Chapter 2 The relationships between sugars, protein and oil of ‘Hass’
2. Materials and Methods
2.1. Fruit Material
Export grade 'Hass' fruit (236-265 g) were harvested from a commercial orchard near Tzaneen, South Africa (23°43'S, warm subtropical climate) on 18 May, 1 June and 15 June (27.0, 28.6 and 30.3% dry matter, respectively) and from a commercial orchard near Howick (29°27'S, cool subtropical climate) on 12 July, 7 August and 31 August 2007 (28.5, 29.9 and 36.7% DM). Fruit were harvested from the same orchard on each farm to reduce variability between harvest dates and seasons. Twenty fruit were harvested from the same orchard on each sampling date. Fruit from Tzaneen reached the laboratory within two days of harvest, using refrigerated transport (~15°C). Fruit from Howick were delivered to the laboratory on the day of harvest. Fruit were ripened at 21 ± 2°C. Dry matter was determined on a subset of 100 fruit.
2.2. Sampling
Avocado fruit are highly heterogeneous, so to reduce sampling variation, two cores 15 mm in diameter were taken from each fruit on each sampling day, according to Kanellis et al.
(1989). The sampled area of the fruit was immediately sealed with warm petroleum jelly to prevent oxidation. Fruit from Tzaneen were sampled two, five, eight, 11, 13 and 15 days after harvest. Fruit from Howick were sampled on the day of harvest and three, seven, 10 and 13 after harvest. The sampled mesocarp (~6.5 g fresh mass) was flash frozen in liquid nitrogen, lyophilised, hand-milled and stored at -20°C until further use.
2.3. Firmness
Firmness was measured according to Köhne et al (1998) using a hand-held densimeter (5 mm tip) (Bareiss, Oberdischingen, Germany). For comparison, a firmometer (300 g weight) has a scale of 0 (hard) to 120 (soft), a 5 mm densimeter has equivalent readings of 85-90 (hard) to 55-60 (soft). Readings were taken on the area to be sampled immediately before sampling. Fruit were deemed ripe when the reading on the densimeter was less than 60 and removed from the experiment if the reading was below 50 or if there were symptoms of fungal infection.
Fast and slow ripening fruit were defined as ripening in 6-7 days and 12-13 days
postharvest, respectively.
2.4. Respiration rate
The concentration of CO2 was measured using an infrared gas analyser (EGM-1, PP Systems, Hitchin, UK). Individual fruit were incubated in a 1 L container for 15 min. The headspace CO2 concentration was converted to respiration rate taking into account fruit mass, fruit volume, free space in the jar and the ambient CO2 concentration.
2.5. Ethylene production rate
Individual fruit were incubated in a 1 L container for 30 min, with a 20 mL autosampler vial enclosed. Ethylene was measured using GC-FID (DANI 1000, DANI Instruments, Monzese, Italy). The GC was fitted with a stainless steel column packed with alumina-F1 stationary phase. The injector, column and detector were set at 160°C, 100°C and 180°C, respectively. The mobile phase was instrument grade nitrogen gas at 35 kPa. The autosampler injected 1 mL from the 20 mL vial. The ethylene production rate (EPR) was calculated taking into account fruit mass, fruit volume and free space in the jar.
2.6. Sugar Analysis
Sugars were analysed according to Liu et al. (1999a), with slight modifications. A 10 mL aliquot of 80% (v.v-1) ethanol was added to 100 mg of mesocarp, heated in an 80°C water bath for 60 min and then incubated at 4°C for 24 h. Samples were then filtered through glass wool and dried in a vacuum concentrator (Savant, Farmingdale, NY). Dried samples were re- suspended in 2.0 mL ultrapure water, placed in a sonic bath for 10 min, and filtered through a 0.45 μm nylon syringe filter. The analysis was performed using an HPLC (Shimadzu, Kyoto, Japan) equipped with a differential refractometric detector (RID–10A) and a Phenomenex column (Rezex RCM-Monosaccharide, 200 mm × 780 mm × 8 μm). The elution was isocratic, using ultrapure water as the mobile phase. Individual sugars were identified by co-elution with standards of glucose, fructose, sucrose (Sigma-Aldrich, St Louis, MO), mannoheptulose and perseitol (Glycoteam, Hamburg, Germany). Sugars were quantified by applying a standard curve for each sugar. Samples were analysed twice and the mean taken.
2.7. Protein Analysis
Protein extraction was according to Kanellis and Kalaitzis (1992), with slight modifications. Milled tissue (100 mg) was thawed in 5 mL extraction buffer containing 50 mM Tris-HCl (pH 7.4), 0.2 M NaCl, 20 mM MgSO4,, 1 mM EDTA, 5 mM β-mercaptoethanol, 0.5 mM PMSF, 10 μM leupeptin and 10% (v.v-1) glycerol. The mixture stood on ice for 15 min with gentle shaking, was filtered through Miracloth and stored at -75°C. Protein quantification was done using the micro-assay of Bradford (1976), in duplicate, and a calibration curve using bovine serum albumin applied.
2.8. Oil analysis
Oil was quantified according to Meyer and Terry (2008), with slight modifications.
Hexane (9.0 mL) was added to 300 mg mesocarp and the test tubes placed into a sonic bath for 10 min. The supernatant was filtered under vacuum and another 6 mL hexane added to the sample test tube. This was left for 5 min and the tube emptied into the Büchner funnel. The test tube was then rinsed with 3 mL hexane. The 18 mL of hexane was combined and dried using a vacuum concentrator (Savant, Farmingdale, NY). The oil was weighed and converted to percent dry mass (% DM).
2.9. Statistical analysis
Single fruit replications were used, with 20 fruit per treatment. The eight fruit closest to the mean respiration, ethylene and firmness values were used for further analysis. Statistical analysis was conducted using Genstat 12.1 (VSN International, Hemel Hempsted, UK) using a repeated measures REML analysis, using harvest and time after harvest as factors, at a 95%
confidence level. Fruit from the two production areas were analysed separately because of the additional effect of time between harvest and sampling. Means were separated using individual LSDs. Data are presented as the mean ± standard error of the mean (SEM). Tests for correlation were done using Spearman’s rank correlation (rs).