CN114380926B - 从柑橘加工水中回收柑橘果胶的方法及其用途 - Google Patents
从柑橘加工水中回收柑橘果胶的方法及其用途 Download PDFInfo
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Abstract
本发明公开了一种从柑橘加工水中回收柑橘果胶的方法,包括以下步骤:将柑橘加工水浓缩,得浓缩液;将浓缩液与无水乙醇混合、静置、离心,离心所得沉淀用去离子水溶解;所得的沉淀溶液用木瓜蛋白酶结合Sevag试剂进行脱蛋白处理,然后于去离子水中透析,Mw截流2000Da,接着浓缩、冷冻干燥,得到粗柑橘果胶(CCP);将粗柑橘果胶(CCP)进行分离纯化,分别得到CP0、CP1和CP3这三种果胶组分。上述组分具有抗氧化、降血脂等活性。
Description
技术领域
本发明属于食品工程技术领域,涉及从柑橘加工水中回收果胶,提高柑橘加工产品附加值,并解决加工水造成的环境污染问题。
背景技术
果胶广泛存在于高等植物的细胞壁中,是一类结构非常复杂的天然酸性杂多糖,主要由三种结构组成,即同型半乳糖醛酸聚糖(HG)、鼠李半乳糖醛酸聚糖-I(RG-I)和鼠李半乳糖醛酸聚糖-II(RG-II)。一般来说,RG-I结构域含量高的果胶具有良好的生物学功能。果胶可以从多种来源制备,不同来源的果胶的结构和功能有很大差异。商品果胶,通常由至少65%的半乳糖醛酸单体组成,其以HG为主,还含有少量RG-I结构域。
柑橘在许多国家都广泛种植,产量很大。柑橘加工的副产品(如果皮等)富含果胶,是生产果胶的良好来源。另外,在柑橘罐头加工中产生的大量加工水含有丰富的果胶多糖。
因此从这些加工水中回收果胶,不仅能提高柑橘产品的附加值,而且能解决加工水造成的环境污染问题。
柑橘罐头加工工艺中产生的酸液:pH为1左右,含有总固形物约0.7%,果胶含量约0.1~0.3%,总黄酮含量约0.07%;柑橘罐头加工工艺中产生的碱液:pH为13左右,含有总固形物约1.3%,果胶含量约0.2~0.5%,总黄酮含量约0.003%。
CN106188335A的发明《柑橘罐头加工工艺中同步提取果胶及多酚的方法与系统》告知了:柑橘罐头加工工艺中产生的酸、碱液依次进行pH值调整,低温浓缩,高分子果胶萃取,回收萃取液,一级分离,低分子果胶提取,除盐、重金属,多酚提取,单糖提取。
CN103122039A的发明《一种柑橘罐头生产酸排液提取果胶的工艺》以及CN103122038A的发明《一种柑橘罐头生产碱排液提取果胶的工艺》告知:柑橘罐头生产中的酸排液/碱排液调节pH值、过滤、浓缩、喷雾干燥后得果胶粉。
发明内容
本发明要解决的技术问题是提供一种从柑橘加工水中回收柑橘果胶的方法,采用该方法能从柑橘加工水中回收制备性能优良的果胶组分,该果胶组分具有抗氧化、降血脂活性。
为了解决上述技术问题,本发明提供一种从柑橘加工水中回收柑橘果胶的方法,包括以下步骤:
1)、将柑橘加工水浓缩,得浓缩液;
2)、将所述浓缩液与无水乙醇混合,于4±1℃静置12±2h,离心(9000±1000rpm,15±5分钟),离心所得用去离子水溶解,得沉淀溶液;
浓缩液:无水乙醇=1:4~6的体积比;
沉淀:去离子水=1:20~30的重量比;
3)、将步骤2)所得的沉淀溶液用木瓜蛋白酶(0.1%,w/v)结合Sevag试剂进行脱蛋白处理,然后于去离子水中透析72±6小时,Mw截流2000Da,接着浓缩、冷冻干燥,得到粗柑橘果胶(CCP);
所述Sevag试剂为氯仿:正丁醇=4:1体积比的混合剂。
作为本发明的从柑橘加工水中回收柑橘果胶的方法,还包括如下的步骤4):
将粗柑橘果胶(CCP)进行分离纯化,分别得到CP0、CP1和CP3这三种果胶组分。
作为本发明的从柑橘加工水中回收柑橘果胶方法的进一步改进,步骤4)为:
将粗柑橘果胶(CCP)加去离子配制成浓度为20~25mg/mL的CCP溶液,离心(6000±1000rpm,15±5分钟),所得的上清液用于后续的上样;
选用纤维素DEAE-52柱,上清液上样(即,取上清液上样于已处理好的纤维素DEAE-52柱);
先用去离子水洗脱,再依次用0.1M、0.3M、0.5M、0.7M和0.9M的NaCl溶液分别进行洗脱,流速为1mL/min;上述去离子水、0.1M、0.3M、0.5M、0.7M和0.9M的NaCl溶液的用量均为2~3.5倍柱体积;
将去离子水对应所得的洗脱液减压浓缩、冷冻干燥,得到果胶组分CP0;
将0.1M和0.3M NaCl对应所得的两种洗脱液分别进行如下处理:减压浓缩、透析脱盐、冷冻干燥,对应得到果胶组分CP1和CP3。
说明:
用苯酚-硫酸法测定洗脱液中果胶的含量;由于0.5M、0.7M和0.9M NaCl的洗脱液中果胶含量较少,因此不予进一步研究。
将纤维素DEAE-52处理后装柱(柱子规格为60cm×Φ2.6cm,有效长度为32cm,有效柱床体积为176mL),得纤维素DEAE-52柱;取8~10mL CCP溶液所得的上清液用于上样。
作为本发明的从柑橘加工水中回收柑橘果胶方法的进一步改进,步骤3)为:
按照0.1g/100ml的比例,将木瓜蛋白酶加入至沉淀溶液中,于50±5℃水浴保温3±0.5小时,灭酶活后,离心(9000±1000rpm,15±5分钟),取上清液;
将上清液用Sevag试剂进行处理:即,按上清液:Sevag试剂=4~6:1(优选5:1)的体积比,在上清液中加入Sevag试剂搅拌混匀后静置,直至分层;取分层所得的上层溶液反复用Sevag试剂进行处理(即,上层溶液替代上清液用Sevag试剂进行处理),直至不再出现蛋白层;所得称为样品溶液;
将所得的样品溶液装入2000Da透析袋中再置于去离子水中进行透析,透析时间为72±6小时,将所得的透析液浓缩(于50±5℃的条件下旋转浓缩至为原体积的1/4),再冷冻干燥(-40~-60℃冷冻干燥24小时),得到粗柑橘果胶(CCP)。
作为本发明的从柑橘加工水中回收柑橘果胶方法的进一步改进,所述步骤1):
将柑橘加工水(总碳水化合物含量约10mg/mL)50±5℃于旋转蒸发器中浓缩至为原体积的1/3~1/5(优选1/4),得浓缩液。
本发明还同时提供了利用上述方法所得的粗柑橘果胶(CCP)、果胶组分CP1、果胶组分CP3的用途:制备具有抗氧化、降血脂活性的制品。即,制备具有降血脂功效的功能性食品基料。
经细胞毒实验,CCP、CP1、CP3均判定成细胞无毒性,而400和800μg/mL的CP0组细胞活力低于80%,认为有一定的细胞毒性。因此,CP0不进相应的性能活性检测。
作为本发明用途的改进:制备食品增稠剂。
本发明具有如下技术优势:
1.本发明所得的柑橘果胶CCP及组分CP1和CP3具有良好的流变特性,因此可用作食品增稠剂。
2.本发明所得的柑橘果胶CCP及组分CP1和CP3具有良好的抗氧化和降血脂活性,因此可用作相关功能性食品的基料。
综上,本发明获得了活性好的高RG-I果胶。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细说明。
图1是按实验1方法测定实施例1得到的果胶粗品(CCP)和实施例2得到的果胶分离组分CP0、CP1和CP3的表观黏度。
图2是按实验1方法测定实施例1得到的CCP和实施例2得到的CP0、CP1和CP3的动态粘弹特性。
图3是实施例1得到的CCP和实施例2得到的CP0、CP1和CP3,按实验2方法进行DPPH自由基清除能力的测试结果。
图4是实施例1得到的CCP和实施例2得到的CP0、CP1和CP3,按实验3方法进行羟基自由基清除能力的测试结果。
图5是实施例1得到的CCP和实施例2得到的CP0、CP1和CP3,按实验4方法进行测定的对H2O2-诱导的氧化应激的HepG2细胞中活性氧(ROS)水平的影响结果;
注:#表示模型组与空白组相比P<0.05;*表示与模型组相比P<0.05同组内标有不同小写字母的表示同一组分不同浓度具有显著性差异(P<0.05);组间标有不同大写字母的表示同一浓度不同组分具有显著性差异(P<0.05)。
图6是实施例1得到的CCP和实施例2得到的CP0、CP1和CP3,按实验5方法进行测定的对H2O2-诱导的氧化应激的HepG2细胞中超氧化物歧化酶(SOD)活性的影响结果;
注:#表示模型组与空白组相比P<0.05;*表示与模型组相比P<0.05同组内标有不同小写字母的表示同一组分不同浓度具有显著性差异(P<0.05);组间标有不同大写字母的表示同一浓度不同组分具有显著性差异(P<0.05)。
图7是实施例1得到的CCP和实施例2得到的CP0、CP1和CP3,按实验6方法进行测定的对5种胆酸盐的结合能力的测试结果。消胆胺为阳性对照;
注:大写字母表示与阳性对照消胆胺相比样品是否有显著性差异(P<0.05),小写字母表示每种胆酸盐组内不同样品是否有显著性差异(P<0.05)。
图8是由本发明施例1得到的CCP和实施例2得到的CP0、CP1和CP3,按实验7方法进行测定的对油酸诱导的高脂肪HepG2细胞甘油三酯(TG)含量的影响结果;
注:#表示模型组与空白组相比P<0.05;*表示与模型组相比P<0.05同组内标有不同小写字母的表示同一组分不同浓度具有显著性差异(P<0.05);组间标有不同大写字母的表示同一浓度不同组分具有显著性差异(P<0.05)。
图9是实施例1得到的CCP和实施例2得到的CP0、CP1和CP3,按实验8方法进行测定的对油酸诱导的高脂肪HepG2细胞内总胆固醇(TC)含量的影响结果;
注:#表示模型组与空白组相比P<0.05;*表示与模型组相比P<0.05同组内标有不同小写字母的表示同一组分不同浓度具有显著性差异(P<0.05);组间标有不同大写字母的表示同一浓度不同组分具有显著性差异(P<0.05)。
具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此。
柑橘加工水为将柑橘罐头加工工艺中产生的酸液、碱液混合调配至pH6,而后进行常规的减压浓缩至总碳水化合物含量约10mg/mL。
本发明所用的木瓜蛋白酶的酶活为2000unit/mg。
实施例1、从柑橘加工水中回收的柑橘果胶的方法,依次进行以下步骤:
1)、将柑橘加工水
将柑橘加工水于50℃在旋转蒸发器中浓缩至约原体积的1/4,得浓缩液;
2)、将上述浓缩液与无水乙醇按照1:5的体积比混合,于4℃静置12h,离心(9000rpm,15分钟)后取用沉淀,用少量去离子水溶解,得沉淀溶液;
即,沉淀:去离子=1:25的重量比;
3)、将木瓜蛋白酶按0.1%(w/v)的比例加入至沉淀溶液中,50℃水浴保温3小时后,再100℃水浴加热15分钟使酶失活,待溶液冷却至室温,离心(9000rpm,15分钟),取用上清液。
上述0.1%(w/v),即,木瓜蛋白酶:沉淀溶液=0.1g/100ml;
将上清液用Sevag试剂进行处理:将上清液与Sevag试剂按5:1的体积比混匀,剧烈搅拌30分钟,静置直至分层,取上层溶液反复多次用Sevag试剂处理(即,上层溶液替代上清液用Sevag试剂进行处理),直至不再出现蛋白层(至少静置3小时);所得称为样品溶液。
所述Sevag试剂为氯仿:正丁醇=4:1(v/v)的混合剂;
将100ml柑橘加工水对应所得的样品溶液装入2000Da透析袋中再置于1L的去离子水中进行透析,透析时间为72小时,将所得的透析液浓缩(于50℃的条件下旋转浓缩至原透析液体积的约1/4),于-60℃冷冻干燥24小时,得到柑橘果胶粗品(CCP)约0.82g。
CCP的组成及分子量见表1,总糖含量用苯酚-硫酸法测定;蛋白质含量采用考马斯亮蓝法测定;单糖组成采用高效阴离子色谱法测定;高效凝胶过滤色谱法(HPGFC)测定。由表1可见,CCP主要由RG-I结构域组成(占73.4%),主要组分(含量达68.88%)的分子量较小,为31.8kDa,因此CCP为高RG-I的优质果胶。
表1、CCP的组成和分子量
注:Ara、Gal、Man、Glc、Xyl、Rha和GalA分别代表阿拉伯糖、半乳糖、甘露糖、葡萄糖、木糖、鼠李糖和半乳糖醛酸。HG=GalA–Rha,RG-I=[GalA–HG]+Rha+Ara+Gal.
实施例2、
按照常规方式,将纤维素DEAE-52处理后装柱(柱子规格为60cm×Φ2.6cm,有效长度为32cm,有效柱床体积为176mL)。
纤维素DEAE-52处理例如具体为:先将DEAE-52阴离子交换剂干粉浸泡于离子水中24h使其充分溶胀,去除杂质;再在0.5N的HCl溶液中浸泡1~2h,再用无离子水或蒸馏水洗至pH值中性或pH 4以上,并将其在抽滤漏斗中抽干;将抽干的离子交换剂浸泡在0.5N的NaOH溶液中1~2h,再用无离子水或蒸馏水将其洗至中性。
称取实施例1制备获得的柑橘果胶粗品(CCP)200mg,溶于8.5mL的去离子水中,离心(6000rpm,15分钟),取上清液,均匀的加入到层析柱填料上表面。依次用去离子水(500ml)、0.1mol/L(600ml)、0.3mol/L(500ml)、0.5mol/L(400ml)、0.7mol/L(500ml)、0.9mol/L(400ml)的NaCl溶液分别进行洗脱,控制流速1mL/min,每管10mL,用自动收集器收集,以硫酸-苯酚法隔管检测其在490nm处的紫外吸收值,并绘制洗脱曲线。由于0.5mol/L、0.7mol/L、0.9mol/L NaCl的洗脱液中糖含量较少,不予进一步研究。
将用去离子水、0.1mol/L和0.3mol/L NaCl洗脱得到的三种含糖洗脱液分别各自合并,经减压浓缩(于50℃用旋转蒸发仪浓缩至为原体积的约10%)、透析脱盐(用截留分子量为2000Da的透析袋透析,每隔一段时间---约4小时换水,直至水中无氯离子检出)(注:其中去离子水的洗脱液对应的浓缩液不需脱盐)、冷冻干燥(-50~-60℃浓缩干燥至恒重),得到柑橘果胶的三个分离组分,分别标记为CP0、CP1和CP3,得率分别为15.61%、44.29%和17.01%。
得率(%)=(分离果胶组分重量/上柱CCP重量)×100。
表2、果胶分离组分CP0、CP1和CP3的组成和分子量
Note:Rha,Ara,Gal,Glc,Xyl,Man和GalA分别代表鼠李糖、阿拉伯糖、半乳糖、葡萄糖、甘露糖、木糖和半乳糖醛酸。HG=GalA–Rha,RG-I=[GalA–HG]+Rha+Ara+Gal.
实验1、
流变特性测定:配制10mg/mL的CCP、CP0、CP1、CP3各样品溶液,采用TA RHEOMETERDiscovery HR-2流变仪对四种样品进行稳态剪切测试和动态黏弹测试。平板直径为40mm,狭缝距离为0.5mm,测试温度为25℃。稳态测试时剪切速率为0.01-100S-1,动态测试时角频率为0.1-100rad/s。
由图1可知,当果胶样品(10mg/mL)的剪切速率在0.01-100(1/s)范围内,随着剪切速率的升高,果胶溶液呈剪切稀化现象,表现出非牛顿流体的特征。当剪切速率达到1(1/s)以后,其对果胶的黏度影响趋于平缓,果胶溶液的黏度已基本稳定,CCP、CP0、CP1和CP3的稳定黏度分别在36、8、15、21mPa.s左右。
在角速度为0.1—100rad/s的范围内确定不同果胶溶液的储能模量(G’)和损耗模量(G”)。G’代表样品的弹性性能,G”代表样品的黏性性能。从图2各样品的频率扫描图可以观察到,CCP、CP0、CP1和CP3的G’和G”都随着频率的增加而增加。损耗正切值tanδ=G”/G’表现了黏性和弹性对材料流变特性的贡献。tanδ>1时,体系属于溶胶体系;tanδ<1时,体系属于凝胶体系。G”和G’的值若有交叉点出现,表明果胶具有良好的黏弹性,交叉值越低,弹性贡献越大。CCP、CP1和CP3三种果胶样品都在1.5rad/s处观察到交点,LCP0在1rad/s处观察到交点,说明样品具有较好的黏弹性。
图1和图2的结果表明CCP、CP0、CP1、CP3四种果胶样品为典型的剪切稀释非牛顿流体,具有良好的粘弹性,因而可作为食品增稠剂改进其流变性能。
实验2、
采用文献(MJ Shi,XY Wei,J Xu,BJ Chen,DY Zhao,S Cui,TZhou.Carboxymethylated Degraded Polysaccharides from Enteromorpha prolifera:Preparation and in Vitro Antioxidant Activity.Food Chemistry 2017,215,76–83.)报道的方法,将实施例1得到的CCP和实施例2得到的CP0、CP1和CP3进行DPPH自由基清除能力的测试。由图3可见,四种果胶对DPPH自由基都有较强的清除能力,IC50分别为:2.68、18.89、6.363、6.722mg/mL。清除能力为CCP>CP1>CP3>CP0。
实验3、
采用文献(MJ Shi,XY Wei,J Xu,BJ Chen,DY Zhao,S Cui,TZhou.Carboxymethylated Degraded Polysaccharides from Enteromorpha prolifera:Preparation and in Vitro Antioxidant Activity.Food Chemistry 2017,215,76–83.)报道的方法,将实施例1得到的CCP和实施例2得到的CP0、CP1和CP3进行羟基自由基清除能力的测试。由图4可见,四种果胶对羟基自由基都有较强的清除能力,IC50分别为:3.91、21.797、5.181、2.819mg/mL。清除能力为CP3>CCP>CP1>CP0。
实验4、
细胞内活性氧(ROS)水平的测定:采用文献(YZ Zhu,JM Zhou,W Liu,XE Pi,QQZhou,P Li,T Zhou,Q Gu.Effects of exopolysaccharide from Lactobacillusrhamnosus on human gut microbiota in in vitro fermentation model.LWT-FoodScience and Technology,2021,139,110524.)进行HepG2细胞培养。将处于指数生长期的HepG2细胞调整浓度为5×104个/mL,以每孔100μL接种于96黑板,培养24h后,吸弃培养基,加入100μL不同浓度(100、200、400μg/mL)的CCP、CP1和CP3的样品溶液。将细胞继续培养24h后,吸弃原有培养基,用无菌PBS清洗1遍细胞,向每孔中加入含有25μL DCFH-DA的PBS溶液100μL,置于培养箱中孵育1h后,吸弃孔内液体,加入100μL 4mM的H2O2继续孵育1h,取出孔板,吸弃孔内液体,用无菌PBS洗涤细胞后,用酶标仪检测每孔的荧光强度(激发波长485nm,发射波长525nm)。荧光强度反映了细胞内ROS水平。
Normal为:HepG2没有用H2O2和果胶处理的正常细胞组;Model为:HepG2没有用果胶处理,而用H2O2处理的模型细胞组。
由图5可见,经过H2O2处理以后,模型组细胞内ROS的水平显著增加(P<0.05),达到了正常对照的154.96%,表明细胞内已经产生过量ROS,细胞自身的防御系统不能有效地清除ROS,机体的氧化与抗氧化机制的平衡被打破,细胞处于氧化损伤状态。CCP、CP1、CP3的浓度达到200μg/mL和400μg/mL时,细胞内ROS水平与模型组相比有显著下降(P<0.05),表明三种果胶样品对细胞的保护作用显著。在浓度为400μg/mL的情况下,CCP、CP1、CP3组细胞内ROS水平较模型细胞分别下降了44.38%和36.45%和54.55%。
实验5、
细胞内SOD活性的测定:将调整后的密度为2×105个/mL的均匀HepG2细胞悬液接种于6孔板,每孔接种2mL细胞液。置于37℃,5%二氧化碳的培养箱中培养24h后,吸弃旧培养基,向正常组和模型组中每孔加入2mL培养基,向实验组中分别加入2mL不同浓度(100、200、400μg/mL)的CCP、CP1和CP3的样品溶液。培养24h后,吸弃孔内原有培养基,实验组和模型组加入2mL用无血清配制的4mM的H2O2继续孵育1h,正常组用不含H2O2且不含血清的培养基进行相同处理。到孵育时间后,吸弃培养基,用预冷的无菌PBS洗涤1遍细胞,再向每孔中加入150μL含有1mM PMSF的Triton X-100细胞裂解液后,置于冰上裂解30min。将裂解液吸吹均匀以后分别转移至1.5mL离心管。按照碧云天总SOD活性检测试剂盒(WST-8法)说明书测定计算细胞内SOD活性。
由图6可知,与正常组相比,经过H2O2诱导的细胞处于氧化应激状态以后,其SOD活力显著降低(P<0.05)。但是在经过CCP、CP1和CP3的保护处理之后,SOD含量显著升高(P<0.05)。
实验6、
采用文献(何平伟.三种不同谷物可溶性(1→3)(1→4)-β-D-葡聚糖结构与束缚胆汁酸特性的对比研究[D].华南理工大学,2016.)报道方法,以消胆胺为阳性对照,分别对实施例1得到的CCP和实施例2得到的CP0、CP1和CP3结合胆酸钠、鹅去氧胆酸钠、甘氨胆酸钠、牛磺胆酸钠、脱氧胆酸钠的能力进行测定。
由图7,针对胆酸钠来说,CP1和CP3的结合率较好,分别为3.83和3.74(μmol/100mg),分别达到了消胆胺的84.26%和82.20%;CCP和CP0对牛磺胆酸钠的结合率分别达到了消胆胺的78.96%和78.47%;CP3对鹅去氧胆酸钠其的结合率达到了85.37%;CCP和CP3对甘氨胆酸的结合率分别为85.77%和82.69%。四种果胶样品对脱氧胆酸钠结合效果都比较好,其中CCP和CP3的结合效果较好。四种果胶样品相对阳性对照对不同胆酸盐的结合率如表3所示,这些结果表明本发明制备的果胶具有较好的降血脂活性。
表3.不同样品相对阳性对照对不同胆酸盐的结合率
实验7、
将HepG2细胞浓度调整为2×105个/mL,接种至24孔板,每孔0.5mL。置于细胞培养箱中培养24h。正常组(Normal)将原培养基更换为含1%FBS的DMEM培养基,模型组(Model)、实验组和阳性对照组(Sim)均更换0.5mM的油酸造模剂,将细胞板继续放置培养箱中孵育24h后,正常组和模型组换用1%FBS的DMEM培养基,实验组分别加入0.5mL不同浓度(100、200、400μg/mL)的CCP,CP1和CP3的样品溶液,阳性对照组加入0.5mL浓度为20μg/mL的辛伐他汀(Sim)溶液,继续培养24h。弃去24孔板中的培养基,用预冷的PBS将细胞洗涤一遍,再向每孔中加入40μL含有1mM PMSF的Triton X-100细胞裂解液,将孔板置于冰上裂解30min,期间要不时摇动,保证孔内细胞都能被裂解。将裂解液吸吹均匀以后转移至1.5mL离心管,用于细胞内甘油三酯(TG)和总胆固醇(TC)含量的测定。TG和TC的含量分别按照南京建成甘油三酯(TG)GPO-PAP酶法和南京建成总胆固醇(T-CHO)COD-PAP法试剂盒说明书进行检测和计算。
见图8可知,三种果胶样品都能够一定程度地减少细胞内甘油三酯的含量,且呈浓度依赖性关系。这些果胶样品在200和400μg/mL浓度下均能显著改善细胞内TG水平(P<0.05),其中当CCP在400μg/mL下,TG含量为0.148mmol/g prot,与正常组相近吗,且低于阳性对照;与模型组相比,下降了49.9%。
由图9可知,HepG2细胞经过油酸诱导24h之后,胆固醇含量显著增加(P<0.05)。而细胞经过不同浓度的三种果胶样品处理之后,胆固醇含量显著下降。当CCP和CP3的浓度为200和400μg/mL时,细胞内的胆固醇含量低于正常组。在三个样品中,降胆固醇能力最高的是CCP,在200以及400μg/mL的作用浓度下,胆固醇含量与模型组相比显著下降(P<0.05),分别使其降低了49.03%和50.30%。
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
Claims (2)
1.粗柑橘果胶CCP、果胶组分CP1、果胶组分CP3在制备具有胆酸结合活性的制品中的应用,其特征是:粗柑橘果胶CCP、果胶组分CP1、果胶组分CP3的制备方法为包括以下步骤:
1)、将柑橘加工水浓缩,得浓缩液:
柑橘加工水为将柑橘罐头加工工艺中产生的酸液、碱液混合调配至pH6,而后进行减压浓缩至总碳水化合物含量为10 mg/mL,
将柑橘加工水浓缩至原体积的1/4,得浓缩液;
2)、将所述浓缩液与无水乙醇混合,于4±1℃静置12±2 h,离心,离心所得沉淀用去离子水溶解,得沉淀溶液;
浓缩液:无水乙醇=1:4~6的体积比;
沉淀:去离子水=1:20~30的重量比;
3)、将步骤2)所得的沉淀溶液用木瓜蛋白酶结合Sevag试剂进行脱蛋白处理,然后于去离子水中透析72±6小时,Mw截流2000 Da,接着浓缩、冷冻干燥,得到粗柑橘果胶CCP;
所述Sevag试剂为氯仿:正丁醇=4:1体积比的混合剂;
4)、将粗柑橘果胶CCP进行分离纯化,分别得到CP0、CP1和CP3这三种果胶组分:
将粗柑橘果胶CCP加去离子水配制成浓度为20~25 mg/mL的CCP溶液,离心,所得的上清液用于后续的上样;
选用纤维素 DEAE-52柱,上清液上样;
先用去离子水洗脱,再依次用0.1 M、0.3 M、0.5 M、0.7 M和0.9M的NaCl溶液分别进行洗脱,流速为1 mL/min;上述去离子水、0.1 M、0.3 M、0.5 M、0.7 M和0.9M的NaCl溶液的用量均为2~3.5倍柱体积;
将去离子水对应所得的洗脱液减压浓缩、冷冻干燥,得到果胶组分CP0;
将0.1 M和0.3 M NaCl对应所得的两种洗脱液分别进行如下处理:减压浓缩、透析脱盐、冷冻干燥,对应得到果胶组分CP1和CP3。
2.根据权利要求1所述的应用,其特征是所述步骤3)为:
按照0.1g/100ml的比例,将木瓜蛋白酶加入至沉淀溶液中,于50±5℃水浴保温3±0.5小时,灭酶活后,离心,取上清液;
将上清液用Sevag试剂进行处理:按上清液:Sevag试剂=4~6:1的体积比,在上清液中加入Sevag试剂搅拌混匀后静置,直至分层;取分层所得的上层溶液反复用Sevag试剂进行处理,直至不再出现蛋白层;所得称为样品溶液;
将所得的样品溶液装入2000 Da透析袋中再置于去离子水中进行透析,透析时间为72±6小时, 将所得的透析液浓缩,再冷冻干燥,得到粗柑橘果胶CCP。
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