CN108719454A - 一种葡萄保鲜剂及其使用方法 - Google Patents
一种葡萄保鲜剂及其使用方法 Download PDFInfo
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- CN108719454A CN108719454A CN201810402234.7A CN201810402234A CN108719454A CN 108719454 A CN108719454 A CN 108719454A CN 201810402234 A CN201810402234 A CN 201810402234A CN 108719454 A CN108719454 A CN 108719454A
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- grape
- antistaling agent
- bacillus subtilis
- hydrolysis liquid
- fermentation
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Abstract
本发明公开了一种葡萄保鲜剂,所述葡萄保鲜剂包括枯草芽孢杆菌(CGMCC编号:1.8801)发酵水解液和水溶性壳聚糖。本发明还公开了一种葡萄保鲜剂的使用方法。使用本发明的保鲜剂的葡萄表面微生物数量比未使用保鲜剂的葡萄成指数倍降低,果粒硬度高,葡萄梗的颜色更为鲜绿,在葡萄贮藏期间,葡萄营养物质流失率小,外观新鲜度和口感得到很好的保持,整体葡萄的保鲜期要延长7~10天。本发明的葡萄保鲜剂具有高效、安全和环保等特点,有广阔的应用前景。
Description
技术领域
本发明涉及食品生物技术领域,具体涉及一种葡萄保鲜剂及其使用方法。
背景技术
枯草芽孢杆菌(Bacillus subtilis)是一类好氧内生芽孢的革兰氏阳性细菌,是对人畜无毒无害的益生菌,该菌不仅对人与动物无毒害作用,且对环境友好,具有显著的抗菌活性和抗逆性,已应用于农业。枯草芽孢杆菌生长快、营养简单,能产生耐热、抗逆的芽孢,其芽孢具有抗逆性强、利于保藏的特点,又能忍受极端的外部环境而长期存活,国内外学者在枯草芽孢杆菌防治植物病害方面进行了大量研究,一些枯草芽孢可对于采收前大田玉米,小麦,辣椒,黄瓜等,表现出明显的防治病害的作用。目前对枯草芽孢杆菌用于大田作物防霉的专利申请有:CN105505836A、CN106544304A等。可是对于果蔬采后保鲜的研究及应用,鲜见报道。
葡萄营养丰富,外观与风味俱佳,是我国的六大水果之一,葡萄含糖量高、水分多、果皮薄、果肉柔软而极易受机械损伤和病原菌的侵染,造成葡萄采后或贮藏期间落粒、萎蔫和腐烂,严重影响葡萄的营养品质和感官品质;而目前葡萄主要采用熏硫保鲜,可能会严重影响葡萄的安全品质。
随着科技的发展,人们生活水平的提高,人们对于不同地区不同季节生产的水果的需求量逐渐加大,而葡萄不耐贮藏,这使得人们对葡萄的保鲜贮存和运输提出了越来越高的要求,有关研究人员在冰温保鲜、气调贮藏、臭氧、短时高浓度CO2处理、涂膜保鲜、高压静电场处理、缓释/控释保鲜剂等非硫或降硫保鲜措施方面进行深入研究,并取得了一定的进展。但迄今为止,葡萄保鲜的最有效和主要的手段仍然是硫制保鲜剂处理,可有效地控制灰霉病菌的生长。但过量SO2会对果实漂白伤害,以及引发环境的污染,甚至会对人类健康造成危害,这就对葡萄贮藏保鲜提出了更高的食用安全要求,研究更安全、高效的葡萄保鲜技术尤为重要。
发明内容
为了解决现有技术存在的问题,本发明的目的在于提供一种采用蛋白酶对枯草芽孢杆菌发酵液进行水解所得到的水解液,与水溶性壳聚糖复配制成的葡萄保鲜剂,该葡萄保鲜剂安全且高效,解决葡萄保鲜中存在化学残留及对环境污染等一些问题,同时具有良好的保鲜效果,可显著延长葡萄的保鲜期。
为实现上述目的,本发明的第一方面提供了一种葡萄保鲜剂,所述葡萄保鲜剂包括枯草芽孢杆菌(CGMCC编号:1.8801)发酵水解液和水溶性壳聚糖。
进一步地,所述枯草芽孢杆菌(CGMCC编号:1.8801)发酵水解液的重量百分含量为85~95%。
进一步地,所述枯草芽孢杆菌发酵水解液的制备方法包括如下步骤:
1)菌种种子液制备:将枯草芽孢杆菌在NA平板培养基上活化,取单菌落划线于NA培养基上,封胶倒置于33℃培养箱培养24h,得到活化的菌株,用移菌环将活化的菌株转移至LB培养基中,在33~50℃,180~300r/min的条件下,培养24~50h,制得的菌种种子液于-1~4℃保存备用;
2)发酵液的制备:将步骤1)备好的菌种种子液,以3~6%(V/V)的接种量接种于NB培养基的三角瓶中,在33~50℃、180~300r/min条件下,培养24~48h,获得菌悬液,将菌悬液经10000r/min离心后取上清液,用微孔滤膜过滤除菌,得到发酵液于4℃保存备用;
3)发酵水解液的制备:用蛋白酶在50~60℃、pH为7.5~9.2、以酶量为2.5~5.0%的条件下水解步骤2)制得的发酵液10~20h,得到发酵水解液。
进一步地,NA平板培养基或NA培养基的组成为:1000ml蒸馏水、3.0g牛肉浸膏、7.0g蛋白胨、10.0g葡萄糖、15.0g琼脂;LB培养基的组成为:1000ml蒸馏水、10.0g氯化钠、5.0g酵母提取物、10.0g胰蛋白胨;NB培养基的组成为:1000ml蒸馏水、3.0g牛肉浸膏、7.0g蛋白胨、10.0g葡萄糖。
进一步地,步骤2)中,以5%(V/V)的接种量接种于NB培养基的三角瓶中,在33℃、180r/min的条件下,培养48h,获得菌悬液。
进一步地,步骤3)为:用蛋白酶在50℃、pH为8.2、以酶量为2.5%的条件下水解步骤2)得到的发酵液10h,得到发酵水解液。
进一步地,所述蛋白酶为胰蛋白酶、碱性蛋白酶、木瓜蛋白酶中的任一种。
进一步地,所述葡萄为采后鲜食葡萄,包括巨峰、玫瑰香、夏黑、青提中的任一种。
本发明的第二方面提供了一种上述的葡萄保鲜剂的使用方法,所述葡萄保鲜剂按照10~30ml/kg的用量,均匀的喷洒在葡萄表面,待葡萄表面干燥后,于-1~4℃下保存。
本发明具有如下优点:
1、本发明通过将具有明显抑菌效果的生防细菌枯草芽孢杆菌的天然发酵水解液与水溶性壳聚糖进行复配,得到葡萄保鲜剂,其中,枯草芽孢杆菌水解液主要是利用枯草芽孢杆菌的代谢产物对葡萄易生的霉菌的生长进行抑制,因其具有高效、安全和环保等特点,有广阔的应用前景。
2、通过试验得到:在葡萄贮藏期间,已使用本发明的保鲜剂的葡萄表面微生物数量比未使用保鲜剂的葡萄成指数倍降低,果粒硬度高,葡萄梗的颜色更为鲜绿,葡萄营养物质流失率小,外观新鲜度和口感得到很好的保持,整体葡萄的保鲜期要延长7~10天。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
本发明使用的枯草芽孢杆菌(CGMCC编号:1.8801),购自中国普通微生物菌种保藏管理中心。
实施例1
一种葡萄保鲜剂,按照重量百分含量计,包括枯草芽孢杆菌发酵水解液85%、水溶性壳聚糖15%。
该葡萄保鲜剂的制备方法包括以下步骤:
1)NA平板培养基或NA培养基:在1000ml蒸馏水中加入牛肉浸膏3.0g、蛋白胨7.0g、葡萄糖10.0g、琼脂15.0g,混合制成;LB培养基:在1000ml蒸馏水中加入氯化钠10.0g、酵母提取物5.0g、胰蛋白胨10.0g,混合制成;NB培养基:在1000ml蒸馏水中加入牛肉浸膏3.0g、蛋白胨7.0g、葡萄糖10.0g,混合制成;
2)菌种种子液制备:将枯草芽孢杆菌在NA平板培养基上活化,取单菌落划线于NA培养基上,封胶倒置于33℃培养箱培养24h,得到活化的菌株,用移菌环将菌株转移至LB培养液,在33℃,180r/min的条件下,培养24h,制得的菌种种子液于4℃保存备用;
3)发酵液的制备:将备好的菌种种子液,以5%(V/V)的接种量接种于NB培养基的三角瓶中,在33℃、180r/min条件下培养48h,获得菌悬液,将菌悬液经10000r/min离心后取上清液,用0.22μm微孔滤膜过滤除菌,得到发酵液于4℃保存备用;
4)发酵水解液的制备:用胰蛋白酶水解发酵液,在50℃、pH为8.2、以酶量为2.5%的条件下水解10h,得到发酵水解液,经检测,发酵水解液含有的肽序列(如序列表中SEQ IDNO:1~36所示)见表1;
5)将水溶性壳聚糖15重量份数加入到85重量份数的发酵水解液中,混合均匀,即制得葡萄保鲜剂。
表1
按照牛津杯法对步骤3)得到的发酵液与步骤4)得到的发酵水解液的抑菌效果进行测定,结果见表2
表2
实施例2
一种葡萄保鲜剂,按照重量百分含量计,包括枯草芽孢杆菌发酵水解液95%、水溶性壳聚糖5%。
该葡萄保鲜剂的制备方法包括以下步骤:
1)培养基的准备:同实施例1;
2)菌种种子液制备:将枯草芽孢杆菌在NA平板培养基上活化,取单菌落划线于NA培养基上,封胶倒置于33℃培养箱培养24h,得到活化的菌株,用移菌环将菌株转移至LB培养液,在40℃,300r/min的条件下,培养24h,制得的菌种种子液于4℃保存备用;
3)发酵液的制备:将备好的菌种种子液,以3%(V/V)的接种量接种于NB培养基的三角瓶中,在50℃、180r/min条件下培养24h,获得菌悬液,将菌悬液经10000r/min离心后取上清液,用0.22μm微孔滤膜过滤除菌,得到发酵液于4℃保存备用;
4)发酵水解液的制备:用木瓜蛋白酶水解发酵液,在60℃、pH为7.6、以酶量为4.0%的条件下水解18h,得到发酵水解液,经检测,发酵水解液含有的肽序列同表1;
5)将水溶性壳聚糖5重量份数加入到95重量份数的发酵水解液中,混合均匀,即制得葡萄保鲜剂。
实施例3
一种葡萄保鲜剂,按照重量百分含量计,包括枯草芽孢杆菌发酵水解液90%、水溶性壳聚糖10%。
该葡萄保鲜剂的制备方法包括以下步骤:
1)培养基的准备:同实施例1;
2)菌种种子液制备:将枯草芽孢杆菌在NA平板培养基上活化,取单菌落划线于NA培养基上,封胶倒置于33℃培养箱培养24h,得到活化的菌株,用移菌环将菌株转移至LB培养液,在50℃,240r/min的条件下,培养36h,制得的菌种种子液于4℃保存备用;
3)发酵液的制备:将备好的菌种种子液,以6%(V/V)的接种量接种于NB培养基的三角瓶中,在42℃、260r/min条件下培养24h,获得菌悬液,将菌悬液经10000r/min离心后取上清液,用0.22μm微孔滤膜过滤除菌,得到发酵液于4℃保存备用;
4)发酵水解液的制备:用碱性蛋白酶水解发酵液,在55℃、pH为9.2、以酶量为3.0%的条件下水解12h,得到发酵水解液,经检测,发酵水解液含有的肽序列同表1;
5)将水溶性壳聚糖10重量份数加入到90重量份数的发酵水解液中,混合均匀,即制得葡萄保鲜剂。
实施例4
一种葡萄保鲜剂,按照重量百分含量计,包括枯草芽孢杆菌发酵水解液88%、水溶性壳聚糖12%。
本实施例的葡萄保鲜剂的制备方法同实施例1。
实施例5
一种葡萄保鲜剂,按照重量百分含量计,包括枯草芽孢杆菌发酵水解液92%、水溶性壳聚糖8%。
本实施例的葡萄保鲜剂的制备方法同实施例1。
应用例1
准备好干净无霉变空间,并摆放足够数量的储物架,室温为25~30℃,贮存期为20天,将实施例1制得的葡萄保鲜剂按照用量10ml/kg喷洒在葡萄表面,在第十天,观察发现喷洒过保鲜剂的葡萄表面的霉菌比未喷洒过的葡萄明显少很多,微生物的数量分别为1.08×103cfu/g,8.91×103cfu/g,硬度分别为5.8N,3.2N,并且保鲜时间比未喷洒过的葡萄保鲜时间延长5~10天。
应用例2
准备干净整洁、无异味、无霉变的储存空间,温度在0~1℃,准备充足的储物架,将实施例2制得的葡萄保鲜剂按照用量30ml/kg均匀喷洒在葡萄表面,待到表面几乎已干之后,移到冷藏室,在贮藏期为20天时观察到,喷洒保鲜剂的葡萄表观比未喷洒过的要新鲜,梗鲜绿,硬度几乎与刚采摘时没有变化,维持在7N左右,微生物数量很少,在100~150cfu/g之间,保鲜效果明显。
应用例3
准备干净整洁、无异味、无霉变的储存空间,温度在0~1℃,准备充足的储物架,将实施例4制得的葡萄保鲜剂按照用量15ml/kg均匀喷洒在葡萄表面,在贮藏期第20天,可以观察到喷洒过的保鲜剂的葡萄外观品质比未喷洒过的葡萄高很多,但是微生物数量比应用例2喷洒过的葡萄要多,在200~260cfu/g之间。硬度维持很好,在6.0-6.8N之间,外观品质总体保鲜期延长8~12天。
虽然,上文中已经用一般性说明及具体实施例对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 北京林业大学
北京农学院
<120> 一种葡萄保鲜剂及其使用方法
<130> 201804
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Claims (9)
1.一种葡萄保鲜剂,其特征在于,所述葡萄保鲜剂包括枯草芽孢杆菌(CGMCC编号:1.8801)发酵水解液和水溶性壳聚糖。
2.根据权利要求1所述的葡萄保鲜剂,其特征在于,所述枯草芽孢杆菌(CGMCC编号:1.8801)发酵水解液的重量百分含量为85~95%。
3.根据权利要求1所述的葡萄保鲜剂,其特征在于,所述枯草芽孢杆菌发酵水解液的制备方法包括如下步骤:
1)菌种种子液制备:将枯草芽孢杆菌在NA平板培养基上活化,取单菌落划线于NA培养基上,封胶倒置于33℃培养箱培养24h,得到活化的菌株,用移菌环将活化的菌株转移至LB培养基中,在33~50℃,180~300r/min的条件下,培养24~50h,制得的菌种种子液于-1~4℃保存备用;
2)发酵液的制备:将步骤1)备好的菌种种子液,以3~6%(V/V)的接种量接种于NB培养基的三角瓶中,在33~50℃、180~300r/min条件下,培养24~48h,获得菌悬液,将菌悬液经10000r/min离心后取上清液,用微孔滤膜过滤除菌,得到发酵液于4℃保存备用;
3)发酵水解液的制备:用蛋白酶在50~60℃、pH为7.5~9.2、以酶量为2.5~5.0%的条件下水解步骤2)制得的发酵液10~20h,得到发酵水解液。
4.根据权利要求3所述的葡萄保鲜剂,其特征在于,NA平板培养基或NA培养基的组成为:1000ml蒸馏水、3.0g牛肉浸膏、7.0g蛋白胨、10.0g葡萄糖、15.0g琼脂;LB培养基的组成为:1000ml蒸馏水、10.0g氯化钠、5.0g酵母提取物、10.0g胰蛋白胨;NB培养基的组成为:1000ml蒸馏水、3.0g牛肉浸膏、7.0g蛋白胨、10.0g葡萄糖。
5.根据权利要求3所述的葡萄保鲜剂,其特征在于,步骤2)中,以5%(V/V)的接种量接种于NB培养基的三角瓶中,在33℃、180r/min的条件下,培养48h,获得菌悬液。
6.根据权利要求3所述的葡萄保鲜剂,其特征在于,步骤3)为:用蛋白酶在50℃、pH为8.2、以酶量为2.5%的条件下水解步骤2)制得的发酵液10h,得到发酵水解液。
7.根据权利要求3所述的葡萄保鲜剂,其特征在于,所述蛋白酶为胰蛋白酶、碱性蛋白酶、木瓜蛋白酶中的任一种。
8.根据权利要求1所述的葡萄保鲜剂,其特征在于,所述葡萄为采后鲜食葡萄,包括巨峰、玫瑰香、夏黑、青提中的任一种。
9.一种权利要求1所述的葡萄保鲜剂的使用方法,其特征在于,所述葡萄保鲜剂按照10~30ml/kg的用量,均匀的喷洒在葡萄表面,待葡萄表面干燥后,于-1~4℃下保存。
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