A Fast, Simple, and Reliable Method to Predict Pasta Yellowness B. X. Fu,1,2 L. Schlichting,1 C. J. Pozniak,3 and A. K. Singh4 ABSTRACT
Cereal Chem. 88(3):264–270
Pasta yellowness depends on the semolina carotenoid content, carotenoid degradation by lipoxygenase (LOX), and pasta processing conditions. In breeding programs, early generation lines are selected for high grain yellow pigment content with the intent to improve pasta color. This approach has been successful in increasing the grain yellow pigment of Canadian durum wheat in the last few decades. In recent years, however, a weak relationship between pasta yellowness (b*) as measured by a Minolta spectrophotometer and semolina yellow pigment content (r = 0.19–0.52) was noted in the Canadian durum wheat lines. Thus, total semolina yellow pigment content cannot effectively predict the yellowness of its pasta product. Therefore, a fast and simple method was developed to predict pasta yellowness by measuring semolina dough sheet
color at different time intervals after sheeting (0.5, 2.0, and 24 hr). Spaghettis were processed from the semolina samples at two drying temperature cycles (70 and 90°C). There were significant correlations between dough sheet b* values at all three times and spaghetti b* values at both drying temperatures (r = 0.87–0.94). Semolina dough sheet can be easily prepared in 15 min and requires only 30 g of material. Shortly after sheeting (30 min), dough sheet b* values can be used to predict pasta yellowness without producing the end product (involving mixing, extrusion, and drying). In this study, we also found that dough sheet b* values increased significantly with time over the sampling intervals after sheeting for those breeding lines with superior pasta color. DNA analysis revealed that all those lines lacked the Lpx-B1.1 duplication.
One of the most important quality factors in durum wheat is the potential of producing pasta products with bright yellow color (Troccoli et al 2000). Pasta yellowness is affected by various factors: the inherent carotenoid pigment content of seeds, which is largely a varietal characteristic (Clarke et al 2006); the residual pigment content after the storage of grain and milling (Borrelli et al 2008); the oxidative degradation of pigments by lipoxygenase (LOX) during pasta processing (Irvine and Winkler 1950; Irvine and Anderson 1953; McDonald 1979; Borrelli et al 1999; Carrera et al 2007); and the processing conditions such as drying temperature, extrusion die design and type (Baroni 1988; De Stefanis and Sgrulletta 1990; Dexter and Marchylo 2001; Borrelli et al 2003). Carotenoids consist of two classes of molecules: the carotenes, which are strictly hydrocarbons, and xanthophylls, which contain oxygen and are more polar. The pigments in durum endosperm consist of primarily xanthophylls and a small amount of carotenes. Lutein represents >80% of total carotenoids in durum semolina, with the remaining being zeaxanthin, β-carotene, and other unidentified compounds (Ramachandran et al 2010). In durum wheat breeding programs, selection for elevated grain yellow pigment concentration is practiced in early generations (Clarke 2001). Durum breeders typically screen for color by measuring total grain yellow pigment content or semolina color. Direct measurement of pasta color is usually not performed until later generations, where breeding lines are grown in multiple environments over years. Several analytical procedures have been developed to measure pigment content or evaluate yellowness in durum whole meal or semolina (Fratianni et al 2005). The methods most commonly used are based on light reflectance measurement (colorimetric method), spectrophotometric determination after pigment extraction with organic solvents, and near-infrared reflectance spectroscopy (NIR) estimation (Pozniak et al 2007). Methods based on HPLC have been developed to allow the separation and identification of individual carotenoid components (Hentschel et al 2002; Panfili et al 2004; Abdel-Aal et al 2007; Ramachandran et
al 2010). In the durum and pasta industry, color of semolina and pasta is commonly measured based on the CIE 1976 L*a*b* color space system. In durum wheat, positive correlations were found between CIE b* (yellowness) measured using a Minolta chroma meter (Humphries et al 2004), total yellow pigment content (Ramachandran et al 2010), and lutein concentration quantified by HPLC in durum wheat. The cost and time to produce semolina for early-generation screening has prompted investigations of flour or whole meal for prediction of semolina color. Konzak et al (1975) utilized a micromill capable of milling ≈1,000 samples (2–5 g) per day. Color of the crude semolina was then scored visually or with a color meter to distinguish between samples that were visually similar. Johnston et al (1981) found that color of whole meal correlated well (r = 0.85–0.90) with semolina color in a set of 10 durum genotypes. They suggested that color measurement of 1 or 2 g of whole meal sample offered speed and efficiency for prediction of semolina color. McCaig et al (1992) found that NIR can easily and accurately measure pigment content in a diverse group of durum genotypes. Application of NIR instruments for prediction of semolina color would be desirable and widely usable in breeding programs due to simplicity and low cost. Sgrulletta and DeStefanis (1997) found that b* of whole meal could be predicted with a scanning NIR instrument. Standard methods (ICC 152, AACC 14-50.01) for yellow pigment determination are still based on the extraction of total pigments with water-saturated 1-butanol and subsequent spectrophotometric measurement. Similar in principle to the official methods, Beleggia et al (2010) developed a micro-method for the determination of yellow pigment content in durum wheat. A significant proportion of the carotenoid pigments are lost during pasta processing (Irvine and Winkler 1950; Matsuo et al 1970). This reduction in yellow pigments during pasta processing is mainly due to LOX activity (McDonald 1979; Borrelli et al 1999; Yemenicioglu and Ercan 1999). LOX are non-haem, ironcontaining dioxygenases that catalyze oxidation of polyunsaturated fatty acids (Siedow 1991). Radical forms produced during the intermediate states of substrate peroxidation are responsible for the degradation of carotenoid pigments. Relatively few breeding programs routinely attempt to measure LOX to eliminate lines that have potential for high color loss during processing. This is principally due to the difficulty in measurement of LOX activity. Borrelli et al (1999) demonstrated that lipoxygenase activity of some cultivars was more important in determination of pasta color than was pigment content of the whole grain. They reported that two isozymatic forms of LOX (LOX-2 and LOX-3) were responsible for loss of color during pasta production. Enzyme activity
1 Grain
Research Laboratory, Canadian Grain Commission, 1404-303 Main Street, Winnipeg, MB, Canada. Publication number 1037, Grain Research Laboratory, Canadian Grain Commission, 1404-303 Main St. Winnipeg, MB, Canada R3C 3G7. 2 Corresponding author. E-mail address:
[email protected] 3 Crop Development Centre, University of Saskachewan, Saskatoon, SK, Canada. 4 Semiarid Prairie Agricultural Research Centre, Agriculture and Agri-Food Canada, Swift Current, SK, Canada. doi:10.1094 / CCHEM-12-10-0173 © 2011 AACC International, Inc.
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was highly correlated to loss of β-carotene. Manna et al (1998) compared mRNA levels for LOX activity in nine durum cultivars with LOX activity and β-carotene levels. LOX mRNA levels showed a strong negative correlation with semolina β-carotene content and yellow index (r = –0.98 and –0.96). Carrera et al (2007) reported the existence of duplication at the Lpx-B1 locus on chromosome 4B of durum wheat and deletion of an Lpx-B1.1 copy was associated with a 4.5-fold reduction in LOX activity and improved pasta color (P < 0.0001) but not semolina color, suggesting reduced pigment degradation during pasta processing. Trono et al (1999) showed that semolina LOX reaction may be inhibited by the carotenoids, and consistently lower semolina bleaching was observed in the samples with a higher carotenoid content. Semolina LOX may be inhibited by endogenous carotenoids, making a high carotenoid content of semolina desirable because it gives a good yellow-amber color and also, perhaps, prevents carotenoid degradation during pasta processing. It would be useful to develop early-generation screening procedures to select against factors that adversely affect pasta color. This is especially true in the case of LOX activity, given the difficulty of measurement by conventional biochemical techniques. Over the past few decades, efforts have extensively focused on the genetics of carotenoid accumulation and on devising strategies to improve the yellow color of durum semolina in durum breeding programs (Pozniak et al 2007; Singh et al 2009; Ramachandran et al 2010). This approach has resulted in the release of cultivars with high pigment levels (Clarke 2001). Although breeders have been successful in improving semolina color, weak relationships between pasta yellowness (b*) as measured by Minolta spectrophotometer and semolina yellow pigment content among the durum wheat lines were noted in advanced durum wheat lines evaluated in Canadian Durum Wheat Variety Registration Trials in the last few years. For example, pasta yellowness was less than predicted based on semolina pigment levels in some breeding lines, or vice versa. Thus, selection for improved pasta color cannot rely solely on yellow pigment content. It is not practical, however, to produce pasta for routine color evaluation due to the limitation of sample size, processing facilities, and time required. Prediction of pasta color during early-generation evaluation is challenging because of the large number of breeding lines and the limited amount of seed available for evaluation. As such, a new method must be developed to predict pasta color effectively. The method should take into consideration both the content of pigment and the degradation of pigment by LOX, and be simple and fast. Dough sheet color measurement is widely used to assess the degree of darkening induced by polyphenol oxidase in Asian noodles, including alkaline noodles made from durum wheat fine flour (Fu et al 2006). The main objective of this study was to examine whether the dough sheet method can be used to predict pasta yellowness from semolina dough sheet color. To that end, we made a semolina dough sheet in which the oxidative enzymes were activated, and then we measured dough sheet yellowness at different time intervals after sheeting. In addition, semolina will be used to produce spaghetti. Correlation between dough sheet b* values at different time intervals and spaghetti b* values will be evaluated to determine whether semolina dough sheet color is a good predictor of spaghetti color. The total yellow pigment contents of each semolina sample are measured to establish and confirm the relationships among pigment content, semolina color, dough sheet color, and spaghetti color. MATERIALS AND METHODS Durum Wheat Samples, Milling, and DNA Analysis Advanced durum wheat lines evaluated in the 2007, 2008, and 2009 Canadian Durum Wheat Variety Registration Trials were used in this study. Each trial included ≈25 samples with five check cultivars and experimental durum lines in the first, second, and
third year of testing. Five check cultivars (Avonlea, AC Morse, AC Navigator, Strongfield, and Commander) were included in all three trial years. Check cultivars and test lines were grown in various stations in Saskatchewan, Alberta, and Manitoba. Grain harvested at stations with high levels of disease damage (e.g., fusarium, midge) were excluded from composites. The amount of wheat from each remaining station was chosen based on protein content and crop grade to prepare 6-kg composites for quality testing of each check cultivar or test line. Most samples were graded as No. 1 or No. 2 Canada Western Amber Durum (CWAD), with a few graded as No. 3 CWAD. Durum wheat was milled on a four-stand Allis-Chalmers laboratory mill in conjunction with a laboratory purifier according to the mill flow described by Dexter et al (1990). All lines were milled in duplicate 2-kg batches. The mill room was controlled for temperature (21°C) and humidity (60% rh). Samples were conditioned before milling to 16.5% moisture overnight. Semolina of 65% extraction was prepared by combining selected streams derived from the break, sizing, and purifier systems. Each of the lines evaluated in the 2007-2009 trials were evaluated for the duplication of LOX genes at the Lpx-B1 locus. The DNA from each line was extracted from three-day-old leaves as described previously (Pozniak et al 2007). Polymerase chain reaction (PCR) analysis was performed on 100 ng of DNA using the primers LOXB-L/R and PCR conditions described previously (Carrera et al 2007). PCR amplicons were digested with HaeII before gel electrophoresis on a 1.5% (w/v) agarose gel. Spaghetti Processing and Color Measurement Spaghetti was prepared using the microprocessing technique of Matsuo et al (1972) and dried using 70 and 90°C drying cycles in a computer-controlled Afrem laboratory-scale dryer (Clextral, Lyon, France). Two temperatures are necessary to simulate the high temperature (HT) and ultra high temperature (UHT) drying cycles commonly used in today’s pasta industry; temperature influences the color of finished products. Extruded samples were immediately placed into the drying cabinet at 40°C and 92% rh and held at these conditions for 10 min after the last sample was placed in the cabinet before applying one of two drying cycles. In the HT cycle, cabinet temperature was rapidly increased to 70°C and maintained at this temperature for most of the cycle. Relative humidity was rapidly decreased at the onset of the cycle and continued to decrease at a more gradual rate when the maximum temperature (70°C) was reached. One hour before the end of the cycle, cabinet temperature and relative humidity were linearly decreased to ambient conditions. Total drying cycle time was 11 hr. In the UHT drying cycle, cabinet temperature was briefly raised to 90°C, and decreased to 85°C for the remainder of the cycle. Relative humidity was decreased rapidly as cabinet temperature reached the maximum temperature; however humidity was maintained at a high level for most of the cycle. One hour before the end of the cycle, temperature was dropped to room conditions together with a more gradual decrease in humidity. Total drying time was 5 hr. Tristimulus color measurements were performed with a spectrophotometer (CM-525i, Minolta Canada, Mississauga, ON) on spaghetti strands mounted on white cardboard. Color readings were expressed on the CIE 1976 color space system for L* (lightness), a* (red-green), and b* (yellow-blue). Semolina Dough Sheet Preparation and Color Measurement Semolina samples of 30 g each were mixed in a 50-g Farinograph bowl (C.W. Brabender, Hackensack, NJ) maintained at 45°C by circulating water. The bowl recovered from an old farinograph was powered by a stronger motor (¼ horsepower) to accommodate the stiff low moisture dough. Water absorption, mixing speed, and mixing time were 35%, 90 rpm, and 3.5 min, respectively. After mixing, the dough was sheeted in a pair of Vol. 88, No. 3, 2011
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sheeting rolls with a gap setting of 3.0 mm. The resulting dough sheet was folded twice and sheeted, followed by three sheetings without folding. After the sheeting process, the smooth dough sheet was transferred to a resealable plastic bag and stored in a closed drawer at room temperature (23 ± 1°C). The color of the semolina dough sheet surface was measured at 0.5, 2, and 24 hr after sheeting using the spectrophotometer.
procedure) were used to examine the general relationships between total yellow pigment, semolina b*, dough sheet color, and dried spaghetti yellowness (b*). A general linear model (PROC GLM procedure) was fitted to the data and Fisher’s least significant difference (LSD) test at α = 0.05 was used to determine significant differences between genotype means. RESULTS AND DISCUSSION
Semolina Color Measurement and Pigment Analysis Dry semolina CIE b* value was quantified using the spectrophotometer. Semolina was spooned into a small cup (5 cm diameter, 2 cm deep), leveled, and covered with nonreflective glass before measurement. For each sample, b* value was an average of two separate measurements. The effect of semolina particle size on CIE b* values was minimized because all durum samples were milled under the same conditions by the same miller. Total yellow pigment in semolina was assessed using Approved Method 14-50.01 (AACC International 2010). Water-saturated nbutanol (40 mL) was added to 8.0 g of semolina (14% mb), shaken, and extracted overnight. Extracts were filtered through Whatman No. 1 filter paper, and absorbance measured at 436 nm using a UV/Visible Ultrospec 3000 (Pharmacia Biotech, Cambridge, England). Two individual absorbance measurements from extracts per sample were recorded and values were averaged and converted to yellow pigment using the coefficient for β-carotene. Statistical Analysis Durum wheat milling, semolina TYP content, and semolina, spaghetti, and dough sheet b* measurement were done randomly in duplicate. Statistical analyses were performed (v.9.1, SAS Institute, Cary, NC). Data from each year were analyzed separately because test lines, growing locations, and composite makeup varied between years. Pearson correlation coefficients (PROC CORR
Semolina Pigment Content and Spaghetti Color Bright yellow color is valued in the semolina and pasta industry for its consumer appeal and for the potential health benefits associated with antioxidant activity of carotenoids. Therefore, elevated yellow pigment concentration is the target of durum breeding programs worldwide. Total grain yellow pigment (TYP) content and yellowness (b*) of semolina and spaghetti yellowness (b*) have been important parameters in evaluation of advanced durum wheat breeding lines for registration in Canada. TYP content is a highly heritable trait controlled by additive gene effects, which makes it appropriate to select for in early-generation breeding programs (Clarke et al 2006). Tables I, II, and III show the correlations between TYP content and b* values of semolina and spaghetti dried using 70 and 90°C cycles produced from the check cultivars and advanced breeding lines in 2007-2009 registration trials in Canada. The TYP content has been improved significantly in the last few decades as most of the CWAD cultivars released before 1990 had TYP contents 8.0 mg/kg, with some lines well over 10.0 mg/kg. The strong correlation between TYP and semolina yellowness were clearly demonstrated in all three trial years. These results are in agreement with those of Humphries et al (2004), who identified b* value as a
TABLE I Semolina Pigment Content and Color in Relation to Spaghetti Color (2009 Trial)
Checks and Test Lines
DNAa Lpx-B1.1
DT661-Avonlea 0 DT484-ACMorse 0 DT673-ACNavigator 1 DT712-Strongfield 0 DT722-Commander 1 DT801 0 DT809 0 DT557 0 DT561 0 DT562 0 DT813 0 DT815 1 DT818 0 DT820 1 DT563 0 DT565 0 DT828 1 DT829 0 DT830 0 DT832 0 DT833 1 DT834 0 DT835 0 – LSDc Correlation with TYP – Correlation with spaghetti b* 70°C (90°C) a
Spaghetti
Semolina TYPb (mg/kg)
b*
8.3 8.0 9.3 8.7 9.8 9.2 11.6 9.3 10.9 11.1 8.5 8.6 8.5 8.5 11.0 12.4 10.6 8.3 9.2 9.9 10.1 10.5 8.6 0.07 –
33.5 32.6 34.3 33.7 35.3 34.0 37.1 34.2 37.0 37.1 33.2 33.3 33.1 33.7 36.4 39.2 35.7 32.9 34.6 35.4 35.4 35.8 33.5 0.43 0.98d –
b* (70°C) 61.4 57.0 66.8 59.9 69.3 64.4 61.3 63.7 63.8 60.8 61.0 65.5 61.1 64.5 60.0 64.9 68.6 60.6 61.8 62.3 69.2 62.7 61.4 2.00 0.27nse –
Dough Sheet Color
b* (90°C)
b* (0.5 hr)
b* (2 hr)
60.9 58.4 66.4 57.3 66.6 63.3 61.3 63.6 61.3 61.5 61.2 64.3 60.3 65.3 58.7 64.0 68.7 61.9 62.1 62.3 67.7 62.1 61.9 2.76 0.19nse –
29.3 27.5 33.0 28.8 34.6 32.0 30.9 32.0 30.0 30.8 27.1 33.2 29.7 31.5 28.0 34.0 36.1 29.3 29.4 29.4 38.0 28.6 29.4 1.63 – 0.90 (0.87)d
29.9 28.0 36.0 29.6 37.5 33.0 32.1 32.7 31.7 32.0 27.9 36.2 31.1 34.2 28.8 35.0 38.7 30.3 31.3 31.4 40.7 30.0 30.5 1.24 – 0.94 0.91)d
Score 0 means existence of duplication at the Lpx-B1 locus; score 1 means a deletion of the Lpx-B1.1 copy. Total yellow pigment. α = 0.05. d Significant at P < 0.0001. e Not significant at P < 0.01. b c
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b* (24 hr) 31.2 29.7 45.0 31.6 45.5 35.7 33.3 33.0 34.5 35.4 31.2 43.1 33.2 42.1 31.4 35.1 45.7 32.3 33.8 33.8 45.2 31.3 31.8 1.45 – 0.90 (0.88)d
useful diagnostic for rapid screening of durum wheat cultivars for lutein content. However, the relationship between pasta b* and TYP content was weak for all three trial years. The correlation coefficients between pigment content and yellowness of spaghetti dried at 70 and 90°C were 0.27, and 0.19 for samples of the 2009 trial, and 0.34, and 0.30 for samples of the 2008 trial, and 0.52 and 0.38 for samples of the 2007 trial. These results indicated that the yellow pigment content of semolina was not effective in predicting spaghetti color, and that possibly factors other than TYP content are influencing variation in pasta yellowness among the genotypes currently in Canadian durum wheat breeding programs. Thus breeders cannot rely solely on breeding for elevated yellow pigment as this does not guarantee the yellowness of pasta. Semolina Dough Sheet Color and Spaghetti Color Spaghetti processing at laboratory-scale involves milling to produce semolina, mixing, extrusion, and drying. Due to the limitation of sample size and time requirement, it is not practical to produce spaghetti in breeding programs to select and evaluate breeder lines for good spaghetti color. Development of new protocols is necessary to serve as selection tools available to breeding programs to improve pasta color. Here we report a protocol designed to evaluate the color of a dough sheet prepared by mixing water and semolina and subjecting it to a sheeting process. Smooth and uniform dough sheets were prepared to eliminate the impact of surface property on color reading. The dough sheet color was monitored at 0.5, 2.0, and 24 hr after sheeting. The results are summarized in Tables I, II, and III for the 2009, 2008, and 2007 trials. The semolina dough sheet color appeared to be genotype dependent. For example, the dough sheet b* values of AC Navigator and Commander cultivars were much higher than the other
three check cultivars in all three trials. Furthermore, there were significant correlations between dough sheet b* values at 0.5, 2, and 24 hr and spaghetti b* values at drying temperatures of 70 and 90°C. The three time intervals measuring dough sheet color are equally effective in predicting pasta color. This can provide some flexibility in scheduling dough sheet color measurement in a practical laboratory. Dough sheet color measured at 0.5 hr after sheeting is sufficient and effective for this purpose. Color of dough sheet after extended storage (e.g., 24 hr) did not show any better prediction for pasta color, as indicated by correlation coefficients. Measurement of dough sheet color immediately after sheeting is not recommended because some resting is required for the dough sheet to have an evenly hydrated surface; it is important to have reproducible color measurements. Resting after 0.5 hr was sufficient for this purpose because the dough sheet color measurement was very reproducible (coefficient of variation ≤2.5% for all resting times over three years). The correlation coefficients between dough sheet b* values and semolina pigment contents and b* values are shown in Table IV. Results of all three trial years indicated that variations in dough sheet color were independent of semolina pigment content. This is in agreement with the observation that pigment content can be a poor indicator of pasta color in Canadian durum wheat breeding programs (Tables I, II, III). Semolina dough sheet color, which is the result of both pigment content and its degradation by oxidation, is a much better predictor of pasta yellowness. The oxidation of polyunsaturated fatty acids (mostly linoleic and linolenic) by LOX present in wheat gives rise to hydroperoxides. Carotenoids are very reactive to hydroperoxides. The result of the reaction is yellow color loss during pasta processing (Borrelli et al 1999). Water is required to activate LOX and incorporation of
TABLE II Semolina Pigment Content and Color in Relation to Spaghetti Color (2008 Trial)
Checks and Test Lines
DNAa Lpx-B1.1
DT661-Avonlea 0 DT484-ACMorse 0 DT673-ACNavigator 1 DT712-Strongfield 0 DT722-Commander 1 DT787 0 DT800 0 DT801 0 DT809 0 DT557 0 DT558 0 DT560 0 DT561 0 DT562 0 DT813 0 DT814 1 DT815 1 DT816 0 DT817 0 DT818 0 DT819 0 DT820 1 DT822 0 DT823 0 DT824 0 – LSDc Correlation with TYP – Correlation with spaghetti b* 70°C (90°C)
Spaghetti
Semolina TYPb
(mg/kg)
7.8 7.7 8.8 8.3 9.2 9.7 10.0 8.5 10.8 8.4 8.3 8.0 9.7 10.2 8.3 8.9 7.9 7.9 8.7 8.2 11.4 7.9 8.4 8.7 7.9 0.05 –
b* 32.4 32.2 33.6 33.2 34.2 34.9 35.7 33.2 36.1 33.3 33.4 31.6 35.7 35.7 32.8 33.6 32.9 31.6 33.3 32.4 37.1 32.2 33.3 33.8 32.2 0.45 0.97d –
Dough Sheet Color
b* (70°C)
b* (90°C)
b* (0.5 hr)
b* (2 hr)
b* (24 hr)
61.6 59.5 67.8 59.5 68.8 58.4 63.2 62.8 63.0 61.6 63.0 55.1 61.5 64.0 62.8 68.5 66.6 57.6 62.1 60.2 66.8 65.0 61.6 58.6 60.1 1.49 0.34nse –
59.2 60.0 64.4 60.0 65.9 58.2 59.4 61.9 61.4 61.5 62.4 58.2 61.1 62.4 61.2 66.6 64.0 58.2 61.0 60.5 65.8 64.0 60.7 59.5 58.5 1.65 0.31nse –
27.8 28.1 34.3 27.1 34.8 28.6 30.8 30.5 27.6 29.1 30.6 24.1 27.0 28.4 27.7 35.6 34.9 26.3 27.6 26.8 32.0 31.8 28.6 26.1 26.8 1.18 – 0.91 (0.86)d
27.5 27.8 36.6 26.8 37.1 27.6 30.4 30.1 27.3 29.0 30.0 23.7 26.7 28.4 27.9 37.5 36.8 27.0 27.4 26.7 32.8 34.4 28.1 25.9 26.7 1.02 – 0.91 (0.87)d
24.2 26.7 39.2 25.3 42.8 24.8 28.6 28.8 26.3 27.5 28.2 23.3 25.7 27.5 27.5 39.9 38.9 25.3 25.7 25.5 33.3 38.6 26.1 25.1 26.2 1.79 – 0.88 (0.87)d
a
Score 0 means existence of duplication at the Lpx-B1 locus; score 1 means a deletion of the Lpx-B1.1 copy. Total yellow pigment. α = 0.05. d Significant at P < 0.0001. e Not significant at P < 0.01. b c
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TABLE III Semolina Pigment Content and Color in Relation to Spaghetti Color (2007 Trial) DNAa Lpx-B1.1
Checks and Test Lines
DT661-Avonlea 0 DT484-ACMorse 0 DT673-ACNavigator 1 DT712-Strongfield 0 DT722-Commander 1 DT773 0 DT776 0 DT550 0 DT787 0 DT795 1 DT555 0 DT799 0 DT800 0 DT801 0 DT802 0 DT803 0 DT804 0 DT805 0 DT806 0 DT809 0 DT810 1 DT811 1 DT812 1 DT933 1 c – LSD Correlation with TYP – Correlation with spaghetti b* 70°C (90°C)
Spaghetti
Semolina TYPb
(mg/kg)
8.0 8.4 9.3 8.3 9.6 9.3 8.6 9.2 9.8 9.8 9.6 9.5 10.9 8.8 8.9 9.4 8.5 9.1 9.7 10.5 10.5 11.2 10.3 8.2 0.06 –
b*
b* (70°C)
33.5 33.9 34.8 34.0 35.4 35.3 34.1 35.3 36.2 35.9 36.1 35.9 37.6 34.7 35.0 35.8 34.0 35.7 35.7 36.8 36.4 37.7 36.7 34.1 – 0.97d –
63.6 62.4 67.7 61.8 70.4 65.6 64.6 64.9 63.5 66.8 64.3 65.8 67.8 65.0 66.2 62.7 62.0 63.4 63.9 64.1 68.8 71.1 65.4 68.2 2.08 0.52e –
Dough Sheet Color
b* (90°C)
b* (0.5 hr)
b* (2 hr)
b* (24 hr)
63.1 61.6 67.4 61.7 69.1 64.8 63.8 63.6 63.0 66.5 62.4 64.3 63.8 64.5 65.7 63.6 62.1 62.8 61.8 62.0 67.9 69.1 65.7 66.2 2.39 0.38e –
28.4 28.4 35.4 27.6 37.0 30.8 30.2 30.1 29.2 33.2 29.6 28.7 31.2 31.1 31.0 29.6 27.8 28.3 28.7 27.9 37.2 39.2 32.7 35.8 0.96 – 0.92 (0.95)d
27.6 27.5 37.1 26.7 39.0 30.2 30.2 29.4 28.1 33.6 28.5 28.2 30.9 30.8 30.7 28.7 27.8 28.2 28.3 27.4 38.7 41.2 32.8 37.4 0.89 – 0.92 (0.95)d
25.3 25.4 40.9 24.1 43.9 26.9 28.2 27.2 24.4 34.5 25.3 26.9 28.3 27.9 28.0 25.8 26.0 25.8 27.0 25.8 40.1 45.3 33.2 39.6 1.90 – 0.89 (0.93)d
a
Score 0 means existence of duplication at the Lpx-B1 locus; score 1 means a deletion of the Lpx-B1.1 copy. Total yellow pigment. c α = 0.05. d Significant at P < 0.0001. e Significant at P < 0.01. b
TABLE IV Correlation Coefficientsa Between Dough Sheet b* and Total Yellow Pigment (TYP) in Semolina Semolina TYP
Semolina b*
Semolina Dough Sheet b*
2009
2008
2007
2009
2008
2007
0.5 hr 2.0 hr 24.0 hr
0.33 0.28 0.11
0.18 0.12 0.08
0.43 0.40 0.37
0.32 0.27 0.10
0.22 0.15 0.09
0.33 0.29 0.26
a
Not significant at P < 0.001.
oxygen is necessary to facilitate the LOX-catalyzed oxidation of polyunsaturated fatty acids that can lead to oxidation of carotenoid pigments by a coupled reaction. The color of a semolina and water mixture will be the result of total yellow pigments and their degradation by LOX. This might explain the poor relationship between pasta yellowness and semolina yellow pigment content observed in this study. The difficulty and cost associated with the measurement of LOX prevent it from being a selection tool in most durum breeding programs (Clarke 2001). Association of Dough Sheet Color Gain, Spaghetti Color, and Deletion of Lpx-B1.1 Gene It is interesting to note that all the lines with a significant increase in dough sheet color during the 24-hr storage period had superior pasta yellowness (Tables I, II, III). DNA analysis revealed those lines lacked the Lpx-B1.1 duplication. In durum wheat, two Lpx-1 genes have been identified on chromosome 4B, Lpx-B1.1 and Lpx-B1.2 and evidence has been reported that the deletion of Lpx-B1.1 is associated with a strong reduction in LOX activity in semolina (Carrera et al 2007). The variability in carotenoid content and LOX activity of durum wheat germplasm was recently assessed in a large collection (De Simone et al 2010). That study confirmed the correlation between high/low LOX activity levels 268
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in semolina and the presence/absence of the Lpx-B1.1 locus. The increased use of germplasm with Lpx-B1.1 deletion in Canadian durum wheat breeding programs might be responsible for the poor power of pigment content for predicting pasta color. It appears that the dough sheet method can effectively identify the lines with Lpx-B1.1 deletion. A recent study (Verlotta et al 2010) revealed that the distribution of the Lpx-B1 genes and alleles among the genotypes defines three different haplotypes (I, II, and III) with high, intermediate, and low LOX activity in mature grains, respectively. Information on the genetics of these Lpx-B1 haplotypes, coupled with functionality evaluation such as dough sheet color will allow a precise selection of genotypes with very low LOX activity and superior color in end products. Based on total yellow pigment contents and spaghetti color, the durum wheat lines in the Canadian durum wheat breeding programs can be classified into four groups: high pigment with good pasta color; high pigment with average pasta color; average pigment with good pasta color; and average pigment with average pasta color. Examples of the four types are given in Table V. All the lines with superior pasta color were characterized by a significant increase in dough sheet color during dough sheet resting regardless of TYP content in semolina, while little change in dough sheet color was observed for those lines with average pasta color.
TABLE V Durum Genotypes Based on Pigment Content and Spaghetti Color Spaghetti
Semolina TYPa
Types/Lines High pigment with good pasta yellowness DT673-ACNavigator DT722-Commander High pigment with average pasta yellowness DT809 DT563 Average pigment with good pasta yellowness DT815 DT820 Average pigment with average pasta yellowness DT813 DT835 a b
(mg/kg)
9.3db 9.8c
b*
b* (70°C)
Dough Sheet Color
b* (90°C)
b* (0.5 hr)
b* (2 hr)
b* (24 hr)
34.3d 35.3c
66.8b 69.3a
66.4a 66.6a
33.0ab 34.6a
36.0a 37.5a
45.0ab 45.5a
11.6a 11.0b
37.1a 36.4b
61.3c 60.0c
61.3bc 58.7c
30.9bc 28.0c
32.1bc 28.8d
33.3c 31.4d
8.6e 8.5e
33.3e 33.7e
65.5b 64.5b
64.3ab 65.3a
33.2ab 31.5b
36.2a 34.2b
43.0b 42.1b
8.5e 8.6e
33.2e 33.5e
61.0c 61.4c
61.2bc 61.9bc
27.1c 29.4c
27.9d 30.5cd
31.2d 31.8cd
Total yellow pigment. Mean values followed by the same letter in a column are not significantly different (P < 0.05).
It is evident that those lines with superior pasta color were lacking the Lpx-B1.1 gene or low in LOX activity. However, the biochemical basis of the dough sheet color increases in relation to Lpx-B1.1 deletion or low LOX activity remains to be investigated. The yellowness of the dough sheet would not be expected to increase, even if no LOX activity can be detected. Other mechanisms might be involved in color loss or development once LOX is reduced at a very low level. The antioxidants such as phenolic acid can protect the carotenoids from oxidation as recently reported by Fares et al (2010). We can also speculate that dough sheet color is the balance between color gain and color loss by oxidation over time. To the best of our knowledge, this is the first report of a color increase in any products made from durum semolina. It would be useful to identify the physical or biochemical factors responsible for the color gain in dough sheet over time. This finding might lead to a new direction to improve pasta color of durum wheat. Work is in progress to examine the quantitative and qualitative changes in pigments in dough sheets with different time intervals. CONCLUSIONS There is limited relationship between semolina yellow pigment content and pasta yellowness in advanced durum wheat lines evaluated in Canadian Durum Wheat Variety Registration Trials. Breeders cannot rely solely on breeding for elevated yellow pigment as this does not guarantee the yellowness of pasta. It is clear that breeding programs will have to include selection for pasta color. A fast, simple, and reliable method was developed that can be used for selection in durum wheat breeding programs. The protocol developed in this study based on semolina dough sheet color appears to be promising in predicting pasta color. The dough sheet color is the result of pigment content and its degradation by LOX. There were significant correlations between dough sheet b* values and spaghetti b* values. Semolina dough sheet can be easily prepared in 15 min and requires only 30 g of material. Shortly after sheeting (30 min), dough sheet b* values can be used to predict pasta yellowness without making the end product, which involves mixing, extrusion, and drying. Up to 25 samples can be evaluated for one technician in a day. When compared with pastamaking for analyzing color characteristics of durum breeder lines in late generations, the semolina dough sheet method has higher throughput and requires smaller quantities of semolina. This can reduce labor and accelerate durum wheat breeding by conducting pasta color selection at earlier generations. The dough sheet method is now in use at Canadian Grain Commission for the routine durum wheat quality assurance programs (harvest survery, cargo monitoring, and plant breeder line evaluation) and other research projects.
While TYP content is still important for the improvement of pasta color, the effectiveness can be further improved by selection of dough sheet color or a deletion at the Lpx-B1.1 locus. In this study, all the lines with superior pasta color were characterized by the lack of Lpx-B1.1 and a significant increase in dough sheet color during dough sheet resting. The biochemical basis of the increase in dough sheet b* values for those lines and its relationship to pasta color remain to be investigated. ACKNOWLEDGMENTS We acknowledge the technical assistance of W. Aarts, D. Taylor, D. Turnock, and N. Edward and D. W. Hatcher for helpful discussions. LITERATURE CITED AACC International. 2010. Approved Methods of Analysis, 11th Ed. Method 14-50.01. Available online only. AACC International: St. Paul, MN. Abdel-Aal, E. M., Young, J. C., Rabalski, I., Hucl, P., and Fregeau-Reid, J. 2007. Identification and quantification of seed carotenoids in selected wheat species. J. Agric. Food Chem. 55:787-794. Baroni, D. 1988. Manufacture of pasta products. III. Pasta drying. Pages 203-210 in: Durum Chemistry and Technology. G. Fabriani and C. Lintas, eds. AACC International: St. Paul, MN. Beleggia, R., Platani, C., Nigro, F., and Cattivelli, L. 2010. A micromethod for the determination of yellow pigment content in durum wheat. J. Cereal Sci. 52:106-110. Borrelli, G. M., Troccoli, A., DiFonzo, N., and Fares, C. 1999. Durum wheat lipoxygenase activity and other quality parameters that affect pasta color. Cereal Chem. 76:335-340. Borrelli, G. M., De Leonardis, A. M., Fares, C., Platani, C., and Di Fonzo, N. 2003. Effects of modified processing conditions on oxidative properties of semolina dough and pasta. Cereal Chem. 80:225-231. Borrelli, G. M., DeLeonardis, A. M., Platani, C., and Troccoli, A. 2008. Distribution along durum wheat kernel of the components involved in semolina colour. J. Cereal Sci. 48:494-502. Carrera, A., Echenique, V., Zhang, W., Helguera, M., Manthey, F., Schrager, A., Picca, A., Cervigni, G., and Dubcovsky, J. 2007. A deletion at the Lpx-B1 locus is associated with low lipoxygenase activity and improved pasta color in durum wheat (Triticum turgidum ssp. Durum). J. Cereal Sci. 45:67-77. Clarke, J. M. 2001. Improvement of durum wheat grain quality: Breeding. Pages 27-54 in: Proc. Int. Workshop on Durum Wheat, Semolina and Pasta Quality: Recent Achievements and New Trends. P. Feillet, ed. Institute National de la Recherche: Montpellier, France. Clarke, F. R., Clarke, J. M., McCaig, T. N., Knox, R. E., and DePauw, R. M. 2006. Inheritance of yellow pigment concentration in seven durum wheat crosses. Can. J. Plant Sci. 86:133-141. De Simona,V., Menzo, V., De Leonardis, A. M., Ficco, D. B. M., Trono, D., Cattivelli, L., and De Vita, P. 2010. Different mechanisms control lipoxygenase activity in durum wheat kernels. J. Cereal Sci. 52:121-128. De Stefanis, E., and Sgrulletta, D. 1990. Effect of high-temperature dryVol. 88, No. 3, 2011
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[Received December 2, 2010. Accepted March 16, 2011.]
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