CN113265439A - 一种制备反式虾青素的方法 - Google Patents
一种制备反式虾青素的方法 Download PDFInfo
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- CN113265439A CN113265439A CN202110636442.5A CN202110636442A CN113265439A CN 113265439 A CN113265439 A CN 113265439A CN 202110636442 A CN202110636442 A CN 202110636442A CN 113265439 A CN113265439 A CN 113265439A
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Abstract
本发明公开了一种制备反式虾青素的方法。属于生物制剂领域；具体步骤：以葡萄糖、氨水及硫酸铵作为原料，法夫酵母或大肠杆菌微生物作为生产菌种进行发酵，代谢合成虾青素，在合成过程中添加诱导剂进行细胞催化促使菌体合成反式虾青素；对发酵液进行浓缩并收集菌体，得含有菌体的浓缩液；再进行细胞破壁，得到破壁溶液；向破壁溶液中添加预胶化淀粉、倍他环糊精、普鲁兰多糖、明胶或羟丙甲纤维素，用乳化机进行均质乳化，进行包埋处理；对包埋过的混有虾青素的菌泥进行干燥成粉，获得反式虾青素。本发明具有生产工艺简单，生物利用率高，生产规模容易放大，生产成本低的优点，同时，本发明所获得的的虾青素为全反式结构，生物活性高，应用范围广。
Description
技术领域
本发明属于生物制剂领域，涉及一种制备反式虾青素的方法，特别是一种采用生物法制备反式虾青素的方法。
背景技术
虾青素(Astaxanthin)即3,3′-二羟基-4,4′-二酮基-β,β′-胡萝卜素，为萜烯类不饱和化合物，化学分子式为C40H52O4，分子结构中有两个β-紫罗兰酮环，11个共轭双键。虾青素广泛存在于自然界，如大多数甲壳类动物和鲑科鱼类体内，植物的叶、花、果，以及火烈鸟的羽毛中等。虾青素具有多种生理功效，如在抗氧化性、抗肿瘤、预防癌症、增强免疫力、改善视力等方面都有一定的效果。目前，国际上虾青素的生产方式主要有化学合成和天然产物提取两种。
1)、化学合成：虾青素是类胡萝卜素合成的终产物，由β-胡萝卜素转变为虾青素需加上2个酮基和2个羟基，人工化学合成比较困难、工艺复杂，价格昂贵，产物大多为顺式结构，而只有反式结构的虾青素才具有生物活性。
2)、天然产物提取法：①从水产品加工的下脚料中提取虾青素：传统方法是将虾蟹壳粉碎，酸解用有机溶剂丙酮、石油醚等提取。近年来，CO2超临界萃取法、酶提取法和复合工艺提取法也广泛应用于虾青素的提取。国外采用聚合系统从鳌虾的废弃物中提取虾青素、虾青素酯和虾红素，其产率每克可达153μg。②从藻类中提取虾青素：现阶段只有雨生红球藻可用于工业化生产虾青素。雨生红球藻干品中虾青素含量可达0.2～2％，优良的雨生红球藻藻体中虾青素占类胡萝卜素总量的90％以上，但其培养周期长、生产工艺复杂。国内的雨生红球藻培养技术大多还处于实验室研究阶段，因产量低导致产品市场的占有率不高。③从细菌中提取虾青素:红法夫酵母、粘红酵母、南极酵母、乳酸分枝杆菌病和一些海洋细菌在合适条件下都可以产生虾青素，其中红法夫酵母是虾青素含量最丰富的酵母，也是目前进行虾青素工业化的主要生产菌。
虾青素的其他提取方法:可利用细菌、原生动物、农作物体内的β-胡萝卜素作为前体物质，通过转基因技术将合成虾青素的酶转入相应的生物中合成虾青素，目前这种方法还处于实验研究阶段，如等通过代谢工程，将虾青素合成的关键酶基因β-胡萝卜素酮化酶基因和β-胡萝卜素羟化酶基因转入花生，使其在花生中高效表达，在花生种皮中生物合成虾青素。
化学合成的虾青素，在结构、功能、应用、安全性和经济性等方面明显逊色于天然虾青素。
发明内容
发明目的：本发明的目的是提出了一种制备反式虾青素的方法，其通过生物法合成全反式虾青素，并通过破壁、包埋、干燥等方法生产饲料级虾青素。
技术方案：本发明所述的一种制备反式虾青素的方法，具体操作步骤如下：
(1.1)、以葡萄糖、氨水及硫酸铵为原料，以法夫酵母或大肠杆菌微生物为生产菌种进行发酵，通过代谢合成虾青素，在合成过程中添加诱导剂进行细胞催化促使菌体合成反式虾青素，发酵结束得发酵液；
(1.2)、利用陶瓷膜、管式膜或离心机对发酵液进行浓缩并收集菌体，即得含有菌体的浓缩液；
(1.3)、通过高压均质机或乳化机对收集到的浓缩液中的菌体进行细胞破壁，使胞内虾青素进行释放，从而得到含有虾青素的破壁溶液；
(1.4)、向破壁溶液中添加预胶化淀粉、倍他环糊精、普鲁兰多糖、明胶或羟丙甲纤维素，用乳化机进行均质乳化，对混有虾青素的菌泥进行包埋；
(1.5)、采用闭式或开式离心喷雾干燥或压力式喷雾干燥对包埋过的混有虾青素的菌泥进行干燥成粉，即获得反式虾青素。
进一步的，在步骤(1.1)中，所述葡萄糖、氨水及硫酸铵按照碳氮比3:1-6:1进行配比；
所述氨水和硫酸铵的质量比为2:1-5:1；
所述法夫酵母或大肠杆菌微生物的接种量为发酵液体积的5-10％。
进一步的，在步骤(1.1)中，所述通过代谢合成虾青素的发酵温度是27-35℃；发酵的时间是60-80h。
进一步的，在步骤(1.1)中，所述添加的诱导剂为异丙基-β-D-硫代半乳糖苷。
进一步的，在步骤(1.2)中，所述发酵液进行浓缩的浓缩倍数为2-5倍。
进一步的，在步骤(1.3)中，所述采用高压均质机进行破壁的工作压力是1000-1400Bar，其破壁次数是2次。
进一步的，在步骤(1.4)中，所述预胶化淀粉、倍他环糊精、普鲁兰多糖、明胶或羟丙甲纤维素的添加比例为所述破壁溶液容积的2-6％。
进一步的，在步骤(1.5)中，所述干燥的工艺条件为进风温度180-220℃，出风温度60-90℃。
有益效果：本发明与现有技术相比，本发明具有生产工艺简单，生物利用率高，生产规模容易放大，生产成本低的优点，同时，本发明所获得的的虾青素为全反式结构，生物活性高，应用范围广。
附图说明
图1为本发明的操作流程图。
具体实施方式
下面对本发明的实施方法做进一步的说明。
本发明所述的一种制备反式虾青素的方法，具体操作步骤如下：
(1.1)、以葡萄糖、氨水及硫酸铵为原料，以法夫酵母或大肠杆菌微生物为生产菌种进行发酵，通过代谢合成虾青素，在合成过程中添加诱导剂进行细胞催化促使菌体合成反式虾青素，发酵结束得发酵液；
(1.2)、利用陶瓷膜、管式膜或离心机对发酵液进行浓缩并收集菌体，即得含有菌体的浓缩液；
(1.3)、通过高压均质机或乳化机对收集到的浓缩液中的菌体进行细胞破壁，使胞内虾青素进行释放，从而得到含有虾青素的破壁溶液；
(1.4)、向破壁溶液中添加预胶化淀粉、倍他环糊精、普鲁兰多糖、明胶或羟丙甲纤维素，用乳化机进行均质乳化，对混有虾青素的菌泥进行包埋；
(1.5)、采用闭式或开式离心喷雾干燥或压力式喷雾干燥对包埋过的混有虾青素的菌泥进行干燥成粉，即获得反式虾青素。
进一步的，在步骤(1.1)中，所述葡萄糖、氨水及硫酸铵按照碳氮比3:1-6:1进行配比；
所述氨水和硫酸铵的质量比为2:1-5:1；
所述法夫酵母或大肠杆菌微生物的接种量为发酵液体积的5-10％。
进一步的，在步骤(1.1)中，所述通过代谢合成虾青素的发酵温度是27-35℃；发酵的时间是60-80h。
进一步的，在步骤(1.1)中，所述添加的诱导剂为异丙基-β-D-硫代半乳糖苷(IPTG)。
进一步的，在步骤(1.2)中，所述发酵液进行浓缩的浓缩倍数为2-5倍。
进一步的，在步骤(1.3)中，所述采用高压均质机进行破壁的工作压力是1000-1400Bar，其破壁次数是2次。
进一步的，在步骤(1.4)中，所述预胶化淀粉、倍他环糊精、普鲁兰多糖、明胶或羟丙甲纤维素的添加比例为所述破壁溶液容积的2-6％(V/V)。
进一步的，在步骤(1.5)中，所述干燥的工艺条件为进风温度180-220℃，出风温度60-90℃。
实施案例1
1、以葡萄糖、氨水及硫酸铵按照质量比12:3:1配制原料，，采用法夫酵母为生产菌，接种量为初始发酵液体积的10％，经过27-35℃条件下经过种子培养、发酵72小时，过程中加入专用诱导剂进行细胞催化，转化生成全反式虾青素；发酵终止，采用夹套进汽，温度上升至60-80℃保温灭活60分钟，进行菌体灭活；
2、采用50-200nm的陶瓷膜进行浓缩，浓缩倍数4-5倍；
3、采用高压均质机进行破壁，工作压力1000-1400Bar，破壁2次，使菌体细胞完全破壁；
4、向破壁溶液中添加3-5％(V/V)的预胶化淀粉，用乳化机均质乳化后，准备喷雾干燥；
5、采用离心式喷雾干燥机，干燥工艺条件为进风温度180-220℃，出风温度60-90℃，喷雾干燥，即得虾青素成品；成品含量可达到0.5-1.2％。
实施案例2
1、以葡萄糖、氨水及硫酸铵按照质量比9:2:1配制原料，采用大肠杆菌为生产菌，接种量5％，在27～30℃条件下经过种子培养、发酵80小时，发酵过程中加入诱导剂进行细胞催化，转化为全反式虾青素；发酵终止，采用夹套进汽，温度上升至60-80℃保温灭活60分钟，进行菌体灭活；
2、采用50-200nm的陶瓷膜进行浓缩，浓缩倍数2-3倍；
3、采用高压均质机进行破壁，工作压力800-1200Bar，破壁2次，使菌体细胞完全破壁；
4、向破壁溶液中添加4-6％的预胶化淀粉，用乳化机均质后，准备喷雾干燥；
5、采用离心式喷雾干燥机，干燥工艺条件为进风温度180-220℃，出风温度60-90℃，喷雾干燥，即得虾青素成品；成品含量可得到全反式虾青素0.5-1.2％，干燥过程成品收得率可达到85-90％。
实施案例3
1、以葡萄糖、氨水及硫酸铵按照质量比36:5:1配制原料，采用大肠杆菌为生产菌，接种量10％，在30-35℃条件下经过种子培养、发酵60小时，发酵过程中加入诱导剂进行细胞催化，转化生成全反式虾青素；发酵终止，不进行菌体灭活；
2、采用50～200nm的陶瓷膜进行浓缩，浓缩倍数为3倍；
3、采用高压均质机进行破壁，工作压力900-1200Bar，破壁2次，使菌体细胞完全破壁；
4、向破壁溶液中添加3％的倍他环糊精，用乳化机均质后，准备喷雾干燥；
5、采用离心式喷雾干燥机，干燥工艺条件为进风温度180-220℃，出风温度60-90℃，喷雾干燥，即得虾青素成品。成品含量可得到全反式虾青素0.5～1.2％，干燥过程成品收得率可达到86～90％。
上述过程还可以采取添加普鲁兰多糖、明胶或羟丙甲纤维素等材料进行包埋后喷雾干燥。
Claims (8)
1.一种制备反式虾青素的方法，其特征在于：具体操作步骤如下：
(1.1)、以葡萄糖、氨水及硫酸铵为原料，以法夫酵母或大肠杆菌微生物为生产菌种进行发酵，通过代谢合成虾青素，在合成过程中添加诱导剂进行细胞催化促使菌体合成反式虾青素，发酵结束得发酵液；
(1.2)、利用陶瓷膜、管式膜或离心机对发酵液进行浓缩并收集菌体，即得含有菌体的浓缩液；
(1.3)、通过高压均质机或乳化机对收集到的浓缩液中的菌体进行细胞破壁，使胞内虾青素进行释放，从而得到含有虾青素的破壁溶液；
(1.4)、向破壁溶液中添加预胶化淀粉、倍他环糊精、普鲁兰多糖、明胶或羟丙甲纤维素，用乳化机进行均质乳化，对混有虾青素的菌泥进行包埋；
(1.5)、采用闭式或开式离心喷雾干燥或压力式喷雾干燥对包埋过的混有虾青素的菌泥进行干燥成粉，即获得反式虾青素。
2.根据权利要求1所述的一种制备反式虾青素的方法，其特征在于：在步骤(1.1)中，所述葡萄糖、氨水及硫酸铵按照碳氮比3:1-6:1进行配比；
所述氨水和硫酸铵的质量比为2:1-5:1；
所述法夫酵母或大肠杆菌微生物的接种量为发酵液体积的5-10％。
3.根据权利要求1所述的一种制备反式虾青素的方法，其特征在于：在步骤(1.1)中，所述通过代谢合成虾青素的发酵温度是27-35℃；发酵的时间是60-80h。
4.根据权利要求1所述的一种制备反式虾青素的方法，其特征在于：在步骤(1.1)中，所述添加的诱导剂为异丙基-β-D-硫代半乳糖苷。
5.根据权利要求1所述的一种制备反式虾青素的方法，其特征在于：在步骤(1.2)中，所述发酵液进行浓缩的浓缩倍数为2-5倍。
6.根据权利要求1所述的一种制备反式虾青素的方法，其特征在于：在步骤(1.3)中，所述采用高压均质机进行破壁的工作压力是1000-1400Bar，其破壁次数是2次。
7.根据权利要求1所述的一种制备反式虾青素的方法，其特征在于：在步骤(1.4)中，所述预胶化淀粉、倍他环糊精、普鲁兰多糖、明胶或羟丙甲纤维素的添加比例为所述破壁溶液容积的2-6％。
8.根据权利要求1所述的一种制备反式虾青素的方法，其特征在于：在步骤(1.5)中，所述干燥的工艺条件为进风温度180-220℃，出风温度60-90℃。
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