New emerging markets such as Japan and the United States require cold sterilisation of
South African citrus fruit as a phytosanitary standard against fruit fly. However, citrus fruit
are chilling susceptible, with lemons being the second-most chilling susceptible after
grapefruit. Chilling injury is a physiological rind disorder; the occurrence of which is despite
its prevalence in horticultural commodities, not well understood. Therefore, the aim of this
study was to investigate physiological compounds regulating chilling susceptibility or
resistance in citrus fruit, with special emphasis on lemons. Furthermore, the potential of hot
water dips or “molybdenum soaks” to maintain a certain level of physiological compounds
which determine manifestation of chilling injury symptoms in citrus fruit was investigated.
Moreover, it was attempted to create an understanding of the order in which physiological
compounds mitigate chilling injury.
Lemon fruit from different farms known to be chilling susceptible or resistant were
obtained during the 2007 and 2008 harvest season. Thereafter, fruit were treated by soaking for 30 min in 1μM NaMo04.2H20 solution followed by a 2 min HWD 47 or 53°C. Treated fruit were waxed, weighed and stored at -0.5°C for up to 28 days and sampled for chilling injury
evaluation 7, 14, 21, or 28 days into cold storage. A second evaluation was carried out five
days after withdrawal from cold storage to allow development of chilling injury symptoms as
a shelf-life simulation. After the second evaluation fruit were peeled, peel freeze-dried, milled
using mortar and pestle and stored at -21°C for further physiological analysis. Freeze-dried
peel was analysed for soluble sugars (glucose, fructose, sucrose), vitamin C (ascorbic acid),
vitamin E (α-tocopherol), β-carotene, polyamines (putrescine, spermine, spermidine),
specific flavanones (naringin and hesperidin) using HPLC-UV-Vis detector and proline, total
antioxidant assays (FRAP, ABTS, DPPH), total phenolics, total flavonoids, lipid peroxidation
using spectrophotometry, as well as for the heat shock protein (HSP70) using electrophoresis
and silver-staining.
Chilling susceptibility of lemon fruit varied with fruit source; those sourced from Ukulinga
and Eston Estates were chilling resistant, while fruit from Sun Valley Estates showed chilling
injury symptoms after 28 days of cold storage plus five days shelf-life. Furthermore, hot
water dips (HW) 53°C, 1 μM Molybdenum (Mo) and 10 μM Mo plus HW 53°C significantly
reduced chilling injury symptoms compared with the control and HW 47°C. In addition, Sun
Valley Estates fruit also showed higher fruit weight loss compared with non-chilling resistant
lemons. The alignment of higher fruit weight loss during storage with chilling susceptibility
ascertains the use of weight loss as a non-destructive parameter for chilling susceptibility.
With respect to flavedo sugars, glucose was found to be the dominant soluble sugar with
multi-functional roles during cold storage. This plays a significant role in mitigating cellular
stress. Chilling susceptible lemons from Sun Valley Estates had low flavedo glucose
concentrations and, therefore, little conversion of glucose to ascorbic acid was possible
resulting in a low antioxidant capacity. However, treatments with HW 53°C and Mo soaks
seemed to enhance the enzymatic conversion of glucose to ascorbic acid leading to a higher
antioxidant capacity in the flavedo of such treated fruit. Furthermore, glucose also feeds into
the pentose phosphate pathway which is coupled with the shikimate pathway synthesizing
secondary metabolites, especially of the phenolics group. The decrease in glucose was
aligned to the levels of total phenolics, but not to that of β-carotene, naringin and hesperidin
through 28 days into cold storage period. Moreover, as glucose also feeds into shikimate
pathway, simultaneously an increase in proline flavedo concentration was observed. Proline
is an antioxidant synthesized from glutamate; as cellular glucose decreases so does the total antioxidant capacity during cold storage.
Ascorbic acid is a dominant and potent antioxidant in lemon flavedo as proven with the FRAP, ABTS and DPPH assays. Chilling resistant fruit have significantly higher ascorbic acid conversion. Furthermore, ascorbic acid also acts to generate the α-tocopheroxy radical to further important membrane-bound antioxidant, vitamin E (α-tocopherol equivalent).
Furthermore, the DPPH assay was found to be effective in quantifying total antioxidants in lemon flavedo since it detects both lipophilic and hydrophilic antioxidants compared with the ABTS and FRAP assays which are bias to the estimation of liphophilic or hydrophilic antioxidants, respectively. The hot water and molybdenum treatments increased total
antioxidants (DPPH assay) with reduced lipid peroxidation 7 days into cold storage and
therefore, reduced chilling symptoms in fruit from Sun Valley Estates.
The capacity of antioxidant to scavenge reactive oxygen species (ROS) was increased
during cold storage and membrane stability significantly improved. Furthermore, putrescine
as low valency polyamine was reduced as such compound acted as precursor to the synthesis
of the high valency polyamines, spermine and spermidine. Chilling susceptible lemons from Sun Valley Estates showed increased soluble-conjugated polyamines as a response to stress. Furthermore, HW 53°C, 1 μM Mo and 10 μM Mo plus HW 53°C significantly increased the
protein concentration and, therefore, likely also the occurrence of proteins with 70kDa (as estimator of HSP70). Additionally, the concentration of conjugated high valency polyamines
was also increased, resulting in reduced chilling injury symptoms.
The effect of ROS has only been viewed as damaging, while recently their role has also
been viewed as stress acclamatory signalling compounds when produced concentrations
below critical damaging threshold. Therefore, hot water dips seems to signals synthesis of
total protein which include HSPs which then act throughout cold stress to protect other protein and channel other damaged proteins towards proteolysis. While molybdenum increased ROS production below damaging critical threshold, with ROS signalling stress acclimation by further signalling production of bioactive compound with antioxidant properties. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/9859 |
| Date | 01 November 2013 |
| Creators | Mathaba, Nhlanhla. |
| Contributors | Bertling, Isa., Bower, John P. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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