Postharvest Quality of Leafy Vegetables in relation to Nitrate and Nitrite Concentration

Azimah bt Hamidon GS 30608 Supervisory committee Chairman: Prof. Madya Dr. Siti Hajar Ahmad Member: Prof. Madya Datin Dr. Rosenani Abu Bakar INTRODUCTION More than 80-90% of the nitrogen absorbed by plants from fertilizer or organic material which essential to protein synthesis is in the form of nitrates that are present naturally in soils, water and plant materials as a result of nitrogen fixation (Chung et al., 2003; Meah et al., 1994; Santamaria et al., 1999). However, nitrates can turn to nitrites exogenously or endogenously (Gangolli at al., 1994) and nitrites are harmful to human health because they react with hemoglobin and form methemoglobin which caused blue baby syndrome (Ellis et al., 1998). Also, nitrites lead to formation of carcinogenic nitrosoamines (Du et al., 2007; Bartsch et al., 1988). Nitrite concentrations in vegetables may increase to elevated levels due to bacterial transformation of nitrate and nitrite, when vegetables are stored in room temperature and poor sanitation can increase the nitrite concentrations to potentially hazardous levels (Chung et al., 2003) LITERATURE REVIEW Leafy greens vegetables has rich source of vitamin C, K, E, and B, as well as iron, calcium, and fiber, phytochemicals and antioxidant which are essential for good health (Jahangir et al., 2009; Podsedek, 2007; Fahey, 2003). It also contribute to the higher demand of good quality of leafy vegetables by Malaysian consumer in response to their incomes rise (Tey et al., 2009). The presence of nitrates in vegetables, as in water and other food products, is a serious threat to human health, not so much due to their toxicity, which is low, but for the compounds they induced in the organism. De Martin and Restani (2001) showed that leafy green vegetables accumulate high amounts of nitrates, concentrations reaching up to 6000mg/ kg. Nitrites, produced by nitrate reduction, induce methaemoglobinemia, or it can form nitrosamines and nitrosamides by reacting with amines and amides, whose carcinogenic action are well known (Kross, 1992; Funn Burning, 1991; Shank, 1975). However, some components of vegetables (e.g ascorbic acid, phenol, etc) have been reported to inhibit the negative effects of nitrites (Walker, 1990). Since chlorophyll mostly consisted of nitrogen containing enzymes and other organic compounds (Chapman and Barreto, 1997), there is a positive correlation between the nitrogen and chlorophyll concentrations of leaves (Tuncay, 2011). The darker the colour of leafy vegetables, the higher is nitrate content, as the excessive fertilizer contributes to the greenness of vegetables colour. Park (1998) reported that there a high nitrate content in organic grown vegetables, like kale, lettuce, and cabbage, as it probably came from a wide range of organic fertilizers. Differences in nitrate accumulation in plants may be due to several factors such as harvesting period, amount and composition of fertilizers applied, amount and types of nutrients present in soil, and temperature and light intensity (Tuson and Ustun, 2004; Zhou et al., 2000). Nitrite concentrations are usually very low in fresh vegetables, but under adverse postharvest storage condition nitrite levels can increase in vegetables (Ayaz et al., 2007). Storage of vegetable salads, raw purees and juices at room temperature can increase the nitrite concentrations to potentially hazardous levels (Chung et al., 2003). JUSTIFICATION Unregulated application of nitrogen fertilizers would result in luxury consumption of nitrogen uptake by the plant which would result in increasing of nitrate uptake in leaves. After harvest, the conversion of nitrate to nitrite to nitrosamines happens during storage. It is known that nitrosamines is carcinogenic, and eventually will reducing the quality of the produce. There are tendency for growers to produce very intense green colour of leafy vegetables as they said consumers like the vegetables better. Commonly growers used higher rate of nitrogen fertilizer to produce dark green leafy vegetables. Despite the health benefits of organic vegetables, high nitrate and nitrite level may pose a safety problem. Information on nitrate and nitrite levels in leafy vegetables in Malaysia is currently lacking as most of the available data are from temperate climates. Biochemical system in tropical plant cell is different from temperate because of nitrogen assimilation coupling and photosynthetic electron transport in

leaves as it implies that light intensity, heat and water stress tolerance are the key factor in determining nitrate concentration in the plant. OBJECTIVES Thus, the objectives of the study are : 1. To obtain N fertilization data from three organic and three conventional leafy vegetable (LV) farms, 50 km radius from UPM 2. To determine nitrate, nitrite and chlorophylls concentrations, nitrate reductase activity and leaf colour of three freshly harvested organic and conventional LV and correlations between parameters. 3. To determine effects of cropping systems (organic and conventional) and storage duration on nitrate, nitrite concentrations, and postharvest quality characteristics of the LV. METHODOLOGY Collection of historical N fertilization background data of farm. Years of farming the land Method of fertilizer application Types of soil Rates of fertilizer used Types of fertilizer used Types, amount and frequency of compost used. Frequancy of the fertilization Planting systems (rotation, intercropping, shelter or open field) 2. Determining nitrate, nitrite and chlorophylls concentrations, nitrate reductase activity and leaf colour of three freshly harvested organic and conventional leafy vegetables and correlations between parameters. Three types of vegetables (bayam, sawi and leaf lettuce), at 7 weeks after planting, will be harvested from the selected organic and conventional farms. Immediately after harvest, the vegetables will be taken to the Postharvest Lab, Faculty of Agriculture, UPM, where the vegetables will be analise. The parameters that analised were: a) Leaf color will be measured using a chromameter to get colour values of L*, C*, h b) Nitrate and nitrite concentration Nitrate were determined according to the method of Sumiko and Masako (1993) and Tsuji et. al., (1993). The analysis will be carried out using Ion Chromatograph. Nitrite contents will be determined according to the Diazo method (Sumiko et. al., 1993). c) Nitrate reductase activity (NRA) Nitrate reductase activity were determined according to the method of Zou (1995) and Li (2000). The analsis of the samples were measured by using a spectrophotometer (Model S1200, Spectrawave, England) with absorbance readings at 540 nm. The nitrite released to the medium was expressed as mol NO2hg FW (Snell and Snell 1955). d) Chlorophyll concentration Chlorophyll was extracted in 80% acetone from fresh leaves according to the methods of Arnon (1949) and Coombs et al. (1985). The amount of chlorophyll present in the leaves was calculated, taking the OD of the extract at 645 and 663 nm and using the formula, mg chlorophyll/g of tissue. 3. Experimental design. 1.

For historical fertilization background data of farm, the experimental design used were purposive sampling method where there are two cropping system (organic and conventional) and three types of vegetables (bayam, sawi and leaf lettuce) for each cropping system. Data presented and summarized using frequency histogram and percentages.
Determining nitrate, nitrite, chlorophylls concentrations, nitrate reductase activity and leaf colours (L*, C* and h) of three freshly harvested organic and conventional leafy vegetables and correlations between parameters. Factorial experiment was arranged in a complete randomized design with five replications and 5 plant per rep.

RESULTS AND DISCUSSION From the 3 conventional and 3 organic farm background studies, generally conventional farm has the longest farming period than organic farm as the averages of farming period of organic and conventional are 13.6 years and 17 years respectively. This might be because Malaysian consumer just known the importance of organic vegetables in recent 10 to 15 years. However, farm sizes of organic farms are bigger than conventional

farms, 32 acre to 13.8 acre correspondingly. Most of organic farms apply their own compost as their fertilizer and their own ways to control pest, disease and weeds. Whereas most conventional farms are using NPK blue 15:15:15 and mostly using pesticides, insecticides and herbicides to control pest, disease and weeds in their farm. The essential studies required in this experiment is to determine the highest nitrate and nitrite content among 6 farms (3 organic and 3 conventional). There were significant differences (P0.05) in nitrate content between 6 farms, Ladybird organic farm, GK Gan organic, Yee Bio Farm, Hulu Yam, Serendah and Kundang. The highest nitrate content in bayam is 1617.04 mg/kg from Hulu Yam. As it is grown conventionally and used a lot of chicken dung and chemical fertilizer, it is expected to have higher nitrate content. However, the average of Koreas amaranth family that grown conventionally was 2174.2 mg/kg (Kim et al., 2011) which means more higher than our locals. The highest nitrate content in salad and sawi were both planted at Ladybird organic farm with 1563 mg/kg and 7389 mg/kg respectively. Whereas, according to Kim et al., nitrate concentration for Korean lettuce was 2620.4 mg/kg and green mustard was 4233.7 mg/kg. There were also significant differences (P0.05) in nitrite content between 6 farms included both organic and conventional farms. The highest nitrite content in bayam and sawi was from Hulu Yam, while GK Gan Organic farm has the highest nitrite content in salad. Moreover, nitrate content was found to be positively correlate with chromaticity (C*) of leaf colour and nitrite. According to Tuncay (2011) there is a positive correlation between the nitrogen and chlorophyll concentrations of leaves. However, the correlation coefficient between nitrate content and chlorophyll content were significantly negative in this study. This might be because of the conversion of nitrogen to nitrate that take place in the vacuole of the cell. Nitrite content was also found to be positively correlate with chlorophyll content and negatively correlate with lightness (L*) of leaf colour. FUTURE STUDY Nitrate content verification using colour chart in fresh leafy vegetables should be determine to assist supervision of fertilizer application in the future. Furthermore, the possible method to decrease nitrate content in leafy vegetables should be determined as information on nitrate and nitrite levels in leafy vegetables in Malaysia is currently lacking and most available data are from temperate climates.
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