The work in this chapter contributed to the following publication which is bound into the back of this thesis:
Luke Bell, Hanis Nadia Yahya, Omobolanle Oluwadamilola Oloyede, Lisa Methven and Carol Wagstaff. 2017. Changes in rocket salad phytochemicals within the commercial supply chain: Glucosinolates, isothiocyanates, amino acids and bacterial load increase significantly after processing. Food Chemistry 221: 521–534.
6.1 Introduction
Members of Brassicaceae including rocket are known to contain high concentrations of glucosinolates (GLSs). Apart from being responsible for the hot and peppery flavour of rocket, glucosinates were reported to possess biological activity against the growth of cancer cells (Ombra et al., 2017), bacteria and fungal pathogens (Blazevic et al., 2010; Khoobchandani et al., 2010; Ombra et al., 2017) and insects (Wittstock et al., 2004). The formation of GLS in plants is triggered by stress factors and influenced by cultural and environmental factors such as fertilizers and salts, temperature and radiation, as well as by postharvest handling and storage conditions (Jahangir et al., 2009).
To be converted into an active form, GLS must be hydrolysed in the presence of the enzyme myrosinase. Under intact cellular conditions, GLS and myrosinase are physically
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separated into different cellular compartments; both GLS and myrosinase are stored in vacuole but they are physical separated. Myrosinase is stored as myrosin grains while glucosinolates are stored in adjacent separate ‘cells’ (Koroleva et al., 2000).
The two substances are mixed when the cells are disrupted upon various physical and mechanical processes such as wounding, chewing and cutting, as well as after exposure to high temperature (Blazevic et al., 2010). Following hydrolysis of GLS products such as isothiocyanates, nitriles, oxazolidinethiones, thiocyanates and epithionitriles are formed. These newly formed products are actually responsible for the complex aroma of brassicas and various biological activities that are linked to GLS described earlier.
Within the same genus and species, the content of total GLS and its individual GLS or their derivatives varied among accessions, as reported by Bell et al. (2016) for rocket and Charron et al. (2004) for broccoli. In rocket, variation in individual and total GLS content of six varieties of rocket used in this study are given in Table 6.1. Therefore, the biological activity against bacterial growth amongst varieties of rocket containing different concentration and composition of GLS was expected to differ. Ludwig-Muller et al. (1997) for example noticed that cabbage with a high content of aliphatic glucosinolates were more sensitive to club root disease caused by Plasmodiophora brassicae, whereas those resistant to the disease contained high concentrations of aromatic glucosinolates. Charron et al. (2004) evaluated the impact of glucosinolate content in broccoli on the growth of Pseudomonas marginalis, a causal agent of bacterial soft rot. They reported that in an in vitro assay, there was a linear relationship between total GLS and the percentage of suppression of P. marginalis with a R2 of 0.48. Based on this finding, it is
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hypothesized that the extracts of rocket leaves containing high concentration of GLS would have lower bacteria loads.
The study reported in this chapter examined the changes in the naturally occurring bacterial population residing in leaves of different varieties of rockets with varying compositions of GLS and subjected to different handling and storage conditions. In addition, the antimicrobial activity of the leaf extracts obtained from different varieties of rocket was also examined.
6.2 Materials and Methods
6.2.1 Microbial loads in fresh RTE rocket of different varieties containing varying concentrations of glucosinolates
6.2.1.1 Plant materials and experimental treatments
Five gene bank accessions of Eruca sativa and one commercial variety (Diplotaxis tenuifolia var. Torino) with different glucosinolate contents (Bell et al., 2017a, Table 6.1) were used in this study. Total plate count (TPC) of the plant materials were determined at nine different processing points. The details of the processing points are given in Table 6.2. TPC analysis for commercial rocket (wild rocket) were only determined at the point of prewashed until two days after display until date. The wild rocket samples were commercially produced in Italy and supplied in the UK by Alresford Salads.
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Table 6.1. Rocket varieties with different concentrations of individual and total glucosinolates (mg.g-1 DW sinigrin hydrate equivalents).
Variety Gluco- erucin Gluco- raphanin Digluco- thiobeinin Gluco- sativin Gluco- iberverin DMB Total GLS ERU16 0.938 1.447 0.021 5.314 0 1.76 9.658 ERU18 0 1.424 0.011 3.113 0.011 0.397 4.956 ERU140 0 0.736 0 3.083 0 2.172 5.991 ERU154 1.665 2.045 0.011 2.898 0 0.698 7.315 CGN24247 0.357 1.26 0.012 6.09 0.011 0.021 7.751 Torino 0.48 1.275 0 7.568 0 2.201 11.524
174 Samples were taken in three biological replicates.
Table 6.2. Sampling points and descriptions of activity for determination of microbial loads of six varieties of rocket.
Date of activities Processing points Code Descriptions 16 July 2014 10-day-old plants
10 days Leaves of 10-day old, field grown rocket was sampled at The Watercress Company farm (TWC), UK. (During sampling, the temperature was 23 °C). About 30g for each variety were sampled at random and put in two different zip- lock plastic bags. Each bag is considered as one replicate.
25 July 2014 Harvest Day
Harvest day
The rocket was harvested as normal farm practices and the leaves were stored at TWC for three days at 4°C
28 July 2014 Alresford Salad Factory
Alresford After three days of storage at TWC, the rocket was transported to factory at Alresford in a refrigerated truck (5 °C). Journey took around two hours. The leaves were sampled immediately upon arrival at the factory.
29 July 2014 Prewash Prewash The rocket was kept at 4 °C overnight before processing began. The samples were taken just prior to washing.
29 July 2014 Post wash Post wash The rocket was washed using potable water and spun in a salad spinner to remove excess water. The working room temperature was between 10 -12 °C. The rocket was sampled prior to packaging. 30 July 2014 Samples were transported to University of Reading (Reading/D ay 0).
Reading Samples were transported from Alresford Salad Factory in a refrigerated truck (4 °C) to Reading University. The journey took around one hour. Samples from each variety were taken in three replicates for microbial analysis and kept at 4 °C.
1 Aug. 2014 Day 2 D2 TPC was performed on day two on samples stored at 4 °C
6 Aug. 2014 Display until date (DUD)
DUD TPC was performed on DUD date (on 6 days after Day 0)
8 Aug. 2014 Two days after DUD
DUD2 TPC was performed on two days after DUD date (eight days after day 0)
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6.2.1.2 Microbial count analysis
As described in section 3.2.1.2 but without the 13 and 25 oC incubator in apparatus
section.
6.2.1.3 Data analysis
As described in Section 3.2.1. 3
6.2.2 Antimicrobial properties of extracts of different of rocket variety
6.2.2.1 Plant Materials
Leaves of five rocket salads (Eruca sativa) (coded as CGN 24247, ERU 16, ERU 18, ERU 140, and ERU 154 and one variety of Diplotaxis tenuifolia var. Torino) were used in the study. The leaves were sampled on eight days of storage (DUD2) as the leaves were found to contain the concentration of glucosinolates at the stage.
6.2.2.2 Extraction of glucosinolates
As described in section 3.4. Rocket extracts were sterilised using 0.22 µm filter disc with a low protein binding Durapore polyvinylidene fluoride (PVDF) membrane (Millex, EMD Millipore, Billerica, MA, USA).