CN111961698A - 一种利用短乳杆菌产酶降解西番莲果皮多糖的制备方法及其应用 - Google Patents
一种利用短乳杆菌产酶降解西番莲果皮多糖的制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种利用短乳杆菌产酶降解西番莲果皮多糖的酶解修饰工艺及其应用，属于药用食品研发领域。本发明取西番莲果皮制备成西番莲果皮多糖，以短乳杆菌为实验菌株，以葡萄糖为碳源，培养产出粗酶液与多糖混匀，进行酶解得到酶解修饰后的西番莲果皮多糖。结果显示经短乳杆菌产酶降解后西番莲果皮多糖具有更好的抗氧化活性，在抗氧化类保健品和化妆品中具有广阔的应用前景。
Description
技术领域
本发明涉及一种利用短乳杆菌产酶降解西番莲果皮多糖的酶解修饰工艺及其在制备抗氧化剂中应用，属于药用食品研发领域。
背景技术
西番莲是西番莲科西番莲属的草质藤本植物，它具有多种水果的综合香味，所以也称为百香果、鸡蛋果等。因其果汁显橙黄色，色泽鲜艳，气味尤为芳香，因此享有“果汁之王”的美誉。西番莲在温暖或者高温湿润的气候适合生长，不适宜寒冷地带，在热带和亚热带地区比较多见。
西番莲是药食同源的高品质水果，具有健脾开胃、引气止痛等功能，可用于治疗多种疾病。西番莲果皮的主要功能成分是多糖，具有抗炎、抗肿瘤、抗氧化和降血脂等多种生物活性。天然多糖已经在食品，医药以及化妆品等行业进行研究开发应用，且发挥引导性作用。从西番莲果皮中提取的粗多糖，其分子量相对来说比较大，粘度也比较高，在一定程度上影响了其最大效果的发挥，所以通常会采用降解的方法获得小分子量的多糖。多糖的生物活性与许多因素有关，如溶解度和分子量大小。降解会增加多糖的亲水基，使多糖的溶解性增加，随之进一步提高多糖的活性水平。
常用的降解方法有自由基氧化降解法、超声降解法、酶解法、酸降解法等。研究显示，超声降解法等物理降解法效果不明显；酸降解是传统的化学降解方法，与其他方法比较具有快速简便的优点，而且成本相对较低，但其反应条件比较剧烈，很难控制，容易出现硫酸基脱落等副反应。酶降解法是利用酶降解多糖，得到较低分子量多糖的生物降解法，此法存在许多优点，其酶降解的特异性较强，整个降解工艺也比较环保、反应相对温和、能耗较低、降解效率较高、降解后的产物结构和活性都能得到最大限度的保护，酶催化还具有高效性和专一性。用适当方法对多糖酶降解可以增加其亲水性和溶解性，并对其抗氧化性、抗炎有很大提高，且能增强甚至产生新的生物活性。本发明利用短乳杆菌产酶降解西番莲果皮多糖，确定其结构特征，比较研究了酶降解前后多糖的抗氧化活性。关于利用短乳杆菌所产酶降解植物性多糖研究目前还未见相关报道。
发明内容
本发明的目的是提供一种利用短乳杆菌产酶降解西番莲果皮多糖的制备方法，并获得降解前后多糖抗氧化作用结果。为西番莲果皮的综合开发利用提供参考依据。
一种利用短乳杆菌产酶降解西番莲果皮多糖的制备方法。具体包括：西番莲果皮多糖的制备和短乳杆菌产酶降解西番莲果皮多糖的制备。先取西番莲果皮制备成西番莲果皮多糖粉末，以短乳杆菌为实验菌株，以葡萄糖做为碳源的MRS培养基，培养产出粗酶液，将粗酶液与多糖混匀，进行酶解得到酶解修饰后的西番莲果皮多糖。
实现本发明目的技术方案是：
一种利用短乳杆菌产酶降解西番莲果皮多糖，并获得降解前后多糖抗氧化作用。
本发明通过以下技术方案达到上述目的：
本发明提供了一种利用短乳杆菌产酶降解西番莲果皮多糖的制备方法，
(1)取西番莲果皮洗净，烘干，粉碎过20目筛；
(2)称取适量上述原料，按照料液比(1:20g/ml)加入75％乙醇，冷凝回流提取3次，每次2h，过滤，干燥，即得去除色素、酚类等物质的粉末；
(3)称取适量上述粉末，按照料液比(1:16g/ml)加入蒸馏水，100℃，3h提取两次，合并提取液进行浓缩；
(4)浓缩液离心(4000rpm 15min)，取上清液醇沉，至乙醇浓度为70％，静置48h，离心，取沉淀冷冻干燥得西番莲果皮多糖粉末；
(5)以短乳杆菌为实验菌株，接种于平板上培养；挑取长势较好的单菌落纯化。活化2次，接种斜面保存；以葡萄糖做为碳源的MRS培养基，37℃，120rpm的振荡培养箱中进行培养，将基础MRS培养基微热溶解后，将1％比例的菌种悬液接入高压灭菌后的培养基中，37℃，120rpm振荡培养。
(6)每隔4h取样，4℃，8000rpm低温离心10min，上清液即粗酶液，分离得到粗酶液；
(7)精确称取(4)中多糖粉末溶入蒸馏水中，以短乳杆菌所产酶作为水解酶，与西番莲果皮多糖混合进行酶解反应，得到酶解修饰后的西番莲果皮多糖。
附图说明
图1为实施所述不同培养时间短乳杆菌所产粗酶液酶活曲线
图2为实施所述不同降解反应时间对还原糖含量影响
图3为实施所述短乳杆菌产酶降解前后多糖对DPPH自由基清除作用
图4为实施所述短乳杆菌产酶降解前后多糖对还原性能力作用
图5为实施所述短乳杆菌产酶降解前后多糖对羟基自由基清除作用
具体实施方式
以下通过实施例对本发明的技术方案做进一步说明。
(1)西番莲果皮多糖的制备
西番莲果皮先用75％乙醇去除色素、酚类等物质。用蒸馏水浸没，料液比为1：16g/ml，100℃，3h提取两次，合并提取液进行浓缩，浓缩液4000rpm离心15min，上清液醇沉，至乙醇浓度为70％，再次离心，取沉淀冷冻干燥，收集干燥后样品即西番莲果皮多糖。
(2)短乳杆菌产酶降西番莲果皮
以短乳杆菌为实验菌株，以葡萄糖做为碳源的MRS培养基。37℃，120rpm的振荡培养箱中进行培养。分离粗酶液，与西番莲果皮多糖混合进行酶解反应即得。
菌种活化：短乳杆菌接种于平板上培养；挑取长势较好的单菌落纯化。活化2次，接种斜面保存。
(3)酶活力的测定
将基础MRS培养基微热溶解后，将1％比例的菌种悬液接入高压灭菌后的培养基中，37℃，120rpm振荡培养。每隔4h取样，4℃，8000rpm低温离心10min，上清液即粗酶液，测定酶活力，绘制曲线(图1)。根据测出的酶活曲线，确定出菌种的最佳培养时间，并分离粗酶液。将粗酶液与1mg的WPEP混合后放入40℃，120rpm振荡培养箱，每隔1h测定混合液中还原糖的含量，绘制曲线，根据趋势图确定最佳反应时间(图2)。
本实施例制得的酶解修饰西番莲果皮多糖与未修饰多糖应用于抗氧化类保健品和化妆品,具体对比效果如下：
1、经短乳杆菌产酶降解前后西番莲果皮多糖对DPPH自由基清除作用
酶降解前后西番莲果皮多糖对DPPH自由基清除能力均随着样品液浓度的增加而增大(图3)，且酶降解后西番莲果皮多糖(WPEP-E)清除率的升高情况明显高于未降解西番莲果皮多糖(WPEP)，由此可以表明WPEP-E的抗氧化能力更强，且西番莲果皮多糖经酶解修饰对DPPH自由基清除率最高达到81.51％，说明酶解修饰有利于西番莲果皮多糖清除DPPH自由基。
2、经短乳杆菌产酶降解前后西番莲果皮多糖对还原性能力作用
以抗坏血酸作为对照，同一浓度下，经短乳杆菌产酶降解的西番莲果皮多糖的还原性能力明显大于未降解的西番莲多糖(图4)，说明西番莲果皮多糖的酶解修饰有利于其还原能力。
3、经短乳杆菌产酶降解前后西番莲果皮多糖对羟基自由基清除作用
随着多糖浓度的增大，短乳杆菌产酶降解前后西番莲果皮多糖对羟基自由基清除能力也增大(图5)。且酶解修饰后西番莲果皮多糖的清除率增加趋势明显高于未降解西番莲果皮多糖，降解后清除率最高为39.09％，说明短乳杆菌产酶降解修饰西番莲果皮多糖有利于其对羟基自由基清除能力的增强。
Claims (4)
1.一种西番莲果皮多糖酶降解产物的制备方法，其特征在于具体步骤为：
(1)取西番莲果皮洗净，烘干，粉碎过20目筛；
(2)称取适量上述原料，按照料液比为1:20g/ml的比例加入75％乙醇，冷凝回流提取3次，每次2h，过滤，干燥，即得去除色素、酚类物质的粉末；
(3)称取适量上述粉末，按照料液比为1:16g/ml的比例加入蒸馏水，100℃，3h提取两次，合并提取液进行浓缩；
(4)浓缩液4000rpm离心15min，取上清液醇沉，至乙醇浓度为70％，静置48h，离心，取沉淀冷冻干燥得西番莲果皮多糖粉末；
(5)以短乳杆菌为实验菌株，接种于平板上培养；挑取长势较好的单菌落纯化，活化2次，接种斜面保存；以葡萄糖做为碳源的MRS培养基，37℃，120rpm的振荡培养箱中进行培养，将基础MRS培养基微热溶解后，将1％比例的菌种悬液接入高压灭菌后的培养基中，37℃，120rpm振荡培养；
(6)每隔4h取样，选取酶活力较强时间段分离出粗酶液，4℃，8000rpm低温离心10min，上清液即粗酶液；
(7)精确称取(4)中多糖粉末溶入蒸馏水中，以短乳杆菌所产酶作为水解酶，与西番莲果皮多糖混合进行酶解反应，得到酶解修饰后的西番莲果皮多糖。
2.根据权利要求1所述的一种西番莲果皮多糖酶降解产物的制备方法，其特征在于：步骤(5)所述实验菌株为短乳杆菌，且根据菌落长势可以活化2次及2次以上。
3.根据权利要求1所述的一种西番莲果皮多糖酶降解产物的制备方法，其特征在于：步骤(6)中根据酶活力确定出菌种的最佳培养时间为24h以后分离短乳杆菌所产粗酶液。
4.根据权利要求1所述的制备方法制备的酶降解后西番莲果皮多糖的应用，其特征在于该酶解西番莲果皮多糖应用于抗氧化类保健品与化妆品。
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