CN115093493A - 一种从白星花金龟提取制备甲壳素和壳聚糖的方法 - Google Patents
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Abstract
本发明涉及甲壳素和壳聚糖制备技术领域,且公开了一种从白星花金龟提取制备甲壳素和壳聚糖的方法,S1原料预处理;S2脱脂处理;S3脱钙处理;S4脱蛋白处理;S5脱盐处理;S6脱色处理;S7酶处理;S8脱乙酰基处理,传统酸碱法使用大量的酸碱,不仅消耗能源,还造成环境污染,本发明在传统方法基础上改进,采用有机酸混合溶液替代盐酸,同时增加脱脂步骤去除虫体内大量脂肪,可降低甲壳素提取过程中对环境造成的污染,避免提取甲壳素发生变化,进一步提高产品的得率和纯度,白星花金龟成虫野外易获取,也易于人工养殖,不受季节和环境的限制。将该害虫作为工业化提取甲壳素和壳聚糖的来源,可有效降低生产成本,减轻虫害损失,变废为宝。
Description
技术领域
本发明涉及甲壳素和壳聚糖制备技术领域,具体为一种从白星花金龟提取制备甲壳素和壳聚糖的方法。
背景技术
甲壳素(chitin)化学名为β-(1,4)-2-乙酰氨基-2-脱氧-D-葡萄糖,分子式为(C8H15NO6)n,被誉为“人类第六生命要素”,是自然界中仅次于纤维素的第二大生物资源。壳聚糖(chitosan)是甲壳素(chitin)经脱乙酰化处理后的产物,是甲壳素最基本、最重要的衍溶生物。因具有良好的吸湿性、生物降解性、安全无毒、吸附性、生物相容性、易成膜成纤等诸多优良特性,甲壳素、壳聚糖及其衍生物被广泛应用在环保材料、纺织造纸、医药保健、食品加工、工农业、生物工程等方面。目前,目前工业化生产甲壳素的原料主要是虾、蟹壳等水产品,来源单一有限,且灰分、钙、无机盐等含量较高造成提取成本较高,使其应用受到一定限制。此外,提取甲壳素、壳聚糖大多采用强酸强碱法,造成能源浪费、环境污染。因此开发新的甲壳素替代资源、改良提取方法具有重要意义。
昆虫种类多、生物量大、繁殖速度快,富含甲壳素(20%~60%),且无机盐、灰分、重金属含量相比虾、蟹壳较低,具有良好的资源潜力和开发优势。近年来,国内外针对蝉蜕、蝇蛆、松毛虫、蚕蛹、黄粉虫等几种昆虫的甲壳素提取进行了深入研究,但将昆虫作为原材料进行工业生产的报道却不多。
白星花金龟Protaetia brevitarsis(Lewis)属鞘翅目(Coleoptera)、金龟总科(Scarabaeoidea)、花金龟科(Cetoniidae)、星花金龟属(Protaetia),在我国广泛分布。其成虫具有杂食性,是农林果蔬害虫;幼虫具有腐食性,一般不危害活的植物根系。白星花金龟幼虫和成虫甲壳素含量为23.3%~28.3%,是虾蟹壳的数倍,明显超出了现在甲壳素常规生产的原料中所含有的量。同时,白星花金龟体壳中提取的甲壳素品质要优于虾蟹壳来源的甲壳素。因此可以替代虾、蟹壳作为新的提取原料,对其合理资源化开发利用可变害为益、变废为宝。
发明内容
(一)解决的技术问题
针对现有技术的不足,本发明提供了一种从白星花金龟提取制备甲壳素和壳聚糖的方法。
(二)技术方案
为实现上述目的,本发明提供如下技术方案:一种从白星花金龟提取制备甲壳素和壳聚糖的方法,其特征在于:
S1:原料预处理:将白星花金龟置于塑料密封袋中闷死后清洗干净,放入纱布袋中高温烘干后收集粉碎;
S2:脱脂处理:选取两种有机溶剂通过不同的比例混合,加入S1中粉碎虫粉,水浴抽提去除脂肪,高温烘干;
S3:脱钙处理:将S2中虫粉按一定比例添加EDTA饱和溶液,室温处理至无气泡产生,水洗后烘干;
S4:脱蛋白处理:将S3中虫粉按一定比例加入NaOH溶液,高温水浴处理后废弃碱液,水洗烘干;
S5:脱盐处理:将S4中虫粉按一定比例加入有机酸混合溶液,室温搅拌处理后废弃酸液,水洗烘干;
S6:脱色处理:将S5中虫粉按一定比例加入H2O2溶液,高温水浴浸泡至脱色;
S7:酶处理:将S6溶液冷却至室温,加入酶处理多余H2O2,过滤烘干后制得白色粉状甲壳素;
S8:脱乙酰基处理:将S7中甲壳素经高浓度NaOH溶液高温水浴处理,废弃碱液,水洗烘干后可制得壳聚糖。
优选的,所述S1中选取白星花金龟3龄幼虫或成虫,于80~100℃鼓风干燥箱烘至虫体硬脆,经小型粉碎机粉碎后过30~40目筛备用。
优选的,所述S2中有机溶剂为二氯甲烷、石油醚Ⅰ(30~60℃),按质量比(w:w)1:1~3:1混合,粉碎虫粉与混合有机溶剂固液比为1:(15~20)(g/mL),65~85℃水浴抽提3~5h,于80~100℃鼓风干燥箱烘干至恒重。
优选的,所述S3采用10~20%EDTA饱和溶液室温下浸泡脱脂虫粉2~4h,固液比为1:(20~30)(g/mL),蒸馏水洗涤至中性,80~100℃鼓风干燥箱烘干至恒重。
优选的,所述S4采用质量分数为6~10%的NaOH,虫粉与碱液的固液比为1:(10~15)(g/mL),60~80℃水浴处理4~6h,废弃碱液,蒸馏水洗至pH值=7.0~7.5,80~100℃鼓风干燥箱烘干至恒重。
优选的,所述S5采用柠檬酸、乙酸、水杨酸按照质量比(w:w)1:1:1混合至1.0~2.0g/L,虫粉与有机酸固液比为1:(10~20)(g/mL),室温采用磁力搅拌机处理3~5h,废弃酸液,蒸馏水洗涤至pH值=6.5~7.0,80~100℃鼓风干燥箱烘干至恒重。
优选的,所述S6采用10~20%H2O2溶液,按虫粉与脱色液比例为1:(10~20)(g/mL),40~60℃水浴浸泡1~3h脱色。
优选的,所述S6采用H2O2酶,去除溶液中多余H2O2,采用蛋白膜过滤多余的酶,于80-100℃鼓风干燥箱中烘干,可制得白色粉状甲壳素。
优选的,所述S7采用40~60%NaOH,于60~100℃水浴处理4~6h,废弃碱液,蒸馏水洗涤至pH值=7.0~7.5,80~100℃鼓风干燥箱烘干至恒重,可制得壳聚糖。
(三)有益效果
与现有技术相比,本发明提供了一种从白星花金龟提取制备甲壳素和壳聚糖的方法,具备以下有益效果:
1、该从白星花金龟提取制备甲壳素和壳聚糖的方法,相比虾、蟹壳,白星花金龟甲壳素含量较高,且无机盐、灰分、重金属含量较低,提取甲壳素和壳聚糖的纯度相对较高。
2、该从白星花金龟提取制备甲壳素和壳聚糖的方法,白星花金龟成虫野外易获取,也易于人工养殖,不受季节和环境的限制。将该害虫作为工业化提取甲壳素和壳聚糖的来源,可有效降低生产成本,减轻虫害损失,变废为宝。
3、该从白星花金龟提取制备甲壳素和壳聚糖的方法,传统酸碱法使用大量的酸碱,不仅消耗能源,还造成环境污染,本发明在传统方法基础上改进,采用有机酸混合溶液替代盐酸,同时增加脱脂步骤去除虫体内大量脂肪,可降低甲壳素提取过程中对环境造成的污染,避免提取甲壳素发生变化,进一步提高产品的得率和纯度。
附图说明
图1为本发明白星花金龟提取制备甲壳素和壳聚糖的方法流程图;
图2为本发明不同提取方法甲壳素和壳聚糖收率及其它指标的影响表格。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
请参阅图1-2
实施例一
将白星花金龟成虫置于塑料密封袋中闷死后清洗干净,放入纱布袋中于80~100℃鼓风干燥箱烘至虫体硬脆,经小型粉碎机粉碎后过30~40目筛备用。采用二氯甲烷:石油醚Ⅰ(30~60℃)按质量比(w:w)3:1混合,加入粉碎虫粉脱脂,固液比为1:15(g/mL),65℃水浴抽提4h,于80~100℃鼓风干燥箱烘干至恒重;采用15%EDTA饱和溶液室温下浸泡脱脂虫粉3h脱钙,固液比为1:25(g/mL),蒸馏水清洗后于80~100℃鼓风干燥箱烘干至恒重;采用8%NaOH脱蛋白处理,虫粉与碱液的固液比为1:10,60℃水浴5h,3000~5000r/min离心5~10min,弃去上清液,蒸馏水洗至pH值=7.0~7.5,80~100℃鼓风干燥箱烘干至恒重;采用柠檬酸、乙酸、水杨酸按照质量比(w:w)1:1:1混合至1.5g/L,虫粉与有机酸固液比为1:15(g/mL),室温采用磁力搅拌机处理4h,3000~5000r/min离心5~10min,弃去上清液,蒸馏水洗涤至pH值=6.5~7.0,80~100℃鼓风干燥箱烘干至恒重;采用20%H2O2溶液,按虫粉与脱色液比例为1:15(g/mL),50℃水浴浸泡2h脱色;溶液冷却至室温后,采用H2O2酶,去除溶液中多余H2O2,采用蛋白膜过滤多余的酶,于80~100℃鼓风干燥箱中烘干,可制得白色粉状甲壳素;采用50%NaOH,于80℃水浴处理5h,3000~5000r/min离心5~10min,弃去上清液,蒸馏水洗涤至pH值=7.0~7.5,80~100℃鼓风干燥箱烘干至恒重,可制得壳聚糖。
实施例二
将白星花金龟3龄幼虫置于塑料密封袋中闷死后清洗干净,放入纱布袋中于80~100℃鼓风干燥箱烘至虫体硬脆,经小型粉碎机粉碎后过30~40目筛备用。采用二氯甲烷:石油醚Ⅰ(30~60℃)按1:1混合,加入粉碎虫粉脱脂,固液比为1:20(g/mL),85℃水浴抽提3h,于80~100℃鼓风干燥箱烘干至恒重;采用10%EDTA饱和溶液室温下浸泡脱脂虫粉4h,固液比为1:30(g/mL),蒸馏水清洗后于80~100℃鼓风干燥箱烘干至恒重;采用6%NaOH脱蛋白处理,虫粉与碱液的固液比为1:15,80℃水浴4h,3000~5000r/min离心5~10min,弃去上清液,蒸馏水洗至pH值=7.0~7.5,80~100℃鼓风干燥箱烘干至恒重;采用柠檬酸、乙酸、水杨酸按照质量比(w:w)1:1:1混合至2.0g/L,虫粉与有机酸固液比为1:20(g/mL),室温采用磁力搅拌机处理5h,3000~5000r/min离心5~10min,弃去上清液,蒸馏水洗涤至pH值=6.5~7.0,80~100℃鼓风干燥箱烘干至恒重;采用10%H2O2溶液,按虫粉与脱色液比例为1:20(g/mL),40℃水浴浸泡3h脱色;溶液冷却至室温后采用H2O2酶,去除溶液中多余H2O2,采用蛋白膜过滤多余的酶,于80~100℃鼓风干燥箱中烘干,可制得白色粉状甲壳素;采用40%NaOH,于60℃水浴处理6h,3000~5000r/min离心5~10min,弃去上清液,蒸馏水洗涤至pH值=7.0~7.5,80~100℃鼓风干燥箱烘干至恒重,可制得壳聚糖。
对照例1
将白星花金龟成虫置于塑料密封袋中闷死后清洗干净,放入纱布袋中于80~100℃鼓风干燥箱烘至虫体硬脆,经小型粉碎机粉碎后过30~40目筛备用。采用8%NaOH脱蛋白处理,虫粉与碱液的固液比为1:10,60℃水浴5h,3000~5000r/min离心5~10min,弃去上清液,蒸馏水洗至pH值=7.0~7.5,80~100℃鼓风干燥箱烘干至恒重;采用柠檬酸、乙酸、水杨酸按照质量比(w:w)1:1:1混合至1.5g/L,虫粉与有机酸固液比为1:15(g/mL),室温采用磁力搅拌机处理4h,3000~5000r/min离心5~10min,弃去上清液,蒸馏水洗涤至pH值=6.5~7.0,80~100℃鼓风干燥箱烘干至恒重;采用20%H2O2溶液,按虫粉与脱色液比例为1:15(g/mL),50℃水浴浸泡2h脱色;溶液冷却至室温后,采用H2O2酶,去除溶液中多余H2O2,采用蛋白膜过滤多余的酶,于80~100℃鼓风干燥箱中烘干,可制得白色粉状甲壳素;采用50%NaOH,于80℃水浴处理5h,3000~5000r/min离心5~10min,弃去上清液,蒸馏水洗涤至pH值=7.0~7.5,80~100℃鼓风干燥箱烘干至恒重,可制得壳聚糖。
对照例2
将白星花金龟成虫置于塑料密封袋中闷死后清洗干净,放入纱布袋中于80~100℃鼓风干燥箱烘至虫体硬脆,经小型粉碎机粉碎后过30~40目筛备用。采用二氯甲烷:石油醚Ⅰ(30-60℃)按质量比(w:w)3:1混合,加入粉碎虫粉脱脂,固液比为1:15(g/mL),65℃水浴抽提4h,于80~100℃鼓风干燥箱烘干至恒重;采用15%EDTA饱和溶液室温下浸泡脱脂虫粉3h脱钙,固液比为1:25(g/mL),蒸馏水清洗后于80~100℃鼓风干燥箱烘干至恒重;采用8%NaOH脱蛋白处理,虫粉与碱液的固液比为1:10,60℃水浴5h,3000~5000r/min离心5~10min,弃去上清液,蒸馏水洗至pH值=7.0~7.5,80~100℃鼓风干燥箱烘干至恒重;采用柠檬酸、乙酸、水杨酸按照质量比(w:w)1:1:1混合至1.5g/L,虫粉与有机酸固液比为1:15(g/mL),室温采用磁力搅拌机处理4h,3000~5000r/min离心5~10min,弃去上清液,蒸馏水洗涤至pH值=6.5~7.0,80~100℃鼓风干燥箱烘干至恒重;采用4%KMnO4溶液,按虫粉与脱色液比例为1:15(g/mL),50℃水浴浸泡2h脱色;溶液冷却至室温后采用1%维生素C溶液,去除溶液中多余KMnO4,于80~100℃鼓风干燥箱中烘干,可制得白色粉状甲壳素;采用50%NaOH,于80℃水浴处理5h,3000~5000r/min离心5~10min,弃去上清液,蒸馏水洗涤至pH值=7.0~7.5,80-100℃鼓风干燥箱烘干至恒重,可制得壳聚糖。
分别采用实施例1、实施例2、对照例1、对照例2提取白星花金龟中甲壳素和壳聚糖,每个试验3组重复。试验结束后,测定氮量和灰分含量,计算甲壳素收率。收率=甲壳素样品质量(g)/昆虫原料(g)×100%;测定脱乙酰度和黏度,计算壳聚糖收率。收率=壳聚糖样品质量(g)/甲壳素样品质量(g)×100%。数据如下表所示。
图2中数据为平均值±标准误,同一列数据后不同小写字母代表经ANOVA单因素方差分析和Turkey’s多重比较后差异显著,p<0.05。
从上表可以看出,实施例1和实施例2甲壳素和壳聚糖收率、黏度显著高于对照例1,而灰分含量显著降低,达到食品级标准(<1%),表明采用本发明中的脱脂脱钙方法可提高甲壳素和壳聚糖收率和品质;实施例1中甲壳素和壳聚糖收率显著高于实施例2,表明白星花金龟成虫中甲壳素含量高于幼虫;实施例1中甲壳素白度值显著高于对照例2,表明采用H2O2溶液脱色效果优于KMnO4。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (9)
1.一种从白星花金龟提取制备甲壳素和壳聚糖的方法,其特征在于:
S1原料预处理:将白星花金龟置于塑料密封袋中闷死后清洗干净,放入纱布袋中高温烘干后收集粉碎;
S2脱脂处理:选取两种有机溶剂通过不同的比例混合,加入S1中粉碎虫粉,水浴抽提去除脂肪,高温烘干;
S3脱钙处理:将S2中虫粉按一定比例添加EDTA饱和溶液,室温处理至无气泡产生,水洗后烘干;
S4脱蛋白处理:将S3中虫粉按一定比例加入NaOH溶液,高温水浴处理后废弃碱液,水洗烘干;
S5脱盐处理:将S4中虫粉按一定比例加入有机酸混合溶液,室温搅拌处理后废弃酸液,水洗烘干;
S6脱色处理:将S5中虫粉按一定比例加入H2O2溶液,高温水浴浸泡至脱色;
S7酶处理:将S6溶液冷却至室温,加入酶处理多余H2O2,过滤烘干后制得白色粉状甲壳素;
S8脱乙酰基处理:将S7中甲壳素经高浓度NaOH溶液高温水浴处理,废弃碱液,水洗烘干后可制得壳聚糖。
2.根据权利要求1所述的一种从白星花金龟提取制备甲壳素和壳聚糖的方法,其特征在于:所述S1中选取白星花金龟3龄幼虫或成虫,于80~100℃鼓风干燥箱烘至虫体硬脆,经小型粉碎机粉碎后过30~40目筛备用。
3.根据权利要求1所述的一种从白星花金龟提取制备甲壳素和壳聚糖的方法,其特征在于:所述S2中有机溶剂为二氯甲烷、石油醚Ⅰ(30~60℃),按质量比(w:w)1:1~3:1混合,粉碎虫粉与混合有机溶剂固液比为1:(15~20)(g/mL),65~85℃水浴抽提3~5h,于80~100℃鼓风干燥箱烘干至恒重。
4.根据权利要求1所述的一种从白星花金龟提取制备甲壳素和壳聚糖的方法,其特征在于:所述S3采用10~20%EDTA饱和溶液室温下浸泡脱脂虫粉2~4h,固液比为1:(20~30)(g/mL),蒸馏水洗涤至中性,80~100℃鼓风干燥箱烘干至恒重。
5.根据权利要求1所述的一种从白星花金龟提取制备甲壳素和壳聚糖的方法,其特征在于:所述S4采用质量分数为6~10%的NaOH,虫粉与碱液的固液比为1:(10~15)(g/mL),60~80℃水浴处理4~6h,废弃碱液,蒸馏水洗至pH值=7.0~7.5,80~100℃鼓风干燥箱烘干至恒重。
6.根据权利要求1所述的一种从白星花金龟提取制备甲壳素和壳聚糖的方法,其特征在于:所述S5采用柠檬酸、乙酸、水杨酸按照质量比(w:w)1:1:1混合至1.0~2.0g/L,虫粉与有机酸固液比为1:(10~20)(g/mL),室温采用磁力搅拌机处理3~5h,废弃酸液,蒸馏水洗涤至pH值=6.5~7.0,80~100℃鼓风干燥箱烘干至恒重。
7.根据权利要求1所述的一种从白星花金龟提取制备甲壳素和壳聚糖的方法,其特征在于:所述S6采用10~20%H2O2溶液,按虫粉与脱色液比例为1:(10~20)(g/mL),40~60℃水浴浸泡1~3h脱色。
8.根据权利要求1所述的一种从白星花金龟提取制备甲壳素和壳聚糖的方法,其特征在于:所述S6采用H2O2酶,去除溶液中多余H2O2,采用蛋白膜过滤多余的酶,于80-100℃鼓风干燥箱中烘干,可制得白色粉状甲壳素。
9.根据权利要求1所述的一种从白星花金龟提取制备甲壳素和壳聚糖的方法,其特征在于:所述S7采用40~60%NaOH,于60~100℃水浴处理4~6h,废弃碱液,蒸馏水洗涤至pH值=7.0~7.5,80~100℃鼓风干燥箱烘干至恒重,可制得壳聚糖。
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Application publication date: 20220923 |