CN111088317A - 一种小杂粮粗多酚抑菌活性测定方法及应用 - Google Patents
一种小杂粮粗多酚抑菌活性测定方法及应用 Download PDFInfo
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- CN111088317A CN111088317A CN201911398892.4A CN201911398892A CN111088317A CN 111088317 A CN111088317 A CN 111088317A CN 201911398892 A CN201911398892 A CN 201911398892A CN 111088317 A CN111088317 A CN 111088317A
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Abstract
本发明属于小杂粮粗多酚抑菌活性测定技术领域,公开了一种小杂粮粗多酚抑菌活性测定方法及应用,进行原料预处理,杂粮粗多酚溶液制备;菌种活化;谷物粗多酚抑菌活性测定;谷物粗多酚MIC确定。本发明以青稞、紫米、苦荞为材料浸提谷物多酚,选定金黄色葡萄球菌、李斯特菌、沙门氏菌为目标菌种,以打孔法测定谷物多酚的抑菌圈直径,对半稀释法测定其最小抑菌浓度,比较三种谷物抑菌效果。实验表明,青稞对金黄色葡萄球菌的抑菌作用最强,抑菌圈直径为1.2cm,MIC值为1.0mg/mL;紫米、苦荞无较明显抑菌效果。青稞多酚具有作为抑菌剂应用的前景,本发明为青稞资源的开发利用提供了理论依据。
Description
技术领域
本发明属于小杂粮粗多酚抑菌活性测定技术领域,尤其涉及一种小杂粮粗多酚抑菌活性测定方法及应用。
背景技术
目前,最接近的现有技术:小杂粮是小宗粮豆作物的俗称,分布范围广,生育期较短,且含有多种微量营养物质,是人们喜爱的主要食品之一,在食品生产和加工方面具有重要的地位和作用。小杂粮在种植地域及面积上呈现广而大的特点,在种类及品种方面体现了资源丰富的特点,其中包括了青稞、苦荞、紫米、荞麦等。小杂粮一年四季在各地均有种植,主要以春播和秋播为主。小杂粮虽然品种较多、分布较为广泛,但是其产区和品种分布相对集中。近年来,随着人们饮食观念的改变,带来的是拉动了小杂粮的需求,且小杂粮在种植方式上有间套种等栽种优势,使得我省小杂粮的种植面积在逐年上升中。种植面积占全国6.1%,产量占全国7%-11.7%,在粮食产业上有较大的发展潜力。小杂粮的生长特点为耐寒耐贫瘠,而气候的寒冷却导致其长势的矮小,因此小杂粮多与主粮进行间作套种;生长周期过于短造就了其可以很好的利用时间上的茬口,在种植业的资源合理配置及灾年补救粮食生产的主要粮作物之一。
谷物在中国的传统饮食文化中占据地位。2500多年前《黄帝内经》中有“五谷为养”之说,即谷物不仅可以满足我们的口腹之欲,而且是我们身体基本营养物质的补给。谷物中不仅含有我们人体所需的基础营养物质,各营养物质的含量还很高,如裸燕麦中的蛋白质、矿物质总量及不饱和脂肪酸含量均居谷物之首;荞麦中蛋白质含量远高于大米、小麦,且含有的八种必须氨基酸含量最为丰富;绿豆中富含氨基酸及矿物质,是一种高蛋白,低脂肪的食物原料。除此之外,还含有诸如β-胡萝卜素、β-葡聚糖、α-维生素E等其他微量化学物质。分析表明,谷类食品可以在一定程度上降低某些疾病的发生,如心血管疾病、II型糖尿病、肥胖和一些癌症。实验分析表明,小杂粮谷物中含有大量的不饱和脂肪酸,其中很多为人体必需脂肪酸,且脂肪酸对于细菌抑制及促进细胞生成和调节脂质代谢等方面有起到作用。说明了小杂粮对改善人们健康有着重要意义。
小杂粮中除了含有人体所需的基本营养素以外,还含有大量功能性营养成分诸如多酚和膳食纤维等。膳食纤维被称为第七大营养素的一类物质,是植物中类似碳水化合物的可食用部分。膳食纤维中主要包括的有β-葡聚糖、木质素,纤维素和半纤维素,这些物质对于降低心脏病、II型糖尿病和结肠癌有较好的预防作用,在控制体重及三高风险上也有较好作用。大量的实验分析表明多酚类物质与人体健康存在密切关系。谷物中含有多酚类物质,其中酚酸类物质最为突出,大部分的酚类在体内可以清除多余的自由基。进而减少机体的细胞的损伤延缓衰老。并且酚类物质在抗菌、降血压、降血糖、抗炎、抗肿瘤等方面有着重要作用和意义。
酚类化合物是指在芳香烃中苯环上的氢原子被羟基取代所生成的一大类含有酚羟基的化合物,自然界中的酚类大多从植物中来,其主要存在形式有酚酸、类黄酮、木质素和芪类四种。在果蔬及谷物中最为常见的为酚酸,酚酸又可细分为羟基苯甲酸和羟基肉桂酸等。在食物中羟基肉桂酸极易与葡萄糖等发生简单酯化,其常见形式有咖啡酸和阿魏酸等。而羟基苯甲酸在食物中通常以葡糖苷的形式存在,如苯甲酸及原儿茶素是其最常见的存在形式。由两个苯丙素聚合而成的是木质素,其常见形式有五味子甲素和五味子乙素等,具有很强的抗氧化能力且在治疗肿瘤、癌症、炎症等方面啊有一定意义。芪类是以C6-C2-C6为碳骨架的1,2-二苯乙烯,主要包括白藜芦醇、紫檀芪等。黄酮类是由三个碳原子及两个芳香环组合而成的化合物,主要包含花青素、黄烷醇、黄酮、黄烷酮和黄酮醇五类化合物,类黄酮在自然界中广泛存在,其中被人类已经发现的就有5000余种。通常以糖基化或酯化的缀合物形式存在于果蔬、谷物和茶叶等许多可食性植物中。
谷物中含有丰富的多酚,如青稞、紫米和苦荞等谷物中含有大量的多酚类物质,对于分析抗氧化性能有显著前景。谷物多酚主要包括酚酸、类黄酮、单宁和原花青素等。酚酸中含量最为丰富的是阿魏酸,阿魏酸对于抗菌、抗氧化、抑制血小板聚集、抗病毒等方面有较好作用。类黄酮则以结合态或者自由态的形式大多存在于果蔬、豆类和茶叶中,在人类饮食中是最为常见的一种,而在谷物中其多存在于谷物的果皮之中。单宁是一种水溶性的小分子酚类化合物,在植物界中广泛存在,是一种极其重要的次级代谢产物,在高粱、红小米和大麦等谷物中均有存在。分析发现荞麦和高粱中酚酸类物质可媲美果蔬,且具有较强的生物活性。
现今多酚抑菌分析方向大多为茶多酚抑菌,其抑菌方面有抗菌谱广和对革兰氏阴性菌及阳性菌均有较好的抑制作用的特点,EGCG(表没食子儿茶素没食子酸酯,Epigallocatechin gallate)被发现是最有效的抑菌成分,且革兰氏阳性菌相对于阴性菌对EGCG更为敏感。在谷物中多酚抑菌分析方向多为主食类作物,如玉米须及玉米苞叶多酚提取的抑菌分析,玉米苞叶提取物总黄酮及玉米须提取物没食子酸对于部分细菌均有一定的抑制作用。前人分析发现,小杂粮中含有较高的多酚,具有一定的抗氧化活性,但是关于小杂粮的抑菌活性分析鲜有报道,因此本发明以云南特色小杂粮为原材料,提取小杂粮多酚,分析对大肠杆菌、李斯特菌和金黄色葡萄球菌的抑菌活性,进一步为小杂粮的多方面开发利用提供科学依据。
综上所述,现有技术存在的问题是:现有技术关于小杂粮的抑菌活性分析鲜有报道。不能为小杂粮多酚的进一步应用提供依据。
解决上述技术问题的难度:小杂粮多酚不稳定,在提取过程中易被氧化分解,提取过程需要进行严格的低温避光操作。其次,实验过程中所用菌均为制病菌,在操作过程中需要严格遵守实验要求,注意人身安全。
解决上述技术问题的意义:明确小杂粮多酚的抑菌活性,为小杂粮多酚的进一步开发和利用给提供理论依据。
发明内容
针对现有技术存在的问题,本发明提供了一种小杂粮粗多酚抑菌活性测定方法及应用。本发明利用小杂粮多酚进行抑菌活性分析,目前关于小杂粮多酚的抑菌活性分析未见报道。
本发明是这样实现的,一种小杂粮粗多酚抑菌活性测定方法,所述小杂粮粗多酚抑菌活性测定方法包括以下步骤:
步骤一,原料预处理:将谷物籽粒进行杂质筛选处理后,置于磨粉机中磨粉,得到面粉过筛,备用。
步骤二,杂粮粗多酚溶液的制备:依次进行杂粮粗多酚的提取、杂粮粗多酚的纯化、扩散溶剂的选择以及多酚含量的测定。
步骤三,菌种活化:取冻存菌液并加入到培养液中,将培养瓶置于恒温振荡培养箱中恒温培养,备用。
步骤四,谷物粗多酚抑菌活性测定:将活化好的菌液置于液体培养基中恒温振荡培养后,加入多酚溶液进行抑菌活性测定。
步骤五,谷物粗多酚MIC确定:将各浓度的多酚溶液及70%乙醇溶液加入到恒温振荡培养后的菌液中,进行MIC确定。
进一步,步骤一中,所述原料预处理的方法为:
将采买来的谷物籽粒进行杂质筛选处理,挑除霉变粒、破损粒。将取挑选好的谷物籽粒于磨粉机中磨粉,得到面粉将其过50目筛备用。
进一步,步骤二中,所述杂粮粗多酚提取的方法为:
取谷物面粉与75%乙醇以料液比为1:20(g:mL)混匀;首先将谷物面粉溶液装入适宜的容器内,置于超声仪中,超声25min,每隔5min摇晃溶液,再将超声完成的谷物面粉溶液,置于离心机,室温下4000r/min离心10min;以上重复操作3次,合并离心上清,将上清液旋转蒸发,得到浓缩液。最后用冷冻干燥将浓缩液冻干至成粉末状,取出冲氮气置于-20℃低温下冷藏备用,标记为1号粉,之后3种谷物1号粉均由此方法提取得到。
进一步,步骤二中,所述杂粮粗多酚的纯化方法为:
取谷物面粉与75%乙醇以料液比为1:20(g:mL)混匀;首先将谷物面粉溶液装入适宜的容器内,置于超声仪中,超声25min,每隔5min摇晃溶液,将清洗完成的谷物面粉溶液,置于离心机,室温下4000r/min离心10min;以上步骤重复操作3次,合并离心上清,将上清液旋转蒸发,旋转浓缩得到浓缩液。再将旋转蒸发得的上清浓缩液与90%的乙醇以料液比为1:4(v:v)混匀,冲氮后置于-20℃冰箱醇沉48h;最后将醇沉好的谷物多酚溶液置于旋转蒸发仪中旋转浓缩,将浓缩液置于冷冻干燥机,冻干至其成粉末状,取出冲氮气置于低温下-20℃冷藏备用,标记为2号粉,之后3种谷物1号粉均由此方法提取得到。
进一步,步骤二中,所述扩散溶剂的选择方法为:
为了使谷物多酚冻干粉更易被溶解和在抑菌实验中更易扩散,因此对多酚冻干粉溶解液进行选择。称取3份质量各1g的阿魏酸标样粉,按1:10(g:mL)的料液比将阿魏酸标样粉分别溶于70%乙醇、50%甲醇和蒸馏水溶剂中,三种溶液分别标号a,b和c,混合后得到浓度为0.1g/mL的3种阿魏酸溶液。再分别将3种溶液进行对半稀释,a,b,c各取其体积的1/2依次加入同等体积70%乙醇、50%甲醇、蒸馏水,得到浓度为0.05g/ml的阿魏酸溶液,重复以上步骤,得到0.025g/ml的阿魏酸溶液。3种溶液将作为多酚抑菌扩散溶液的选择。
进一步,步骤二中,所述多酚含量测定方法为:
吸取多酚化合物粗提液2mL置于离心管中,加入Folin-Ciocalteau试剂,摇匀后,加入饱和碳酸钠溶液,充分混匀,室温下避光反应35min,取蓝色上清液于725nm下测定吸光度。用粗多酚溶剂替代样品提取液,相同条件下作为空白进行调零。以阿魏酸为标准品建立回归方程为:y=0.0066x-0.003(R2=0.998),得每克样品所含总多酚相当于阿魏酸的量,转化单位为μg FAE/mL。
进一步,步骤三中,所述菌种活化的方法为:
配置牛肉膏蛋白胨液体培养基并将其灭菌,首先取冻存菌液各1mL,加入到100mL培养液中,最后将培养瓶置于恒温振荡培养箱中以150r/min 37℃恒温培养4h备用。
进一步,步骤四中,所述谷物粗多酚抑菌活性的测定方法为:
配制牛肉膏蛋白胨液体培养基及牛肉膏蛋白胨固体培养基于高压灭菌锅设置温度为121℃时间为15min将其灭菌;将活化好的菌液吸取1mL于液体培养基中;将培养瓶置于恒温振荡培养箱中,150r/min 37℃恒温振荡培养4h;取CFU为107-108的菌液100μL与20mL的固体培养基溶液于试管中混匀;将混匀后的培养基立即倒入平板中,同时对培养基进行打孔,然后在每个孔中打入100μL各浓度的多酚溶液,在同一平板内打孔70%乙醇溶液作对照。设置空白实验既不打孔也不打任何溶液,将空白培养基倒放,置于4℃泡沫盒中静置1h后放到37℃恒温培养箱中培养24h。
进一步,步骤五中,所述谷物粗多酚MIC的确定方法为:
培养基处理如定性实验,将7.9mg/mL的多酚溶液浓度按照对半稀释法,将提取得到多酚溶液与70%的乙醇溶液以1:1(v/v)形式混合,取多酚提取物溶于70%乙醇溶液,得到溶液混匀后取其1/2与相同体积的70%乙醇混合,重复9次以上步骤得到10个不同浓度梯度的粗多酚溶液,分别是7.9、3.9、1.9、1.0、0.5、0.2、0.1、0.06、0.03、0.01mg/mL,将提前活化好的菌液吸取1mL于液体培养基中;再将培养瓶置于恒温振荡培养箱中,150r/min 37℃恒温振荡培养4h;取CFU为107-108的菌液100μL与20mL的固体培养基溶液于试管中混匀;将混匀后的培养基立即倒入平板中,同时对培养基进行打孔,然后在每个孔中打入100μL各浓度的多酚溶液及70%乙醇溶液。设置空白实验既不打孔也不打任何溶液,将空白培养基倒放,实验培养基不倒,置于4℃泡沫盒中静置1h后放到37℃恒温培养箱中培养24h,24h后观察出现最小抑菌浓度的谷物粗多酚溶液。
本发明另一目的在于提供一种利用所述小杂粮粗多酚抑菌活性测定方法测定的小杂粮粗多酚抑菌剂,所述小杂粮粗多酚抑菌剂为青稞谷物粗多酚提取物。
综上所述,本发明的优点及积极效果为:本发明提供的小杂粮粗多酚抑菌活性测定方法,以青稞、紫米、苦荞为材料浸提谷物多酚,选定金黄色葡萄球菌、李斯特菌、沙门氏菌(上述菌种均为市购)为目标菌种,以打孔法测定谷物多酚的抑菌圈直径,对半稀释法测定其MTC(最小抑菌浓度,minimum inhibitory concentration),比较三种谷物的抑菌效果。实验结果表明,青稞对金黄色葡萄球菌的抑菌作用最强,抑菌圈直径为1.2cm,MIC值为1.0mg/mL。紫米、苦荞无较明显抑菌效果。因此,青稞多酚具有作为抑菌剂应用的前景,本发明为青稞资源的开发利用提供了理论依据。
本发明通过对青稞、紫米、苦荞三种谷物进行谷物多酚的浸提、抑菌活性比较,结果显示青稞谷物粗多酚提取物抑菌效果为优,且在革兰氏阳性菌中损伤更明显。本发明表明不同种类谷物中谷物多酚含量与其抑菌效果有关。金黄色葡萄球菌、李斯特菌、沙门氏菌都是食品加工、储存过程中常见的病源菌种,在自然界中分布也都较为广泛,谷物的抑菌活性分析,对于谷物资源的有效利用及天然植物抑菌剂的开发具有一定的实际意义,为人们深入分析谷物保健功能及抑菌机理分析提供了实验依据。
附图说明
图1是本发明实施例提供的小杂粮粗多酚抑菌活性测定方法流程图。
图2是本发明实施例提供的3种溶剂扩散效果示意图。
图中:图(A)是50%甲醇溶解扩散效果;图(B)是70%乙醇溶解扩散效果;图(C)是蒸馏水溶解扩散效果。
图3是本发明实施例提供的75%乙醇浸提青稞多酚抑菌平板示意图。
图4是本发明实施例提供的谷物抑菌效果示意图;
图中:图(A)是苦荞抑菌效果;图(B)是紫米抑菌效果。
图5是本发明实施例提供的青稞粗多酚抑菌效果示意图;
图中:图(A)是金黄色葡萄球菌;图(B)是沙门氏菌;图(C)是李斯特菌,1和2的浓度分别为7.9和3.9mg/ml。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
针对现有技术存在的问题,本发明提供了一种小杂粮粗多酚抑菌活性测定方法,下面结合附图对本发明作详细的描述。
如图1所示,本发明实施例提供的小杂粮粗多酚抑菌活性测定方法包括以下步骤:
S101,原料预处理:将谷物籽粒进行杂质筛选处理后,置于磨粉机中磨粉,得到面粉过筛,备用。
S102,杂粮粗多酚溶液的制备:依次进行杂粮粗多酚的提取、杂粮粗多酚的纯化、扩散溶剂的选择以及多酚含量的测定。
S103,菌种活化:取冻存菌液并加入到培养液中,将培养瓶置于恒温振荡培养箱中恒温培养,备用。
S104,谷物粗多酚抑菌活性测定:将活化好的菌液置于液体培养基中恒温振荡培养后,加入多酚溶液进行抑菌活性测定。
S105,谷物粗多酚MIC确定:将各浓度的多酚溶液及70%乙醇溶液加入到恒温振荡培养后的菌液中,进行MIC确定。
下面结合实施例对本发明作进一步描述。
实施例1
1、材料与方法
1.1实验时间、地点
本实验于2016年11月-2018年11月期间在云南农业大学食品科学技术学院实验室完成。
1.2实验材料
1.2.1实验原材料
实验原材料使用的云黑青稞、紫米、苦荞分别购于云南省迪庆藏族自治州香格里拉市、云南省普洱市墨江县、云南省红河哈尼族彝族自治州泸西县。
1.2.2实验菌种
实验三种菌种均由云南农业大学食品科学与技术学院提供,菌种如表1所示。
表1供试菌种
1.2.3实验试剂
实验试剂如表2所示。
表2实验试剂
1.2.4实验仪器和设备
实验主要仪器和设备如表3所示。
表3实验主要仪器和设备
1.3实验方法
1.3.1原料的预处理
将采买来的谷物籽粒进行杂质筛选处理,挑除霉变粒、破损粒等不宜用于实验的谷物籽粒,取挑选好的谷物籽粒于磨粉机中磨粉,得到面粉将其过50目筛备用。
1.3.2杂粮粗多酚溶液的制备
1.3.2.1杂粮粗多酚提取
取谷物面粉与75%乙醇以料液比为1:20(g:mL)混匀;首先将谷物面粉溶液装入适宜的容器内,置于超声仪中,超声25min,每隔5min摇晃溶液,再将超声完成的谷物面粉溶液,置于离心机,室温下4000r/min离心10min;以上重复操作3次,合并离心上清,将上清液旋转蒸发,得到浓缩液。最后用冷冻干燥将浓缩液冻干至成粉末状,取出冲氮气置于-20℃低温下冷藏备用,标记为1号粉,之后3种谷物1号粉均由此方法提取得到。
1.3.2.2杂粮粗多酚的纯化
取谷物面粉与75%乙醇以料液比为1:20(g:mL)混匀;首先将谷物面粉溶液装入适宜的容器内,置于超声仪中,超声25min,每隔5min摇晃溶液,将清洗完成的谷物面粉溶液,置于离心机,室温下4000r/min离心10min;以上步骤重复操作3次,合并离心上清,将上清液旋转蒸发,旋转浓缩得到浓缩液。再将旋转蒸发得的上清浓缩液与90%的乙醇以料液比为1:4(v:v)混匀,冲氮后置于-20℃冰箱醇沉48h;最后将醇沉好的谷物多酚溶液置于旋转蒸发仪中旋转浓缩,将浓缩液置于冷冻干燥机,冻干至其成粉末状,取出冲氮气置于低温下-20℃冷藏备用,标记为2号粉,之后3种谷物1号粉均由此方法提取得到。
1.3.2.3扩散溶剂的选择
为了使谷物多酚冻干粉更易被溶解和在抑菌实验中更易扩散,因此对多酚冻干粉溶解液进行选择。称取3份质量各1g的阿魏酸标样粉,按1:10(g:mL)的料液比将阿魏酸标样粉分别溶于70%乙醇、50%甲醇和蒸馏水溶剂中,三种溶液分别标号a,b和c,混合后得到浓度为0.1g/mL的3种阿魏酸溶液。再分别将3种溶液进行对半稀释,a,b,c各取其体积的1/2依次加入同等体积70%乙醇、50%甲醇、蒸馏水,得到浓度为0.05g/ml的阿魏酸溶液,重复以上步骤,得到0.025g/ml的阿魏酸溶液。3种溶液将作为多酚抑菌扩散溶液的选择。
1.3.2.4多酚含量测定
吸取多酚化合物粗提液2mL置于离心管中,加入Folin-Ciocalteau试剂,摇匀后,加入饱和碳酸钠溶液,充分混匀,室温下避光反应35min,取蓝色上清液于725nm下测定吸光度。用粗多酚溶剂替代样品提取液,相同条件下作为空白进行调零。以阿魏酸为标准品建立回归方程为:y=0.0066x-0.003(R2=0.998),得每克样品所含总多酚相当于阿魏酸的量,转化单位为μg FAE/mL。
1.3.3菌种活化
配置牛肉膏蛋白胨液体培养基并将其灭菌,首先取冻存菌液各1mL,加入到100mL培养液中,最后将培养瓶置于恒温振荡培养箱中以150r/min 37℃恒温培养4h备用。
1.3.4谷物粗多酚抑菌活性
配制牛肉膏蛋白胨液体培养基及牛肉膏蛋白胨固体培养基于高压灭菌锅设置温度为121℃时间为15min将其灭菌;将活化好的菌液吸取1mL于液体培养基中;将培养瓶置于恒温振荡培养箱中,150r/min 37℃恒温振荡培养4h;取CFU为107-108的菌液100μL与20mL的固体培养基溶液于试管中混匀;将混匀后的培养基立即倒入平板中,同时对培养基进行打孔,然后在每个孔中打入100μL各浓度的多酚溶液,在同一平板内打孔70%乙醇溶液作对照。设置空白实验既不打孔也不打任何溶液,将空白培养基倒放,置于4℃泡沫盒中静置1h后放到37℃恒温培养箱中培养24h。
1.3.5谷物粗多酚MIC确定
培养基处理如定性实验,将7.9mg/mL的多酚溶液浓度按照对半稀释法,将提取得到多酚溶液与70%的乙醇溶液以1:1(v/v)形式混合,取多酚提取物溶于70%乙醇溶液,得到溶液混匀后取其1/2与相同体积的70%乙醇混合,重复9次以上步骤得到10个不同浓度梯度的粗多酚溶液,分别是7.9、3.9、1.9、1.0、0.5、0.2、0.1、0.06、0.03、0.01mg/mL,将提前活化好的菌液吸取1mL于液体培养基中;再将培养瓶置于恒温振荡培养箱中,150r/min 37℃恒温振荡培养4h;取CFU为107-108的菌液100μL与20mL的固体培养基溶液于试管中混匀;将混匀后的培养基立即倒入平板中,同时对培养基进行打孔,然后在每个孔中打入100μL各浓度的多酚溶液及70%乙醇溶液。设置空白实验既不打孔也不打任何溶液,将空白培养基倒放,实验培养基不倒,置于4℃泡沫盒中静置1h后放到37℃恒温培养箱中培养24h,24h后观察出现最小抑菌浓度的谷物粗多酚溶液。
2、结果
2.1结果与分析
2.1.1阿魏酸扩散溶剂选择
用不同溶剂溶解阿魏酸粉末得到的阿魏酸溶液进行抑菌定性实验时,发现70%乙醇以料液比为1:10(m:v)溶解再进行2次对半稀释上均可以完全溶解阿魏酸,且无沉淀;50%甲醇1:10(m:v)及1:20(m:v)的比例下溶液略有沉淀;蒸馏水溶解液则不能完全溶解阿魏酸其效果最差。在抑菌实验中,发现在同等条件下用70%乙醇溶解下的阿魏酸的抑菌效果更为明显,如图2所示,扩散能力也较强,50%甲醇与70%乙醇有显著差异,且阴性对照实验表明,溶剂对于抑菌效果无较为明显的促进作用,结果如表4所示。
表4 3种溶剂不同扩散效果的抑菌效果
注:实验数据为3次重复得到结果,α=0.05,“_”表示无明显抑菌效果,“*”表示阳性对照,直径单位为mm。
2.1.2青稞提取物1号粉及2号粉抑菌比较
在青稞粗多酚提取中,用75%提取液进行抑菌定性实验,得到的多酚对3种病源微生物无明显抑制效果,且与培养基内其它部分相比,加入该提取液的孔周围明显菌落长势密集且厚,孔内沉积物较多;用90%乙醇醇溶48h后的提取液对3种病源微生物则有较为明显的抑菌效果。相比于75%乙醇提取液而言,90%乙醇提取物抑菌实验中平板中孔周围的菌无明显密集,抑菌结果如表5及图3所示。
表5不同浸提抑菌比较
注:实验数据为3次重复得到结果,“_”表示无明显抑菌效果,直径单位为mm。
2.1.3阿魏酸及3种谷物抑菌定性比较
同等条件下对阿魏酸及3种谷物粗多酚的抑菌结果进行定性,结果见表6。把4个实验对象的浓度定为4..8mg/mL时,阿魏酸有一定的抑菌效果,在实验中发现3种谷物中只有青稞提取物有一定的抑菌效果,而苦荞和紫米在此次的实验中没有表现出较为明显的抑菌圈,如图4所示,相较于紫米而言,在苦荞抑菌定性实验中平板中孔周围的菌不密集。
表6阿魏酸及3种谷物多酚抑菌比较
注:实验数据为3次重复得到结果,α=0.05,“_”表示无明显抑菌效果,直径单位为mm。
2.1.4青稞提取物MIC结果
青稞粗多酚提取物的最小抑菌浓度见表7。青稞提取物MIC经实验可得,沙门氏菌、金黄色葡萄球菌、李斯特氏MIC值基本为1.0mg/mL,青稞多酚提取物对3种供试菌株均有一定程度的抑制作用,其中抑菌效果最好的是金黄色葡萄球菌,其次沙门氏菌抑制作用相对来说较弱。青稞多酚提取物的抑菌效果如图5所示。
表7青稞多酚最小抑菌浓度
实验数据为3次重复得到结果,α=0.05,“_”表示无明显抑菌效果,直径单位为mm。
2.2结果:
在选择溶剂过程中,抑菌活性依赖于溶剂与Nunes R等人的分析基本一致,与50%甲醇与蒸馏水相比较70%乙醇的抑菌活性及在培养基中的扩散程度更强,阿魏酸其物理性质为微溶于冷水且在水溶液中稳定性差,易溶于乙醇、甲醇。从实验中可看出,相对于50%甲醇溶解阿魏酸来说,70%乙醇对于阿魏酸的溶解效果更好,当抑菌性物质被全部溶解时,溶剂可以更易扩散开来从而达到更为理想的抑菌效果。
在青稞粗多酚溶液进行处理时发现,青稞粗多酚溶液的纯净度影响着病原菌的生长情况,经过二次醇溶后的粗多酚溶液,前者经混匀后直接作为样品液和后者混匀后离心作为样液注入孔内。前者菌的长势明显比后者好。同时发现,虽然离心后利于病源微生物生长的大大分子物质降落在底部。但是,二次醇溶后的青稞提取物依然可能存在着促进病原菌生长的物质。且本次实验所采用的多酚为粗多酚,提取剂为乙醇,乙醇作为一种有机溶剂可以用于多种有机物及其无机物的提取与分离,75%的乙醇提取物对于所选取的三种有害菌抑菌效果不明显,90%乙醇醇溶后得到的提取物对于所选取的三种有害菌有较为明显的抑制效果,浓度较低的乙醇溶液更易保留更多利于病源微生物生长的物质,对于抑菌来说可能是不利的。
青稞抑菌效果明显低于阿魏酸。经由90%乙醇醇溶后得到的青稞粗多酚,其对所选择的3种有害菌含有某种支持抗菌活性的物质,表面看此次的实验结果苦荞及紫米并没有可以支持抗菌活性的物质存在。且从文献中看出紫米、苦荞中的碳水化合物含量远高于青稞,但此次实验中的多酚均为粗多酚,紫米和苦荞中经测定发现其含有阿魏酸,但是其在此次实验中并未有明显抑菌效果,紫米和苦荞中的促进成分可能高于抑制成分。
在抑菌活性实验中,细菌细胞壁所具有的特性可能会影响青稞提取物抑制被测细菌的生长能力,沙门氏菌为革兰氏阴性菌,革兰氏阴性菌其细胞壁以肽聚糖的形式被膜结构包围,且其含有脂多糖或内毒素成分,可以阻止抗菌剂进入细胞,脂多糖层特殊的选择体系可以对陌生物质进行较好的选择并阻止,革兰氏阴性菌比革兰氏阳性菌对陌生物质的抗性更强,金黄色葡萄球菌和李斯特菌为革兰氏阳性菌,较沙门氏菌来看李斯特菌与金黄色葡萄球菌的抑菌效果更好,这与Mzid M等人的分析基本一致。
通过对青稞、紫米、苦荞三种谷物进行谷物多酚的浸提、抑菌活性比较,结果显示青稞谷物粗多酚提取物抑菌效果为优,且在革兰氏阳性菌中损伤更明显。本发明表明不同种类谷物中谷物多酚含量与其抑菌效果有关。金黄色葡萄球菌、李斯特菌、沙门氏菌都是食品加工、储存过程中常见的病源菌种,在自然界中分布也都较为广泛,谷物的抑菌活性分析,对于谷物资源的有效利用及天然植物抑菌剂的开发具有一定的实际意义,为人们深入分析谷物保健功能及抑菌机理分析提供了实验依据。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种小杂粮粗多酚抑菌活性测定方法,其特征在于,所述小杂粮粗多酚抑菌活性测定方法包括以下步骤:
步骤一,原料预处理:将谷物籽粒进行杂质筛选处理后,置于磨粉机中磨粉,得到面粉过筛,备用;
步骤二,杂粮粗多酚溶液的制备:依次进行杂粮粗多酚的提取、杂粮粗多酚的纯化、扩散溶剂的选择以及多酚含量的测定;
步骤三,菌种活化:取冻存菌液并加入到培养液中,将培养瓶置于恒温振荡培养箱中恒温培养,备用;
步骤四,谷物粗多酚抑菌活性测定:将活化好的菌液置于液体培养基中恒温振荡培养后,加入多酚溶液进行抑菌活性测定;
步骤五,谷物粗多酚MIC的确定:将各浓度的多酚溶液及70%乙醇溶液加入到恒温振荡培养后的菌液中,进行MIC确定。
2.如权利要求1所述的小杂粮粗多酚抑菌活性测定方法,其特征在于,步骤一中,所述原料预处理的方法为:
将采买来的谷物籽粒进行杂质筛选处理,挑除霉变粒、破损粒;将取挑选好的谷物籽粒于磨粉机中磨粉得到面粉,并过50目筛备用。
3.如权利要求1所述的小杂粮粗多酚抑菌活性测定方法,其特征在于,步骤二中,所述杂粮粗多酚提取的方法为:
(1)取谷物面粉与75%乙醇以料液比为1:20(g:mL)混匀;
(2)将谷物面粉溶液装入适宜的容器内,置于超声仪中,超声25min,每隔5min摇晃溶液,再将超声完成的谷物面粉溶液,置于离心机,室温下4000r/min离心10min;
(3)将步骤(2)重复操作3次,合并离心上清,将上清液旋转蒸发,得到浓缩液;
(4)最后用冷冻干燥将浓缩液冻干至成粉末状,取出冲氮气置于-20℃低温下冷藏备用,标记为1号粉,之后3种谷物1号粉均由此方法提取得到。
4.如权利要求1所述的小杂粮粗多酚抑菌活性测定方法,其特征在于,步骤二中,所述杂粮粗多酚的纯化方法为:
(Ⅰ)取谷物面粉与75%乙醇以料液比为1:20(g:mL)混匀;
(Ⅱ)将谷物面粉溶液装入适宜的容器内,置于超声仪中,超声25min,每隔5min摇晃溶液,将清洗完成的谷物面粉溶液,置于离心机,室温下4000r/min离心10min;
(Ⅲ)将步骤(Ⅱ)重复操作3次,合并离心上清,将上清液旋转蒸发,旋转浓缩得到浓缩液;再将旋转蒸发得的上清浓缩液与90%的乙醇以料液比为1:4(v:v)混匀,冲氮后置于-20℃冰箱醇沉48h;
(Ⅳ)最后将醇沉好的谷物多酚溶液置于旋转蒸发仪中旋转浓缩,将浓缩液置于冷冻干燥机,冻干至成粉末状,取出冲氮气置于低温下-20℃冷藏备用,标记为2号粉,之后3种谷物1号粉均由此方法提取得到。
5.如权利要求1所述的小杂粮粗多酚抑菌活性测定方法,其特征在于,步骤二中,所述扩散溶剂的选择方法为:
1)称取3份质量各1g的阿魏酸标样粉,按1:10(g:mL)的料液比将阿魏酸标样粉分别溶于70%乙醇、50%甲醇和蒸馏水溶剂中,三种溶液分别标号a,b和c,混合后得到浓度为0.1g/mL的3种阿魏酸溶液;
2)再分别将3种溶液进行对半稀释,a,b,c各取其体积的1/2依次加入同等体积70%乙醇、50%甲醇、蒸馏水,得到浓度为0.05g/ml的阿魏酸溶液;
3)重复以上步骤,得到0.025g/ml的阿魏酸溶液;3种溶液将作为多酚抑菌扩散溶液的选择。
6.如权利要求1所述的小杂粮粗多酚抑菌活性测定方法,其特征在于,步骤二中,所述多酚含量测定方法为:
吸取多酚化合物粗提液2mL置于离心管中,加入Folin-Ciocalteau试剂,摇匀后,加入饱和碳酸钠溶液,充分混匀,室温下避光反应35min,取蓝色上清液于725nm下测定吸光度;
用粗多酚溶剂替代样品提取液,相同条件下作为空白进行调零;以阿魏酸为标准品建立回归方程为:y=0.0066x-0.003(R2=0.998),得每克样品所含总多酚相当于阿魏酸的量,转化单位为μgFAE/mL。
7.如权利要求1所述的小杂粮粗多酚抑菌活性测定方法,其特征在于,步骤三中,所述菌种活化的方法为:
配置牛肉膏蛋白胨液体培养基并将其灭菌,首先取冻存菌液各1mL,加入到100mL培养液中,最后将培养瓶置于恒温振荡培养箱中以150r/min 37℃恒温培养4h备用。
8.如权利要求1所述的小杂粮粗多酚抑菌活性测定方法,其特征在于,步骤四中,所述谷物粗多酚抑菌活性的测定方法为:
①配制牛肉膏蛋白胨液体培养基及牛肉膏蛋白胨固体培养基于高压灭菌锅设置温度为121℃时间为15min将其灭菌;
②将活化好的菌液吸取1mL于液体培养基中;将培养瓶置于恒温振荡培养箱中,150r/min 37℃恒温振荡培养4h;
③取CFU为107-108的菌液100μL与20mL的固体培养基溶液于试管中混匀;将混匀后的培养基立即倒入平板中,同时对培养基进行打孔,然后在每个孔中打入100μL各浓度的多酚溶液,在同一平板内打孔70%乙醇溶液作对照;
④设置空白实验既不打孔也不打任何溶液,将空白培养基倒放,置于4℃泡沫盒中静置1h后放到37℃恒温培养箱中培养24h。
9.如权利要求1所述的小杂粮粗多酚抑菌活性测定方法,其特征在于,步骤五中,所述谷物粗多酚MIC的确定方法为:
(a)将7.9mg/mL的多酚溶液浓度按照对半稀释法,将提取得到多酚溶液与70%的乙醇溶液以1:1(v/v)形式混合,取多酚提取物溶于70%乙醇溶液,得到溶液混匀后取其1/2与相同体积的70%乙醇混合;
(b)重复9次步骤(a)得到10个不同浓度梯度的粗多酚溶液,分别是7.9、3.9、1.9、1.0、0.5、0.2、0.1、0.06、0.03、0.01mg/mL,将提前活化好的菌液吸取1mL于液体培养基中;
(c)再将培养瓶置于恒温振荡培养箱中,150r/min 37℃恒温振荡培养4h;取CFU为107-108的菌液100μL与20mL的固体培养基溶液于试管中混匀;将混匀后的培养基立即倒入平板中,同时对培养基进行打孔,然后在每个孔中打入100μL各浓度的多酚溶液及70%乙醇溶液;
(d)设置空白实验既不打孔也不打任何溶液,将空白培养基倒放,实验培养基不倒,置于4℃泡沫盒中静置1h后放到37℃恒温培养箱中培养24h。
10.一种利用权利要求1~9任意一项所述小杂粮粗多酚抑菌活性测定方法测定的小杂粮粗多酚抑菌剂,其特征在于,所述小杂粮粗多酚抑菌剂为青稞谷物粗多酚提取物。
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