Research Group on Microbiology and Quality of Fruit and Vegetables, Food Science and Technology Department, CEBAS-CSIC, PO Box 164, E-30100, Espinardo, Spain.
Research Group on Microbiology and Quality of Fruit and Vegetables, Food Science and Technology Department, CEBAS-CSIC, PO Box 164, E-30100, Espinardo, Spain.
Food Microbiol. 2023 Feb;109:104155. doi: 10.1016/j.fm.2022.104155. Epub 2022 Oct 6.
Some water disinfection treatments, such as chlorine and chlorine dioxide, used in the fresh-cut industry to maintain the microbiological quality of process water (PW), inactivate bacterial cells in the water but they also lead to the induction of an intermediate state between viable and non-viable known as viable but non-culturable (VBNC) state. Viable cells can participate in cross-contamination events but the significance of VBNC cells in PW, transfer to the product and potential resuscitation capacity during storage is unclear. The present study aims to determine first, if VBNC cells present in PW can cross-contaminate leafy greens during washing and secondly its potential revival during shelf-life. Process water characterized by a high chemical oxygen demand, due to the presence of high levels of organic matter, was inoculated with Listeria monocytogenes or Escherichia coli O157:H7. Inoculated PW was then treated for 1 min with chlorine dioxide (3 mg/L) or chlorine (5 mg/L) to generate VBNC cells. Absence of culturable cells was confirmed by plate count and VBNC cells by viability quantitative polymerase chain reaction (v-qPCR) complemented with two dyes, ethidium (EMA) and propidium (PMAxx) monoazide. Cross-contamination of shredded lettuce was demonstrated by monitoring the VBNC cells after washing the product for 1 min in the contaminated PW and during shelf life (15 days at 7 °C). In the case of L. monocytogenes, considering the total concentration of L. monocytogenes VBNC cells present in the PW, only a low proportion of cells were able to cross-contaminate the product during washing. VBNC L. monocytogenes cells were able to resuscitate on the product during shelf life, although levels of cultivable bacteria, close to the limit if detection (0.7 ± 0.0 log CFU/g), were only detected at the end of storage. On the other hand, VBNC cells of E. coli O157:H7 present in PW were not able to cross-contaminate shredded lettuce during washing. Moreover, when shredded lettuce was artificially inoculated with VBNC E. coli O157:H7, resuscitation of the VBNC cells during storage (15 days at 7 °C) was not observed. Based on the results obtained, injured L. monocytogenes cells present in the PW are able to be transferred to the product during washing. If VBNC L. monocytogenes cells present in leafy greens (shredded lettuce and baby spinach), they can resuscitate, although cultivable numbers remained very low. Taking all the results together, it could be concluded that under industrial conditions, VBNC cells can be transferred from water to product during washing, but their capacity to resuscitate in the leafy greens during storage is low.
一些水消毒处理方法，如氯和二氧化氯，用于鲜切行业以维持工艺水（PW）的微生物质量，它们可以杀死水中的细菌细胞，但也会导致细菌进入一种介于存活和非存活之间的中间状态，即存活但非可培养（VBNC）状态。存活细胞可能参与交叉污染事件，但 PW 中的 VBNC 细胞、转移到产品中以及在储存期间潜在复苏的能力尚不清楚。本研究旨在首先确定 PW 中存在的 VBNC 细胞是否会在清洗过程中污染叶类蔬菜，其次是在货架期内其潜在的复苏能力。PW 的特点是化学需氧量高，因为存在高水平的有机物，用单核细胞增生李斯特菌或大肠杆菌 O157:H7 进行接种。然后用二氧化氯（3mg/L）或氯（5mg/L）处理接种 PW 1 分钟，以产生 VBNC 细胞。通过平板计数确认无可培养细胞的存在，通过定量聚合酶链反应（v-qPCR）结合两种染料，即乙锭（EMA）和吖啶（PMAxx）单叠氮来确认 VBNC 细胞的存在。通过在污染 PW 中清洗产品 1 分钟以及在货架期（7°C 下 15 天）监测 VBNC 细胞，证明了切碎生菜的交叉污染。对于单核细胞增生李斯特菌，考虑到 PW 中存在的单核细胞增生李斯特菌 VBNC 细胞的总浓度，只有一小部分细胞能够在清洗过程中污染产品。VBNC 单核细胞增生李斯特菌细胞能够在货架期内复苏，但在储存结束时仅检测到接近检测极限（0.7±0.0 log CFU/g）的可培养细菌水平。另一方面，PW 中存在的大肠杆菌 O157:H7 的 VBNC 细胞在清洗过程中不能污染切碎的生菜。此外，当切碎的生菜人工接种 VBNC 大肠杆菌 O157:H7 时，在储存期间（7°C 下 15 天）未观察到 VBNC 细胞的复苏。根据获得的结果，PW 中存在的受伤单核细胞增生李斯特菌细胞能够在清洗过程中转移到产品中。如果叶类蔬菜（切碎的生菜和婴儿菠菜）中存在 VBNC 单核细胞增生李斯特菌细胞，它们可以复苏，尽管可培养数量仍然很低。综合所有结果，可以得出结论，在工业条件下，VBNC 细胞可以在清洗过程中从水转移到产品中，但它们在储存期间在叶类蔬菜中复苏的能力很低。
