CN113812583A - 一种水母提取物的制备方法 - Google Patents
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Abstract
本申请公开一种水母提取物的制备方法,包括以下步骤:(1)取洗净后的水母触手,组织匀浆,冷冻干燥得到冻干粉;(2)将冻干粉和水混合,搅拌,分散均匀得到分散液;(3)调节分散液的pH至7.5~8.0,升温至45~50℃,加入胰蛋白酶,搅拌使之酶解,酶解后使酶失活;(4)向酶解后的分散液中乙醇和氯化钠,搅拌,在20~25℃下保温60~120s,离心取滤液;(5)取离心后的滤液,加入EDTA二钠,搅拌反应除重金属;(6)过滤:将去重金属后的滤液用0.5μm滤膜真空抽滤,取滤液;(7)浓缩、干燥,过程控温、控量等,具有抑制ACE的优点。
Description
技术领域
本发明涉及一种水母提取物的制备方法。
背景技术
白色霞水母俗称:麻蛰、海蜇。白色霞水母(Cyanea nozakii Kishinouye)隶属于腔肠动物门,刺胞亚门(Coelenterata),钵水母纲(Class Scyphomedusae),旗口水母目(Semaeostomeae),霞水母科(Cyaneidae),霞水母属,是一类海洋大型浮游动物。白色霞水母的资源丰富、分布范围广,是中国沿海大陆架地区大型水母中的优势种群。
目前国内外关于霞水母的研究主要集中在霞水母的形态结构、生长规律、渔业生态学及对海洋渔业资源的危害等方面。而有关霞水母药用价值的研究主要集中于水母毒素方面,霞水母中富含胶原蛋白,但一直以来仅以初加工食品为主,市场上仅见有食品类的即食产品,未见有下游深加工产品的开发。
因此,研发出一款水母提取物具有重要的意义。
发明内容
本发明的目的是提供一种水母提取物的制备方法,具有抑制ACE的优点。
本发明的上述技术目的是通过以下技术方案得以实现的:
一种水母提取物的制备方法,包括以下步骤:
(1)取洗净后的水母触手,组织匀浆,冷冻干燥得到冻干粉;
(2)将冻干粉和水混合,搅拌,分散均匀得到分散液;冻干粉和水的投入质量比为1:8~12;
(3)调节分散液的pH至7.5~8.0,升温至45~50℃,加入胰蛋白酶,搅拌使之酶解,酶解时间为1.5~2.5hr,酶解后升温至95~100℃并保温12~20min使酶失活;分散液和胰蛋白酶的质量比为100:0.4~0.6;
(4)向酶解后的分散液中乙醇和氯化钠,搅拌,在20~25℃下保温60~120s,离心取滤液;分散液、乙醇和氯化钠的质量比为1:0.04~0.06:0.002~0.004;
(5)取离心后的滤液,加入EDTA二钠,搅拌反应除重金属;离心后的滤液和EDTA二钠的质量比为100:0.01~0.03;
(6)过滤:将去重金属后的滤液用0.5μm滤膜真空抽滤,取滤液;
(7)浓缩、干燥。
优选的,水母选用白色霞水母;步骤(3)包括以下两个部分:
①调节分散液的pH至7.5~8.0,升温至45~50℃,加入胰蛋白酶,搅拌使之酶解,酶解时间为1.5~2.5hr,酶解后升温至95~100℃并保温12~20min使酶失活;分散液和胰蛋白酶的质量比为100:0.4~0.6;
②调节分散液的pH至6.8~7.2,升温至38~42℃,加入脂肪酶,搅拌使之酶解,酶解时间为2~4hr,酶解后升温至85~95℃并保温4~5min使酶失活;分散液和脂肪酶的质量比为100:0.1~0.3。
优选的,步骤(2)还增加有碳酸二氢钙、碳酸氢钠、微晶纤维素,冻干粉、碳酸二氢钙、碳酸氢钠、微晶纤维素和水的投入质量比为1:0.02~0.03:0.04~0.05:0.01~0.03:8~12。
优选的,步骤(5)中,除了向滤液中加入EDTA二钠外,步骤(5)中还添加有微晶纤维素和乙酸乙酯;步骤(5)中,离心后的滤液、EDTA二钠、微晶纤维素和乙酸乙酯的质量比为100:0.01~0.03:0.07~0.09:10~12。
优选的,步骤(6)除了抽滤外,还将滤液进行以下处理:将抽滤后得到的滤液和氯化钙按质量比1:0.06~0.10混合,用醋酸调节pH至6.5~7.0,在40~45℃下搅拌反应40~50min。
优选的,包括以下步骤:
(1)取洗净后的白色霞水母触手,组织匀浆,冷冻干燥得到冻干粉;
(2)将冻干粉、碳酸二氢钙、碳酸氢钠、微晶纤维素和水混合,搅拌,分散均匀得到分散液;冻干粉、碳酸二氢钙、碳酸氢钠、微晶纤维素和水的投入质量比为1:0.02:0.04:0.02:10;
(3)酶解:
①调节分散液的pH至7.8,升温至45℃,加入胰蛋白酶,搅拌使之酶解,酶解时间为2.0hr,酶解后升温至100℃并保温15min使酶失活;分散液和胰蛋白酶的质量比为100:0.5;
②调节分散液的pH至7.0,升温至40℃,加入脂肪酶,搅拌使之酶解,酶解时间为3hr,酶解后升温至90℃并保温5min使酶失活;分散液和脂肪酶的质量比为100:0.2;
(4)向酶解后的分散液中乙醇和氯化钠,搅拌,在25℃下保温90s,离心取滤液;分散液、乙醇和氯化钠的质量比为1:0.05:0.003;
(5)取离心后的滤液,加入EDTA二钠、微晶纤维素和乙酸乙酯,搅拌反应除重金属;离心后的滤液、EDTA二钠、微晶纤维素和乙酸乙酯的质量比为100:0.02:0.08:10;
(6)过滤:将去重金属后的滤液用0.5μm滤膜真空抽滤,取滤液;将抽滤后得到的滤液和氯化钙按质量比1:0.08混合,用醋酸调节pH至6.8,在42℃下搅拌反应45min;
(7)真空浓缩至固含量为50wt%,喷雾干燥。
本发明技术效果主要体现在以下方面:
白色霞水母中富含胶原蛋白,通过本申请的方式来获得小分子肽类物质,能有效抑制血管紧张素转化酶(ACE),从而达到降低血压的作用,且因来源于食物,安全性高;
在处理过程中利用EDTA二钠进行重金属处理,降低产品中重金属含量,提高食品安全性;在处理过程中由于重金属的存在,过程中的多肽和重金属存在螯合的可能性,从而导致产品中存在螯合的重金属,利用碳酸氢钙在碱性条件下和重金属的作用推动、并结合微晶纤维素对其分散和螯合的阻碍,从而减少重金属和多肽的螯合,使重金属处于游离态,以便其与EDTA二钠作用,提高重金属去除效果,也减少多肽的损失;
在酶解后得到的酶解液中乳化稳定性较好,分离不理想,影响分离时间和产品质量,在本申请中通过脂肪酶酶解脂肪,破坏蛋白质和脂肪相互作用,降低乳液的稳定性,从而使得油水分离,并通过乙酸乙酯和微晶纤维素的作用,提高分离效果,加快工艺进程,提高蛋白回收率;
在酶解后通过乙醇沉降、将过滤液和氯化钙螯合作用,提高产品的稳定性,适于室温保存。
具体实施方式
实施例1:一种水母提取物的制备方法,包括以下步骤:
(1)取洗净后的白色霞水母触手,组织匀浆,冷冻干燥得到冻干粉;
(2)将冻干粉、碳酸二氢钙、碳酸氢钠、微晶纤维素和水混合,搅拌,分散均匀得到分散液;冻干粉、碳酸二氢钙、碳酸氢钠、微晶纤维素和水的投入质量比为1:0.02:0.04:0.02:10;
(3)酶解:
①调节分散液的pH至7.8,升温至45℃,加入胰蛋白酶,搅拌使之酶解,酶解时间为2.0hr,酶解后升温至100℃并保温15min使酶失活;分散液和胰蛋白酶的质量比为100:0.5;
②调节分散液的pH至7.0,升温至40℃,加入脂肪酶,搅拌使之酶解,酶解时间为3hr,酶解后升温至90℃并保温5min使酶失活;分散液和脂肪酶的质量比为100:0.2;
(4)向酶解后的分散液中乙醇和氯化钠,搅拌,在25℃下保温90s,离心取滤液;分散液、乙醇和氯化钠的质量比为1:0.05:0.003;
(5)取离心后的滤液,加入EDTA二钠、微晶纤维素和乙酸乙酯,搅拌反应除重金属;离心后的滤液、EDTA二钠、微晶纤维素和乙酸乙酯的质量比为100:0.02:0.08:10;
(6)过滤:将去重金属后的滤液用0.5μm滤膜真空抽滤,取滤液;将抽滤后得到的滤液和氯化钙按质量比1:0.08混合,用醋酸调节pH至6.8,在42℃下搅拌反应45min;
(7)真空浓缩至固含量为50wt%,喷雾干燥。
实施例2:步骤(2)对水母提取物的制备方法的影响
测试对象:实施例1、对照组1-4。
对照组1:参照实施例1,与实施例1的区别在于,步骤(2)中,冻干粉、碳酸二氢钙、碳酸氢钠、微晶纤维素和水的投入质量比为1:0.03:0.05:0.03:10。
对照组2:参照实施例1,与实施例1的区别在于,步骤(2)中冻干粉中未添加碳酸二氢钙、碳酸氢钠或微晶纤维素,仅添加水且冻干粉和水的投入质量比为1:10。
对照组3:参照实施例1,与实施例1的区别在于,步骤(2)中冻干粉中未添加微晶纤维素,冻干粉、碳酸二氢钙、碳酸氢钠和水的投入质量比为1:0.02:0.04:10。
对照组4:参照实施例1,与实施例1的区别在于,步骤(2)中,冻干粉、碳酸二氢钙、碳酸氢钠、微晶纤维素和水的投入质量比为1:0.05:0.07:0.06:10。
测试内容:分别对测试对象进行粗蛋白的测定以及铅、镉、铬这三类重金属总量的测定。
粗蛋白的测定:凯氏定氮法测定,标准为GB5009.5-2010。蛋白回收率(%)=产品蛋白含量×100%/原料蛋白含量,产品蛋白含量即步骤(7)喷雾干燥后得到的产品中的蛋白含量,而原料蛋白含量则为步骤(1)获得的冻干的蛋白含量。
重金属测定:参照GB15618-1995,测试对象为步骤(7)喷雾干燥后得到的产品。
测试结果如表1所示。表1显示:按实施例1和对照组1的步骤(2)进行,蛋白回收率高,重金属含量低,实施例1为最佳。
表1
实施例3:步骤(3)和步骤(5)的影响
测试对象:实施例1、对照组5-7。
对照组5:参照实施例1,与实施例1的区别在于,步骤(3)酶解步仅包括以下一个部分:调节分散液的pH至7.8,升温至45℃,加入胰蛋白酶,搅拌使之酶解,酶解时间为2.0hr,酶解后升温至100℃并保温15min使酶失活;分散液和胰蛋白酶的质量比为100:0.5。
对照组6:参照实施例1,与实施例1的区别在于,步骤(3)包括:
①调节分散液的pH至7.0,升温至52℃,加入中性蛋白酶,搅拌使之酶解,酶解时间为6hr,酶解后升温至80℃并保温10min使酶失活;分散液和中性蛋白酶的质量比为100:0.3;
②调节分散液的pH至7.0,升温至40℃,加入脂肪酶,搅拌使之酶解,酶解时间为3hr,酶解后升温至90℃并保温5min使酶失活;分散液和脂肪酶的质量比为100:0.2。
对照组7:参照实施例1,与实施例1的区别在于,步骤(5)向离心后的滤液中仅添加EDTA二钠,未添加微晶纤维素或乙酸乙酯。
测试内容:分别对测试对象进行抽真空过滤耗时的测定,即取1kg步骤(5)处理后的滤液,用0.5μm滤膜真空抽滤,抽滤压强为0.1MPa抽滤温度为25℃,从开始抽滤起计时,至滤液全滤过,记录总耗时。
粗蛋白的测定:凯氏定氮法测定,标准为GB5009.5-2010。蛋白回收率(%)=产品蛋白含量×100%/原料蛋白含量,产品蛋白含量即步骤(7)喷雾干燥后得到的产品中的蛋白含量,而原料蛋白含量则为步骤(1)获得的冻干粉的蛋白含量。
测试结果如表2所示。表2显示,相比在步骤(3)中未进行二步处理的对照组5和①中用中性蛋白酶处理的对照组6,本申请的实施例1抽真空过滤耗时更快,蛋白回收率更高;相比在步骤(5)中未添加乙酸乙酯或微晶纤维素的对照组7,本申请的实施例1抽真空过滤耗时更快,蛋白回收率更高。
表2
抽真空过滤耗时(min) | 蛋白回收率(%) | |
实施例1 | 7 | 74.3 |
对照组5 | 56 | 51.0 |
对照组6 | 31 | 50.3 |
对照组7 | 36 | 47.5 |
实施例4:ACE活性测试
测试对象:实施例1、对照组8-9、对照组6。
对照组8:参照实施例1,与实施例1的区别在于,未设置步骤(4)。
对照组9:参照实施例1,与实施例1的区别在于,步骤(6)仅包括过滤:将去重金属后的滤液用0.5μm滤膜真空抽滤,取滤液。
ACE抑制率的测定:取测试对象,溶于0.1mol/L硼酸缓冲液(pH=8.3,含0.3mol/LNaCl)制成相应的样品供试液。HHL(马尿酰-组氨酰-亮氨酸)、ACE(血管紧张素转化酶)分别用0.1mol/L硼酸缓冲液(pH=8.3,含0.3mol/L NaCl)配成5mmol/L HHL溶液和100Mu/mLACE溶液。取30μl HHL与10μl样品供试液(空白组采用硼酸缓冲溶液代替样品供试液)混匀后,置于37℃下预热5min,之后加入20μl的ACE溶液启动反应,混匀,置于37℃下反应1hr,迅速加入70μl 1mol/L HCl终止反应,用高效液相色谱仪分析结果。
色谱条件:Xbridge-C18柱(4.6×250mm,5μm,美国water公司);流动相:A为0.05%三氟乙酸水溶液,B为乙腈,梯度洗脱条件:0~30min、B流动相10~60%(体积),流速0.8mL/min;检测波长228nm;柱温30℃;进样量10μl。
将样品密封保存在玻璃瓶内,分别放置在0℃、25℃下60d,进行ACE抑制率测试。
表3
当然,以上只是本发明的典型实例,除此之外,本发明还可以有其它多种具体实施方式,凡采用等同替换或等效变换形成的技术方案,均落在本发明要求保护的范围之内。
Claims (6)
1.一种水母提取物的制备方法,其特征是,包括以下步骤:
(1)取洗净后的水母触手,组织匀浆,冷冻干燥得到冻干粉;
(2)将冻干粉和水混合,搅拌,分散均匀得到分散液;冻干粉和水的投入质量比为1:8~12;
(3)调节分散液的pH至7.5~8.0,升温至45~50℃,加入胰蛋白酶,搅拌使之酶解,酶解时间为1.5~2.5hr,酶解后升温至95~100℃并保温12~20min使酶失活;分散液和胰蛋白酶的质量比为100:0.4~0.6;
(4)向酶解后的分散液中乙醇和氯化钠,搅拌,在20~25℃下保温60~120s,离心取滤液;分散液、乙醇和氯化钠的质量比为1:0.04~0.06:0.002~0.004;
(5)取离心后的滤液,加入EDTA二钠,搅拌反应除重金属;离心后的滤液和EDTA二钠的质量比为100:0.01~0.03;
(6)过滤:将去重金属后的滤液用0.5μm滤膜真空抽滤,取滤液;
(7)浓缩、干燥。
2.根据权利要求1所述的一种水母提取物的制备方法,其特征是,水母选用白色霞水母;步骤(3)包括以下两个部分:
①调节分散液的pH至7.5~8.0,升温至45~50℃,加入胰蛋白酶,搅拌使之酶解,酶解时间为1.5~2.5hr,酶解后升温至95~100℃并保温12~20min使酶失活;分散液和胰蛋白酶的质量比为100:0.4~0.6;
②调节分散液的pH至6.8~7.2,升温至38~42℃,加入脂肪酶,搅拌使之酶解,酶解时间为2~4hr,酶解后升温至85~95℃并保温4~5min使酶失活;分散液和脂肪酶的质量比为100:0.1~0.3。
3.根据权利要求2所述的一种水母提取物的制备方法,其特征是,步骤(2)还增加有碳酸二氢钙、碳酸氢钠、微晶纤维素,冻干粉、碳酸二氢钙、碳酸氢钠、微晶纤维素和水的投入质量比为1:0.02~0.03:0.04~0.05:0.01~0.03:8~12。
4.根据权利要求3所述的一种水母提取物的制备方法,其特征是,步骤(5)中,除了向滤液中加入EDTA二钠外,步骤(5)中还添加有微晶纤维素和乙酸乙酯;步骤(5)中,离心后的滤液、EDTA二钠、微晶纤维素和乙酸乙酯的质量比为100:0.01~0.03:0.07~0.09:10~12。
5.根据权利要求4所述的一种水母提取物的制备方法,其特征是,步骤(6)除了抽滤外,还将滤液进行以下处理:将抽滤后得到的滤液和氯化钙按质量比1:0.06~0.10混合,用醋酸调节pH至6.5~7.0,在40~45℃下搅拌反应40~50min。
6.根据权利要求5所述的一种水母提取物的制备方法,其特征是,包括以下步骤:
(1)取洗净后的白色霞水母触手,组织匀浆,冷冻干燥得到冻干粉;
(2)将冻干粉、碳酸二氢钙、碳酸氢钠、微晶纤维素和水混合,搅拌,分散均匀得到分散液;冻干粉、碳酸二氢钙、碳酸氢钠、微晶纤维素和水的投入质量比为1:0.02:0.04:0.02:10;
(3)酶解:
①调节分散液的pH至7.8,升温至45℃,加入胰蛋白酶,搅拌使之酶解,酶解时间为2.0hr,酶解后升温至100℃并保温15min使酶失活;分散液和胰蛋白酶的质量比为100:0.5;
②调节分散液的pH至7.0,升温至40℃,加入脂肪酶,搅拌使之酶解,酶解时间为3hr,酶解后升温至90℃并保温5min使酶失活;分散液和脂肪酶的质量比为100:0.2;
(4)向酶解后的分散液中乙醇和氯化钠,搅拌,在25℃下保温90s,离心取滤液;分散液、乙醇和氯化钠的质量比为1:0.05:0.003;
(5)取离心后的滤液,加入EDTA二钠、微晶纤维素和乙酸乙酯,搅拌反应除重金属;离心后的滤液、EDTA二钠、微晶纤维素和乙酸乙酯的质量比为100:0.02:0.08:10;
(6)过滤:将去重金属后的滤液用0.5μm滤膜真空抽滤,取滤液;将抽滤后得到的滤液和氯化钙按质量比1:0.08混合,用醋酸调节pH至6.8,在42℃下搅拌反应45min;
(7)真空浓缩至固含量为50wt%,喷雾干燥。
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