CN111544603A - 一种银杏果制品的功能评价方法 - Google Patents
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Abstract
本发明公开了一种银杏果制品的功能评价方法，包括以下步骤：(1)取菌液进行涂布，然后培养；(2)选择秀丽隐杆线虫，转移至上述培养的菌中进行培养，在线虫处于产卵期时，将亲代和子代分开；(3)对上述培养的线虫给予银杏果制品，检测羟自由基清除能力、总抗氧化能力和超氧阴离子清除能力，以评价银杏果制品的体外抗氧化活性；(4)对上述培养的线虫给予银杏果制品，检测SOD与GSH‑PX酶活性指标，以评价银杏果制品的的体内抗衰老能力。相对于现有技术，本发明采用特定的方法对银杏果制品的功能进行评价，为银杏种仁的加工利用和营养保健食品的开发提供理论依据。
Description
技术领域
本发明属于银杏果制品功能评价技术领域，具体涉及一种银杏果制品的功能评价方 法。
背景技术
银杏(Ginkgo biloba L.)又称白果树，为裸子植物门银杏科银杏属植物，雌雄异株， 是世界上最古老的孑遗树种之一，集食用、药用、观赏、科研等价值于一体。银杏种实核果状，椭圆形，径2cm左右，熟时呈淡黄色或橙黄色，外被白粉；外种皮肉质，有 臭味；中种皮白色，骨质；内种皮膜质；胚乳肉质味甘微苦，子叶2。银杏种仁制粉由 银杏种仁脱毒、磨浆、干燥制备而来，主要成分是淀粉、蛋白质、脂肪和多糖。银杏蛋 白质能清除活性氧和抑制生物氧化活性，多糖可抑制肝癌细胞BEL7402和胰腺癌细胞 PANC-1，酚酸类物质可抑制葡萄球菌、链球菌、白喉杆菌、伤寒杆菌等。银杏种仁制 粉现作为营养品在市场上进行推广，如银杏粉除直接冲饮纯粉外，还可以作为营养配料 添加到其他食品中。将银杏种仁深加工为银杏果粉，提高了银杏种实的实用价值，拓宽 了种实原料的应用范围，为进一步证明银杏种仁的营养价值提供便利。
目前银杏白果的开发利用仍然属于粗放式、小作坊式生产，得不到市场的推广。同时，银杏特殊成分且相关的检测方法和标准不一致，也是引起我国银杏市场发展滞后的 重要原因。通过对银杏种仁制粉的功能进行探究，会带动银杏产业进入良性循环，也会 带动了银杏产业地区的经济发展，又可引导银杏产业向模式化、规范化、集团化方向发 展。目前的生产上缺乏对银杏全粉制品可食用安全、功能性的系统分析。
发明内容
发明目的：针对上述技术问题，本发明提供了一种银杏果制品的功能评价方法，以有效验证银杏果制品的抗氧化、抗衰老作用。
技术方案：为了达到上述发明目的，本发明所采用的技术方案如下：
一种银杏果制品的功能评价方法，包括以下步骤：
(1)取菌液进行涂布，然后培养；
(2)选择秀丽隐杆线虫，转移至上述培养的菌中进行培养，在线虫处于产卵期时，将亲代和子代分开；
(3)对上述培养的线虫给予银杏果制品，检测羟自由基清除能力、总抗氧化能力和超氧阴离子清除能力，以评价银杏果制品的体外抗氧化活性；
(4)对上述培养的线虫给予银杏果制品，检测SOD与GSH-PX酶活性指标，以 评价银杏果制品的的体内抗衰老能力。
作为优选：
步骤(1)中所述菌液中的菌选自E.coil OP50。
步骤(1)中所述培养，是在37℃培养12h，使菌长成薄薄的一层乳白色即可使用。
步骤(2)中所述秀丽隐杆线虫，选用雌雄同体的野生型线虫C.elegans BristolN2。
步骤(2)中将培养中的L4期线虫转移至上述培养的菌中进行培养。
步骤(3)中所述银杏果制品为银杏果粉水提物。
步骤(4)中所述银杏果制品为银杏果粉水提物。
更具体的方法，包括以下步骤：
(1)在NGM平板上加适量的菌液，用涂布棒均匀地将菌液涂布在NGM平板上， 菌液边缘距培养皿边缘0.5cm左右。涂布好细菌的NGM平板吹风至干透，放入恒温箱 中37℃培养12h，使菌长成薄薄的一层乳白色即可使用。
(2)选用雌雄同体的野生型线虫C.elegans Bristol N2，将培养中的L4期线虫转移 至已经预先涂满E.coil OP50的NGM平板，于20℃培养箱中培养。在线虫处于产卵 期时，将亲代线虫每天转移至新板，以便将亲代和子代分开，之后每天对线虫进行观察。
(3)采用羟自由基清除能力、总抗氧化能力、超氧阴离子清除能力测定不同浓度的银杏种仁制粉水提物的体外抗氧化活性。
(4)利用秀丽隐杆线虫这一动物模型在不同浓度的银杏种仁制粉水提物处理下测定 其生理生化指标从而分析体内抗衰老能力。
有益效果：相对于现有技术，本发明具有以下优势：
(1)该方法操作方便，简单易行，响应市场需求从而适合在生产上进行推广。
(2)与其他生物材料相比，C.elegans培养方便，成本低，繁殖能力强；C.elegans中存在的抗氧化酶与人体的抗氧化酶十分相似，是真正的多细胞生物(相比细胞系)， 用线虫做的研究是真正的体内实验。
(3)该方法讨论了银杏果粉可能的作用，对于干扰衰老进程会具有重要的现实意义，并且可以为银杏种仁的加工利用和营养保健食品的开发提供理论依据。
附图说明
图1不同浓度的银杏果粉水提物对自由基清除能力的情况，其中：A羟自由基清 除率；B总抗氧化能力；C超氧阴离子清除率(P<0.05)；
图2不同浓度的银杏果粉水提物对线虫生理指标的影响，其中：A银杏果粉水提物对线虫育雏能力的影响；B银杏果粉水提物对线虫运动能力的影响；C不同浓度的银 杏果粉对线虫爬行距离的影响(P<0.05)；
图3不同浓度的银杏果粉水提物对线虫生化指标的影响，其中：A银杏果粉水提 物对秀丽线虫超氧化物歧化酶(SOD)活力的影响；B银杏果粉水提物对秀丽线虫谷胱 甘肽过氧化物酶(GSH-PX)活力的影响(P<0.05)。
具体实施方式
下面通过具体实施例对本发明所述的技术方案给予进一步详细的说明。
实施例1
1、实施方法
步骤一：秀丽隐杆线虫在不同浓度的银杏种仁制粉处理下的培养
(1)试验地点的选择
试验地点选择江苏省扬州市扬州大学园艺与植物保护学院园艺系果树学科实验室 (经度120.86，纬度32.74)。实验室干净整洁，定时杀菌消毒，培养箱内外温度相差 不大，便于实验操作。
(2)试验材料的选择
试验材料选择东台捷尔银杏科技有限公司生产的的银杏果粉，混合多品种银杏种仁 制粉，具有综合性，干燥储藏。
(3)试验材料的处理
在NGM平板上加适量的菌液，用涂布棒均匀地将菌液涂布在NGM平板上，菌液 边缘距培养皿边缘0.5cm左右。涂布好细菌的NGM平板吹风至干透，放入恒温箱中 37℃培养12h，使菌长成薄薄的一层乳白色即可使用。
选用雌雄同体的野生型线虫C.elegans Bristol N2，将培养中的L4期线虫转移至已 经预先涂满E.coil OP50的NGM平板，于20℃培养箱中培养。在线虫处于产卵期时， 将亲代线虫每天转移至新板，以便将亲代和子代分开，之后每天对线虫进行观察。
银杏种仁制粉浓度选择5、10、20、40mg/ml，秀丽隐杆线虫置于20℃的恒温培养 箱进行繁衍。
(4)后期管理
后期的管理避免线虫受真菌、细菌污染，定时拖地、开紫外灯杀菌。
(5)相关指标测定
在无FUDR的NGM平板上培养放入一只L4期线虫进行生育实验，计算每条虫 的子代总数，计算每种浓度下的平均子代数；线虫的生育力测定在第6天停止。从第0 天开始，每隔1天测定实验组和对照组线虫的头部摆动次数和身体弯曲次数。记录1分 钟内头部摆动的次数作为头部摆动频率指标，记录线虫在20秒内身体弯曲的次数作为 身体弯曲频率指标。挑取给药5d和10d的线虫，每个给药组和空白组各5只，在体视 镜下每次跟踪一只计时一分钟，统计吞咽次数。将对照组与处理组的线虫组织匀浆液进 行SOD与GSH-PX酶活性比较。所有数据都进行3次重复，采用Microsoft Excel 2016 整理和分析数据，GraphPad Prism 7与Adobe illustrator绘制图表。
步骤二：不同银杏果粉浓度的筛选
(1)不同银杏果粉浓度自由基清除能力分析
如图1所示，数据统计发现，5、10、20、40mg/ml银杏果粉水提物对羟自由基、 总抗氧化能力、超氧阴离子均具有一定的清除能力。羟自由基清除率、总抗氧化能力随 浓度升高而显著增加，具有浓度依赖性。5mg/ml银杏果粉水提物超氧阴离子清除率高 达52.63％，其他三种浓度的清除率为29.58-51.42％，不具有线性关系。
(2)线虫生理指标分析
如图2所示，数据统计发现，适当的银杏果粉水提物可增强线虫的运动能力与育雏能力。银杏果粉水提物的加入提高了线虫在前期阶段的孵化能力，增强了其生殖能力。 在处理第4天时，浓度之间的效果差异最明显，以40mg/ml的处理组繁殖总数最高。 与对照组相比，不同浓度的银杏果粉水提物给药以后，线虫在测定的时间后期的运动轨 迹更为活跃，呈现浓度依赖性。数据结果说明，适当的银杏果粉水提物可以适当地提高 线虫的运动能力，有效地保护机体。
(3)线虫生化指标分析
如图3所示，将对照组与处理组的线虫组织匀浆液进行SOD与GSH-PX酶活性， 处理组线虫的SOD酶活性比对照组高出0.42-0.67倍，GSH-PX酶活性比对照组高出 0.03-0.14倍，间接反映了通过喂养银杏果粉水提物提高了线虫体内抗氧化能力。
Claims (7)
1.一种银杏果制品的功能评价方法，其特征在于，包括以下步骤：
(1)取菌液进行涂布，然后培养；
(2)选择秀丽隐杆线虫，转移至上述培养的菌中进行培养，在线虫处于产卵期时，将亲代和子代分开；
(3)对上述培养的线虫给予银杏果制品，检测羟自由基清除能力、总抗氧化能力和超氧阴离子清除能力，以评价银杏果制品的体外抗氧化活性；
(4)对上述培养的线虫给予银杏果制品，检测SOD与GSH-PX酶活性指标，以评价银杏果制品的的体内抗衰老能力。
2.根据权利要求1所述的银杏果制品的功能评价方法，其特征在于，步骤(1)中所述菌液中的菌选自E.coilOP50。
3.根据权利要求1所述的银杏果制品的功能评价方法，其特征在于，步骤(1)中所述培养，是在37℃培养12h，使菌长成薄薄的一层乳白色即可使用。
4.根据权利要求1所述的银杏果制品的功能评价方法，其特征在于，步骤(2)中所述秀丽隐杆线虫，选用雌雄同体的野生型线虫C.elegans Bristol N2。
5.根据权利要求1所述的银杏果制品的功能评价方法，其特征在于，步骤(2)中将培养中的L4期线虫转移至上述培养的菌中进行培养。
6.根据权利要求1所述的银杏果制品的功能评价方法，其特征在于，步骤(3)中所述银杏果制品为银杏果粉水提物。
7.根据权利要求1所述的银杏果制品的功能评价方法，其特征在于，步骤(4)中所述银杏果制品为银杏果粉水提物。
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