CN116463385A - 普鲁兰酶PulY103B在制备豌豆抗性淀粉及方便米线中的应用 - Google Patents
普鲁兰酶PulY103B在制备豌豆抗性淀粉及方便米线中的应用 Download PDFInfo
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Abstract
本发明公开了一种普鲁兰酶PulY103B在制备豌豆抗性淀粉及方便米线中的用途，普鲁兰酶PulY103B的氨基酸序列如SEQ ID NO:1所示，普鲁兰酶PulY103B能在低温25‑40℃下酶解制得具有高持水特性、较高的结晶度，相对较低溶解性和溶胀度，独特晶体结构的豌豆抗性淀粉，进一步的豌豆抗性淀粉用在制备干制方便米线中，制得的米线具有高吸水、低蒸煮损失、低断条率，复水速度快等特性，具有优良蒸煮特性的同时具有低糖释放的特性，本发明米线品质提升的同时具有作为减肥食品的潜质。
Description
技术领域
本发明属于食品生物技术领域，具体涉及一种普鲁兰酶PulY103B在制备豌豆抗性淀粉中的应用，以及豌豆抗性淀粉进一步在制备方便米线中的应用。
背景技术
抗性淀粉（RS）定义为健康人小肠内剩余的不被消化吸收的淀粉及其降解产物的总称，它不受健康人胃肠道消化酶的作用，并具有与传统膳食纤维相似的生理功效。目前关于抗性淀粉的制备主方式要有物理法（热处理、超声波处理）、化学法（酸处理）和酶法（脱支处理）。基于环境友好和经济高效的诉求，抗性淀粉制备通常采用物理法，酶法以及两者相结合的方法，目前通常采用压热法和酶法相结合的方式，在以往的酶法工艺中通常用到α-淀粉酶和普鲁兰酶，根据酶的反应条件需要调节反应温度和pH（市售的α-淀粉酶反应温度为105℃，pH为6.0；普鲁兰酶反应温度为60℃，pH为4.5），随着研究的深入，发现抗性淀粉的制备可以通过压热法和普鲁兰酶脱支的方法进行。为了节约能耗，达到环境友好，由此温和普鲁兰酶（温度在40℃左右，pH在6.0左右）尤其是I型普鲁兰酶开发势在必行（I型专一性强，在抗性淀粉制备过程中具有较大优势）。
制备抗性淀粉的原材料种类较多，目前最为常用的是玉米淀粉，然而不同原材以及不同工艺制备出的抗性淀粉特性也有所不同。豌豆是世界上最主要的豆类作物之一，我国豌豆每年总产量为500万t，占世界总产量的40.7%。豌豆营养价值很高，其组成主要为48%-52%的淀粉和24%的蛋白质。豌豆淀粉作为工业蛋白质提取过程中的副产品，比玉米、马铃薯和小麦淀粉更加便宜。豌豆淀粉中直链淀粉含量较高，是生产制备RS3型抗性淀粉的最佳原料之一。根据目前报道，制备所得的豌豆抗性淀粉含量差异较大，主要原因是原料不同（原始抗性淀粉含量不同），在已有的酶法制备豌豆抗性淀粉的报道中，抗性淀粉含量增长率均小于1.2。同时目前还未有用温和普鲁兰酶制备豌豆抗性淀粉的报道。
抗性淀粉可以分为五类，即物理包埋淀粉（RS1）、抗性淀粉颗粒（RS2）、回生淀粉（RS3）、化学改性淀粉（RS4）和直链淀粉-脂质复合物（RS5）。抗性淀粉因组成不同，其品质及控糖能力会有较大的差异。抗性淀粉在功能性食品尤其是减肥食品和糖尿病食品中发挥积极的作用。同时抗性淀粉在提升食品特质方面也具有其优势。例如高热稳定性、以及低溶解度和膨胀度。
目前减肥食品和糖尿病食品品类包括面包、饼干以及意大利面等，但仍无法满足市场需求，尤其是亚洲市场，亚洲人喜好米类制品，米线便是代表之一。研究报道中不乏提升米线品质及提升米线功能的研究。目前已有用含抗性淀粉高的天然淀粉（RS2）与米线基料混合来达到以上需求。但米线吸水性方面仍存在缺陷，同时在添加RS3的方式现在仍是空白。
发明内容
本发明提供了一种采用普鲁兰酶PulY103B制备豌豆抗性淀粉的方法，所述普鲁兰酶PulY103B的氨基酸序列如SEQ ID NO:1所示。
为了实现本发明的上述目的，本发明提供了如下的技术方案：
1、普鲁兰酶PulY103B制备
取含有重组质粒PulY103B-PET23b的BL21单菌落，接种于10mL含50µg/mL氨苄青霉钠盐的LB液体培养基中，于37℃摇床以250 rmp的转速培养6-7h，将诱导种子液以1%的接种量转入含50µg/mL氨苄青霉钠盐的LB液体培养基中，于37℃摇床以250rmp的转速培养至菌液浓度OD600为0.8左右（生长对数期）时，加入诱导剂IPTG使其终浓度达到0.6 mM，于16 ℃恒温摇床180rmp诱导8 h；10000g冷冻离心10min收集培养基中的细胞，用含10mM咪唑、500mM NaCl的20mM pH 7.4磷酸缓冲液重悬细胞沉淀，以10000g冷冻离心10min，重复两到三次，超声法破碎细胞，10000 g冷冻离心20 min，收集上清液，即为普鲁兰酶PulY103B酶液。
2、豌豆抗性淀粉的制备
以豌豆原淀粉（PS-N）为原料，用50mM、pH6.5的磷酸缓冲液配制质量体积浓度8-10%的淀粉乳，淀粉乳在80-90℃下磁力搅拌预糊化20-30min后，冷却至25-40℃，按60-100U/g干淀粉的比例，添加普鲁兰酶PulY103B酶解10-14h，酶解完后取出放入高压灭菌锅中，在121℃下压热20-30min，取出后冷却至室温，放置于4℃下老化回生18-30h，老化完成后置于50℃烘箱烘干至恒重，用打粉机粉碎并过100目筛，取筛下物装袋备用，即得豌豆抗性淀粉PS-PA。
3、豌豆抗性淀粉特性分析
本发明采用差示扫描量热仪（DSC）方法测定其热稳定性，采用核磁共振（XRD）检测晶体结构、结晶度，采用Feng等人的方法测定溶解性和溶胀度（Feng, Y., Juliet, I. C.,Qin, W., Duan, Y., Gao, X., Liu, Q., ... & Ma, H. (2022). Rheology, In VitroDigestion and Functional Properties of Sagittaria sagittifolia L. ResistantStarch as Affected by Different Preparation Methods. Starch‐Stärke, 74(7-8),2100181.）；结果显示本发明制得的豌豆抗性淀粉具有较高的结晶度，相对较低的溶解性和溶胀度，具有与以往报道豌豆抗性淀粉不同的晶体结构。
本发明另一目的是将上述方法制得的豌豆抗性淀粉应用在干制方便米线制备中，豌豆抗性淀粉的添加量为米粉质量的6-10%。
本发明的优点和技术效果：
本发明提供了一种温和条件下利用普鲁兰酶高效制备豌豆抗性淀粉的方法，所制备的抗性淀粉具有高持水特性，良好的、较高的结晶度，相对较低的溶解性和溶胀度，制得的方便米线具有高吸水、低蒸煮损失、低断条率，复水速度快等特性，具有优良蒸煮特性的同时具有低糖释放的特性，本发明方便米线在品质提升的同时具有作为减肥食品的潜质。
附图说明
图1为豌豆淀粉样品扫描电镜，PS-N，豌豆原淀粉；PS-A，压热法豌豆抗性淀粉；PS-PA，酶法/压热法豌豆抗性淀粉；
图2为豌豆淀粉样品核磁共振图谱，PS-N，豌豆原淀粉；PS-A，压热法豌豆抗性淀粉；PS-PA，酶法/压热法豌豆抗性淀粉。
实施方式
下面通过实施例对本发明作进一步详细说明，但本发明的内容并不局限于此，本实施例中方法如无特殊说明的均按常规方法操作，所用试剂如无特殊说明的均采用常规试剂或按常规方法配置的试剂；
实施例中方便米线的复水时间检测方法为白宝兰等研究方法（白宝兰, & 高巍.(2012). 荞麦米线加工工艺研究. 食品研究与开发, 33(1), 102-105.），吸水率、蒸煮损失、断条率检测采用Qiao等人的方法（Qiao, F., Wang, L., Guan, C., Long, X., Lin,Q., & Liu, C. (2023). Effects of soybean 7S protein on the quality anddigestibility of dry rice noodles under twin‐screw extrusion process.International Journal of Food Science & Technology, 58(1), 463-472.）。葡萄糖释放采用Srikaeo等人的方法检测（Srikaeo, K., & Sangkhiaw, J. (2014). Effects ofamylose and resistant starch on glycaemic index of rice noodles. LWT-Food Science and Technology, 59(2), 1129-1135.）。
以下实施例同时以压热法制备的豌豆抗性淀粉（PS-A）为对照，压热法制备工艺是：以豌豆原淀粉（PS-N）为原料，用50mM pH 6.5的PB缓冲液分别配制成10%的淀粉乳，放入85℃的恒温水浴锅中磁力搅拌预糊化30min，取出样品放入高压灭菌锅中，在121℃的条件下压热20-30min，取出后冷却至室温，放置于4℃冰箱老化回生24 h，老化完成后放置于50℃烘箱烘干至恒重，用打粉机粉碎并过100目筛，取筛下物装袋备用。
实施例1：以豌豆原淀粉（PS-N）为原料，用50mM、pH6.5的PB缓冲液配制质量体积浓度8%的淀粉乳，淀粉乳放入85℃的恒温水浴锅中磁力搅拌预糊化20min，冷却至25℃，按60U/g干淀粉的比例添加普鲁兰酶PulY103B酶解14 h后，取出样品放入高压灭菌锅中，在121℃下压热20 min，取出后冷却至室温，放置于4℃冰箱中老化回生18 h，老化完成后放置于50℃烘箱烘干至恒重，用打粉机粉碎并过100目筛，取筛下物装袋备用，得样品PS-PA1，抗性淀粉含量23.5%，与PS-N相比，抗性淀粉增长率为1.9（见表1）；
本实施例豌豆抗性淀粉扫描电镜见图1，相比于PS-N、PS-A从图中可以看出PS-PA1颗粒较小，棱角更尖锐，这样的外观结构对其抗酶解特性有增强作用。
豌豆抗性淀粉的晶体结构特征见图2，X-衍射数据表明本实施例酶法制备的豌豆抗性淀粉（PS-PA）具有与以往报道不同的晶体结构B+V型，典型衍射角：5.9°, 17.0°,19.2°, 21.9°以及23.9°，以往报道的豌豆抗性淀粉19°附近没有衍射角。
实施例2：以豌豆原淀粉（PS-N）为原料，用50mM、pH6.5的PB缓冲液配制质量体积浓度8%的淀粉乳，淀粉乳放入85℃的恒温水浴锅中磁力搅拌预糊化20 min，冷却至30℃，按80U/g干淀粉的比例添加普鲁兰酶PulY103B酶解12 h后，取出样品放入高压灭菌锅中，在121℃下压热20min，取出后冷却至室温，放置于4℃冰箱中老化回生18 h，老化完成后放置于50℃烘箱烘干至恒重，用打粉机粉碎并过100目筛，取筛下物装袋备用，得样品PS-PA2，抗性淀粉含量27.5%，与PS-N相比，抗性淀粉增长率为2.35（见表1）；
实施例3：以豌豆原淀粉（PS-N）为原料，用50 mM、pH6.5的PB缓冲液配制质量体积浓度10%的淀粉乳，淀粉乳放入85℃的恒温水浴锅中磁力搅拌预糊化25min，冷却至35℃，按80 U/g干淀粉的比例添加PulY103B，酶解12 h，取出样品放入高压灭菌锅中，在121℃下压热25 min，取出后冷却至室温，放置于4℃冰箱老化回生24 h，老化完成后放置于50℃烘箱烘干至恒重，用打粉机粉碎并过100目筛，取筛下物装袋备用，得样品PS-PA3；抗性淀粉含量30.0%，与PS-N相比，抗性淀粉增长率为2.66（见表1）。
实施例4：以豌豆原淀粉（PS-N）为原料，用50 mM、pH6.5的PB缓冲液配制质量体积浓度12%的淀粉乳，淀粉乳放入85℃的恒温水浴锅中磁力搅拌预糊化30min，冷却至40℃，按100U/g干淀粉的比例添加PulY103B，酶解10h，取出样品放入高压灭菌锅中，在121℃下压热30 min，取出后冷却至室温，放置于4℃冰箱老化回生30 h，老化完成后放置于50℃烘箱烘干至恒重，用打粉机粉碎并过100目筛，取筛下物装袋备用，得样品PS-PA4，抗性淀粉含量28.1%，与PS-N相比，抗性淀粉增长率为2.43（见表1）。
上述实施例制得的豌豆抗性淀粉的特性见表1，从表1可以看出酶法制备的豌豆抗性淀粉（PS-PA）具有高持水特性（与以往报道不同），低溶解度及膨胀度，同时具有良好的热稳定性，这些物性预示其在改善淀粉类食品品质上具有良好的潜力；这些潜力与其淀粉分子特征（图1）以及晶体结构特征（图2）一致；
表1 豌豆抗性淀粉特性
。
上述实施例制得的豌豆抗性淀粉在制备方便米线中的应用
1、称取籼米粉500g，用400mL蒸馏水浸泡2h，搅拌均匀，然后加入85℃加热糊化后的抗性淀粉（加入籼米粉质量6%的豌豆抗性淀粉PS-PA3），混匀，混合物经米线机挤压制得米线，收集米线，老化4 h，置于烘箱中50℃热风干燥40 min即为干制方便米线（CRN1）；
2、称取500g籼米粉，用400mL蒸馏水浸泡3h，搅拌均匀，然后加入85℃加热糊化后的抗性淀粉（加入籼米粉质量8%的豌豆抗性淀粉PS-PA3），混匀，混合物经米线机挤压制得米线，收集米线，老化4h，置于烘箱中55℃热风干燥40min即为干制方便米线（CRN2）；
3、称取500g籼米粉，用400mL蒸馏水浸泡4h，搅拌均匀，然后加入85℃加热糊化后的抗性淀粉（加入籼米粉质量10%的豌豆抗性淀粉PS-PA3），混匀，混合物经米线机挤压制得米线，收集米线，老化4h，置于烘箱中60℃热风干燥40min即为干制方便米线（CRN3）；
同时只采用籼米粉传统配方制得的米线为对照；
制得的方便米线的特性见表2，从表2可以看出复配豌豆抗性淀粉PS-PA制备的方便米线相较于对照具有较短的复水时间，较高的吸水能力（与以往报道不同，以往报道混合其它淀粉原料米线吸水能力普遍存在下降趋势），较低的蒸煮损失和断条率；同时通过体外消化实验发现相对于对照复配豌豆抗性淀粉PS-PA的方便米线糖释放低于对照，为对照的85.8%、80.9%、78.7%，同等条件下低于现有报道，由此可见本发明所述的方便米线具有作为减肥及糖尿病食品的潜质；
表2方便米线特性
。
Claims (5)
1.普鲁兰酶PulY103B在制备豌豆抗性淀粉中的应用，所述普鲁兰酶PulY103B的氨基酸序列如SEQ ID NO:1所示。
2.根据权利要求1所述的应用，其特征在于：普鲁兰酶PulY103B在25-40℃下酶解制备豌豆抗性淀粉。
3.根据权利要求1所述的应用，其特征在于：豌豆抗性淀粉的晶体结构为B+V型，典型衍射角为5.9°、17.0°、19.2°、21.9°、23.9°。
4.权利要求1所述应用中制得的豌豆抗性淀粉在干制方便米线制备中的应用。
5.根据权利要求4所述的应用，其特征在于：豌豆抗性淀粉的添加量为米粉质量的6-10%。
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| CN117106833B (zh) * | 2023-08-29 | 2024-03-19 | 江西省科学院微生物研究所(江西省流域生态研究所) | 一种利用酶解协同压热处理制备的高益生性莲子抗性淀粉及其制备方法和应用 |
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