郭俊杰,馬喬治,康海岐,連喜軍
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含醇溶蛋白小麥回生抗性直支鏈淀粉性質(zhì)分析
郭俊杰1,馬喬治1,康海岐2,連喜軍1※
(1. 天津商業(yè)大學(xué),生物技術(shù)與食品科學(xué)學(xué)院,天津市食品生物技術(shù)重點(diǎn)實(shí)驗(yàn)室,天津 300134;2. 四川省農(nóng)業(yè)科學(xué)院作物研究所,成都 610066)
為研究含醇溶蛋白小麥回生抗性直支鏈淀粉性質(zhì),該文采用醇溶法從小麥粉中提取醇溶蛋白,采用回生-酶解法分離得到小麥直、支鏈淀粉。通過可見光譜、紅外光譜、X-射線衍射、差熱掃描等方法分析研究醇溶蛋白對(duì)小麥直、支鏈淀粉回生的影響。結(jié)果表明,在凝膠化及回生過程中醇溶蛋白與淀粉相互作用,導(dǎo)致淀粉回生率增加。紅外光譜研究表明,直鏈淀粉與醇溶蛋白在高壓糊化后干燥或回生的條件下,醇溶蛋白的酰胺II鍵伸縮振動(dòng)從1 546 cm?1降低至1 539 cm?1,即直鏈淀粉與醇溶蛋白通過氫鍵結(jié)合。X-射線衍射圖譜顯示在2衍射角為17°,19°,22°等的衍射峰沒有發(fā)生明顯變化,表明添加醇溶蛋白后,直、支鏈淀粉的晶型未發(fā)生明顯改變。DSC結(jié)果顯示直鏈淀粉與醇溶蛋白之間的氫鍵是在共同回生過程中產(chǎn)生的,樣品中多晶結(jié)構(gòu)和雙螺旋結(jié)構(gòu)共存。研究結(jié)果表明,淀粉中空間位阻小的6位碳原子上的羥基與醇溶蛋白中的脯氨酸和谷氨酰胺通過氫鍵結(jié)合,這種類型的氫鍵阻礙了淀粉酶對(duì)淀粉的解離,即醇溶蛋白通過與淀粉形成新型氫鍵而促進(jìn)了淀粉的回生。該研究提供了一種提高小麥淀粉的回生率的新技術(shù),為進(jìn)一步深入研究醇溶蛋白促進(jìn)淀粉回生的機(jī)理提供理論支撐。
淀粉;農(nóng)產(chǎn)品;品質(zhì)控制;醇溶蛋白;回生機(jī)理;表征
淀粉是植物儲(chǔ)備的碳水化合物,小麥中含有65%~70%的淀粉,11%~13%的蛋白以其他營(yíng)養(yǎng)組分[1]?;厣剐缘矸踇2](resistant starch 3, RS3)不被健康人體的小腸消化吸收,但能在大腸中發(fā)酵降解,該類淀粉具有廣泛的應(yīng)用:可以顯著提高食品的膨脹系數(shù)、增加耐泡性、耐煮性、保持食品松脆[3];黏度穩(wěn)定、持水力低、流變性高,常用作食品增稠劑,并可提高乳制品的益生菌活力,延長(zhǎng)貨架期[4];在體內(nèi)代謝較慢,持續(xù)提供身體能量,從而增強(qiáng)人體的工作耐力[5];能與一些膳食纖維組分相互作用,促進(jìn)類脂代謝、礦物質(zhì)吸收,抑制肥胖[6-7];可以延緩血糖升高、激活腸道免疫系統(tǒng)、控制總膽固醇量,對(duì)于糖尿病、腸道疾病及心腦血管疾病等均具有良好預(yù)防效果[8-10]。因此每日攝入適量抗性淀粉對(duì)人體健康尤為重要[11]。
回生抗性淀粉主要通過回生法制備,淀粉回生的本質(zhì)是糊化淀粉分子由高能無序態(tài)向有序態(tài)轉(zhuǎn)化的過程,即糊化后的淀粉分子借助氫鍵相互吸引,排列成序、形成高度致密的、結(jié)晶化的不溶性分子微束[12]。目前制備回生淀粉的方法主要有酸解、擠壓、壓熱處理、酶解和晶種促進(jìn)等幾種方法。其中酸解法可以顯著降低淀粉的粘度,增加結(jié)晶性,從而提高回生率[13];擠壓法和壓熱處理均使得淀粉顆粒完全破裂,直鏈淀粉分子更易于相互作用形成氫鍵,從而明顯增加回生淀粉量[14];酶解法處理淀粉會(huì)生成很多游離的直鏈淀粉,直鏈淀粉分子在冷卻老化過程中重新纏繞形成新的晶體,從而提高回生淀粉量,酶解法也是目前研究回生淀粉較多的一種方法[15]。但這些方法均不能使淀粉的回生率超過50%,低回生率也降低了該類產(chǎn)品的競(jìng)爭(zhēng)優(yōu)勢(shì),限制了其產(chǎn)業(yè)化和市場(chǎng)化,至今中國(guó)尚未有該類產(chǎn)品面世。
據(jù)報(bào)道[16-17],小麥蛋白中的醇溶蛋白促進(jìn)小麥淀粉的回生,谷蛋白卻對(duì)淀粉回生產(chǎn)生抑制作用,而醇溶蛋白與淀粉作用的機(jī)理非常復(fù)雜[18]。通過現(xiàn)代表征方法探究淀粉回生的機(jī)理一直是近年研究的熱點(diǎn)[19-21]。本文通過可見光譜、紅外光譜、X-射線衍射和差熱掃描量熱法等手段研究和表征了小麥直、支鏈淀粉與醇溶蛋白混合前后的結(jié)構(gòu)變化,探索醇溶蛋白促進(jìn)小麥淀粉回生的可能機(jī)制。
小麥粉為市售;NaCl,NaOH,I2,KI和CH3CH2OH均為分析純,由天津富?;瘜W(xué)品公司提供;高溫-淀粉酶、酸性、堿性和中性蛋白酶由天津市諾奧科技發(fā)展有限公司提供。
YXQG02手提式電熱壓力蒸汽消毒器,山東安德醫(yī)療科技有限公司;DH-101-3BS電熱恒溫鼓風(fēng)干燥箱,天津市中環(huán)實(shí)驗(yàn)電爐有限公司;BCD-229KB海爾冰箱,青島海爾股份有限公司;L535-1離心沉淀機(jī),湖南湘儀離心機(jī)儀器有限公司;Bio-Rad FES135紅外分光光度計(jì),美國(guó)Bio-Rad公司;島津UV-2450/2550紫外可見分光光度計(jì);D/max-2500 X-射線衍射分析儀,日本理學(xué)公司;Q20 差熱掃描量熱器,美國(guó)TA公司。
1.2.1 小麥面筋的制備
參照Lian等[16]的試驗(yàn)方法,將小麥面粉和水按質(zhì)量比(1:3)充分混合揉搓成團(tuán),將面團(tuán)在30℃下發(fā)酵30 min(添加0.5%的酵母),揉搓5 min后,再次于30℃發(fā)酵30 min,然后再揉搓10 min,靜置10 min。最后用水充分沖洗除去面團(tuán)中的淀粉,得到淺黃灰色面筋。
1.2.2 醇溶蛋白的提取
面筋蛋白冷凍干燥后粉碎,用95%的乙醇浸泡3 d,充分?jǐn)嚢?,將上清液中?倍體積的水,析出醇溶蛋白,充分水洗后離心分離、冷凍干燥后粉碎備用。
1.2.3 小麥直、支鏈淀粉的分離
將水洗法[22]制備的小麥淀粉按照質(zhì)量分?jǐn)?shù)10%~15%,用水分散后,于95 ℃水浴鍋中攪拌糊化2 h至透明,120 ℃進(jìn)一步高壓(0.2 MPa)糊化40 min。冷卻至常溫后放冰箱中老化2 d,高溫淀粉酶酶解、水洗后得到回生淀粉。將得到的回生淀粉中加入3倍體積的正丁醇,充分?jǐn)嚢韬箅x心分離得小麥直鏈淀粉。小麥支鏈淀粉制備采用鹽洗法,用0.15 mol/L NaOH分散400 g小麥淀粉,手動(dòng)攪拌40 min后,加入質(zhì)量分?jǐn)?shù)為5%的NaCl溶液3 600 mL,充分?jǐn)嚢韬箪o置5 min,用HCl調(diào)pH值至7.0、靜置24 h,離心分離(4 000 r/min,5 min)、鹽洗(碘液測(cè)量變?yōu)樽霞t色),離心分離后烘干(65 ℃,3 d)。
1.2.4 小麥直、支鏈淀粉與醇溶蛋白的共同回生
將得到的小麥直、支鏈淀粉用脂肪酶和堿性蛋白酶先后酶解純化后,按1.2.3法回生得回生后的小麥直、支鏈淀粉。
直、支鏈淀粉與醇蛋白共同回生過程如下:分別將干燥后的小麥直鏈和支鏈淀粉10 g與0.4 g醇溶蛋白混合,加入20 mL 蒸餾水,95℃手動(dòng)攪拌、糊化1.5 h,進(jìn)一步高溫高壓(0.2 MPa,120℃)糊化30 min,干燥后得到醇溶蛋白與淀粉直接糊化后的樣品。重復(fù)上述步驟,將所得糊化淀粉4℃老化24 h后干燥,得到醇溶蛋白與淀粉共同回生的樣品。
1.2.5 測(cè)試方法
淀粉紫外可見吸收測(cè)定:將淀粉溶于4.0 mol/L的KOH溶液中,再用鹽酸調(diào)節(jié)pH值至7.0,加1滴碘液[23],靜置30 min后,用島津UV-2450/2550紫外可見分光光度計(jì)測(cè)定最大吸收波長(zhǎng);紅外分析:將樣品用光譜純KBr壓片,在27℃下用紅外分光光度計(jì)Bio-Rad FES135測(cè)定淀粉紅外吸收,掃描范圍4 000~400 cm-1;X-射線測(cè)試采用D/max-2 500 X-射線衍射分析儀,銅靶,測(cè)試范圍2:4~50°,步長(zhǎng)0.05°;DSC測(cè)試采用Q20 差熱掃描量熱器,掃描范圍30~240℃,5 ℃/min。
碘分子可嵌入淀粉的雙螺旋結(jié)構(gòu)內(nèi)部而生成藍(lán)紫色絡(luò)合物[24-26],絡(luò)合物的最大吸收波長(zhǎng)取決于淀粉分子的重復(fù)單元數(shù)目[27]。在高溫糊化過程中,小麥直鏈淀粉的碘吸收峰消失(圖1a),表明其雙螺旋結(jié)構(gòu)被破壞,在小麥直鏈淀粉和醇溶蛋白之間形成了新的雙螺旋結(jié)構(gòu)。而在回生過程中,醇溶蛋白和小麥直鏈淀粉分離,淀粉雙螺旋結(jié)構(gòu)重新生成。小麥支鏈淀粉(圖1b)與醇溶蛋白高溫糊化及回生后,都沒有碘吸收峰出現(xiàn),表明小麥支鏈淀粉之間不能再形成雙螺旋結(jié)構(gòu)了。
圖1 不同方式處理直鏈和支鏈淀粉的可見光譜圖
圖2為不同方式處理的小麥直、支鏈淀粉的紅外光譜圖。3 400 cm?1附近處的吸收峰為淀粉分子內(nèi)和分子間O-H的伸縮振動(dòng)。2 930 cm?1附近吸收峰代表淀粉中6位碳原子亞甲基C-H鍵的伸縮振動(dòng)[28-29],直鏈及支鏈淀粉回生后,O-H和C-H的伸縮振動(dòng)移向低場(chǎng)(如羥基的伸縮振動(dòng)從3 417 cm?1移動(dòng)到3 412 cm?1,3 431 cm?1移動(dòng)到3 407 cm?1,亞甲基的吸收從2 927 cm?1移動(dòng)到2 926 cm?1,2 930 cm?1移動(dòng)到2 926 cm?1)。
小麥直鏈淀粉與醇溶蛋白在高壓糊化后干燥或回生的條件下,酰胺II鍵從1 546 cm?1(醇溶蛋白的伸縮振動(dòng))降低至1 539 cm?1,表明小麥直鏈淀粉在高壓糊化后干燥或回生的條件下都可以和醇溶蛋白結(jié)合。由于2 929 cm?1處的吸收未改變,這種結(jié)合不會(huì)是淀粉6位碳原子上的羥基與醇溶蛋白結(jié)合,應(yīng)該是二者之間形成了雙螺旋結(jié)構(gòu)。小麥支鏈淀粉在1 546 cm?1在處的吸收表明,高壓糊化后干燥或者回生,小麥支鏈淀粉均可以與醇溶蛋白結(jié)合。由于回生后的支鏈淀粉比直接干燥的吸收峰弱,表明回生過程會(huì)導(dǎo)致小麥支鏈淀粉與醇溶蛋白脫離。由于小麥支鏈淀粉的空間位阻,淀粉中所含的蛋白不能被完全被酶解,所以在小麥支鏈淀粉及回生的小麥支鏈淀粉的中1 547 cm?1處均有弱的吸收峰。
圖2 不同方式處理小麥直鏈和支鏈淀粉的紅外光譜圖
圖3為不同方式處理的小麥直、支鏈淀粉的X-射線衍射圖譜。一般來說,特定的淀粉晶型具有特定的X-射線衍射峰[30]。直鏈淀粉樣品在2衍射角為17°處出現(xiàn)最強(qiáng)衍射峰,2衍射角為19°、22°一些小衍射峰以及5.6°的特征峰的出現(xiàn)說明直鏈淀粉是A型和B型的混合晶型。在回生過程中,2衍射角為19°的衍射峰幾乎消失,表明回生使得有些晶面消失。與醇溶蛋白共同高溫糊化后干燥或者回生,直鏈淀粉晶體形貌均未發(fā)生改變。
回生后的支鏈淀粉,沒有明顯的衍射峰,說明回生使得支鏈淀粉變成無定型結(jié)構(gòu)。當(dāng)與醇溶蛋白高溫糊化并干燥后,支鏈淀粉的晶面增加,而回生后晶面反而減少了。2衍射角為5.2°衍射峰的出現(xiàn),表明支鏈淀粉與醇溶蛋白共同回生后,變?yōu)锽型結(jié)構(gòu)。
圖4和表1為不同方式處理小麥直鏈和支鏈淀粉的差熱掃描圖和熱性能特征數(shù)據(jù)。小麥支鏈淀粉具有長(zhǎng)于直鏈淀粉的分子鏈[31-32],所以支鏈淀粉的熔點(diǎn)(圖4b)高于直鏈淀粉(圖4a)。鏈長(zhǎng)越長(zhǎng),分子間的氫鍵越多[33],熔點(diǎn)越高。
在小麥直鏈淀粉及回生小麥直鏈淀粉中(圖4a),出現(xiàn)了代表雙螺旋結(jié)構(gòu)的低溫峰,也出現(xiàn)了代表多晶結(jié)構(gòu)的高溫峰,說明雜多晶結(jié)構(gòu)更加穩(wěn)定。醇溶蛋白與小麥直鏈淀粉結(jié)合后,熔點(diǎn)降低,結(jié)合的蛋白越多,熔點(diǎn)降低的也越多?;厣蟮男←溨ф湹矸郏▓D4b)顯示出3種微觀形貌,A型多晶、雙螺旋結(jié)構(gòu)、體積稍大一些的多晶。與醇溶蛋白共同回生后,支鏈淀粉熔點(diǎn)升高,說明在回生過程中醇溶蛋白與支鏈淀粉發(fā)生了分離?;旌洗既艿鞍缀笮←溁厣ф湹矸蹧]有了高溫峰(晶體熔化峰),說明支鏈淀粉附近的醇溶蛋白抑制了較大結(jié)晶的形成。
圖3 不同方式處理直鏈和支鏈淀粉的X-射線衍射圖
圖4 不同方式處理小麥直鏈和支鏈淀粉的差熱掃描圖譜
表1 不同處理方式的小麥直、支鏈淀粉的熱性能
醇溶蛋白富含脯氨酸和谷氨酰胺,高溫糊化會(huì)破壞淀粉之間的氫鍵并打開雙螺旋結(jié)構(gòu)。在回生過程中淀粉和蛋白表面重新形成氫鍵,生成雙螺旋結(jié)構(gòu)(圖5),而導(dǎo)致淀粉回生率增加。淀粉6位碳原子空間位阻較小,羥基與富含酰胺鍵的脯氨酸和谷氨酸結(jié)合形成氫鍵,與紅外研究結(jié)果一致。羥基與氨基酸這種相互作用抑制了淀粉酶酶解淀粉,即醇溶蛋白促進(jìn)淀粉回生。而當(dāng)醇溶蛋白與淀粉全部結(jié)合形成氫鍵后,就不會(huì)再促進(jìn)淀粉回生了。
圖5 醇溶蛋白促進(jìn)小麥淀粉回生機(jī)理
1)將小麥直、支鏈淀粉與小麥醇溶蛋白共混回生,研究回生前后其結(jié)構(gòu)變化??梢姽庾V表明高溫糊化過程中直鏈淀粉雙螺旋結(jié)構(gòu)被破壞,在直鏈淀粉和醇溶蛋白之間形成了新的雙螺旋結(jié)構(gòu);紅外光譜表明,在淀粉回生時(shí)添加醇溶蛋白使得直鏈淀粉之間的氫鍵減少;X-射線衍射圖譜表明,與醇溶蛋白共同高溫糊化后干燥或者回生,直鏈淀粉形貌均未發(fā)生改變;DSC結(jié)果顯示直鏈淀粉與醇溶蛋白之間的氫鍵是在共同回生過程中產(chǎn)生的,樣品中多晶結(jié)構(gòu)和雙螺旋結(jié)構(gòu)共存。
2)醇溶蛋白對(duì)小麥直、支鏈淀粉回生的促進(jìn)機(jī)理為醇溶蛋白與直、支鏈淀粉共同回生過程中,空間位阻小的6位碳原子上的羥基與醇溶蛋白中的脯氨酸和谷氨酰胺通過氫鍵結(jié)合,這種類型的氫鍵阻礙了淀粉酶辨別淀粉鏈的可水解部位,即醇溶蛋白通過與淀粉形成新型氫鍵而在它們表面結(jié)合成雙螺旋結(jié)構(gòu),從而抑制了淀粉的酶水解,增加了淀粉的回生率。
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Property analysis of resistant wheat amylose and amylopectin with wheat gliadin
Guo Junjie1, Ma Qiaozhi1, Kang Haiqi2, Lian Xijun1※
(1.300134; 2.610066)
Starch is the reserve carbohydrate in the plant kingdom. There is about 65 - 70% starch, 11 - 13% protein and some other components in wheat. Resistant starch (RS) has been investigated mainly with regard to colonic effects, glycemic index, cholesterol lowering capability, and losing weight effect. The daily intake of a certain amount of resistant starch is particularly important to human health. Retrogradation is the process of starch recrystallization which is one of the most important methods forthe preparation of RS. Gliadin, accounting for 40%-50% of wheat gluten, promotes the retrogradation of wheat starch while glutelin retards it. The objective of this research is to study the effects of external gliadin in gluten on deferent kinds of wheat starch after purified by lipaseand protease, which is a part of a large research program aimed at gaining an enhanced molecular understanding of the transformations occurring during the processing and storage of starch materials. Gliadin was isolated from wheat flour and its effect on retrogradation of wheat starch was investigated by visible absorbance (starch-iodine), IR, XRD, DSC respectively. The results showed that gliadin probably interacted with starch during the process of gelation and retrogradation, resulting in enhance of starch retrogradation. The IR spectra indicated that the addition of gliadin to wheat starch led to the reduction of hydrogen bonds between amylose. Addition of gliadin in crystal of retrograded wheat starch caused presence of two new lattice planes. The DSC results indicated that the hydrogen bond of amylose and gliadin was formed in the retrogradation progress. The polycrystal structure and the double helix reign were coexisting. The hydroxyl group of C-6 with less steric hindrance can form six-membered ring with carboxyl and acylaminogroup of prolines and glutamine by hydrogen bond respectively. Starch could combine with gliadin by hydrogen bond to form double helix during retrogradation, which resulted in the promotion of short term retrogradation of wheat starch. Gliadin and starch formed double helix in the interface of themselves that inhibit the enzymolysis of starch. This kind of hydrogen bonding might be an inhibitor for-amylase. The gliadin would not promote the retrogradation of starch anymore when all of the aminoacid formed hydrogen bond with starch. In a word, this study has provided some distinct insights into the understanding of the effects of gliadin on the retrogradation of wheat starch.
starch; agricultural products; quality control; gliadin; retrograded mechanism; characterization
2017-01-15
2018-01-31
國(guó)家自然科學(xué)基金項(xiàng)目(31571834);天津市應(yīng)用基礎(chǔ)與前沿技術(shù)研究計(jì)劃項(xiàng)目(14JCYBJC30800);天津市自然科學(xué)基金企業(yè)科技特派員項(xiàng)目(17JCTPJC53800)資助
郭俊杰,博士,副教授,研究方向?yàn)槭称房茖W(xué)。Email:gjjie@tjcu.edu.com
連喜軍,博士,副教授,主要從事回生淀粉研究。Email:lianliu2002@163.com
10.11975/j.issn.1002-6819.2018.04.036
TS231
A
1002-6819(2018)-04-0293-06
郭俊杰,馬喬治,康海岐,連喜軍. 含醇溶蛋白小麥回生抗性直支鏈淀粉性質(zhì)分析[J]. 農(nóng)業(yè)工程學(xué)報(bào),2018,34(4):293-298.doi:10.11975/j.issn.1002-6819.2018.04.036 http://www.tcsae.org
Guo Junjie, Ma Qiaozhi, Kang Haiqi, Lian Xijun. Property analysis of resistant wheat amylose and amylopectin with wheat gliadin[J]. Transactions of the Chinese Society of Agricultural Engineering (Transactions of the CSAE), 2018, 34(4): 293-298. (in Chinese with English abstract) doi:10.11975/j.issn.1002-6819.2018.04.036 http://www.tcsae.org