CAROTENOID RETENTION IN MINIMALLY PROCESSED BIOFORTIFIED GREEN CORN STORED UNDER RETAIL MARKETING

Storing processed food products can cause alterations in their chemical compositions. Thus, the objective of this study was to evaluate carotenoid retention in the kernels of minimally processed normal and vitamin A precursor (proVA)-biofortified green corn ears that were packaged in polystyrene trays covered with commercial film or in multilayered polynylon packaging material and were stored. Throughout the storage period, the carotenoids were extracted from the corn kernels using organic solvents and were quantified using HPLC. A completely factorial design including three factors (cultivar, packaging and storage period) was applied for analysis. The green kernels of maize cultivars BRS1030 and BRS4104 exhibited similar carotenoid profiles, with zeaxanthin being the main carotenoid. Higher concentrations of the carotenoids lutein, β-cryptoxanthin, and β-carotene, the total carotenoids and the total vitamin A precursor carotenoids were detected in the green kernels of the biofortified BRS4104 maize. The packaging method did not affect carotenoid retention in the kernels of minimally processed green corn ears during the storage period.


INTRODUCTION
Much of the world's population thinks that food not only nourishes but also supplies biologically active compounds or elements that provide additional health benefits (Sentanin; Rodriguez-Amaya, 2007).Thus, the concept of functional foods has emerged.Among the bioactive compounds present in food sources are carotenoids, which are natural pigments used as natural food dyes that possess biological activities.
Corn stands out as a source of carotenoids in the diets of populations for which this cereal is considered a staple.Zeaxanthin and lutein, which are xanthophylls, are the main carotenoids present in the kernels of yellow corn, which also contain significant amounts of β-cryptoxanthin and lower levels of α and β-carotenes.The industrial process by which the ears with green corn kernels usually consumed in human diet, as well as those during storage of such products, is of concern, because carotenoids exhibit instability regarding exposure to oxygen, light and oxidative enzymes, give rise to a number of degradation reactions such as the following: isomerization, oxidation and epoxidation (Rodriguez-Amaya, 2001).
Ciênc.Agrotec., Lavras, v. 39, n. 4, p. 363-371, jul./ago., 2015 The possibility of carotenoid oxidation during processing (Rodriguez-Amaya, 1997) has made carotenoid retention a constant concern; moreover, attention has been focused not only on the effects of industrial processing but also on homemade preparation methods that might also cause high losses of the derivatives of biofortified materials (Gama Silos, 2007).However, processing has been demonstrated to have a positive effect on the availability of carotenoids by breaking cellular structures and denaturing proteins that are complexed with pigments, thus facilitating the release of these compounds from the plant-cell matrices (Saunders et al. 2000).
Nutritional and convenience factors continue to be desired by consumers.The latter, when associated with food, relates to the ease of its storage (Sgarbieri, 1986).However, the storage conditions of a product are critical to preserving its nutritional value.Thus, investigating the retention of carotenoids after the processing and during the storage of corn is essential because processed, stored corn is the form of corn product generally consumed.Given the above, the aim of this study was to evaluate the retention of carotenoids in the kernels of normal and vitamin A precursor-biofortified green corn ears that were minimally processed and packed in polystyrene trays with a polyvinyl chloride (18-mm-thick PVC) commercial film covering or in multilayered polynylon packaging under vacuum during storage under retail conditions.

Raw material
The corn was cultivated under controlled conditions of fertilization, irrigation, and pest and disease management.The crops were harvested during the early morning hours, when the kernels were at the milky stage, known as the "green corn point."Ears with the husks were packed in polystyrene boxes containing ice and were transported to the laboratory, where minimal processing was conducted.Two corn cultivars were used for the study, the biofortified variety BRS4104 and the normal variety BRS1030.

Experimental design
The experiment was conducted using a 2x2x4 factorial design including three factors, cultivars BRS1030 and BRS4104, polystyrene packaging and multilayer polynylon, and storage for 0, 3, 6 and 9 days, and three replications were performed.The experimental unit consisted of three ears of corn.

Minimal processing
The ears were processed as shown in the flowchart in Figure 1.
The following were used to package the ears: polystyrene trays (Styrofoam) that were 23.5 cm long x 18.2 cm wide, wrapped in 18-mm-thick polyvinyl chloride (PVC) film (Figure 2A) and multilayered polynylon packaging (20 x 25 cm; TecMaqbrand) suitable for food (Figure 2B).A TM model 150 vacuum sealer was used to seal the latter under vacuum.The packed ears of corn were stored in a BOD at a fixed temperature of 5 °C, with light for nine hours daily, for a period of nine days.The products were distributed randomly within the BOD.To evaluate carotenoid retention, the ears were threshed using a Mecapau brand (model 032 D) autothresher, and the kernels were quartered and then were triturated using a model 1119 Toastmaster coffee grinder for 20 seconds.The carotenoid retention analysis was conducted on days 3, 6 and 9 of storage.

Determination of the carotenoid profile
The carotenoids were extracted from the samples using a series of organic solvents according to the protocol of Rodriguez-Amaya, Kimura and Mieko (2004), with some modifications.The modifications concerned the volume of ethanol used (7 mL), the volume of deionized water used (4 mL), not adding an internal standard, the volume of hexane used (4 mL), replacing dichloroethane with methanol, using acetone (2 mL) to reconstitute the extract for HPLC analysis (sample injected = 40 µL) and not injecting the standard β-apo-8'-carotenal for HPLC analysis.
The carotenoid contents were quantified using high-performance liquid chromatography (HPLC) using an Alliance Waters model e2695 liquid chromatography system equipped with a polymeric YMC C 30 column (3 µm, 4.6 x 250 mm, Waters, Milford, MA, USA) coupled to a diode array detector (Waters Model 2998).Gradient elution was conducted at a flow rate of 0.8 mL/min under gradient conditions that ranged from 80:20 to 20:80 of methanol:tert-butyl methyl ether over 16 minutes, followed by a constant flow of an 80:20 solution for 4 minutes, and finishing with 6 minutes of equilibration.The oven temperature was 30 °C, the detection wavelength was 450 nm, and the injection volume was 40 μL.The laboratory temperature was maintained at 20 °C throughout the process.For compound identification, standards purified from carrots (α-carotene, 94.64% purity) and papayas (β-cryptoxanthin, 92.72% purity) were used, following the protocol of Rodriguez-Amaya, Kimura and Mieko (2004).The carotenoid standards lutein (40 mg lutein, Vision Health) (98.68% purity), zeaxanthin [Swanson ZeaGold zeaxanthin, 4 mg (from paprika (97.99% purity)], and β-carotene [beta carotene (vitamin A), 25,000 IU Supplement, Swanson SW007 (94.57% purity)] were obtained from the respective Swanson-brand capsules.The results of the carotenoid analysis were expressed on a fresh basis.
The concentration of total carotenoids was obtained by summing the values for all of the quantified fractions, including the total lutein, zeaxanthin, β-cryptoxanthin, α-carotene and β-carotene fractions, as described by Murphy, Criner and Gray (1975).
The concentration of carotenoids with provitamin A activity (proVA) was obtained using the following formula: total β-carotene + ½ total α-carotene + ½ total β-cryptoxanthin (mg/g), as described by Murphy, Criner and Gray (1975).The apparent percentage of retention percentage for each variable was calculated using the formula proposed by Murphy, Criner and Gray (1975).This calculation was based on the inherent chemical characteristics of each of these molecules.The chemicals with structures that have at least one β-ionone ring and a chain of eleven carbons exhibit provitamin A activity.β-carotene is formed by two β-ionone rings that give rise to two molecules of retinol, and therefore, this carotenoid has 100% provitamin A activity.β-cryptoxanthin and α-carotene exhibit approximately 50% provitamin A activity because they have only one β-ionone ring.The content of cis isomers was not determined because they do not have significant biological activity.The apparent percentage of retention was calculated according to the formula proposed by Murphy, Criner and Gray (1975), as follows:  The moisture content of the samples was determined using 2 g of ground sample that was dried at 60 °C for 72 hours in a ShelLab SL Model 1350FX oven.The moisture content was calculated according to method 44-15 of the AACC (2000).
The data were subjected to an analysis of variance (ANOVA), and the mean values were compared using the Least Significant Difference test (LSD) (p = 0.05) when evaluating the significance of the differences using the F test.

RESULTS AND DISCUSSION
The average concentrations of the studied carotenoids in the green corn kernels of the two cultivars differed significantly.The concentrations (µg/g) of lutein, zeaxanthin, β-cryptoxanthin, β-carotene, total carotenoids and vitamin A precursor carotenoids in the kernels of minimally processed green corn are shown in Table 1.
The kernels of the BRS4104 corn cultivar had higher concentrations of carotenoids than did those of cultivar BRS1030 (p<0.05),except for the zeaxanthin concentration (p>05).In addition to the average concentration of total carotenoids in the green corn kernels of cultivar BRS4104 (15.96 µg/g) being higher than that of cultivar BRS1030 (11.47 µg/g), this value was higher than the corresponding values for the dried yellow kernels of other commercial cultivars of Brazilian QPM (Quality Protein Maize), such as Assum Preto (11.7 µg/g) and BR 473 (9.17 µg/g), which were determined by Kimura, Rodriguez-Amaya and Nestel (2007).
Cândido (2010) found that the average total carotenoid content of dried kernels of synthetic corn that was also biofortified with provitamin A was 34.49 µg/g in grains, which was higher than that found in the corn biofortified with provitamin A that was studied in the present investigation.This result can be explained by the carotenoid synthesis of the latter being interrupted soon after harvesting by the minimal processing procedure.In this study, the ears were harvested at the milky stage, which is the ideal stage at which to harvest corn and differs from the time of harvest of the cited study.Moreover, the cited study examined dried kernels.Moisture content is another factor that could have affected the results because dried kernels contain an average of 13% moisture, whereas fresh kernels contain 70% to 80% moisture (Nunes, 2013).Therefore, the carotenoids would presumably be more concentrated in the dried corn kernels.
Carotenoid biosynthesis can continue after harvesting, increasing the carotenoid contents of fruits, vegetables and tubers that are maintained fully intact, thereby preserving the enzymatic system responsible for carotenogenesis (Rodriguez-Amaya; Kimura; Amaya-Farfan, 2008).However, minimal processing, even if performed with the recommended care, does not maintain harvested material fully intact.The kernels of the green corn ears of cultivars BRS1030 and BRS4104 exhibited a linear rate of moisture reduction throughout the storage period (Figures 3 and 4), regardless of the packaging used.For cultivar BRS1030, the moisture content of the kernels was higher from the 3 rd day of storing the ears in the multilayered polynylon packaging rather than in the wrapped polystyrene trays.However, the moisture content of the kernels of cultivar BRS4104 green corn ears stored in polystyrene trays was higher throughout the storage period.
To evaluate the apparent retention rate, the average carotenoid content of the kernels of the green corn ears at Day zero expressed in μg/g (wet weight) was considered to be 100%.
Tables 2 and 3 show the levels of retained carotenoids expressed in μg/g (wet weight).A significant effect (p <0.05) of the interaction of the cultivar x the storage period was detected for the variable lutein.The effect of this interaction was not significant (p>0.05) for the variables zeaxanthin, β-cryptoxanthin, β-carotene, total carotenoids or proVA carotenoids.However, we found that the storage period significantly affected the variable zeaxanthin and that the cultivar type significantly affected the variable β-cryptoxanthin (Table 4).
The concentration of α-carotene was below the limit of detection, so this variable was not analyzed.
After the ANOVA was performed regression analysis was applied to the storage period factor for the variable lutein.Regardless of the type of packaging used, the green corn kernels of cultivar BRS1030 retained 67.92% of the lutein on the 3 rd day of storage, whereas the green corn kernels of cultivar BRS4104 retained 105.67% of the lutein, showing no apparent loss of this carotenoid during this period.However, the green corn kernels of cultivar BRS1030 exhibited a higher apparent retention of lutein (124.07%) on the 9 th day of storage, which was significantly higher than that of the synthetic cultivar BRS4104, with a difference of 26% greater retention.The 2 nd -degree polynomial model of the variation in lutein retention in green corn kernels during the storage of ears of the BRS1030 cultivar is shown in Figure 5. Lutein loss apparently occurred by Days 3 (67.92%)and 6 (85.85%), although the loss of moisture may have favored the lutein concentration at the end of the storage period.
For the synthetic BRS4104 cultivar, the apparent lutein retention rate found on the 9 th day of storage was 98.97%, which remained constant throughout storage.
The effect of minimal processing and storage for five days at 5-7 °C on collard led to reduced levels of retained lutein (27%), violaxanthin (20%) and neoxanthin (31%) and an even greater loss of β-carotene.The loss was greatest from Day 0 to Day 1, with no change observed from Day 1 to Day 5 (Azevedo;Rodriguez-Amaya;2005).
The zeaxanthin retained in the immature kernels of minimally processed corn ears stored for nine days significantly increased at the end of the storage period, regardless of the packaging applied to the ears.As previously reported for lutein, an increase in the concentration of this carotenoid was possibly due to moisture loss.
The 2 nd -degree polynomial model of the variation in zeaxanthin retention during storage is presented in Figure 6.The apparent rates of retention of β-cryptoxanthin in the green corn kernels of both cultivars differed significantly.The green kernels of cultivar BRS4104 had a lower average β-cryptoxanthin retention rate, 19.46% less than that of the green corn kernels of the ears of cultivar BRS1030.The apparent β-cryptoxanthin retention rate of cultivar BRS1030 was 109.22%,whereas that of cultivar BRS4104 was 89.76%., Lavras, v. 39, n. 4, p. 363-371, jul./ago., 2015 None of the factors and none of their interactions affected the remaining variables.Thus, there was no significant difference in the apparent retention rates of β-carotene, the total carotenoids and the proVA carotenoids regardless of the cultivar and the types of packaging during storage.The average apparent retention rate of the β-carotene was 106.53%, that of the total carotenoids was 100.86% and that of the proVA carotenoids was 101.62%.
Processing and storing foods may significantly alter the qualitative and quantitative carotenoid composition.However, the retention of proVA carotenoids during storage of processed foods is favored by a low temperature, protection from light and antioxidants that are naturally present or are added to preserve food (Rodriguez-Amaya, 1997).A previous study showed that reducing the processing period and temperature and the interval between peeling, cutting or chopping and processing can significantly improve the retention of carotenoids (Rodriguez-Amaya, 1993).Hussein et al. (2000) studied the effect of two different containers (squeeze and vacuum) on the retention of β-carotene in minimally processed broccoli that was stored for the retail period of this product (10 days) and found that there was no significant difference due to the packaging.The same result was observed in the present study.The type of packaging (polystyrene or multilayered polynylon) did not affect the retention of any of the carotenoids studied, regardless of the cultivar.

CONCLUSIONS
The green corn kernels of cultivars BRS1030 and synthetic BRS4104 had similar carotenoid profiles, with zeaxanthin being the main carotenoid present.The concentrations of lutein, β-cryptoxanthin, β-carotene, the total carotenoids and the total vitamin A precursor carotenoids in the green corn kernels of the synthetic cultivar BRS4104 were higher than those of cultivar BRS1030.Storing minimally processed normal and vitamin A precursor-biofortified ears of green corn for nine days caused a reduction in the moisture content of the kernels but did not affect the apparent retention of β-carotene, the total carotenoids and the proVA carotenoids.

Figure 1 :
Figure 1: Flowchart of the minimal processing procedure for the corn used to evaluate carotenoid retention during the storage period.

Figure 2 :
Figure 2: Image of green corn ears packed in polystyrene (Styrofoam) trays and wrapped with 18-mm-thick polyvinyl chloride (PVC) film (A).Image of green corn ears vacuum-packed in multilayered polynylon (B).

Figure 3 :Figure 4 :
Figure 3: Percentage of moisture in the kernels of green corn ears of cultivar BRS1030 throughout storage

Figure 5 :
Figure 5: Variation of the apparent retention (%) of lutein during the storage of cultivar BRS1030 green corn at 5 °C, with light for 9 h/day.

Figure 6 :
Figure 6: Retention of zeaxanthin in green kernels during the storage of minimally processed corn ears

Table 1 :
Concentration of carotenoids on a fresh basis, expressed in µg/g of kernel samples of minimally processed green corn ears from normal and biofortified cultivars.
1Mean values followed by the same letter in the rows did not differ significantly according to the LSD test at the 5% probability level (p<0,05) -2 SD (standard deviation).

Table 2 :
Mean concentrations of carotenoids in µg/g of kernels (wet weight) at 0, 3, 6 and 9 days of storage of minimally processed corn ears packed in polystyrene trays covered with polyvinyl chloride.

Table 3 :
Mean concentrations of carotenoids in µg/g of kernels (wet weight) at 0, 3, 6 and 9 days of storage of minimally processed corn ears packed in multilayered polynylon packaging.

Table 4 :
Summary of results of an analysis of variance (ANOVA) of the effects of the treatments on the variables.