Abstract/Summary

Bagged fresh ready-to-eat (RTE) vegetables are rich in nutrients, contain high moisture content, have nearly neutral pH and have exposed wounded tissues, making the produce a perfect media for microbes to proliferate. Breaches of cold temperature along the supply chain, could aggravate the situation, thus increasing the risk of foodborne illness and accelerate the rate of food spoilage. A study to evaluate the effects of handling and storage conditions on microbial loads, volatile organic compounds, distribution of naturally occurring bacteria and formation of biofilms, and types of bacteria and moulds present in fresh RTE rocket was conducted. Antimicrobial capability of rocket leaves obtained from different varieties with different glucosinolate (GLS) profiles in rocket leaves (Eruca sativa and Diplotaxis tenuifolia) was additionally evaluated. The abundance of bacteria was also highly dependent on storage temperature and whether the bag was kept sealed (closed) or if it was opened. The produce kept continuously at 4 oC contained low bacterial loads until the end of the study period (day 6 of storage). As the storage temperature was increased from 4 to 13 oC, the bacterial loads remained low up to 4 days of storage when the bags were kept closed. When the produce are kept at an ambient temperature (20 oC), regardless of bag condition (opened or closed), the produce contained low bacterial loads only up to two days of storage. Fresh RTE rocket leaves exposed to 25 oC for 4 h and ‘consumed’ at 24 h after ‘purchase’ did not cause any significant increase in bacterial loads. In contrast, the bacterial loads in leaves that have never been exposed to high temperature but instead kept at ‘display’ temperature (13 oC) for relatively long duration until the date until display and beyond contained high loads of microbes. Heat shock treatment on winter grown rocket increased respiration rate that occurred concurrently with reduced O2 level in the bagged fresh RTE rocket leaves. Organic volatile compounds (VOCs) associated with off-odour of rocket, such as sulphur containing compounds (dimethyl sulfide, dimethyl trisulfide and dimethyl sulfoxide), alcohols (1-butanol, 1-penten-3-ol, (z)-2-penten-1-ol, 1-hexenol and phenethyl alcohol), acetic acid and furan were detected in bagged fresh RTE rocket leaves held at 13 oC. The abundance of the VOCs increased with time duration. Rocket leaves that were purchased early and kept under 4 oC, regardless of whether the bag is opened or closed, and ‘consumed’ on date until display contained the highest concentration of total GLS. Scanning electron microscopy (SEM) images revealed the abundance of bacteria on the rocket leaves was higher on dented areas and stomata, especially along the leaf vein. Long term storage leads to the formation of bacterial biofilms. Whole genome and 16S metagenomic DNA sequencing of the bacteria revealed that the types of bacteria that live on bagged fresh RTE rocket were very diverse. DNA sequencing analysis detected 267 types of bacteria present in RTE rocket leaves. Salmonella enterica was detected in leaves that were heat shocked (4h at 25 oC) followed by keeping at moderate temperature (13 oC) up date until display and in leaves that were continuously kept at 13 oC till date until display. Pseudomonas viriflava and Erwnia sp., and lactic acid bacteria (Lactococcus lactis) were detected in fresh RTE rocket held at 13 oC until date until display. Six strains of toxigenic moulds, Fusarium equiseti, F. chlamydosporum, Alternaria alternata, A. tenuissima, and other two species of Alternaria were present in the RTE rocket leaves exposed to moderate (13 oC) and high (25 oC) temperature. There was no clear relationships exist between GLS content and abundance of naturally occurring bacteria present on leaves of rocket varieties with different GLS profiles. The abundance of E.coli K12 was reduced when the bacteria were cultured in extract of leaves obtained from rocket varieties containing high concentration of GLS. The reduction of bacterial loads was correlated negatively with the concentration of dimeric-4-mercaptobutyl (DMB) in the leaf extracts. The results of the study clearly show that bacterial and mould counts, VOC and GLS abundance, distribution of naturally occurrence bacteria and formation of biofilms in bagged fresh RTE rocket leaves were significantly affected by handling and storage/display conditions of the produce. Analysis of DNA sequencing of the microbes revealed that the species and strains of bacteria and moulds detected residing in the RTE rocket leaves were very diverse, ranging from epiphytes, food spoilage, plant pathogens, as well as human pathogen microbes.