CN109380641A - 一种磁性固定化酶稳定新鲜米糠的方法 - Google Patents

一种磁性固定化酶稳定新鲜米糠的方法 Download PDF

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CN109380641A
CN109380641A CN201811156671.1A CN201811156671A CN109380641A CN 109380641 A CN109380641 A CN 109380641A CN 201811156671 A CN201811156671 A CN 201811156671A CN 109380641 A CN109380641 A CN 109380641A
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papain
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于殿宇
张如春
唐洪琳
陈书曼
吴楠
江连洲
王立琦
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Northeast Agricultural University
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Abstract

本发明提供一种用磁性固定化酶稳定新鲜米糠的方法。首先研究了米糠中易被脂肪酶酶解的底物空间结构，筛选出与底物空间结构相似的木瓜蛋白酶等酶，将木瓜蛋白酶进行磁性固定化，提高了其热稳定性，载酶量达到123.5±1.20mg/g，相对酶活达到2050±20U/g。将其添加到新鲜的米糠中，在米糠含水量24％、磁性固定化木瓜蛋白酶添加量为0.7g、反应时间100min、温度50℃时，脂肪酶酶活力最低(以吸光度记)，吸光度达到0.89，可以有效的钝化米糠中的脂肪酶，避免了底物被酶解。磁性固定化木瓜蛋白酶在重复使用5次后次酶活为78％，其与底物的分离率达到100％，解决了传统游离蛋白酶酶稳定米糠不可重复利用的问题。

Description

一种磁性固定化酶稳定新鲜米糠的方法
技术领域
本发明涉及稻谷加工副产物处理领域，具体涉及磁性固定化酶稳定新鲜米糠的方法。
背景技术
米糠是稻谷加工中主要副产物，是糙米碾白过程中被碾下的皮层及米胚和少量碎米的混合物。据美国农业部研究报告称，稻谷中64％的营养素集中在米糠中，被誉为“天赐营养源”，同时被联合国工业发展组织(UNIDD)称之为“一种极具潜力未被充分利用的资源”。我国是世界第一大稻谷生产国，年产稻谷20,000万t，约占世界总产量29.3％，预计到2020年为20,700万t，米糠产量以8％计，我国年产米糠1600万t以上。
但米糠本身是一种化学性质不稳定的物系，含有脂肪水解酶和过氧化物酶，脂肪水解酶活性很高，碾米后与油脂相互接触，油脂的水解作用迅速发生，产生游离脂肪酸，然后在过氧化物酶、光、热等因素的共同作用下发生脂肪氧化酸败，使米糠酸败变质，米糠的酸价以每小时0.5％～2％的速度递增，数小时后，米糠就呈现不被人接受的霉味，不易储存。
目前国内外有很多稳定米糠的方法，其中酶法稳定米糠由于其作用条件温和且可以更好的保存米糠的营养价值而受到更多的青睐。酶法稳定米糠的作用方式为竞争性抑制，即酶与米糠中脂肪水解酶结构相似，能与底物(米糠中的油脂)竞争脂肪水解酶的活性中心，从而阻碍脂肪水解酶与底物复合物的形成，使脂肪水解酶的活性降低。常用的酶有木瓜蛋白酶、碱性蛋白酶、中性蛋白酶、菠萝蛋白酶等，其中木瓜蛋白酶效果比较理想，但是由于游离酶对反应环境敏感、易变性失活、反应条件不易控制等缺点，导致酶法稳定米糠在实际应用中效果并不明显。同时，游离酶与底物混合后不易分离也是酶法稳定米糠的一大弊端。从现有的信息看，将游离酶进行固定化，可提高它的稳定性和重复利用效率。
本发明首先研究了米糠中易被脂肪水解酶酶解的底物空间结构，筛选出与底物空间结构相似的木瓜蛋白酶、菠萝蛋白酶及碱性蛋白酶等，将木瓜蛋白酶进行磁性固定化，提高了木瓜蛋白酶的热稳定性，将其添加到新鲜的米糠中，磁性蛋白酶可以有效的钝化米糠中的脂肪水解酶，避免了底物被酶解，同时当使用新鲜米糠时，通过磁场将磁性蛋白酶与底物快速分离，提高了酶的重复利用率，降低了生产成本。
该方法同时可以使用菠萝蛋白酶及碱性蛋白酶等，具有同样的效果。
发明内容
本发明为了解决现有酶法稳定米糠时酶的重复利用率低、生产成本大等问题，而提出了一种磁性固定化木瓜蛋白酶稳定米糠的方法。本发明以FeCl3和FeSO4制备磁性Fe3O4纳米粒子，利用Fe3O4纳米粒子和SiOx复合，从而得到Fe3O4/SiOx复合粒子，利用APTS化学改性法修饰Fe3O4/SiOx复合粒子，即引入多官能团，改性后得到Fe3O4/SiOx-g-P(GMA)载体，将木瓜蛋白酶进行固定化，再将其加入米糠中。其主要影响因素为磁性固定化木瓜蛋白酶稳定米糠过程中米糠的含水量、磁性固定化木瓜蛋白酶添加量、酶解时间及酶解温度。
具体实施方式：
具体实施方式一：首先称取200.0g新鲜米糠，将其进行粉碎过100目筛，加水调节米糠含水量为12％-24％，加入一定量磁性载体Fe3O4固定化木瓜蛋白酶，其载酶量达到123.5±1.20mg/g，相对酶活达到2050±20U/g，搅拌使其充分混匀，在反应一定时间及温度后，降温至37℃测量脂肪水解酶活性，然后将磁性固定化木瓜蛋白酶与底物分离，再将其添加到新鲜的米糠中进行重复试验，验证磁性固定化木瓜蛋白酶重复使用次数，测其酶活力和计算分离率。
具体实施方式二：本实施方式与具体实施方式一的不同点在于调节磁性载体固定化木瓜蛋白酶添加量为0.5g-1.0g，其它步骤与具体实施方式一相同。
具体实施方式三：本实施方式与具体实施方式一的不同点在于调节反应时间为40min-120min，其它步骤与具体实施方式一相同。
具体实施方式四：本实施方式与具体实施方式一的不同点在于调节反应温度为40℃-70℃，其它步骤与具体实施方式一相同。
具体实施方式五：本实施方式与具体实施方式一的不同点在于重复将磁性固定化木瓜蛋白酶添加到反应体系中，反应完成后，测定磁性固定化木瓜蛋白酶酶活，其它步骤与具体实施方式一相同。

Claims (5)

1.一种磁性固定化酶稳定新鲜米糠的方法，本发明为了解决现有酶法稳定米糠时酶的重复利用率低、生产成本高等问题，而提出了一种磁性固定化木瓜蛋白酶稳定米糠的方法，本发明以FeCl3和FeSO4制备磁性Fe3O4纳米粒子，利用Fe3O4纳米粒子和SiOx复合，从而得到Fe3O4/SiOx复合粒子，利用APTS化学改性法修饰Fe3O4/SiOx复合粒子，即引入多官能团，改性后得到Fe3O4/SiOx-g-P(GMA)载体，将木瓜蛋白酶进行固定化，再将其加入米糠中，其主要影响因素为磁性固定化木瓜蛋白酶稳定米糠过程中米糠的含水量、磁性固定化木瓜蛋白酶添加量、酶解时间及酶解温度；其特征在于，利用磁性固定化木瓜蛋白酶稳定米糠的方法通过以下步骤实现：步骤一、加工前处理：以FeCl3和FeSO4制备磁性Fe3O4纳米粒子备用，然后将木瓜蛋白酶进行磁性固定化，测量其载酶量及酶活；称取200.0g新鲜米糠，将其进行粉碎过100目筛；步骤二、酶法稳定：加水调节米糠含水量为12％-24％，调节磁性复合载体固定化木瓜蛋白酶添加量为0.5-1.0g，反应时间为30-90min，反应温度为40-80℃；步骤三、脂肪水解酶活性测定：取降温至37℃的米糠1g，加入3mL含0.5％曲拉通的0.1mol/L的磷酸缓冲液中，再加入含有硝基苯酚月桂酸的异丙醇溶液，于37℃水浴4h，煮沸5min，6000r/min离心8min，取上清液于410nm测吸光度；步骤四、将使用后的磁性固定化木瓜蛋白酶重复使用，反应完成后，将磁性固定化木瓜蛋白酶与底物分离，测定其酶活力以及计算其分离率。
2.根据权利要求1所述的一种磁性固定化酶新鲜稳定米糠的方法，其特征在于步骤二中将米糠的含水量调节到12％-24％。
3.根据权利要求1所述的一种磁性固定化酶稳定新鲜米糠的方法，其特征在于步骤二中将磁性复合载体固定化木瓜蛋白酶添加量为0.5g-1.0g。
4.根据权利要求1所述的一种磁性固定化酶稳定新鲜米糠的方法，其特征在于步骤二中将反应时间调节为30min-90min。
5.根据权利要求1所述的一种磁性固定化酶法稳定新鲜米糠的方法，其特征在于步骤二中将反应温度调节到40℃-80℃。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112790326A (zh) * 2021-02-01 2021-05-14 广东省农业科学院蚕业与农产品加工研究所 一种复合酶稳定米糠的方法

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* Cited by examiner, † Cited by third party
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