All chemicals were purchased from Sigma Aldrich®
5.3.1 Determination of ascorbic acid concentration in sweetcorn kernels
The concentration of ascorbic acid was determined in fresh sweetcorn kernels (as described by Boonkasem et al., 2015) with minor adjustments. The determination of ascorbic acid was analysed by sampling kernels from the top, middle and bottom section of the cob. Two rows from five cobs, were chopped into small pieces and weighed out to 2.0 g. The samples were homogenised using in mortar and pestle in 20 ml 3% metaphosphoric acid and transferred into centrifuge tubes, shaken for 30 min and centrifuged at 4000 rpm for 10 min. The supernatant of (1 mL) was transferred into glass tubes followed by addition of 3 mL 0.2 mM DCPIP (dichlorophenolindophenol). The supernatant was mixed for 15 s and immediately read spectrophotometrically (Shimadzu UV-1800 UV-Vis Spectrophotometer, Japan), at 515 nm.
Fig. 5.1 Ascorbic acid calibration curve used for spectrophotometrical determination of ascorbic acid concentration. Ascorbic concentrations (µg/ml sample solution) were calculated using the equation y = 0.005x + 0.5864 with an R2 value of 0.9865, where ‘y’
was absorbance and ‘’x’ the concentration of the sample.
y = -0,005x + 0,5864 R² = 0,9865
-0,10 0,00 0,10 0,20 0,30 0,40 0,50 0,60 0,70
0 20 40 60 80 100 120 140
Absorbance conc. (nm)
Concentration (µg/ml) Ascorbic Acid Calibration Curve
117 5.3.2 Determination of carotenoid and chlorophyll concentrations in sweetcorn
kernels
Chlorophylls and carotenoids were determined in fresh sweetcorn kernels. Fresh kernel material was extracted using acetone 80% solvent. The concentrations of kernel carotenoids and chlorophylls were read spectrophotometrically (Shimadzu UV-1800 UV-Vis Spectrophotometer, Japan), and calculated using the method described by Lichtenthaler and Buschmann, (2001), an improved version of Lichtenthaler, (1987). Two rows of fresh kernels situated in the middle were removed from three cobs and 2.0 g were weighed out. The fresh samples were placed into centrifuge tubes and mixed with 4 ml acetone (80%). Tubes were left to stand on ice covered with aluminium foil for 10 min. Thereafter, the samples were homogenized with mortar and pestle into a very fine paste and transferred to centrifuge tubes.
The paste remaining in the centrifuge tube was rinsed out with an additional 4 ml acetone and supernatant decanted into tubes, subsequently centrifuged for 5 min. Absorbance of supernatant samples were read at 663.2, 646.8, and 470 nm, using 80% acetone as a blank.
Fruit carotenoid and chlorophyll concentrations were calculated using the equations as follows:
Table 5.1 Equations used to determine the concentrations (µg/ml) in fresh sweetcorn kernel tissue where A = sample absorbance chlorophyll a (Ch-a), chlorophyll b (Ch-b), chlorophyll a+b (ch-a+b) and carotenoids (C-x+c) using acetone 80% (Porra et al., 1989;
Lichtenthaler, 1987 and Lichtenthaler and Wellburn, 1983).
Solvent Formulae (µg/mL)
80% acetone (v/v) Ca = 12.25 A663.2 - 2.79 A646.8
Cb = 21.50 A646.8 - 5.10 A663.2
Ca+b = 7.15 A663.2 + 18.71 A646.8
Cx+c = (1000 A470 - 1.82 Ca - 85.02 Cb) / 198
5.3.3 Determination of total soluble solids (TSS) in sweetcorn kernels
The determination of TSS was achieved by weighing approximately 2 g of fresh kernels from the top, middle and bottom parts of fresh cob samples of the treatment and the control. The weighed fresh material was homogenised using mortar and pestle. Each sample was scooped with a spatula onto sterile gauze and juice squeezed on a refractometer (Bellingham +Stanley Digital Refractometer No. BU14006, Kent TN2 3EY, UK) to read TSS (in oBrix).
118 5.3.4 Determination of protein concentration in sweetcorn kernels
Protein was determined on five fresh sweetcorn cobs of the treatment and the control weighing between 99.3 to 158.7 g. Parts analysed were top, middle and bottom parts of the cobs. Five grams of fresh kernel were weighed out and cut into very small pieces, homogenised using mortar and pestle and transferred into the centrifuge tubes. Exactly 30 mL TRIS buffer (100 mM, pH of 7.5), was added to the samples which was centrifuged at 10000 rpm for 15 min at 2oC. The supernatant was decanted and 100 µl subsamples were added to 5 ml Bradford reagent (Bradford, 1976). Samples were measured spectrophotometrically (Shimadzu UV-1800 UV- Vis Spectrophotometer, Japan) at absorbance of 590 nm. Protein concentrations were determined by comparison with a standard curve prepared with bovine serum albumin (BSA) (0.065, 2.125, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 20, 40, 60, 80, 100, 150 and 200 mg) BSA per 100 ml deionised water.
A protein standard curve was plotted using bovine serum albumin (mg/ mL) against absorbance (Fig. 5.2). Protein concentrations (mg/ mL) were determined through Microsoft’s Excel®
software to give an equation of y = 0.2495x - 0.0122 with an R2 of 0.9921. Protein concentration of 0.5 mg/mL was selected as standard for the determination of unknown sample concentration for protein in sweetcorn kernels.
Fig. 5.2 Protein calibration curve using bovine serum albumin.
y = 0,2495x - 0,0122 R² = 0,9921
0,00 0,10 0,20 0,30 0,40 0,50 0,60
0 0,5 1 1,5 2 2,5
Absorbance con. (nm)
Conc. in mg/ml Protein Calibration Curve
119 5.4 Results
Data were statistically analyzed using GenStat®18th edition (VSN International, Hemel Hempstead, UK) and analysis of variance (ANOVA) was performed. Fruit parameters analyzed were: ascorbic acid, total carotenoids, total protein and total soluble solids (TSS).
The differences in the means for these phytochemicals were determined using Fisher’s protected least significant difference test revealed significant difference at (p<0.05 and p<0.01).
Elevated CO2 significantly affected fruit ascorbic acid with CO2 concentration as well as TSS.
Even though protein and carotenoids showed no significant differences, between control and treatment, both parameters in maize kernels of all three cob locations were enhanced under eCO2. The P-values for ascorbic acid, carotenoids, protein and TSS indicate that ascorbic acid was highly significantly affected by locations (P<0.001; figure 4.3). Even though there was a tendency to an increase in carotenoids concentration given with the increase in CO2 (figure 4.4), there were no significant differences (P = 0.101); additionally, protein concentration also tended to increase by the treatment (figure 4.5); however, differences were not significant (P
= 0.544). A positive effect from eCO2 on TSS (figure 4.6) yielded significant differences at (P<0.01).
919,0
731,0
967,0
731,0
976,0
761,0
0,0 200,0 400,0 600,0 800,0 1000,0 1200,0
e C O 2 t o p ke r n e l p a r t
a C O 2 t o p ke r n e l p a r t
e C O 2 mi d d l e ke r n e l p a r t
a C O 2 mi d d l e ke r n e l p a r t
e C O 2 b o t t o m ke r n e l p a r t
a C O 2 b o t t o m ke r n e l p a r t
Conc. (µg/g FM)
400 ppm (aCO2) ; 500 ppm (eCO2) s e d ( C O2 x C o b p a r t ) ( p = 0 .6 23)= 3 4 . 7
120 Fig. 5.3 Ascorbic acid concentration (µg/g FM) in maize kernels from three cob sections that were grown under ~400 ppm (aCO2) and ~500 ppm (eCO2).
Fig. 5.4 Fruit pigments carotenoids and chlorophyll concentrations (mg/g FM) in three sections of fresh sweetcorn kernels grown under ~400 ppm (aCO2) and ~500 ppm (eCO2).
2,03
1,74 1,65
1,91
2,60
2,01
0,00 0,50 1,00 1,50 2,00 2,50 3,00
eCO2 top part aCO2 top part
eCO2 middle part
aCO2 middle part
eCO2 bottom part
aCO2 bottom part
conc. in (µg/g FM)
400 ppm (aCO2); 500 ppm (eCO2) sed(CO2x Cob part) (p=0.166) = 0.3080
560,0
531,0
595,0
454,0 469,0 492,0
0,0 100,0 200,0 300,0 400,0 500,0 600,0 700,0
e C O 2 t o p p a r t
a C O 2 t o p p a r t
e C O 2 mi d d l e
p a r t
a C O 2 mi d d l e
p a r t
e C O 2 b o t t o m
p a r t
a C O 2 b o t t o m
p a r t
Conc. in (µg/g FM)
400 ppm (aCO2) ; 500 ppm (eCO2) s e d ( C O2 x C o b p a r t )( p = 0 . 2 0 3)= 6 3 . 7
121 Fig. 5.5 Protein concentration (µg/g FM) in all of sweetcorn kernels in three sections grown under ~400 ppm (aCO2) and ~500 ppm (eCO2).
Fig. 5.6 TSS concentrations (oBrix) in three sections of fresh sweetcorn kernels grown under ~400 ppm (aCO2) and ~500 ppm (eCO2).
Table 5.2 Ascorbic acid, carotenoids, protein and TSS determined postharvest in sweetcorn kernels of aCO2 and eCO2.
STAR7719
Parameters aCO2 eCO2 Cob part CO2 Cob part x CO2 Mean LSD Ascorbic acid 741 954 0.234ns 0.001*** 0.623ns 847 72.5 Carotenoids 1.884 2.091 0.058ns 0.259ns 0.166ns 1.987 0.643 Proteins 492 541 0.358ns 0.196ns 0.203ns 517 132.8 TSS 13.87 16.11 0.244ns 0.001*** 0.497ns 14.99 1.376
*, **, *** indicate that the parameter is significant at P < 0.05, P < 0.01 or P < 0.001, respectively, where ns= non-significant. Cob part refers to the location of kernels on the maize cob. LSD values indicate significant differences between treatment and control under concentrations ~430 ppm (aCO2) and ~500 ppm (eCO2).
16,8
14,0
15,4
13,7
16,1
13,9
0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 18,0 20,0
eCO2 Top part
aCO2 Top part
eCO2 Middle part
aCO2 Middle part
eCO2 Bottom part
aCO2 Bottom part
TSS conc. (oBrix)
400 ppm (aCO2 ; 500 ppm (eCO2) sed(CO2x Cob part) (p=0.497)= 0.659
122