CN115191480A - 一种超声波联合次氯酸钠和植酸消除鲜切生菜表面大肠杆菌o157:h7及生物膜的方法 - Google Patents
一种超声波联合次氯酸钠和植酸消除鲜切生菜表面大肠杆菌o157:h7及生物膜的方法 Download PDFInfo
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Abstract
本发明公开了一种超声波联合次氯酸钠和植酸消除鲜切生菜表面大肠杆菌O157:H7及其生物膜的方法。本发明所述的方法为：挑选出新鲜的生菜，把叶子切成2～5 cm小段，在超声条件为20～60kHz、100～300W联合浓度为60～100 mg/L的次氯酸处理3～10min，浓度为1～5 g/L植酸处理3～10min。本发明对鲜切生菜表面大肠杆菌O157:H7生物膜有较强的清除效果，清洗前鲜切生菜表面大肠杆菌O157:H7的数量是7.42 log CFU/g，经过超声联合次氯酸钠和植酸处理后，大肠杆菌O157:H7的数量减少了3.80 log CFU/g，杀菌率达到了99.97%。同时保持鲜切生菜的品质，延长货架期。
Description
技术领域
本发明涉及一种杀菌处理工艺，具体涉及利用超声波联合次氯酸钠和植酸消除鲜切生菜表面大肠杆菌O157:H7及生物膜。
背景技术
生菜营养含量丰富，是一种新式速食制品，新鲜生菜经过鲜切加工后，易发生褐变、营养成分流失、质地软化、水分损失和微生物侵染等问题，从而加快了鲜切生菜的品质劣变，缩短了产品货架期。影响鲜切生菜品质的病原菌主要有大肠杆菌O157:H7、李斯特菌、沙门氏菌，其中大肠杆菌O157:H7是引发果蔬品质败坏，导致食源性疾病的重要病原之一。改进现有鲜切产品加工与保鲜技术、研究新型保鲜技术，将对鲜切加工行业的发展具有重要意义。
近年来，鲜切生菜的清洗保鲜技术发展迅速，主要包括物理清洗技术、化学清洗技术和生物清洗技术。物理清洗技术通过如热、超声波和压力等去除鲜切果蔬表面的微生物和流出的汁液，主要包括热水清洗、超声波清洗和机械水力清洗等。物理技术虽能清除部分鲜切生菜表面的大肠杆菌O157:H7，但消除效果不理想，且不能良好的保持生菜品质。化学清洗技术一般采用一定浓度、安全无毒副作用的清洗剂清洗鲜切果蔬。目前，化学清洗技术主要包括臭氧水清洗、酸电解水清洗、有机酸清洗、二氧化氯和次氯酸钠清洗等清洗技术。化学清洗技术能良好的控制鲜切生菜品质，但消除鲜切生菜表面的大肠杆菌O157:H7及生物膜的效果不佳。生物清洗技术主要利用生物体内天然提取物、抗菌素和噬菌体等对鲜切果蔬进行清洗。这种技术清除效果良好，但对于维持鲜切生菜品质方面效果不佳。综上仅采用单一的保鲜技术对鲜切生菜货架期的延长是有限的。因此，本发明将超声波技术与次氯酸钠和植酸联合对鲜切生菜进行处理，抑制鲜切生菜表面大肠杆菌O157:H7及其生物膜的生长，延缓品质衰老劣变，实现货架期延长的目的。
发明内容
本发明提供一种超声波联合次氯酸钠和植酸消除鲜切生菜表面大肠杆菌O157:H7及生物膜的方法。
本发明通过以下步骤实现：
1)选择颜色和大小相同的生菜叶子，将新鲜的生菜用自来水清洗；
2)切掉它们的中脉，把剩下的叶子切成2-2.5 cm宽的条状，然后控制10 g/份；
3)使鲜切生菜在超声条件为40kHz、240W联合浓度为100 mg/L的次氯酸钠处理5min，浓度为3 g/L植酸处理5min。
本发明是利用物理和化学清洗技术相结合的方法，消除鲜切生菜表面大肠杆菌及生物膜，从而达到较好的保鲜效果，延缓鲜切生菜品质劣变。
附图说明
下面结合附图对本发明作进一步说明。
图1不同清洗处理对鲜切生菜表面的大肠杆菌O157:H7杀菌效果对比图（A组：自来水清洗；B组：超声波清洗；C组：微酸性电解水清洗；D组：超声波联合微酸性电解水清洗；E组：超声波联合植酸清洗；F组：次氯酸钠联合植酸清洗；G组：超声联合次氯酸钠和微酸性电解水清洗；H：超声联合次氯酸钠和植酸清洗）。
图2本发明实施例提供的超声联合次氯酸钠和植酸处理前后大肠杆菌 O157:H7的生物膜电镜扫描图（A为对照组， B为水处理， C为超声联合次氯酸钠和植酸处理组）。
图3本发明实施例提供的超声联合次氯酸钠和植酸处理前后大肠杆菌 O157:H7生物膜结晶紫染色图（A为对照组， B为水处理， C为超声联合次氯酸钠和植酸联合处理组）。
图4本发明实施例提供的超声联合次氯酸钠和植酸处理前后对大肠杆菌 O157:H7红外光谱图。
图5本发明实施例提供的超声联合次氯酸钠和植酸处理前后对大肠杆菌 O157:H7红外光谱二阶导数图。
图6本发明实施例提供的超声联合次氯酸钠和植酸处理前后对大肠杆菌 O157:H7的拉曼光谱图。
具体实施方式
下面将结合实施例对本发明作进一步的详细介绍。以下实施用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料均为市售商品。
实施例1超声联合次氯酸钠和植酸处理消除鲜切生菜表面大肠杆菌O157:H7方法
1）选择颜色和大小相同的生菜叶子，将新鲜的生菜用自来水清洗；
2）切掉它们的中脉，把剩下的叶子切成2-2.5 cm宽的条状，然后控制10 g/份；
3）将鲜切生菜在超声条件为40kHz、240W联合浓度为100 mg/L的次氯酸钠处理5min，联合浓度为3 g/L植酸处理5min。
实施例2不同清洗处理消除鲜切生菜表面大肠杆菌O157:H7效果对比
1）选择颜色和大小相同的生菜叶子，将新鲜的生菜用自来水清洗；
2）切掉它们的中脉，把剩下的叶子切成2-2.5 cm宽的条状，然后控制10 g/份；
3）将鲜切生菜分别进行以下处理：A组：自来水清洗10 min；B组：超声波(40 kHz，240 W)清洗10 min；C组：微酸性电解水(pH 5.87 ± 0.04)清洗10 min；D组：超声波联合微酸性电解水清洗10 min；E组：超声波联合3 g/L植酸清0洗10 min；F组：100 mg/L次氯酸钠联合植酸清洗10 min；G组：超声联合次氯酸钠和微酸性电解水处理：超声波联合次氯酸钠清洗5 min，然后超声波联合微酸性电解水清洗5 min。
根据图1得知：清洗前鲜切生菜表面大肠杆菌 O157:H7的数量是7.42 log CFU/g，清洗后鲜切生菜表面大肠杆菌 O157:H7的数量分别为6.93 log CFU/g；6.57 log CFU/g；5.19 log CFU/g；4.85 log CFU/g；4.49 log CFU/g；4.11 log CFU/g；4.26 log CFU/g；3.62 log CFU/g。超声联合次氯酸钠和植酸联合处理对大肠杆菌 O157:H7的抑制作用更显著。大肠杆菌 O157:H7的数量减少了3.80 log CFU/g，杀菌率达到了99.97%。
实施例3超声波联合次氯酸钠和植酸处理大肠杆菌O157：H7生物膜结构的变化
用丙酮浸泡载体片以除去表面的油脂，然后把载体片放入5 mol/L 盐酸溶液中浸泡15 min，再用蒸馏水冲洗干净。最后把载体片放入离心管中，加入45 mL TSB液体培养基，121 ℃灭菌20 min后待用。在含有45 mL TSB液体培养基和载体片的试管中，加入5 mL上述大肠杆菌O157:H7菌液，隔天换培养液，连续培养5 d即可形成稳定生物被膜。
扫描电镜观察：取2片上述的生物被膜载体片，分别置于无菌水中浸泡10 min、超声波结合次氯酸钠中浸泡5 min然后超声波结合植酸中浸泡5 min 后取出，另取1片上述的生物被膜载体片，不作处理，作为对照组。将3组载体片经无菌PBS溶液多次充分漂洗，去掉未附着的细菌，使用2.5% 戊二醛PBS溶液将待测样品固定过夜，依次用50%、70%、80%、90%、100%的乙醇溶液依次对样品进行脱水，每一等级时间为10 min再用乙酸异戊酯置换2次，每次15 min，冷冻干燥后，进行喷金和扫描电镜观察，通过扫描电镜观察处理后的生物被膜清除效果。根据附图2得知：经过超声联合次氯酸钠和植酸处理后，载体片表面上的大肠杆菌O157:H7的数量明显减少，观察不到残留的大肠杆菌O157:H7生物被膜。
结晶紫染色：取2片上述的生物被膜载体片，分别置于无菌水中浸泡10 min、超声波结合次氯酸钠中浸泡5 min然后超声波结合植酸中浸泡5 min 后取出，另取1片上述的生物被膜载体片，不作处理，作为对照组。将3组载体片经无菌PBS溶液多次充分漂洗，去掉未附着的细菌，置于无菌培养皿中，加入10 mL甲醇固定15 min，在每个平皿中加入5 mL结晶紫染色10 min后，自来水冲洗干净，30℃下干燥30 min，再加入5 mL冰醋酸脱色，晾干后光学显微镜下观察。附图3结果表明：经超声联合次氯酸钠和植酸处理后，几乎没有紫色残留物，超声联合次氯酸钠和植酸处理对大肠杆菌O157:H7生物被膜的清除效果优于水处理。
实施例4超声波联合次氯酸钠和植酸处理大肠杆菌O157：H7的傅里叶红外光谱扫描分析
将大肠杆菌O157:H7菌悬液8000 r/min离心10 min后收集菌体，无菌PBS清洗两次，加入1 mL 次氯酸钠在超声波中处理5 min后离心，再加入1 mL植酸在超声波中处理5min，以生理盐水为对照组，处理10 min后，8000 r/min离心10min，弃上清液，收集菌体。
傅里叶红外光谱扫描分析：将上述收集的菌体用PBS冲洗2次后用40倍体积2.5%戊二醛的PBS溶液于4 ℃过夜固定。依次用50%、70%、80%、90%和100%的乙醇溶液梯度脱水，最后用乙酸异戊酯置换乙醇3次，室温干燥。取少量样品粉末，使用傅里叶红外光谱检测仪进行检测分析，检测方法为衰减全反射法，检测波数范围 4000～500 cm−1，分辨率4 cm−1，扫描次数32次。根据附图4、附图5结果表明超声联合次氯酸钠和植酸处理后，大肠杆菌存在氧化反应，使得C=C键断裂，含量减少，从而说明超声联合次氯酸钠和植酸处理对大肠杆菌O157:H7结构产生一定影响。
实施例5超声波联合次氯酸钠和植酸处理对大肠杆菌O157:H7关键受损位点影响
表面增强拉曼光谱（SERS）：用 785nm 激发光收集 300-1750cm-1范围内的SERS信号。激光功率约为 30m W，每次 SERS 测量的曝光时间为 5s。在批量检测中，激光激发光直接聚焦在细菌样品溶液上。每个样品测量三次。4μL细菌样品溶液用于 SERS 检测。将未作处理的对照组大肠杆菌O157:H7和超声联合次氯酸钠和植酸处理后的大肠杆菌O157:H7进行拉曼光谱分析。附图6结果表明经过超声-次氯酸钠-植酸联合处理后，破坏了其络氨酸结构，使得蛋白质的α折叠结构含量增加，同时在1081cm-1处的振动峰减弱，说明超声-次氯酸钠-植酸联合处理使得C=C=C及O-H的官能团含量减少，以上均表明超声-次氯酸钠-植酸联合处理对大肠杆菌O157:H7的组织结构进行了破坏，起到杀菌效果。
Claims (6)
1.一种超声波联合次氯酸钠和植酸消除鲜切生菜表面大肠杆菌O157:H7及生物膜的方法，
该方法通过以超声波技术为主要清洗方法，联合次氯酸钠、植酸对鲜切生菜进行清洗，清洗后的鲜切生菜具有良好的品质。
2.根据权利要求1所述的一种超声波联合次氯酸钠和植酸消除鲜切生菜表面大肠杆菌O157:H7及生物膜的方法，其特征在于，所述的超声条件为20～60kHz、100～300W。
3.根据权利要求1所述的一种超声波联合次氯酸钠和植酸消除鲜切生菜表面大肠杆菌O157:H7及生物膜的方法，其特征在于，所述的次氯酸钠浓度为60～100 mg/L。
4.根据权利要求1所述的一种超声波联合次氯酸钠和植酸消除鲜切生菜表面大肠杆菌O157:H7及生物膜的方法，其特征在于，植酸浓度为1～5 g/L。
5.根据权利要求1所述的一种超声波联合次氯酸钠和植酸消除鲜切生菜表面大肠杆菌O157:H7及生物膜的方法，其特征在于以下步骤：
S1挑选出新鲜的生菜，把叶子切成2～5 cm小段；
S2超声波联合次氯酸钠浸泡清洗3～10min；
S3超声波联合植酸浸泡清洗3～10min。
6.根据权利要求5所述的一种超声波联合次氯酸钠和植酸消除鲜切生菜表面大肠杆菌O157:H7及生物膜的方法的步骤，其步骤构成完整的体系，不可分割、重组。
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| JP2003333987A (ja) * | 2002-05-20 | 2003-11-25 | Katokichi Co Ltd | カット野菜の鮮度保持方法 |
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| JP2003333987A (ja) * | 2002-05-20 | 2003-11-25 | Katokichi Co Ltd | カット野菜の鮮度保持方法 |
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