乳化活性及乳化穩(wěn)定性之間的關(guān)系,利用Origin 7.5對(duì)其關(guān)系進(jìn)行函數(shù)擬合,結(jié)果見圖9、10。
圖9 4種改性條件下SPI柔性與乳化活性的關(guān)系Fig.9 Relatiionships of emulsifying activity with flexibility of soy protein isolate combined with fourmodification conditions
由圖9、10可知,經(jīng)過熱處理、超聲處理、高壓均質(zhì)處理和微波處理以后,隨著柔性的增加,乳化性和乳化穩(wěn)定性呈現(xiàn)遞增趨勢(shì),具有良好的線性相關(guān)關(guān)系,相關(guān)系數(shù)如表1所示。相對(duì)于其他處理?xiàng)l件,121℃熱處理10 min能得到最高的柔性和乳化活性,高壓均質(zhì)處理對(duì)SPI柔性影響小卻對(duì)乳化活性影響大。
圖10 4種改性條件下SPI柔性與乳化穩(wěn)定性的關(guān)系Fig.10 Relatiionships of emulsion stability with flexibility of soy protein isolate combined with fourmodification conditions
表1 4種改性條件下乳化活性、乳化穩(wěn)定性與柔性相關(guān)性系數(shù)Tab.1 Correlation coefficients of emulsifying activity and emulsion stability w ith flexibility of soy protein combined w ith four modification conditions
由表1可知,熱處理、超聲處理?xiàng)l件下SPI柔性與乳化活性、乳化穩(wěn)定性呈極顯著正相關(guān),相關(guān)性系數(shù)分別為0.969、0.950和0.942、0.954。超高壓均質(zhì)處理?xiàng)l件下SPI柔性與乳化活性、乳化穩(wěn)定性呈正相關(guān),相關(guān)系數(shù)分別為0.771、0.720。微波處理?xiàng)l件下SPI柔性與乳化活性呈極顯著正相關(guān),與乳化穩(wěn)定性呈顯著正相關(guān),相關(guān)性系數(shù)分別為0.976、0.862。
TORNBERG[25]認(rèn)為在形成乳液的過程中蛋白質(zhì)分子首先通過擴(kuò)散和/或?qū)α髯饔脧捏w相溶液擴(kuò)散到界面附近,并形成界面層,此過程受蛋白濃度、蛋白粒度、表面疏水性等因素的影響;其次,大部分蛋白質(zhì)分子在界面發(fā)生結(jié)構(gòu)展開;最后,吸附的蛋白質(zhì)分子以更加緊湊的結(jié)構(gòu)在界面上重排。不同的物理改性方法以不同的方式破壞了SPI的剛性結(jié)構(gòu),造成柔性的上升,使SPI更容易在界面展開并快速吸附,從而提高了SPI的乳化性(乳化活性、乳化穩(wěn)定性)。這與KITTIPHATTANABAWON[26]的觀點(diǎn)一致。KREBS等[27]探究了蛋白質(zhì)分子結(jié)構(gòu)特性與界面蛋白構(gòu)象變化的關(guān)系,證實(shí)了柔性蛋白比球蛋白更易在界面上展開和重排。MIN等[28]采用尿素和二硫基蘇糖醇(DTT)分別破壞維系蛋白質(zhì)分子剛性結(jié)構(gòu)的非共價(jià)和二硫鍵,得到更加柔性的蛋白,從而加速了蛋白質(zhì)分子在界面上的展開和重排。
也有理論認(rèn)為,由于能壘的存在,當(dāng)?shù)鞍踪|(zhì)分子到達(dá)界面后,不能立刻發(fā)生吸附,需要克服能壘后才能發(fā)生真實(shí)的吸附,能壘的大小與蛋白質(zhì)分子表面和界面間相互作用的情況相關(guān)[29]。通過外加處理改變SPI柔性也許可以降低吸附過程的能壘,從而提高SPI的乳化性質(zhì)[30]。
(1)各個(gè)改性方式對(duì)SPI柔性和乳化性產(chǎn)生不同的影響且乳化性隨柔性的上升而上升。與其他處理?xiàng)l件相比,121℃熱處理10 min得到最高的柔性和乳化活性,高壓均質(zhì)處理對(duì)SPI柔性影響小但對(duì)乳化活性影響大。
(2)熱處理、超聲處理?xiàng)l件下SPI柔性與乳化活性、乳化穩(wěn)定性呈極顯著正相關(guān),相關(guān)系數(shù)分別為0.969、0.950和0.942、0.954。超高壓均質(zhì)處理?xiàng)l件下SPI柔性與乳化活性、乳化穩(wěn)定性呈正相關(guān),相關(guān)系數(shù)分別為0.771、0.720。微波處理?xiàng)l件下SPI柔性與乳化活性呈極顯著正相關(guān),與乳化穩(wěn)定性呈顯著正相關(guān),相關(guān)性系數(shù)分別為0.976、0.862。
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Effect of Physical Modification on Flexibility and Emulsifying Property of Soy Protein and Its Correlation Analysis
WANG Xibo WANG Jian ZHANG Zeyu CHEN Shuang GAO Tingting JIANG Lianzhou
(College of Food Science,Northeast Agricultural University,Harbin 150030,China)
Effect of different physicalmodification conditions(heating treatment,ultrasound treatment,high-pressure treatment,homogenization treatment and microwave treatment)on emulsifying properties and flexibility characterized by sensitivity to trypsin of soy protein isolatewas investigated.The correlation analysis between flexibility and emulsifying properties(emulsifying activity and emulsion stability)was also investigated.The results suggested that different physicalmodification methods had different effects on flexibility and emulsifying properties of soy protein isolate,and emulsifying activity and emulsion stability were increased with the increase of flexibility.The best flexibility and emulsifying activity were got at121℃ with 10min heating treatment,comparingwith other physicalmodification conditions.Highpressure homogenization treatment had little impact on flexibility but it had great impact on emulsifying activity.Correlation analysis suggested that soy protein isolate flexibility was very significantly and positively correlated with emulsifying activity and emulsion stability under heating and ultrasound treatment conditions,correlation coefficients were 0.969,0.950 and 0.942,0.954,respectively.Flexibility of soy protein isolate was positively correlated with emulsifying activity and emulsion stability under high-pressure treatment conditions,correlation coefficients were 0.771 and 0.720,respectively.Flexibility of soy protein isolate was very significantly and positively correlated with emulsifying activity and emulsion stability under microwave treatment conditions,correlation coefficients were 0.976 and 0.862,respectively.
soy protein isolate;flexibility;emulsifying properties;physical modification;correlation analysis
TS201.2
A
1000-1298(2017)07-0339-06
2016-11-12
2016-12-17
國(guó)家大豆產(chǎn)業(yè)技術(shù)體系項(xiàng)目(CARS-04-PS25)
王喜波(1975—),男,副教授,主要從事糧食、油脂及植物蛋白工程研究,E-mail:wangxibo@neau.edu.cn
江連洲(1960—),男,教授,博士生導(dǎo)師,主要從事糧食、油脂及植物蛋白工程研究,E-mail:jlzname@163.com
10.6041/j.issn.1000-1298.2017.07.043