CN114617268A - 一种促进钙吸收的营养组合物与制备方法及其保健用途 - Google Patents
一种促进钙吸收的营养组合物与制备方法及其保健用途 Download PDFInfo
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- CN114617268A CN114617268A CN202210382906.9A CN202210382906A CN114617268A CN 114617268 A CN114617268 A CN 114617268A CN 202210382906 A CN202210382906 A CN 202210382906A CN 114617268 A CN114617268 A CN 114617268A
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- Prior art keywords
- calcium
- watermelon seed
- peptide
- powder
- parts
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Classifications
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Abstract
本发明公开了一种促进钙吸收的营养组合物与制备方法及其保健用途,属于食品/农产品加工技术领域。该组合物由西瓜籽肽、西瓜籽肽钙螯合物、萝卜缨超微粉以及脱脂奶粉按照一定质量份数混合组成,经过筛、杀菌、封装制得,其中西瓜籽肽是经过超声预处理酶解后,经过胃肠消化后获得,再用该肽制备西瓜籽肽钙螯合物。该营养组合物的组成简单、易于制备、钙吸收利用率高,并具有抗氧化能力,可作为多功能添加剂用于制备保健食品、功能食品或医药品等用途。
Description
技术领域
本发明提供了一种促进钙吸收的营养组合物及其保健用途,属于食品/农产品加工技术领域,可作为功能添加剂用于制备保健食品、功能食品或医药品等用途。
背景技术
钙是人体必需的营养素。钙在维持机体代谢平衡,调节消化内分泌、泌尿、血液循环、免疫、神经、呼吸等系统以及调节细胞内外液浓度中扮演着至关重要的角色。人体缺钙会引发一些列疾病,如老年人骨质疏松、佝偻病机体、手足抽搐等,同时缺钙也会引发高血压、冠心病等慢性疾病。从饮食中摄入钙对人体健康至关重要,是一种主要的钙来源。补钙制剂在市场上占有极大的份额,但以无机钙和有机钙为主的第一、第二代补钙制剂仍有较大缺陷,吸收率和生物利用率低,大多数补钙产品更重视钙的含量,而忽视了人体对钙的吸收率和利用率问题,实际上,在一些情况下,人体缺钙并非是由于摄入量不足,而是由于钙元素不能被人体吸收利用导致的生理性缺钙,此种情况下如过多补充钙制剂,未被机体利用则会引发便秘、胀气和胃酸过多等不良反应,并存在泌尿系统结石的风险。因此,开发高吸收利用率的钙制剂一直是各领域学者研究的重点。
多肽螯合钙作为第三代具有活性结构的生物钙补钙制剂,因稳定性好、抗干扰能力强、吸收效果好、生物利用度高,越来越受到消费者青睐。因此,研究和开发多肽钙螯合型补钙制剂及其营养组合物对于生理性缺钙具有重要的意义。
西瓜籽为双子叶植物葫芦科西瓜的种子。西瓜籽含有丰富蛋白质、脂肪酸、维生素,以及钾、铁、硒、钙、镁等营养元素,有清肺化痰的作用,对咳嗽痰多和咯血等症有辅助疗效。目前关于西瓜籽的资源化利用主要集中在油脂和休闲零食的开发,其油脂加工的残渣的综合利用报道较少,且主要针对蛋白及酶解物的制备及其功能特性研究方法。本课题组研究发现西瓜籽多肽具有优异的抗氧化活性,制备多肽钙螯合物能能显著促进钙吸收。经检索,以西瓜籽蛋白为原料制备肽螯合钙及其营养组合物质尚未见报道。
萝卜缨是萝卜的基生叶,又名萝卜叶、莱菔菜和莱菔叶等,可食用,但在萝卜收获时常常被丢弃于田野,造成资源极度浪费。据报道每100g红萝卜叶含钙350mg,在所有蔬菜中含钙量排在第一位,此外,还含有丰富的维生素C、K和精油等。具有促进胃肠蠕动、治疗胃溃疡、抗氧化、降血压、降血糖等活性。是营养价值极高的蔬菜。
本专利基于西瓜籽多肽螯合钙特性及萝卜缨的多营养特性,开发促进钙吸收的营养组合物,对于改善缺钙引起的诸多疾病具有重要的意义。
发明内容
本发明的目的是为了提供一种促进钙吸收的西瓜籽肽、西瓜籽肽钙螯合物、萝卜缨超微粉营养组合物的制备方法,并用于多种保健功能的用途。该组合物组成简单,易于制备,钙吸收利用率高。
本发明的另一个目的是提供一种促进钙吸收的西瓜籽肽钙螯合物的制备方法。提供不同分子量的肽钙螯合物的制备方法。
本发明提供的一种促进钙吸收的营养组合物,组合物的组成及质量份数为:
西瓜籽肽5-25,
西瓜籽肽钙螯合物3-20,
萝卜缨超微粉10-40,
脱脂奶粉0-50。
本发明提供的一种促进钙吸收的营养组合物的制备方法,按照以下步骤进行:
(1)组合物的组成:西瓜籽肽、西瓜籽肽钙螯合物、萝卜缨超微粉、脱脂奶粉。
(2)组合物质量份数:西瓜籽肽5-25、西瓜籽肽钙螯合物3-20、萝卜缨超微粉10-40、脱脂奶粉0-50,充分混合。
(3)成品:将混合好的粉末过100目筛,紫外灯杀菌30min后,装入密封袋中。
其中所述的西瓜籽肽钙螯合物的制备步骤为:将西瓜籽蛋白配制成20%(w/v)的悬浊液,放于塑料袋中密封,置于40/60kHz发散式双频超声反应器,室温下超声预处理45min后,取出溶液调pH为9.0,加入碱性蛋白酶,酶与蛋白比例为1:30(w/w),混合均匀后,在50℃下酶解4h,调pH为中性,并于沸水中灭酶10min,离心获上清液,脱盐、浓缩后,按照浓缩液干重与人工胃液按照1:20(w/v)混合,在37℃、150rpm/min下震荡消化2h;再调pH 7.5,按1:80(w/v)加入胰酶至混合液中,继续酶解4h,沸水浴灭酶10min;经离心获上清液,脱盐、浓缩,干燥,即得到西瓜籽肽粉末。
其中所述的西瓜籽肽钙螯合物的制备步骤为:将上述获得的西瓜籽肽粉末配制成3%(w/v)的肽溶液,按钙肽比1:6(w/w)加入氯化钙,调溶液pH值8.0、40℃下螯合90min,得到西瓜籽蛋白肽钙螯合液;向螯合液中加入体积比1:8(v/v)的无水乙醇,静置12h,收集醇沉液离心,取沉淀,干燥,即得西瓜籽肽钙螯合物。
其中所述的萝卜缨超微粉的制备步骤为:将新鲜红萝卜缨,经除杂、清洗、低温烘干、粉碎后,过40目筛,得到粗粉,再经气流超微粉碎,获得粒径分布≤1μm的粉体。
本发明的有益效果:
(1)本发明制备的促钙吸收营养组合物,除了西瓜籽肽钙螯合物具有促进钙吸收能力外,西瓜籽肽和萝卜缨中的钙在胃中也能发生螯合,促进萝卜缨中钙的吸收,起到双重促钙吸收的作用,能很好的克服现有钙制剂吸收利用率低的不足,同时又可辅助提高机体抗氧化能力、降血压、促进胃肠蠕动等功效。
(2)本发明中提供的西瓜籽肽及肽钙螯合物的制备方法,其中西瓜籽肽是在超声预处理西瓜籽蛋白后,再经消化道酶解制备的多肽;再将此多肽与钙螯合,制备肽钙螯合物,能显著提高钙的吸收率。
(3)本发明提供的促钙吸收营养组合物中西瓜籽肽和萝卜缨原料,分别为西瓜籽油和萝卜加工的副产物,可为西瓜籽蛋白和萝卜缨的资源再利用提供新思路,对于其资源化开发利用具有重要指导意义。
(4)本发明制备的促钙吸收的营养组合物、西瓜籽肽钙螯合物均可添加到各种形态食品、各种剂型的保健食品、功能食品、医药品等的制备领域,在各领域发挥其多功能的保健用途。
具体实施方式:
实施例中试验涉及到的方法:
1.细胞培养:
小肠吸收转运用模式细胞为Caco-2细胞(购于中国科学院上海细胞生物学研究所)用DMEM高糖培养基(含1%NEAA、10%灭活新生胎牛血清、100μg/mL链霉素和100U/mL青霉素)培养,置于CO2培养箱(37℃、5%CO2、饱和湿度)中,每周更换2次培养基。
2.细胞毒性实验
采用噻唑兰比色法测定,受试物对Caco-2细胞的毒性,取对数生长期的Caco-2细胞,分别按每孔1.0×105个细胞接种于96孔板中,每孔100μL,于37℃、5%CO2及饱和湿度条件下培养24h,吸去培养液,加入终浓度为10-800μg/mL受试物完全培养基继续培养细胞24h,弃上清液,每孔加入1.0mg/mL的噻唑兰溶液100μL,于37℃避光继续孵育4h,弃噻唑兰溶液,加入150μL二甲基亚砜溶解蓝色晶体,使用多功能酶标仪于570nm处读取吸光值。细胞存活率(%)=1-OD样品组/OD空白组×100。
3.钙吸收测定
采用试剂盒测定方法进行(南京建成生物技术有限公司),按照表1添加各种反应液体,混匀后静置5min后,于波长610nm处用酶标仪比色测各孔OD值。钙含量(mmol/L)=(OD测定孔-OD空白孔)/(OD标准孔-OD空白孔)×C标准×N,其中C标准为标准品浓度(1mmol/L),N为样品测试前稀释倍数。
表1钙含量测定
空白孔 | 标准孔 | 测定孔 | |
去离子水(μL) | 10 | - | - |
1mmol/L钙标准液(μL) | - | 10 | - |
样品溶液(μL) | - | - | 10 |
工作液Ι(μL) | 250 | 250 | 250 |
4.ABTS+·清除能力的测定
将受试物溶液与ABTS溶液混合,然后避光反应10min,于734nm测定吸光值,蒸馏水代替样品为空白对照。ABTS+·清除率(%)=1-OD样品吸光度/OD空白吸光度×100,ABTS+·清除能力用IC50值表示。
5.还原力的测定
将受试物溶液和铁氰化钾溶液反应20min,取反应溶液与三氯乙酸混合并摇匀,离心后,取上清液与氯化铁混合,使用紫外分光光度计于700nm处测定吸光值,吸光度值越高表明还原力越强。
6.西瓜籽肽的制备方法
将西瓜籽蛋白(购于西安斯诺特生物科技有限公司,蛋白含量72.65%,多糖含量6.36%、脂肪0.38%、水分2.17%、灰分7.32%)配制成20%(w/v)的悬浊液,将其放于发散式双频超声反应器中,功率密度设为120W/L,频率组合40/60kHz,室温下超声预处理45min后,调溶液pH为9.0,加入酶与蛋白比例为1:30(w/w)的碱性蛋白酶,混合均匀,在50℃和恒定pH下,酶解4h后,调pH为中性,于沸水中灭酶10min,离心获上清液,脱盐、浓缩后,按照浓缩液干重与人工胃液按照1:20(w/v)混合,在37℃、150rpm/min下震荡消化2h;再调pH 7.5,按1:80(w/v)加入胰酶至混合液中,继续酶解4h,沸水浴灭酶10min;经离心获上清液,脱盐、浓缩,干燥,即得到西瓜籽肽粉末。
7.西瓜籽肽钙螯合物的制备方法
将上述获得的西瓜籽肽粉末配制成3%(w/v)的肽溶液,按钙肽比1:6(w/w)加入氯化钙,溶液pH为8.0、40℃下螯合90min,得到西瓜籽蛋白肽钙螯合液;向螯合液中加入体积比1:8(v/v)的无水乙醇,静置12h,收集醇沉液,离心,取沉淀,干燥,即得西瓜籽肽钙螯合物,经计算,钙的螯合量为51.79±0.46mg/g。
8.萝卜缨超微粉的制备方法
新鲜红萝卜缨除杂、清洗、干燥后,经100克手提式高速中药粉碎机(DFT-100)粉碎,过40目网,得到萝卜缨粗粉,再将粗粉吸入气流式超微粉碎机(JCM-T50型)中,控制粉碎压力0.6Mpa,进料速度为50mg/s,得萝卜缨超微粉,经激光粒度分析仪测得粉体粒径≤1μm。
实施例1
取西瓜籽肽5份、西瓜籽肽钙螯合物20份、萝卜缨超微粉40份、脱脂奶粉0份,充分混合,过100目筛,制成促钙吸收营养组合物。
实施例2
取西瓜籽肽25份、西瓜籽肽钙螯合物3份、萝卜缨超微粉10份、脱脂奶粉50份,充分混合,过100目筛,制成促钙吸收营养组合物。
实施例3
取西瓜籽肽15份、西瓜籽肽钙螯合物15份、萝卜缨超微粉30份、脱脂奶粉25份,充分混合,过100目筛,制成促钙吸收营养组合物。
实施例4选择上述实施例1-3组合物进行Caco-2模拟小肠内皮细胞钙吸收
受试物预处理:取组合物干粉、西瓜籽肽、西瓜籽肽钙螯合物各10mg,分别用10mL不完全DMEM培养基溶解(西瓜籽肽钙螯合物中钙浓度相当于1.5mmol/L),磁力搅拌2h,离心、经过滤除菌,用于下述钙吸收测定,。
Caco-2细胞模型建立及钙吸收:按照Caco-2细胞方法连续培养21d后,电阻值>500Ω·cm2,AP侧和BL侧碱性磷酸酶活力比值为3.0左右,即可用于细胞转运试验。将0.5mL预热好的受试物溶液加入到AP侧,BL侧加入1.5mL预热好的HBSS缓冲液,然后将Transwell转运板置于37℃、5%CO2的饱和湿度下孵育,分别于1h、2h、4h吸取BL侧的转运液100μL于EP管中,测定钙含量。细胞内钙含量和基底外侧BL钙含量。钙生物利用度(%)=(细胞内钙含量+基底外侧BL钙含量)/总钙含量×100。
对比例1,采用本实验制备西瓜籽肽钙螯合物所用的氯化钙,取氯化钙用10mL不完全DMEM培养基溶解,配制成1.5mmol/L的钙溶液,并磁力搅拌2h,离心、经过滤除菌,用于钙吸收测定。
对比例2,采用本实验室的发明专利一种具有抗氧化活性的蛋白核小球藻肽螯合钙的制备方法(申请号202110021688.1)中,小球藻肽钙螯合物(分子量1-3KDa的肽与钙螯合物),进行对比钙吸收。取小球藻肽钙螯合物用10mL不完全DMEM培养基溶解,配制成小球藻肽螯合钙中钙浓度相当于1.5mmol/L,磁力搅拌2h,离心、经过滤除菌,用于钙吸收测定,其中。
表2是加入受试物和对比例后,Caco-2细胞活力没有受到影响,表明各组受试物和对比例对细胞均无毒性作用。
表3是各实施例组合物在Caco-2细胞模拟吸收4h后的吸收转运率,从表可见,0.25-2mmol/L各实施例组对均钙具有较好的吸收能力,且随着浓度的延长其吸收转运率增大,当1.5mmol/L的实施例1-3的钙离子吸收转运率分别为50.73%、53.79%和51.83%,较同浓度氯化钙组的转运率(30.64%)分别提高了65.57%、75.38%和69.16%,对比实施例2小球藻肽钙螯合物与实施例2相当(提高了76.44%)。以上结果表明,各组合物均能促进钙离子吸收。
表2各实施例组合物对Caco-2细胞的活力的影响
表3各实施例组合物和对比例在Caco-2细胞模拟吸收4h的钙生物利用率
实施例5:选择上述实施例1-3组合物进行还原力及清除ABTS+·能力
将2.0mg/mL受试物溶液和铁氰化钾溶液(1%,v/v)在55℃的水浴中反应20min,取2.5mL上述溶液与2.5mL的三氯乙酸(10%,v/v)混合并摇匀10min,离心后,取2.5mL的上清液与0.5mL的0.1%氯化铁混合后,于700nm处测定吸光值。吸光度值越高表明还原力越强。从表4看出,各实施例组合物均具有一定的还原能力,尤其实施例2还原能力最强。
分别将0.25、0.50、1.0、2.0、4.0mg/mL的受试物溶液100μL与ABTS溶液混合,然后避光反应10min,于734nm测定吸光值,蒸馏水代替样品为空白对照。结果见表4,各组实施例对ABTS+均具有一定清除能力,其IC50值均在1-3mg/mL之间。
从以上结果表明,本发明的促钙吸收营养组合物具有清除自由基和较强的还原能力,反应该组合物具有一定的抗氧化能力。
表4各实施例组合物的还原力及清除ABTS+·能力
受试物 | 2mg/mL的还原力(A<sub>700</sub>) | 清除ABTS<sup>+·</sup>的IC<sub>50</sub>(mg/mL) |
实施例1 | 0.52±0.02 | 2.69±0.25 |
实施例2 | 0.66±0.02 | 1.98±0.38 |
实施例3 | 0.57±0.01 | 2.46±0.89 |
对比例2 | 0.61±0.02 | 2.32±0.06 |
本发明通过目前被认为是最优选和最实际的实施例描述,应当能被理解的是,本发明不限于公开的实施例。相反,其目的是涵盖随附权利要求精神和范围内的各种修改和等效安排,其范围是给予最广泛的解释,以包含在法律下允许的所有这样的修改和同等结构。
Claims (5)
1.一种促进钙吸收的营养组合物,其特征在于组合物的组成及质量份数为:
西瓜籽肽5-25,
西瓜籽肽钙螯合物3-20,
萝卜缨超微粉10-40,
脱脂奶粉0-50。
2.根据权利要求1所述的一种促进钙吸收的营养组合物的制备方法,其特征在于按照以下步骤进行:
(1)组合物的组成:西瓜籽肽、西瓜籽肽钙螯合物、萝卜缨超微粉、脱脂奶粉;
(2)组合物质量份数:西瓜籽肽5-25、西瓜籽肽钙螯合物3-20、萝卜缨超微粉10-40、脱脂奶粉0-50,充分混合;
(3)成品:将混合好的粉末过100目筛,紫外灯杀菌30min后,装入密封袋中。
3.根据权利要求2所述的一种促进钙吸收的营养组合物的制备方法,其特征在于其中所述的西瓜籽肽钙螯合物的制备步骤为:将西瓜籽蛋白配制成20%(w/v)的悬浊液,放于塑料袋中密封,置于40/60kHz发散式双频超声反应器,室温下超声预处理45min后,取出溶液调pH为9.0,加入碱性蛋白酶,酶与蛋白比例为1:30(w/w),混合均匀后,在50℃下酶解4h,调pH为中性,并于沸水中灭酶10min,离心获上清液,脱盐、浓缩后,按照浓缩液干重与人工胃液按照1:20(w/v)混合,在37℃、150rpm/min下震荡消化2h;再调pH 7.5,按1:80(w/v)加入胰酶至混合液中,继续酶解4h,沸水浴灭酶10min;经离心获上清液,脱盐、浓缩,干燥,即得到西瓜籽肽粉末。
4.根据权利要求2所述的一种促进钙吸收的营养组合物的制备方法,其特征在于其中所述的西瓜籽肽钙螯合物的制备步骤为:将上述获得的西瓜籽肽粉末配制成3%(w/v)的肽溶液,按钙肽比1:6(w/w)加入氯化钙,调溶液pH值8.0、40℃下螯合90min,得到西瓜籽蛋白肽钙螯合液;向螯合液中加入体积比1:8(v/v)的无水乙醇,静置12h,收集醇沉液离心,取沉淀,干燥,即得西瓜籽肽钙螯合物。
5.根据权利要求2所述的一种促进钙吸收的营养组合物的制备方法,其特征在于其中所述的萝卜缨超微粉的制备步骤为:将新鲜红萝卜缨,经除杂、清洗、低温烘干、粉碎后,过40目筛,得到粗粉,再经气流超微粉碎,获得粒径分布≤1μm的粉体。
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