การประชุมทางวิชาการของมหาวิทยาลัยเกษตรศาสตร์ ครัง้ ที่ 52
สาขาอุตสาหกรรมเกษตร
Effect of Storage Conditions on Phenolic Compounds and Antioxidant Capacity of Thai Yam (Dioscorea alata L.) Tubers Tanaboon Sajjaanantakul1, Teerarat Likitwatanasade1, Somsak Idhipong2, Suchirat Sakuanrungsirikul2 and Parichat Hongsprabhas1
ABSTRACT This study investigated the influence of storage conditions, i.e. freezing -18 C for 2 weeks, 4 C or room temperature (27 C) for 5 months on total phenolic compounds, flavonoid and monomeric anthocynanin contents, as well as oxygen radical absorbance capacity (ORAC) and Trolox equivalent antioxidant capacity (TEAC) of methanol extracts from Thai purple yam (Dioscorea alata L.) tubers grown in Khon Kaen province. Freezing yam tubers resulted in enzymatic browning of tubers after thawing, making them unsuitable for further processing to purple yam flour. After storage at 4 C or 27 C, yam tubers contained high contents of total phenolic compounds of 22-27 mg gallic acid equivalent; flavonoid content of 13-16 mg catechin equivalent and monomeric anthocyanin of 17-23 mg in 100 g fresh yam weight. Although the ORAC and TEAC remained unchanged at 24-26 mol Trolox equivalent/g fresh weight and 2.2-2.6 mol Trolox equivalent/g fresh weight, respectively, the types of phenolic compound altered when the storage temperature was increased from 4 C to 27 C.
Key words: antioxidant, Dioscorea, polyphenol; yam e-mail address :
[email protected] 1
2
Department of Food Science and Technology, Faculty of Agro-Industry, Kasetsart University, Chatuchak, Bangkok 10900, Thailand Khon Kaen Field Crop Research Centre, Department of Agriculture, Muang district, Khon Kaen 40002, Thailand
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INTRODUCTION Yam (Dioscorea sp.) is one of the world’s most important starch sources consumed in tropical and sub-tropical countries. Among yam tuber varieties, greater yam, Dioscorea alata L., is the most widely distributed species of the genus Dioscorea in the humid and semi-humid tropics (Jayakody et al., 2007). Not only starch, but also protein, fibers and minerals with low level of lipid, that make yam a good dietary source. They are eaten as staple food in some part of Asia, South America and Pacific islands. Some yam varieties possess antioxidant activity and ability to modify serum lipid levels in humans. Dioscorin, yam storage protein, demonstrated scavenging properties against free radicals, immunomodulatory activity, as well as antihypertensive activities in rats (Chen et al., 2008; Hsu et al., 2002; Liu and Lin 2009). The terpenoid phytochemicals from some varieties such as steroidal saponins like diosgenin and dioscin showed estrogenic, antioxidant, anti-inflammatory and antitumour effects (Chang et al., 2011). In South-East Asia, particularly in the Philippines, yams are commonly known as ube, and are popularly consumed as sweetened food delicacies such as halaya (yam pudding with milk) and sagobe (with parboiled chocolate milk and glutinous rice balls) and rice cake. Ube is also used as flavor and/or filling for ice cream, tarts, bread, cakes etc. (Balangcod and Vallejo, 2013) Thai purple yam, however, is considered underutilized species and received little attention. Thai yam CslA2 gene expression, which related to cellulose synthase like A and glycosyl transferase in other plants, was investigated earlier by our group for conservation purposes. Our preliminary investigation also revealed that Thai yam may have potential use as edible coloured flour for commercial scale production. However, yam tuber can be harvested only once a year. The growth duration of Thai Dioscorea alata L. accession Khon Kaen Field Crop Research Center (acc. KKFCRC) varies from 6 to 7 months, starting from the day of shooting during June until the time of senescence of the leaves and vines (turn yellow and dry up) during December and January. Yam tuber has a dormancy period, which is a physiological rest period with no obvious external signs of biochemical activity after harvesting. The objectives of this study were therefore to investigate the influence of storage conditions of yam tubers on the polyphenolic compounds and their antioxidant capacities after harvesting. The results may be used in controlling the colour and biofunctional properties of polyphenolic compounds in yam after harvesting for commercial production and providing some downstream requirements for yam breeding program in the future.
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MATERIALS AND METHODS 1. Materials Yam tubers (Dioscorea alata L.) acc. KKFCRC were harvested in January, 2009. They were transported from Khon Kaen to the Department of Food Science and Technology, Kasetsart University in Bangkok, 2 days after being harvested. The tubers were kept at -18 C for 2 weeks, 4 C or room temperature (27 C) for 5 months prior to analyses unless stated otherwise. 2. Characteristics of yam flours prepared from fresh and frozen tubers Fresh yam tubers were cleaned with tap water, peeled, sliced, ground with coconut grater, dried in a tray-dryer at 50 C for 8 h, and ground by a Waring blender to obtain dry-milled yam flour. The flour was analysed for moisture, protein, lipid, crude fiber and ash (AOAC, 2000). Dry-milled yam flour was kept at -20 C prior to analysis. Yam tubers (either fresh or frozen) were peeled, sliced and ground with distilled water containing 0.1% sodium azide. Flour suspension was allowed to settle at 4 C for 16 h. The supernatant liquid was discarded, washed with distilled water twice, filtered through sinter glass filter and dried in an oven at 40 C for 16 h and designated as wet-milled flours. The colour of wet-milled flour was determined using CIE colour system (Minolta Spectrophotometer CM3500d, Japan). Colour values; namely brightness (L*), +a* (redness), -a* (greenness), +b* (yellowness) and –b* (blueness) were measured. 3. Determination of phenolic compounds in stored yam tubers Yam tubers stored at 4 C and 27 C were peeled and then cut in 10 x 10 x 10 mm3 cube. Five grams of sample were blended with 20 mL of 100% methanol. The mixture was shaken for 30 min and centrifuged at 3500g for 10 min. The supernatant was filtered through Whatman No. 1 filter paper. The filtrate was made to 15 mL with 100% methanol and kept at 4 oC in amber bottle before analysis for total phenolic content (Liu and Lin, 2009), total flavonoid content (Yang et al., 2004) and total anthocyanin content (Boyles and Wrolstad, 1993). 3.1 Total phenolic contents Total phenolic contents in methanol extracts were determined by mixing 0.1 mL of crude yam extract with 0.1 mL Folin-Ciocalteu’s phenol reagent and 7 % Na2CO3 1.0 mL and 1 mL of distilled water. The reaction was allowed to proceed at room temperature for 90 min using method described by Liu and Lin (2009) with slight modification on dilution. The absorbance at 760 nm was measured by a Genesys 10 UV (Thermo Electron Corporation, WI) and expressed as gallic acid in 100 g of fresh weight. 3.2 Flavonoid content Briefly, 0.25 mL of sample extract in methanol was mixed with 1.25 mL of distilled water and 0.075 mL of 5% NaNO2 solution and allowed to react for 5 min. Then 0.15 mL of 10% aluminum
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chloride was added and allowed to further react for 6 min before 0.5 mL of 1 M sodium hydroxide was added. Distilled water was added to bring the final volume of the mixture to 3 mL. The absorbance of the mixture was measured at 510 nm wavelength against a prepared blank using a Genesys 10 UV spectrophotometer. The flavonoid content was determined by a catechin standard curve and expressed as mg of catechin in 100 g of fresh weight (Yang et al., 2004). 3.3 Anthocyanin content Monomeric anthocyanin content was determined by the pH-differential method described by Boyles and Wrolstad (1993) using two buffer systems: 0.025 M potassium chloride buffer, pH 1.0 and 0.4 M sodium acetate buffer, pH 4.5. Methanol extract (0.25 mL) was mixed with 1 mL methanol to obtain 1.25 mL of solution, which was later mixed with either 1.00 mL of 0.025 M potassium chloride buffer pH 1.0 or 1.00 mL of 0.4 M sodium acetate buffer pH 4.5 and allowed the solutions to equilibrate for 15-30 min. The absorbance was read at 510 and 700 nm using a Genesys 10 UV spectrophotometer. The total monomeric anthocyanin content (mg in 100 g of fresh weight) was calculated on the basis of the following equations: Total monomeric anthocyanins (mg/100g) = ΔA x MW x DF x 100/ ( x 1)………....Eq.1 and ΔA = (A510 - A700) pH1.0 - (A510 - A700) pH4.5…… Eq.2 where A was the absorbance, the MW of cyanidin 3-gluscoside was 449.2, DF was the dilution factor of 5, which was calculated from 1.25 mL/0.25 mL. The molar absorptivity of cyanidin 3-gluscoside () was 26,900. 4. Determination of antioxidant capacity of methanol extracts from stored tubers 4.1 Oxygen radical absorbance capacity (ORAC) The ORAC assay (Prior et al., 2003) was carried out on a BMG Fluostar Optima Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). The reaction was performed at 37 °C in 75 mM phosphate buffer (pH 7.4); the final reaction mixture was 200 L. The methanol extract (20 L; Trolox 2–9 M, or sample 1–3 mg/mL) and fluorescein (120 L; 70 nM final concentration) were loaded into a black 96-well microplate. Fluorescence intensity was measured under the following conditions: the temperature of the incubator was set to 37 °C; a fluorescence filter with an excitation wavelength of 485 nm and an emission wavelength of 520 nm was programmed to record the fluorescence of fluorescein once per cycle. The number of cycles was 50 cycles and each cycle was run for 179 s. During cycle four, 60 L of AAPH was manually injected into the respective wells to give a final AAPH concentration of 12 mM. The plate contents were mixed by shaking for 1 s before reading.
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The final ORACFL values were calculated by using a linear equation between the Trolox standards or sample concentrations and net areas under the fluorescein decay curves. The area under the curve (AUC) was calculated by: AUC = (0.5 + f5/f4 + f6/f4 + f7/f4 +, …, + fi/f4) CT .......Eq.3 where f4 is the initial fluorescence reading at cycle 4, fi is the fluorescence reading at cycle i, and CT is the cycle time in minutes. The net AUC was obtained by subtracting the AUC of the blank from that of a sample. The ORACFL values of samples were then reported as mol Trolox equivalent per mg of protein (mol TE/ g fresh weight). 4.2 Trolox equivalent antioxidant capacity (TEAC) The TEAC assay described by Re et al. (1999) was used to evaluate the relative capacity of the milk peptides in the scavenging of ABTS radical (ABTS•+), compared to the antioxidant potency of Trolox, and reported as mol TE/g fresh weight. The ABTS•+ was generated by mixing 5 mL of 7 mM ABTS with 88 L of 140 mM K2S2O8 in the dark and held for 24 h at room temperature before being used. The ABTS•+ stock solution was diluted to get an absorbance of 0.700 0.020 at 734 nm in phosphate buffer saline (PBS). The methanol extract was diluted with ethanol using the ratio of extract to EtOH of 1:3. A volume of 100 L of diluted sample was mixed with 2 mL of ABTS•+ radical solution. The absorbance at 734 nm was recorded using a Genesys 10 UV spectrophotometer by analysing the decolorisation of the ABTS•+ at 734 nm after 4 min at 30C using PBS as a control. 5. Statistical analysis The experiments were carried out in two separate trials. Each trial was run in duplicate. The data were analyzed by using analysis of variance (ANOVA) at 95% significance level. Significant differences among mean values from ANOVA were determined by Duncan’s multiple range tests. All statistical analyses were performed using SPSS Software Version 12 (SPSS Inc., US).
RESULTS AND DISCUSSION The appearance of yam flesh and its rich purple colour prior to storage is shown in Figure 1. Although yam exhibited inconsistent anthocyanin distribution within the tubers, the purple pigment was quite stable by dry-milling and drying process used in this study. Thai purple yam dry-milled flour contained 2.75% protein, 0.25% lipid, 1.44% crude fiber, 0.60% ash and 85.25% carbohydrate (by difference) on dried weight basis (d.b.) (Table 1).
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Figure 1 Appearance of Thai yam flesh and flour: (a) purple pigment of yam tuber; (b) distribution of purple pigment within a tuber; and (c) purple yam flour after dry-milling and drying process. Table 1 Chemical composition of yam flour prepared by dry-milling and drying process. Constituents Means std. dev. % (d.b.) Moisture Protein Lipid Crude fiber Ash
9.71 0.10 2.75 0.00 0.25 0.02 1.44 0.02 0.60 0.02
However, freezing of yam tubers resulted in severe colour change of yam flesh after thawing (Figure 2). This was likely due to the activity of polyphenoloxidase activated by cellular damages after thawing. The brown colour was apparent and dominated the purple colour of yam flesh, which resulted in discolouration of yam flour (Table 2) even after wet-milling process. Therefore, the frozen yam tubers were excluded in further studies. Table 2 CIE colour values (means s.d.) of yam flour prepared from fresh and frozen tubers. CIE colour system Flour from fresh tuber Flour from frozen tuber L*-value 87.2a 0.2 68.5b 0.4 a*-value 2.4b 0.0 6.8a 0.2 b*-value 5.8b 0.1 12.7a 0.2
Figure 2 Appearance of yam flesh from (a) fresh tuber and (b) frozen and thawed tuber
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The effects of storage temperature on phenolic compounds and antioxidant capacities were summarized in Table 3. Although the antioxidant capacities, measured as ORAC and TEAC, remained unchanged after storage for 5 months at different temperature, the types of phenolic compounds changed as storage temperature increased shown as the reduction in monomeric anthocyanin and an increase in total phenolic compounds. It is apparent that despite dormancy period and no physical changes of tubers, some biochemical changes in tubers still occurred, particularly total phenolic synthesis and degradation of monomeric anthocyanin at room temperature. Table 3 Effect of storage temperature on total phenolic compound, flavonoid, anthocyanin contents and antioxidant capacity (means s.d.) of edible portion of fresh Dioscorea alata tuber. Phenolic constituents and antioxidant capacity of Storage conditions methanol extract 4 C, 5 months 27 C, 5 months Total phenolic compounds (mg gallic acid/100 g fresh weight) Flavonoids (mg catechin/100 g fresh weight) Total monomeric anthocyanin (mg/100 g fresh weight) ORAC (mol TE/g fresh weight) TEAC (mol TE/g fresh weight)
22.18b + 0.84
26.72a + 3.26
16.28a + 2.26 23.92a + 0.05 24.47a + 1.81 2.23a + 0.10
13.54a + 1.50 17.16b + 0.76 26.50a + 1.93 2.59a + 0.45
Means s.d. in the same row followed by different superscripts are significantly different (P < 0.05).
CONCLUSION This study reveals that Thai Dioscorea alata L. showed potential use as the source of caloriebearing tuber rich in polyphenolic compounds and antioxidant capacity. However, phenolic compounds and antioxidant capacities of yam flour were dependent on storage temperature. Therefore, storage stability of such antioxidative compounds needs to be considered if it is to be used at commercial scale all year round. This is because the tubers can be harvested once a year and the dormancy period was around 5 months before germination during rainy season. Insight investigations on the stability of phenolic compounds in dry-milled yam flour and its use in food products are under way.
ACKNOWLEDGEMENTS The financial support from the Department of Agriculture, Ministry of Agriculture and Cooperative is gratefully acknowledged.
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