CN116809033A - 一种磷酸化几丁质复合材料及其制备和应用 - Google Patents
一种磷酸化几丁质复合材料及其制备和应用 Download PDFInfo
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Abstract
本发明公开了一种磷酸化几丁质复合材料及其制备和应用。本发明通过对几丁质进行磷酸化改性,再将磷酸化几丁质复合四氧化三铁微球得到一种复合材料Fe3O4@PCT,该复合材料能特异性吸附几丁质酶。与膨润土和未改性的几丁质比较,该复合材料对几丁质酶的吸附更好,同时提高了果酒中多酚的保留率,有利于提升果酒的质量,在果酒后浑浊防控应用中或在吸附几丁质酶中具有显著的经济价值和应用价值。
Description
技术领域
本发明涉及果酒后浑浊防控领域,具体涉及一种磷酸化几丁质复合材料及其制备和应用。
背景技术
果酒中的后浑浊现象是运输或储存过程中发生的感官质量缺陷,尽管后浑浊可能对味道和风味的影响不大,但会给消费者带来不愉快的体验。目前已有研究表明,导致果酒和饮料中后浑浊形成的蛋白质在植物原料中普遍存在。如文献(Roles ofGrapeThaumatin-like Protein and Chitinase inWhite Wine Haze Formation.JOURNAL OFAGRICULTURAL AND FOOD CHEMISTRY,2021,59(2):733-740))报道,葡萄几丁质酶是未精制葡萄酒中热致浑浊的主要蛋白,几丁质酶聚集的最后阶段被证明是不可逆的并且与可见雾度密切相关。为了防止后浑浊的形成,商业中通常加入膨润土去除蛋白质,然而膨润土处理不仅去除了致浊蛋白,也去除了生理活性成分,如多酚等,导致质量损失。因此,需要一种蛋白质选择性去除技术,控制果酒后浑浊的同时,尽可能保留生理活性成分,提高果酒的质量。
发明内容
本发明的目的是提供一种磷酸化几丁质复合材料及其制备和应用。本发明提供的磷酸化几丁质复合材料能特异性吸附几丁质酶,相比膨润土显著提高了多酚的保留率,在果酒后浑浊防控中具有显著的应用价值。
为了达到上述目的,本发明提供了一种磷酸化几丁质复合材料Fe3O4@PCT的制备方法,包含如下步骤:
1)将几丁质粉末与P2O5、H3PO4、磷酸三乙酯和己醇混合进行反应;
2)将反应所得的混合物在甲醇中沉淀,过滤沉淀并干燥得到磷酸化几丁质;
3)将磷酸化几丁质分散在氢氧化钠、尿素和水的混合溶液中,经冻融工艺得到磷酸化几丁质溶液;
4)将四氧化三铁微球分散到水中,与步骤3)所得的磷酸化几丁质溶液混合,并加入Span 85和异辛烷的混合溶液,将所得的混合体系进行搅拌,再添加Span85和异辛烷继续搅拌;
5)加热步骤4)中的产物以形成磷酸化几丁质复合四氧化三铁微球;
6)用磁铁分离步骤5)所得产物并进行洗涤,将所得的产物进行干燥即得磷酸化几丁质复合材料Fe3O4@PCT。
优选地,上述几丁质粉末与P2O5、H3PO4、磷酸三乙酯和己醇的用量比为1g:2g:10mL:5mL:5mL。
优选地,上述反应的条件为35℃下反应3天。
优选地,上述步骤3)混合溶液中,其氢氧化钠、尿素和超纯水的质量比为2:1:22;上述磷酸化几丁质与该混合溶液的重量比为1:20。
优选地,上述步骤4)中四氧化三铁微球与超纯水的比例为1g:12.5mL。
优选地,上述步骤4)中的搅拌为冰浴下搅拌60min。
优选地,上述步骤5)中的加热条件为为60℃加热。
优选地,上述步骤6)中的洗涤采用水和乙醇进行洗涤。
本发明还提供了一种由上述制备方法制备所得的复合材料Fe3O4@PCT。
本发明提供的复合材料Fe3O4@PCT可应用于果酒后浑浊防控或在吸附几丁质酶中。
优选地,上述的应用包含果酒中致浊蛋白的去除或/和生物活性成分的保留。
本发明的磷酸化几丁质复合材料及其制备和应用,解决了果酒后浑浊防控中常用的膨润土对几丁质酶的吸附效果差、多酚保留率低等问题,具有以下优点:
(1)本发明提供的磷酸化几丁质复合材料制备步骤简单,易于操作,绿色无污染。
(2)本发明将磷酸化几丁质与四氧化三铁磁珠进行连接,连接产物具备了磁效应和更好的固定化性能,是一种高效且成本低廉的吸附材料,具有更显著的应用潜力。
(3)本发明提供的磷酸化几丁质复合材料相比膨润土和未改性的几丁质应用在果酒后浑浊防控中,该复合材料能显著提高几丁质酶的吸附效果,提高酒样中多酚的保留率,在果酒后浑浊防控中具有一定的推动作用,具有显著的经济价值和应用价值。
附图说明
图1为本发明中Fe3O4@PCT和Fe3O4的SEM图。
图2为本发明中Fe3O4@PCT的EDS光谱图,其中图2中的A、B、C、D分别为Fe3O4@PCT中Fe、C、O、P四种元素的EDS光谱。
图3为本发明中几丁质和PCT的FT-IR图。
图4为本发明中Fe3O4、PCT、Fe3O4@PCT(pH=6.5)、Fe3O4@PCT(pH=4.1)的Zeta电位图。
图5为本发明中Fe3O4@PCT的最适吸附温度测定结果。
图6为本发明中Fe3O4@PCT的最适吸附用量测定结果。
图7为本发明中Fe3O4@PCT的最适吸附时间测定结果。
图8为本发明中Fe3O4@PCT吸附蛋白的SDS-PAGE分析结果。
图9为本发明中Fe3O4@PCT吸附的蛋白条带样品质谱分析结果。
图10为本发明中Fe3O4@PCT对蛋白、多酚及花青素的吸附情况测定结果。
图11为本发明中Fe3O4@PCT处理酒样后酒样的热稳定情况测定结果。
具体实施方式
下面将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。
为了进一步提高几丁质对几丁质酶的亲和吸附,本发明采用分子对接软件,在www.pubchem.com网站检索出基于几丁质的化学改性化合物,根据对接得分筛选出磷酸化几丁质作为吸附底物。表面形貌学研究结果也显示,由于磷酸化使得磷酸化几丁质表现出非常多孔和粗糙的结构,进一步加强了几丁质的吸附能力,磷酸化几丁质经过与四氧化三铁磁珠连接后则具备了磁效应和更好的固定化性能,具有更显著的应用潜力。
实施例1复合材料Fe3O4@PCT制备与表征
一种特异选择吸附几丁质酶的复合材料的制备方法,包括以下步骤:
将几丁质粉末(Chitin,5g)添加到圆底烧瓶中,加入P2O5(10g)、H3PO4(50mL)、磷酸三乙酯(25mL)和己醇(25mL)。反应在35℃下连续反应3天。结束后将反应混合物倒入过量的甲醇中沉淀。将沉淀过滤并用甲醇洗涤,然后在37℃的真空烘箱中干燥1天,得到所需的磷酸化几丁质(PCT);
取5g PCT,将其分散到含有氢氧化钠(8g)、尿素(4g)和超纯水(88g)的混合溶液中,使用冻融工艺获得透明的磷酸化几丁质溶液;
通过将0.4g四氧化三铁微球(Fe3O4)分散到5mL超纯水中,超声处理10min,然后在冰浴条件下与上述所得的磷酸化几丁质溶液混合,加入1.1g的Span 85和50g的异辛烷的混合溶液,该混合溶液体系在冰浴下搅拌60min,然后再添加0.6g的Span85和3.5g的异辛烷,继续冰浴下搅拌60min;
搅拌结束后,转入60℃水浴形成磷酸化几丁质复合四氧化三铁微球,使用磁铁分离所得产物,并分别用水和乙醇清洗所得产物3次,将所获得的产物进行干燥,即得复合材料磷酸化几丁质复合四氧化三铁微球-Fe3O4@PCT,收集制备所得的粉状复合材料备用。
对上述制备的复合材料Fe3O4@PCT的表征进行检测,得到Fe3O4@PCT的表征测定结果如图1-图4所示,其中,图1中的A为Fe3O4的SEM图;图1中的B为Fe3O4@PCT的SEM图;图2为Fe3O4@PCT的EDS光谱(其中图2中的A、B、C、D分别为Fe3O4@PCT中Fe、C、O、P四种元素的EDS光谱);图3为几丁质(Chitin)和PCT的FT-IR图;图4为Fe3O4、PCT、Fe3O4@PCT(pH=6.5)、Fe3O4@PCT(pH=4.1)的Zeta电位图。
实施例2复合材料Fe3O4@PCT的最适吸附条件测定
1.最适吸附温度的确定
取5mL果酒于样品瓶中,加入3mg Fe3O4@PCT吸附材料,吸附时间为8h,分别置于不同温度(25、30、35、40、45℃)的恒温振荡器中,吸附结束后用磁铁分离并留取上清液测定其蛋白浓度,计算吸附量,吸附量计算公式如下:
式中:
Qe—吸附容量(mg/g)
C0—初始果酒中蛋白浓度(mg/mL)
C1—吸附后果酒中蛋白浓度(mg/mL)
V—溶液的体积(mL)
m—Fe3O4@PCT的质量(mg)
吸附温度结果如图5,可知,吸附温度为35℃时蛋白吸附量最大,确定35℃为最适温度。
2.最适用量的确定
取5mL酒于样品瓶中,加入不同量的Fe3O4@PCT吸附材料,置于35℃的恒温振荡器中吸附24h,用磁铁分离出材料留取上清,测定上清的蛋白浓度,计算吸附量的方法同上。
最适用量结果如图6所示,可知,当酒样为5mL、复合材料的用量为3mg时,复合材料的单位吸附量最大,确定0.6g/mL酒样中的复合材料添加量为最适用量。
3.最适吸附时间的确定
取5mL果酒于样品瓶中,加入最适用量Fe3O4@PCT吸附材料,置于35℃的恒温振荡器中,吸附不同时间,吸附后用磁铁分离并留取上清液测定其蛋白浓度,计算吸附量的方法同上。
吸附时间结果如图7,可知,吸附8h后蛋白量趋于饱和,确定8h为最适吸附时间。
实施例3Fe3O4@PCT的特异吸附研究
1.SDS-PAGE分析
参照实验例1中所得的最适优化条件,取Fe3O4@PCT加入桑葚果酒中,收集磁铁分离吸附后的Fe3O4@PCT,加入H2O涡旋震荡,收集上清液,然后加入SDS(0.2%)和NaCl溶液涡旋震荡,收集上清液。随后对收集到的上清液进行SDS-PAGE分析。
SDS-PAGE结果如图8所示,其中,泳道1为蛋白质分子量标准,泳道2、3为桑葚果酒原样液(两个重复样),泳道4、5为水洗脱材料回收的上清液(两个重复样),泳道6、7为0.1%SDS和NaCl溶液洗脱的回收上清液(两个重复样)。可知,在桑葚果酒原样液(泳道2、3)中清楚观察到在27-35kDa范围内有明显的条带,根据已有文献报道植物来源的几丁质酶的分子量介于25-35kDa,但在H2O洗脱后(泳道4、5)几乎没有在上清液中检测到蛋白;经SDS和NaCl溶液洗脱后(泳道6、7),在27-35kDa范围内明显出现了两个条带;由于SDS和NaCl破坏了特异吸附的疏水作用力和氢键,使得被特异吸附的蛋白被洗脱下来,结果表明Fe3O4@PCT对蛋白吸附存在特异吸附性。
2.基于nano LC-Q-Exactive-MS/MS技术分析蛋白条带样品
高分辨nano LC-Q-Exactive-MS/MS采集的蛋白条带样品的总离子流图见图9。使用MaxQuant 1.6.14软件对采集到的质谱数据进行处理,利用Andromeda与Uniprot数据库鉴定多肽序列,并归属蛋白来源。最终,从蛋白条带胰酶水解多肽样品中鉴定了136个肽段,来源于68个蛋白。蛋白质表达量强度值得分前20的蛋白质见下表1。其中水解得到的多肽序列分子量为1411.6368,该肽段相对于整个蛋白质的得分是133.71,等于或大于35,经验值可信,蛋白覆盖率为46.4%(20%-50%覆盖率为成功匹配区间),分析结果表明该多肽序列与chitinase Class I(相对分子质量:34.943kDa)有较高相似度。此外该多肽序列与chitinase Class I b(相对分子质量:33.791kDa)的蛋白覆盖率为47.2%,表明与chitinase Class I b有较高的相似度,符合SDS-PAGE分析(图8所示)中两条带的结果。综合SDS-PAGE分析和nano LC-Q-Exactive-MS/MS分析说明Fe3O4@PCT对几丁质酶有很好的特异吸附作用。
表1蛋白胶条中鉴定得分前20的蛋白质
实施例4效果对比验证
参照实验例1中所得的最适优化条件,取适量果酒于样品瓶中,分别加入相同用量的Fe3O4@PCT、几丁质(Chitin)和膨润土(Bentonite),置于恒温振荡器中进行吸附处理。结束后留取上清液,分别测定上清液中蛋白质(Protein)、多酚(Polyphenols)和花青素(Anthocyanin)含量。
测定结果如图10所示,可知,Fe3O4@PCT对蛋白、多酚和花青素的吸附率分别为3.221%、5.09%和3.019%,均显著低于Bentonite和Chitin处理(Bentonite对蛋白、多酚和花青素的吸附率分别为21.24%,14.4%和17.15%;Chitin对蛋白、多酚和花青素的吸附率分别为27.21%,18.98%和25.32%),表明Fe3O4@PCT对蛋白具有选择吸附作用并能显著保留果酒中生理活性物质。
实施例5热稳定性测定
取Fe3O4@PCT、几丁质和膨润土处理后的果酒于样品瓶中,先置于80℃的环境中2h,然后置于-20℃的环境中2h,最后酒样放置至室温,在540nm处测定处理前后吸光度的变化,当酒样加热和未加热之间的吸光度之差(ΔA540nm)>0.02,被认为是蛋白质不稳定的。
测定结果如图11所示,经过膨润土、几丁质和Fe3O4@PCT处理后ΔA540nm均小于0.02,说明Fe3O4@PCT吸附处理后果酒仍具有热稳定性。
综上可知,本发明通过对几丁质进行磷酸化改性,再将磷酸化几丁质复合四氧化三铁微球得到一种复合材料Fe3O4@PCT,该复合材料能特异性吸附几丁质酶,与膨润土和未改性的几丁质比较,该复合材料对几丁质酶的吸附更好,同时提高了果酒中多酚的保留率,有利于提升果酒的质量,在果酒后浑浊防控应用中具有显著的经济价值和应用价值。
尽管本发明的内容已经通过上述优选实施例作了详细介绍,但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后,对于本发明的多种修改和替代都将是显而易见的。因此,本发明的保护范围应由所附的权利要求来限定。
Claims (10)
1.一种磷酸化几丁质复合材料Fe3O4@PCT的制备方法,其特征在于,包含如下步骤:
1)将几丁质粉末与P2O5、H3PO4、磷酸三乙酯和己醇混合进行反应;
2)将反应所得的混合物在甲醇中沉淀,过滤沉淀并干燥得到磷酸化几丁质;
3)将磷酸化几丁质分散在氢氧化钠、尿素和水的混合溶液中,经冻融工艺得到磷酸化几丁质溶液;
4)将四氧化三铁微球分散到水中,与步骤3)所得的磷酸化几丁质溶液混合,并加入Span85和异辛烷的混合溶液,将所得的混合体系进行搅拌,再添加Span85和异辛烷继续搅拌;
5)加热步骤4)中的产物以形成磷酸化几丁质复合四氧化三铁微球;
6)用磁铁分离步骤5)所得产物并进行洗涤,将所得的产物进行干燥即得磷酸化几丁质复合材料Fe3O4@PCT。
2.根据权利要求1所述的制备方法,其特征在于,所述几丁质粉末与P2O5、H3PO4、磷酸三乙酯和己醇的用量比为1g:2g:10mL:5mL:5mL。
3.根据权利要求1所述的制备方法,其特征在于,所述反应的条件为35℃下反应3天。
4.根据权利要求1所述的制备方法,其特征在于,所述步骤3)混合溶液中,其氢氧化钠、尿素和超纯水的质量比为2:1:22;所述磷酸化几丁质与所述混合溶液的重量比为1:20。
5.根据权利要求1所述的制备方法,其特征在于,所述步骤4)中四氧化三铁微球与超纯水的比例为1g:12.5mL。
6.根据权利要求1所述的制备方法,其特征在于,所述步骤4)中的搅拌为冰浴下搅拌60min。
7.根据权利要求1所述的制备方法,其特征在于,所述步骤5)中的加热为60℃下加热。
8.由权利要求1-7中任意一项所述的制备方法制备所得的复合材料Fe3O4@PCT。
9.如权利要求8所述的复合材料Fe3O4@PCT在果酒后浑浊防控的应用或在吸附几丁质酶中的应用。
10.根据权利要求9所述的应用,其特征在于,所述的应用包含果酒中致浊蛋白的去除或/和生物活性成分的保留。
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