CN112877382B - 一种酶法制备磷酸酯淀粉的方法 - Google Patents
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Abstract
本发明公开了一种酶法制备磷酸酯淀粉的方法，属于农产品深加工以及变性淀粉制备方法领域。本发明所述的方法，包括如下步骤：(1)将淀粉和磷酸盐缓冲液混合均匀，得到淀粉乳；(2)将磷酸酶加入磷酸盐缓冲液中溶解，并加入催化离子，混合均匀、活化得到酶液；(3)将酶液分次加入淀粉乳中进行反应；(4)反应结束之后调节体系为中性，之后洗涤、离心、干燥、粉碎，得到磷酸酯淀粉；其中，步骤(1)中淀粉与步骤(2)中酶的质量比为30～300：1；步骤(1)所述淀粉乳的固形物浓度为20％～60％。本发明的方法反应温度低于40℃，反应时间小于5h，反应效率高，而且制备的磷酸酯淀粉可以高达0.806。
Description
技术领域
本发明涉及一种酶法制备磷酸酯淀粉的方法，属于农产品深加工以及变性淀粉制备方法领域。
背景技术
磷酸酯淀粉是淀粉与磷酸盐发生酯化反应得到的淀粉衍生物，由于其具有易糊化、透明度高、粘度高、冻融稳定性好等优良使用特性，在食品添加剂与非食品添加剂中都有广泛应用。磷酸酯淀粉可作为乳化剂、增稠稳定剂应用于多种食品中，如奶油、奶酪、果酱、冷冻食品等，在非食品工业中，磷酸酯淀粉能用作絮凝剂和离子交换剂，也能作为水凝胶和高吸水剂在化妆品、医药、农业中应用。
磷酸酶是一种广泛存在于植物、动物和微生物体内的一种重要调控酶，可催化各种磷酸基团的水解和转移反应；通过控制酶反应条件，酶反应效果可在水解与合成相互转化；与以ATP(三磷酸腺苷)为供体的激酶相比，磷酸酶使用成本较磷酸盐为磷供体，具有一定的经济优势。
发明内容
[技术问题]
目前生产磷酸酯淀粉的方法主要有以下几种：干法、湿法、半干法、挤压法、微波法。其中，湿法生产产品收率低、干法与半干法由于搅拌不易均匀导致产品质量不稳定。此外，化学法生产的淀粉均存在产物复杂、副产物多、分离困难等问题。
[技术方案]
为了解决上述至少一个问题，本发明提供了一种酶法制备磷酸酯淀粉的方法。相较于化学法生产的淀粉磷酸酯，生物酶法提高了反应效率和产率；而且由于酶具有选择性使得得到的产品质量较为均一；此外，酶法反应条件较为温和，工艺简单，反应效率高，使用设备少，具有工业化生产的前景。
本发明的第一个目的是提供一种酶法制备磷酸酯淀粉的方法，包括如下步骤：
(1)将淀粉和磷酸盐缓冲液混合均匀，得到淀粉乳；
(2)将磷酸酶加入磷酸盐缓冲液中溶解，并加入催化离子，混合均匀、活化得到酶液；
(3)将步骤(2)的酶液分次加入步骤(1)的淀粉乳中进行反应；
(4)反应结束之后调节体系为中性，之后洗涤、离心、干燥、粉碎，得到磷酸酯淀粉；
其中，步骤(1)中淀粉与步骤(2)中磷酸酶的质量比为30～300：1；步骤(1)中磷酸盐缓冲液与步骤(2)中磷酸盐缓冲液的体积比为1.0～2.5：1；步骤(1)所述淀粉乳的固形物浓度为20％～60％(m/v)。
在本发明的一种实施方式中，步骤(1)中淀粉与步骤(2)中酶的质量比为30：0.1～0.5。
在本发明的一种实施方式中，步骤(3)所述的反应体系中淀粉的浓度为25-35％(w/w)，进一步优选为30％。
在本发明的一种实施方式中，步骤(1)所述的淀粉包括马铃薯淀粉、普通玉米淀粉、蜡质玉米淀粉、木薯淀粉、大米淀粉、豌豆淀粉、甘薯淀粉、绿豆淀粉。
在本发明的一种实施方式中，步骤(1)和步骤(2)所述磷酸盐缓冲液的摩尔浓度为0.1～1.0M；其中磷酸盐包括正磷酸盐、聚磷酸盐、偏磷酸盐、焦磷酸盐。
在本发明的一种实施方式中，步骤(1)所述的淀粉乳需要保持在20～40℃，这样可以使得其和磷酸酶的最适反应温度一致，便于进行反应。
在本发明的一种实施方式中，步骤(2)所述活化的温度为20～40℃，时间为10～50min。
在本发明的一种实施方式中，步骤(2)中磷酸酶包括酸性磷酸酶、碱性磷酸酶。
在本发明的一种实施方式中，步骤(2)所述的催化离子包括但不限于Cu2+、Zn2+、Fe2 +、Ca2+、Mg2+、Fe3+；所述的催化离子在酶液中的浓度为0.001～1M；进一步优选为所述的催化离子为氯化锌和氯化镁，其在酶液中的浓度分别为0.1M。
在本发明的一种实施方式中，步骤(2)所述的分次是分2～5次加入，多次加入不断促进反应正向进行。
在本发明的一种实施方式中，步骤(3)所述反应的pH为7～10，所述的pH是通过在淀粉乳中加入碱性化合物进行调节，碱性化合物包括但不限于碳酸盐、氢氧化物。
在本发明的一种实施方式中，步骤(3)所述反应的时间为1～5h。
在本发明的一种实施方式中，步骤(3)所述反应的温度为20～40℃。
在本发明的一种实施方式中，步骤(4)所述的调节体系至中性具体为：采用酸溶液中和体系pH为7，其中采用的酸溶液包括但不限于盐酸溶液、碳酸溶液、乙酸溶液等，进一步优选为采用盐酸溶液调节淀粉乳的pH＝7。
在本发明的一种实施方式中，步骤(4)所述的洗涤是采用水洗。
在本发明的一种实施方式中，步骤(4)所述的离心是3500rpm～8000rpm下离心5～20min。
在本发明的一种实施方式中，步骤(4)所述的干燥可以由常用的各种设备完成，可以但不限于冷冻干燥、真空干燥、鼓风干燥、喷雾干燥。
在本发明的一种实施方式中，步骤(4)所述的粉碎可以由磨粉机进行，要求筛分后淀粉能过100目筛。
本发明的第二个目的是本发明所述的方法制备得到的磷酸酯淀粉。
本发明的第三个目的是本发明所述的磷酸酯淀粉在食品、化妆品、医药、造纸、建材、纺织、农业领域等中的应用。
在本发明的一种实施方式中，所述在食品领域的应用包括将磷酸酯淀粉作为乳化剂、增稠稳定剂用于在食品中，所述的食品包括奶油、奶酪、果酱、酸奶、肉制品等。
在本发明的一种实施方式中，所述的应用为将磷酸酯淀粉作为絮凝剂、离子交换剂、高吸水剂应用在建材、纺织、造纸、化妆品、医药、农业领域中。
[有益效果]
(1)与化学法生产工艺相比，本发明的方法反应温度低于40℃，反应时间小于5h，减小了反应过程中的能耗，反应效率高。
(2)本发明制备得到的磷酸酯淀粉取代度高，在磷酸盐供体较为单一且摩尔浓度在1.0M以下时，所制备的磷酸酯淀粉的取代度达到0.011以上，可以高达0.806。
(3)本发明制备得到的磷酸酯淀粉质量稳定，由于酶反应具有选择性，在控制反应温度与反应pH的情况下，酶反应产物相较化学法更简单，且副反应少，便于产品应用。
附图说明
图1为本发明所述的酶法制备磷酸酯淀粉的方法的工艺流程图。
图2为普通玉米原淀粉与实施例1磷酸酯淀粉的糊化特性曲线。
图3为普通玉米原淀粉与实施例2磷酸酯淀粉的糊化特性曲线。
图4为普通玉米原淀粉与实施例3磷酸酯淀粉的糊化特性曲线。
图5为普通玉米原淀粉与实施例4磷酸酯淀粉的糊化特性曲线。
图6为普通玉米原淀粉与实施例5磷酸酯淀粉的糊化特性曲线。
图7为普通玉米原淀粉与实施例6磷酸酯淀粉的糊化特性曲线。
图8为普通玉米原淀粉与实施例7磷酸酯淀粉的糊化特性曲线。
图9为普通玉米原淀粉与对照例1、2磷酸酯淀粉的糊化特性曲线。
具体实施方式
以下对本发明的优选实施例进行说明，应当理解实施例是为了更好地解释本发明，不用于限制本发明。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
测试方法：
1、磷酸酯淀粉取代度测定
结合磷的测定方法：按照国标GB 5009.87-2016中的第一法，钼蓝分光光度法；计算所得的磷含量为结合磷质量分数。
游离磷的测定方法：称取2g试样，用1M的稀盐酸溶解并定容，显色的方法同结合磷的测定方法钼蓝分光光度法。
取代度的计算公式如下：
2、糊化特性测定
使用快速粘度测试仪(RVA)测试样品的糊化特性，在RVA铝盒中配制样品浓度6％(w/w)的悬浮液；按照AACC规定的标准程序2进行测定。
实施例1
一种酶法制备磷酸酯淀粉的方法，工艺流程如图1所示，包括如下步骤：
(1)将30g玉米淀粉加入50g 0.25M焦磷酸钠缓冲液中搅拌均匀制备得到淀粉乳，保持温度37℃；
(2)将0.1g碱性磷酸酶酶粉加入20g 0.25M焦磷酸钠缓冲液中溶解，并加入氯化锌和氯化镁，在37℃下活化30min；得到酶液；其中氯化锌和氯化镁的浓度分别为0.018M；
(3)将步骤(2)的酶液平均分两次加入步骤(1)的淀粉乳中进行反应，反应体系中淀粉的质量浓度为30％，调整pH为8，在37℃下反应2h；
(4)反应结束之后，用盐酸溶液中和反应体系pH为7，水洗，在4000rpm离心10min得到沉淀，之后干燥，粉碎，过100目筛，得到磷酸酯淀粉(糊化特性曲线如图2)。
实施例2
调整实施例1中步骤(3)中“调整pH为8”为“不调整pH”，其他和实施例1保持一致，得到磷酸酯淀粉(糊化特性曲线如图3)。
实施例3
调整实施例2中步骤(2)中碱性磷酸酶酶粉的用量为0.5g，其他和实施例2保持一致，得到磷酸酯淀粉(糊化特性曲线如图4)。
实施例4
调整实施例1中步骤(3)中反应条件为1h，其他和实施例1保持一致，得到磷酸酯淀粉(糊化特性曲线如图5)。
实施例5
调整实施例1中步骤(2)中氯化锌和氯化镁的浓度分别为0.1M，其他和实施例1保持一致，得到磷酸酯淀粉(糊化特性曲线如图6)。
实施例6
调整实施例1中步骤(2)中氯化锌和氯化镁的浓度分别为0.2M，其他和实施例1保持一致，得到磷酸酯淀粉(糊化特性曲线如图7)。
实施例7
调整实施例1中步骤(2)中氯化锌和氯化镁的浓度分别为0.05M，其他和实施例1保持一致，得到磷酸酯淀粉(糊化特性曲线如图8)。
将实施例1-7进行性能测试，测试结果如下：
从表1和表2可以看出：在反应过程中，可以通过控制体系中的pH、加酶量、反应时间、催化离子的浓度控制反应的进行。随取代度的升高。峰值黏度总体出现了先上升后下降的情况，产物中淀粉磷酸酯的组成发生变化。
表1实施例1-4的淀粉的测试结果
| 例 | 实施例1 | 实施例2 | 实施例3 | 实施例4 |
| 取代度 | 0.054 | 0.011 | 0.088 | 0.006 |
| 糊化温度/℃ | 75.4±1.2 | 75.6±0.8 | 75.7±0.4 | 74.7±0.3 |
| 峰值粘度/(mPa·s) | 587±145.8 | 609±11.3 | 442.3±6.1 | 667±22.6 |
| 终值粘度/(mPa·s) | 658.5±176.4 | 742.3±26.6 | 582.3±11.7 | 765±21.2 |
| 崩解值/(mPa·s) | 127.2±43 | 120±5.3 | 105±2.0 | 145±12.7 |
| 回生值/(mPa·s) | 198.8±75.2 | 253.3±21.4 | 245±7.5 | 243±11.3 |
表2实施例5-7的测试结果
| 例 | 实施例5 | 实施例6 | 实施例7 |
| 取代度 | 0.392 | 0.806 | 0.181 |
| 糊化温度/℃ | 74.4±0.14 | 75±0.5 | 73.9±0.1 |
| 峰值粘度(mPa·s) | 473.5±9.2 | 358.5±3.5 | 542±11.3 |
| 终值粘度/(mPa·s) | 678.5±12.0 | 492±4.24 | 742.5±20.5 |
| 崩解值/(mPa·s) | 81±1.4 | 57±1.41 | 114±7.1 |
| 回生值/(mPa·s) | 286±4.2 | 190.5±2.1 | 314.5±16.3 |
对照例1
省略实施例1中步骤(2)，调整步骤(3)为“将步骤(1)的淀粉乳和30mL0.25 M焦磷酸钠缓冲液混合”，其他和实施例1保持一致，得到磷酸酯淀粉。
对照例2
将实施例1中的控制pH＝8替换成pH＝10.5，其他与实施例1保持一致，得到磷酸酯淀粉(糊化特性曲线如图9)。
表3对照例1、2的淀粉的测试结果
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (3)
1.一种酶法制备磷酸酯淀粉的方法，其特征在于，包括如下步骤：
(1)将淀粉和焦磷酸盐缓冲液混合均匀，得到淀粉乳；
(2)将磷酸酶加入焦磷酸盐缓冲液中溶解，并加入催化离子，混合均匀、活化得到酶液；
(3)将步骤(2)的酶液分次加入步骤(1)的淀粉乳中进行反应；
(4)反应结束之后调节体系为中性，之后洗涤、离心、干燥、粉碎，得到磷酸酯淀粉；
其中，步骤(1)中淀粉与步骤(2)中磷酸酶的质量比为300：1；
步骤(2)中磷酸酶为碱性磷酸酶；
步骤(3)所述反应的pH为7～10；反应温度为20～40℃；反应时间为2～5h；
步骤(2)所述的催化离子为氯化锌和氯化镁，两者在酶液中的浓度分别为0.05-0.2M。
2.根据权利要求1所述的方法，其特征在于，步骤(1)和步骤(2)所述焦磷酸盐缓冲液的摩尔浓度为0.1～1.0M。
3.根据权利要求1或2所述的方法，其特征在于，步骤(3)所述的反应体系中淀粉的浓度为25％ -35％(w/w)。
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