CN107259569A - 一种以蔗糖为原料制备新型功能性交替糖的方法 - Google Patents
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- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
本发明公开了一种以蔗糖为原料制备新型功能性交替糖的方法,以蔗糖为原料,利用交替糖蔗糖酶定向受体反应酶催化制备功能性交替糖,即蔗糖经受体催化反应进行生物发酵获取反应液→反应液进行121℃高温灭菌、脱色→离子交换层析分离获取交替糖粗产品→粗产品同步进行浓缩获取交替糖糖浆;本发明以蔗糖为廉价原料,利用交替糖蔗糖酶生物转化制备交替糖,研究规模化过程中,温度、pH、底物浓度、酶和底物的比例、时间及传质传热等对于酶催化反应过程的影响,极大丰富了我国绿色食品添加剂市场,促进功能糖兴化产业发展,通过过程控制优化技术,对于产酶发酵、酶生物转化、分离提纯、结晶精制等过程进行控制优化,降低生产成本,提高产品的质量。
Description
技术领域
本发明涉及一种交替糖的制备方法,特备是一种以蔗糖为原料制备新型功能性交替糖的方法。
背景技术
近年来,随着社会结构的变化,生活节奏的加快,饮食习惯和食物构成改变及社会老龄化,人类疾病普发生了很大的转变,慢性疾病如糖尿病、心血管疾病、肥胖症、高脂血症和高血压等疾病比例不断攀升,已成为目前全球性重大的公共卫生问题。卫生部调查显示我国亚健康者已经超过70%,慢性疾病患病率达到20%,预防上述疾病发生的关键在于科学合理的膳食结构。膳食营养因素对健康的作用仅次于遗传因素,而大于医疗因素,功能食品可及时有效的进行营养干预,及早预防和消除很懂慢性疾病,达到预防效果,同时提高生活质量,减少医药费用的支出,改善人类的健康水平。
交替糖是一种新型的功能性益生元,其为一种白色固体粉末,无色无味无臭,不同于乙醇、丙酮等有机溶剂,易溶于冷水,是一种新型的α—葡聚糖,是一种新型功能性交替糖,具有低热量、耐酸、耐热、抗微生物降解、抗酶解的特性,同时兼具调整肠道菌群平衡、刺激免疫系统提高免疫功能、降低胆固醇、减少心血管疾病、维持饱腹感、持续缓释能量的特性,具有广阔的应用前景。本发明的目的是以蔗糖为原料进行工业化生产制备交替糖,旨在建立高效快速的分离技术纯化交替糖,造福人类健康。
发明内容
为了针对上述提出的问题,本发明提供了一种高效制备交替糖的方法,改善人类膳食结构,提高人类健康水平,其技术方案如下:
一种以蔗糖为原料制备新型功能性交替糖的方法,其特征在于:以蔗糖为原料,利用交替糖蔗糖酶定向受体反应酶催化制备功能性交替糖,具体步骤如下:
(1)蔗糖经受体催化反应进行生物发酵获取反应液;
(2)将上述步骤(1)中的反应液进行121℃高温灭菌、脱色;
(3)将上述步骤(2)中的反应物进行离子交换层析分离获取交替糖粗产品;
(4)将上述步骤(3)中的粗产品同步进行浓缩获取交替糖糖浆。
进一步的,所述步骤(4)中粗产品浓替处理替换成喷雾干燥处理获取交替糖粉末或替换成结晶处理获取交替糖晶体产品。
进一步的,所述交替糖蔗糖酶以柠檬明串珠菌(Leuconostoc citreum)SK24.002进行斜面培养、种子、发酵制备交替糖蔗糖酶,所述发酵后的酶液经离心、细胞超声破碎获取交替糖蔗糖酶液。
进一步的,所述斜面培养其培养基为:葡萄糖1~5g/L,无水氯化钙0.02~0.025g/L,酵母粉5~10g/L ,磷酸氢二钾10~15g/L,琼脂20g/L,七水合硫酸镁0.2~0.5g/L,Vc 0.03~0.05g/L,一水合硫酸锰0.01~0.015g/L ,培养液pH6.9,控制培养温度35℃,培养时间10~18h。
进一步的,所述种子培养基为:葡萄糖5~10g/L,无水氯化钙0.02~0.025g/L,磷酸氢二钾10~15g/L ,酵母粉10~20g/L, Vc 0.03~0.05g/L,七水合硫酸镁0.2~0.5g/L,一水合硫酸锰0.01~0.015g/L,其装液量为100mL三角瓶装液40mL,将斜面培养后的斜面培养基按体积百分比5%~10%接入种子培养基中,培养温度35℃,摇瓶转速160rpm,培养时间8~12h。
进一步的,所述发酵其培养基为:蔗糖20~40/L,无水氯化钙0.02~0.025g/L,磷酸氢二钾10~15g/L ,酵母粉10~20g/L,Vc 0.03~0.05 g/L,七水合硫酸镁0.2~0.5g/L,吐温805~10mL/L,控制pH6.9,将种子培养后的种子培养基按体积百分比5%~10%接入发酵培养基中,培养温度35℃,摇瓶转速160rpm,培养时间12~24h。
本发明与现有技术相比,有如下优点:
以蔗糖为廉价原料,利用交替糖蔗糖酶生物转化制备交替糖,研究规模化过程中,温度、pH、底物浓度、酶和底物的比例、时间及传质传热等对于酶催化反应过程的影响,科学设计与优化酶反应器,突破生物转化过程调控技术、过程工程优化放大等集成研究,通过设备硬件和技术软件的集成化研究,实现交替糖规模化生活参过程总的高效节能与环保的生物转化工艺,解决产业化生产过程中的瓶颈问题,极大丰富了我国绿色食品添加剂市场,促进功能糖兴化产业发展,通过过程控制优化技术,对于产酶发酵、酶生物转化、分离提纯、结晶精制等过程进行控制优化,降低生产成本,提高产品的质量。
具体实施方式
为进一步揭示本发明的技术方案,下面详细说明本发明的实施方式:
一种以蔗糖为原料制备新型功能性交替糖的方法,其特征在于:以蔗糖为原料,利用交替糖蔗糖酶定向受体反应酶催化制备功能性交替糖,具体步骤如下:
(1)蔗糖经受体催化反应进行生物发酵获取反应液;
(2)将上述步骤(1)中的反应液进行121℃高温灭菌、脱色;
(3)将上述步骤(2)中的反应物进行离子交换层析分离获取交替糖粗产品;
(4)将上述步骤(3)中的粗产品同步进行浓缩获取交替糖糖浆。
优选地,所述步骤(4)中粗产品浓替处理替换成喷雾干燥处理获取交替糖粉末或替换成结晶处理获取交替糖晶体产品。
优选地,所述交替糖蔗糖酶以柠檬明串珠菌(Leuconostoc citreum)SK24.002进行斜面培养、种子、发酵制备交替糖蔗糖酶,所述发酵后的酶液经离心、细胞超声破碎获取交替糖蔗糖酶液。
优选地,所述斜面培养其培养基为:葡萄糖1~5g/L,无水氯化钙0.02~0.025g/L,酵母粉5~10g/L ,磷酸氢二钾10~15g/L,琼脂20g/L,七水合硫酸镁0.2~0.5g/L,Vc 0.03~0.05g/L,一水合硫酸锰0.01~0.015g/L ,培养液pH6.9,控制培养温度35℃,培养时间10~18h。
优选地,所述种子培养基为:葡萄糖5~10g/L,无水氯化钙0.02~0.025g/L,磷酸氢二钾10~15g/L ,酵母粉10~20g/L, Vc 0.03~0.05g/L,七水合硫酸镁0.2~0.5g/L,一水合硫酸锰0.01~0.015g/L,其装液量为100mL三角瓶装液40mL,将斜面培养后的斜面培养基按体积百分比5%~10%接入种子培养基中,培养温度35℃,摇瓶转速160rpm,培养时间8~12h。
优选地,所述发酵其培养基为:蔗糖20~40/L,无水氯化钙0.02~0.025g/L,磷酸氢二钾10~15g/L ,酵母粉10~20g/L,Vc 0.03~0.05 g/L,七水合硫酸镁0.2~0.5g/L,吐温80 5~10mL/L,控制pH6.9,将种子培养后的种子培养基按体积百分比5%~10%接入发酵培养基中,培养温度35℃,摇瓶转速160rpm,培养时间12~24h。
实施例1:
首先,进行斜面培养:葡萄糖1 g,酵母粉5g,无水氯化钙0.025g,磷酸氢二钾10g,七水合硫酸镁0.3g,一水合硫酸锰0.01g,Vc 0.05g,琼脂20g,蒸馏水定容至1000mL,调节pH至6.9。30℃下培养12h。种子培养:葡萄糖10 g,酵母粉15g,无水氯化钙0.025g,磷酸氢二钾10g,七水合硫酸镁0.3g,一水合硫酸锰0.01g,Vc 0.05g,蒸馏水定容至1000mL,调节pH至6.9。100mL三角瓶装液40mL,斜面培养基按5%(v/v)接入种子培养基中,用牛皮纸包住,30℃下160rpm,摇床12h。发酵培养:蔗糖20 g,酵母粉15g,无水氯化钙0.025g,磷酸氢二钾10g,七水合硫酸镁0.3g,吐温80 10mL/L,Vc 0.05g,蒸馏水定容至1000mL,调节pH6.9。种子培养基按5%(v/v)接入发酵培养基中,30℃,160rpm,培养17h。测定发酵液的酶活为0.8U/mL。
其次, 交替糖蔗糖酶酶粉的制备
将发酵液在10000g,4℃下离心10min收集上清液和菌体细胞,上清液用分子截留量为30KDa的超滤膜超滤,去除盐与杂蛋白,再用50%的聚乙二醇沉淀,离心得到沉淀1;菌体细胞用pH5.4的醋酸钠缓冲溶液清洗两次,加入10mL pH5.4,8M的脲-醋酸钠缓冲溶液25℃
下振摇3h,经分子截留量为10KDa的透析袋除去残留的脲,用50%的聚乙二醇沉淀使最终聚乙二醇的浓度达到20%,离心得到沉淀2;沉淀1和沉淀2直接冷到干燥,即得交替糖蔗糖酶酶粉,酶活力达到18U/mg。
最后,交替糖的制备,将获得的交替糖蔗糖酶酶粉溶解于pH5.4的醋酸钠缓冲溶液中,使其活力达到8U/mL,以10%的蔗糖为底物,30℃下进行酶反应48h,煮沸灭酶后,离心获得上清液,冷冻干燥得到新型交替糖。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以所述权利要求的保护范围为准。
Claims (6)
1.一种以蔗糖为原料制备新型功能性交替糖的方法,其特征在于:以蔗糖为原料,利用交替糖蔗糖酶定向受体反应酶催化制备功能性交替糖,具体步骤如下:
(1)蔗糖经受体催化反应进行生物发酵获取反应液;
(2)将上述步骤(1)中的反应液进行121℃高温灭菌、脱色;
(3)将上述步骤(2)中的反应物进行离子交换层析分离获取交替糖粗产品;
(4)将上述步骤(3)中的粗产品同步进行浓缩获取交替糖糖浆。
2.根据权利要求1所述的一种以蔗糖为原料制备新型功能性交替糖的方法,其特征在于:
所述步骤(4)中粗产品浓替处理替换成喷雾干燥处理获取交替糖粉末或替换成结晶处理获取交替糖晶体产品。
3.根据权利要求1所述的一种以蔗糖为原料制备新型功能性交替糖的方法,其特征在于:所述交替糖蔗糖酶以柠檬明串珠菌(Leuconostoc citreum)SK24.002进行斜面培养、种子、发酵制备交替糖蔗糖酶,所述发酵后的酶液经离心、细胞超声破碎获取交替糖蔗糖酶液。
4.根据权利要求3所述的一种以蔗糖为原料制备新型功能性交替糖的方法,其特征在于:所述斜面培养其培养基为:
培养液pH6.9,控制培养温度35℃,培养时间10~18h。
5.根据权利要求3所述的一种以蔗糖为原料制备新型功能性交替糖的方法,其特征在于:所述种子培养基为:
其装液量为100mL三角瓶装液40mL,将斜面培养后的斜面培养基按体积百分比5%~10%接入种子培养基中,培养温度35℃,摇瓶转速160rpm,培养时间8~12h。
6.根据权利要求3所述的一种以蔗糖为原料制备新型功能性交替糖的方法,其特征在于:所述发酵其培养基为:
蔗糖20~40/L 无水氯化钙0.02~0.025g/L
磷酸氢二钾10~15g/L 酵母粉10~20g/L
Vc 0.03~0.05g/L 七水合硫酸镁0.2~0.5g/L
吐温80 5~10mL/L,控制pH6.9,将种子培养后的种子培养基按体积百分比5%~10%接入发酵培养基中,培养温度35℃,摇瓶转速160rpm,培养时间12~24h。
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