CN116751256A - 一种源自翅果油树种仁油粕的多肽及辅助抗炎抗氧化组合物 - Google Patents
一种源自翅果油树种仁油粕的多肽及辅助抗炎抗氧化组合物 Download PDFInfo
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- CN116751256A CN116751256A CN202311013875.0A CN202311013875A CN116751256A CN 116751256 A CN116751256 A CN 116751256A CN 202311013875 A CN202311013875 A CN 202311013875A CN 116751256 A CN116751256 A CN 116751256A
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Abstract
本发明提供一种源自翅果油树种仁油粕的多肽及辅助抗炎抗氧化组合物,其中翅果油树种仁油粕蛋白多肽SDCG1,氨基酸序列为FEDADLWYANLYH。利用上述多肽及其产品具有较好的抗炎抗氧化功效,可以有效清除DPPH、ABTS、羟基自由基等自由基,具有结构简单、抗氧化活力强、安全性高的特点,可以作为化学合成抗氧化剂的优良替代,而且具有一定的抗肿瘤活性,为食品、药品及化妆品行业研发新型天然添加剂提供了理论依据和实践参考。
Description
技术领域
本发明属于生物技术领域,具体涉及一种源自翅果油的多肽及辅助降血脂组合物。
背景技术
翅果油树,属胡颓子科胡颓子属落叶大灌木或小乔木,果实质地呈干棉质,多毛,形状接近圆形或椭圆形,种子大多呈纺锤形,有些呈圆形。目前,翅果油树主要分布于山西省南部乡宁县一带,在陕西秦岭北麓的户县也有零星分布。翅果油树不仅具有较高的生物学价值,其叶子、种仁及其油脂还具有较高的药用价值和营养价值等。目前,对翅果油树种仁及叶片的研究,多集中在翅果油树籽油、植物甾醇以及黄酮类化合物、多糖的分离提取等方面。翅果油树种仁中蛋白质的含量可以达到 32%~36%,主要是由水溶蛋白、盐溶蛋白、醇溶蛋白、碱溶蛋白组成,其中碱溶蛋白,即谷蛋白的含量最高,约为65.917%~68.267%。蛋白质中的氨基酸种类丰富且含量较高,其中多种氨基酸的含量是沙棘种子的 2~3倍,是中华猕猴桃果实的 4~5 倍甚至 10倍以上。而脯氨酸含量更为突出,约是沙棘种子的 16~17倍,中华猕猴桃的 350 倍。随着翅果油树籽油利用程度的加大,提油后的副产物-翅果油树种仁油粕的合理开发利用也逐渐引起人们的关注。每年翅果油树种仁油粕产量可达 4 万吨,其大多用于制作肥料、饲料或者被丢弃,产品的附加值低,不仅资源浪费,而且造成环境污染。翅果油树种仁油粕中蛋白质含量丰富,具有较好的应用价值,因此,对翅果油树种仁蛋白这一资源的合理有效利用显得尤为必要。由于翅果油树的分布限制,国内对翅果油树种仁油粕的研究起步较晚,而且缺乏对蛋白的精细研究。
现有技术中针对翅果油或者翅果蛋白的加工还仅仅停留在农产品的粗加工阶段。例如CN109805411A中公开了一种辅助降血脂的保健食品,其中含有翅果油;CN111317047A公开了一种翅果茶饮料及其制备方法,利用翅果油树的叶片制成的茶叶进行粉碎加工;CN105505573A公开了一种超声波辅助提取翅果油树籽油的方法,但还没有对其中的翅果蛋白进行深入研究,难以精确掌握其中的有效成分,因此需要对翅果蛋白进行进一步的深加工,提高加工功能性质,拓宽翅果蛋白的应用领域。众多研究表明翅果蛋白中含有大量的具有抗氧化、抗炎、抗菌、抗血栓形成、抗高血压、免疫调节或金属螯合等生物活性生物生物活性肽,这些生物活性肽为研制相应的产品提供了技术基础。
发明内容
针对现有技术方案的不足,本发明旨在提供一种源自翅果油树种仁油粕的多肽及辅助降血脂组合物。
进一步的,本申请提供一种新的翅果油树种仁油粕蛋白多肽SDCG1,氨基酸序列如SEQ ID NO:1所示,序列为FEDADLWYANLYH。
本发明提供一种新的翅果油树种仁油粕蛋白多肽SDCG1的制备方法,所述制备方法包括如下步骤:
取翅果油树种仁粉末适量,加入正己烷,于室温条件下脱脂抽滤,挥发溶剂得翅果油树种仁油粕。
取脱脂后的样品加入蒸馏水,用NaOH 溶液调 pH至 10.0,超声处理,浸提后离心弃去沉淀;稀盐酸调节上清液pH值至2.5~3.5,离心保留沉淀;再用NaOH水溶液调节pH为中性,透析;
将透析后的翅果油树种仁油粕蛋白用NaOH的水溶液调节pH至8.0,加入酶水解,酶解结束后离心取上清液,得到翅果油树种仁油粕蛋白酶解液,经真空冷冻干燥,得到翅果油树种仁油粕蛋白粉;
阴离子色谱分离纯化:将翅果油树种仁油粕蛋白粉用Tris-盐酸缓冲液配制成溶液, 0.22 μm孔径微滤膜过滤,然后用DEAE-Sepharose离子交换柱层析进行分离纯化,收集最高抗氧化活性的组分,真空冷冻干燥,-20℃低温保存备用。
反相高效液相色谱分离纯化:用去离子水溶解上述组分2冻干粉,经0.22 μm孔径微滤膜过滤后通过Thermo C18色谱柱(4.6 mm×250 mm,5 μm)进行分离纯化,收集峰3真空冷冻干燥。
进一步的,本发明提供翅果油树种仁油粕蛋白多肽SDCG1在制备抗炎抗氧化组合物中的应用,所述应用为将上述翅果蛋白多肽作为活性成分添加到食品中,所述食品具有较好的抗炎抗氧化功效,可以有效清除DPPH、ABTS、羟基自由基等自由基,具有结构简单、抗氧化活力强、安全性高的特点,可以作为化学合成抗氧化剂的优良替代,而且具有一定的抗肿瘤活性。
进一步的,所述组合物为翅果油混合微囊粉。
所述翅果油混合微囊粉的制备方法为:
以翅果油树种仁蛋白多肽和麦芽糊精为壁材,添加翅果油作为芯材,吐温-80和司盘-80为乳化剂,经喷雾干燥制备获得翅果油混合微囊粉。
进一步的,本发明提供翅果油混合微囊粉在制备降血脂组合物中的应用,所述组合物包括:翅果油混合微囊、膳食纤维、大豆磷脂、绿茶粉、硬脂酸镁,辅料。所述辅料包括赤藓糖醇。
进一步的,本发明提供一种抗炎抗氧化制品,包含前述的翅果蛋白多肽和组合物。组合物,优选包括抗性糊精、大豆磷脂、低聚果糖、红曲米粉、绿茶粉;进一步优选,还包括紫苏籽微囊粉和牛磺酸以及复合维生素。其中复合维生素为维生素C、维生素E、维生素B1、维生素B2。
经实验证明,利用上述多肽及其产品具有较好的抗炎抗氧化功效,可以有效清除DPPH、ABTS、羟基自由基等自由基,具有结构简单、抗氧化活力强、安全性高的特点,可以作为化学合成抗氧化剂的优良替代,而且具有一定的抗肿瘤活性,为食品、药品及化妆品行业研发新型天然添加剂提供了理论依据和实践参考。
附图说明
图1:层析图谱采集分析仪HD-A检测获得经DEAE-Sepharose分离纯化后产物的峰图。
图2:反相高效液相色谱图。
具体实施方式
实施例1翅果油树种仁油粕的多肽的提取和纯化
根据本申请人之前的研究成果,从植物蛋白常用的提取方法选择碱溶酸沉法对脱脂后的翅果油树种仁油粕的进行蛋白提取和多肽的筛选。
取翅果油树种仁粉末适量,按料液比 1:8(g/mL)加入正己烷,于室温条件下脱脂3h 后抽滤,将沉淀置通风橱中 12h 以挥发溶剂,得翅果油树种仁油粕。
取脱脂后的样品 1 g,以1g:15~35ml的料液比加入蒸馏水,用 1 mol/L的 NaOH溶液调 pH至 10.0,超声处理20~60min,超声功率为100-200W,45~60℃条件下浸提 30~120min,于 4000 r/min离心 15 min,弃去沉淀;稀盐酸调节上清液pH值至2.5~3.5,7000r/min离心10~15min,保留沉淀;再用NaOH水溶液调节pH为中性,透析;
将上步透析后的翅果油树种仁油粕蛋白加蒸馏水调整成2.5%的蛋白溶液,用1mol/LNaOH的水溶液调节pH至8.0,水解温度为40℃,加入比例1:1的碱性蛋白酶 (CAS号:9014-01-1)和中性蛋白酶(CAS号:9068-59-1))(酶活为2×105U/g,购于索莱宝公司),水解3.5h,在水解过程中通过加入1mol/LNaOH水溶液将反应液pH始终维持在8.0;酶解结束后,将体系置于90℃水浴中20min以终止酶解反应;之后7000r/min条件下离心15min,取上清液,得到翅果油树种仁油粕蛋白酶解液,经真空冷冻干燥,得到翅果油树种仁油粕蛋白粉;
阴离子色谱分离纯化:将翅果油树种仁油粕蛋白粉利用浓度为0.02 M pH值为8.4的Tris-盐酸缓冲液配制成6 mg/mL的溶液,完全溶解后用0.22 μm孔径微滤膜过滤,然后用DEAE-Sepharose离子交换柱层析进行分离纯化,用浓度为0.02 M pH值为8.4的Tris-盐酸缓冲液为平衡液,将已过滤的样品上柱,流速设置为1 mL/min,用含0.2~1 mol/L NaCl 的pH值为8.4的Tris-盐酸缓冲液线性梯度洗脱,层析图谱采集分析仪HD-A检测获得经DEAE-Sepharose分离纯化后产物的峰图(图1),收集4个出峰组分,比较其羟基自由基、DPPH、ABTS自由基清除能力,收集最高抗氧化活性的组分2,真空冷冻干燥,-20℃低温保存备用。
反相高效液相色谱分离纯化:用去离子水溶解上述组分2冻干粉,经0.22 μm孔径微滤膜过滤后通过Thermo C18色谱柱(4.6 mm×250 mm,5 μm)进行分离纯化,样品浓度10mg/mL,进样量20 μL,流动相A:含质量分数0.1%TFA的乙腈,流动相B:含质量分数0.1%TFA的去离子水,流动相流速1.0 mL/min。采用梯度洗脱的方式,洗脱条件是:0-5min,5%-10%流动相A;5-15min,10%-50%流动相A;15-35 min,50%-80%流动相A,检测波长是214 nm,获得组分2的反相高效液相色谱图(图2)。测定各个吸收峰对应的洗脱组分的羟基自由基、DPPH、ABTS自由基清除能力,结果发现洗脱峰3的抗氧化活性最高,收集峰3真空冷冻干燥。
用液相色谱与质谱连用(LC-MS/MS)方法测定洗脱峰3的氨基酸序列,经UniprotKB数据库与NCBI数据库对比发现一种新的翅果油树种仁油粕蛋白多肽SDCG1,氨基酸序列为FEDADLWYANLYH。
实施例2天然抗氧化肽的体外抗氧化活性检测
实施例1中得到的翅果油树种仁油粕蛋白多肽SDCG1进行Fmoc固相合成,将合成后的多肽进行体外抗氧化活性研究。
称取合成多肽样品,用蒸馏水配制成不同浓度的样液。
DPPH自由基清除率:准确称取3.94 mg DPPH,采用体积分数95%的乙醇溶液溶解,定容至100 mL,配成0.1 mmo1/L的DPPH溶液。将1 mL样液与1 mL DPPH溶液充分混匀,室温避光静置30 min,于517 nm测定吸光值;以1 mL体积分数95%的乙醇溶液代替DPPH溶液作为对照组;以1 mL蒸馏水代替样液作为空白对照。样品的DPPH自由基清除率用如下公式计算:
式中:A1—样品组吸光值;A2—对照组吸光值;A3—空白对照组吸光值
ABTS自由基清除活力:配制7 mM的ABTS贮存母液及2.45 mM的过硫酸钾溶液,临用前以1:1的体积比将二者混合,室温放置于暗处16 h后,用pH7.4的5 mM磷酸盐缓冲液稀释至734 nm处吸光值为0.70±0.02,即得到ABTS自由基溶液。将1 mL ABTS自由基溶液与1 mL不同浓度的样品溶液等体积混合,室温反应10 min后,于734 nm下测定吸光值。用蒸馏水代替样液作为空白组。样品的ABTS自由基清除活力按下列公式进行计算:
式中:A1—样品组吸光值;A2—空白组吸光值
羟基自由基清除活性:配置8 mmol/L的硫酸亚铁溶液,3 mmol/L的水杨酸溶液和20 mmol/L的过氧化氢溶液备用。取200 μL样液与60 μL 8 mmol/L硫酸亚铁、200 μL20mmol/L的水杨酸、50 μL 20 mmol/L过氧化氢充分混合,37℃水浴孵育30 min,冷却至室温后加入90 μL去离子水,转速4000 r/min离心30 min。取上清液在510 nm波长下测定反应物的吸光值。用蒸馏水做空白对照。羟基自由基的清除活性按下列公式计算:
式中:A0—空白组吸光值;A1—样品组吸光值
经本实施实例测定,翅果油树种仁油粕蛋白多肽SDCG1对DPPH、ABTS、羟基自由基均有较好的清除率。当该肽浓度达到2 mg/mL时,其DPPH,ABTS,羟基自由基清除率分别为:51.355±0.248%,89.127±0.683%,13.527±0.198%。该翅果油树种仁油粕蛋白多肽SDCG1对DPPH自由基清除率的IC50值为2.429±0.19 mg/mL,对ABTS自由基清除率的IC50值为38.246±0.391 μg/mL,对羟基自由基清除率的IC50值为14.542±0.9 mg/mL。
实施例3 功能性食品验证
利用本领域常规技术将实施例2中固相合成获得的翅果油树种仁蛋白多肽与步骤(2)获得的翅果油混合制备微囊粉(参见:紫苏籽油微囊粉的制备,《食品科技》),2018年 第43卷 第12期,p212-217),概述如下:
以实施例2中步骤(4)获得的翅果油树种仁蛋白多肽和麦芽糊精为壁材,步骤(2)获得的翅果油为芯材,吐温-80和司盘-80为乳化剂,以1.5%的乳化剂(吐温-80和司盘-80质量比1:1)添加量、翅果油与复合壁材翅果油树种仁蛋白多肽与麦芽糊精质量比2:1)芯壁比3:4、总固形物含量为25%、进风温度为160 ℃、进料量3 L/h、转速为18000 r/ min条件下喷雾干燥制备获得翅果微囊粉。
按照质量分数,将制备获得的翅果微囊粉20-40%、抗性糊精40-60%、紫苏籽微囊粉20-40%、大豆磷脂10-20%、低聚果糖10-15%、红曲米粉1-5%、绿茶粉1-5%、牛磺酸0.1-0.5%以及复合维生素(维生素C、维生素E、维生素B1、维生素B2)0.1-0.5%,余份为辅料,将各组分混合均匀后,压片制成片剂。上述其他各成分都为常见商业化产品。
选择同时移植了C-26腺癌的BALB/C小鼠,设环磷酰胺20mg/KG+正常饮食组为阴性对照组(5-FU组),设环磷酰胺20mg/KG+市售肿瘤营养支持产品组为阳性对照(5-FU对照组),设环磷酰胺20mg/KG+翅果组合物(翅果微囊粉30%、抗性糊精50%、紫苏籽微囊粉30%、大豆磷脂15%、低聚果糖10%、红曲米粉2%、绿茶粉2%、牛磺酸0.5%以及复合维生素(0.5%,余份为辅料)为实验组(5-FU实验组),每组8只小鼠,各组营养产品作为常规饲料,饲喂小鼠。饲喂22天后,处死,眼眶采血,计算小鼠生存率、体重及瘤重、脾脏、胸腺指数,测定炎性因子水平。
得到的结果如表1所示
表1各组别死亡率及其各器官重量和炎性因子的影响
从上述结果可以看出,与5-FU+常规饲料组相比,本发明的产品可以明显改善小鼠的体重丢失状况,并且阻止了肿瘤增长的趋势。而且由于常规化疗会造成小鼠的脾增大,胸腺、肾减小,与5-FU常规饲料饮食组相比,本发明的产品可以显著改善化疗造成的脾肿大,同时减轻了5-FU造成的胸腺萎缩的影响。由于炎症和肿瘤的发生、发展与恶化息息相关,身体的慢性炎症,导致DNA的损伤,影响机体的免疫功能,抑制了损伤后的修复,导致肿瘤发生的风险增加;肿瘤病灶特殊的微环境,产生大量的炎症因子,促进血管新生,诱导机体免疫耐受和肿瘤细胞的免疫逃逸,促进了肿瘤的发展和转移。肿瘤治疗过程中的放、化疗,对机体脏器不可避免的产生损伤,导致免疫器官的萎缩或肿大,炎症和感染的风险增加。因而,控制机体的炎症,改善肿瘤患者的炎性体征,对于肿瘤患者的治疗和康复具有不可忽视的积极意义。
以上内容是结合具体的实施方式对本申请所作的进一步详细说明,不能认定本申请的具体实施只局限于这些说明。对于本申请所属技术领域的普通技术人员来说,在不脱离本申请构思的前提下,还可以做出若干简单推演或替换,都应当视为属于本申请的保护范围。
Claims (10)
1.一种翅果油树种仁油粕蛋白多肽SDCG1,其特征在于,所述翅果油树种仁油粕蛋白多肽SDCG1的氨基酸序列如SEQ ID NO:1所示。
2.权利要求1所述的一种翅果油树种仁油粕蛋白多肽SDCG1在制备抗炎抗氧化组合物的应用。
3.一种制备权利要求1所述翅果油树种仁油粕蛋白多肽SDCG1的制备方法,其特征在于,所述制备方法包括如下步骤:
取翅果油树种仁粉末适量,加入正己烷,于室温条件下脱脂抽滤,挥发溶剂得翅果油树种仁油粕;
取脱脂后的样品加入蒸馏水,用NaOH 溶液调 pH至 10.0,超声处理,浸提后离心弃去沉淀;稀盐酸调节上清液pH值至2.5~3.5,离心保留沉淀;再用NaOH水溶液调节pH为中性,透析;
将透析后的翅果油树种仁油粕蛋白用NaOH的水溶液调节pH至8.0,加入酶水解,酶解结束后离心取上清液,得到翅果油树种仁油粕蛋白酶解液,经真空冷冻干燥,得到翅果油树种仁油粕蛋白粉;
阴离子色谱分离纯化:将翅果油树种仁油粕蛋白粉用Tris-盐酸缓冲液配制成溶液,0.22 μm孔径微滤膜过滤,然后用DEAE-Sepharose离子交换柱层析进行分离纯化,收集最高抗氧化活性的组分,真空冷冻干燥,-20℃低温保存备用;
反相高效液相色谱分离纯化:用去离子水溶解上述组分2冻干粉,经0.22 μm孔径微滤膜过滤后通过Thermo C18色谱柱进行分离纯化,收集峰3真空冷冻干燥。
4.一种如权利要求3所述的制备方法制备得到的产品组合物。
5.权利要求4所述的产品组合物在制备抗炎抗氧化组合物中的应用,所述应用为将上述产品组合物作为活性成分添加到食品中。
6.一种含有权利要求1所述翅果油树种仁油粕蛋白多肽SDCG1的组合物,其特征在于所述组合物还包括翅果油混合微囊粉。
7.如权利要求6所述的组合物,所述组合物还包括抗性糊精、大豆磷脂、低聚果糖、红曲米粉、绿茶粉、紫苏籽微囊粉和牛磺酸以及复合维生素。
8.如权利要求7所述的组合物,所述复合维生素为维生素C、维生素E、维生素B1、维生素B2。
9.如权利要求6-8任一所述的组合物在制备抗炎抗氧化组合物中的应用。
10.一种抗炎抗氧化制品,其特征在于,抗炎抗氧化制品中包含有权利要求1所述的一种翅果油树种仁油粕蛋白多肽SDCG1和权利要求6-8任一所述的组合物。
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