The influence of storage condition on nitrite, nitrate and vitamin

Vegetables are the main sources of nitrate and nitrite in food. The presence of nitrate and nitrite at a high level may cause a negative impact on health, because nitrite and nitrate when reduced to nitrite, may react with alkylamine to form carcinogenic nitrosamine. The influence of temperature and time of storage on nitrite, nitrate, and vitamin C contents in vegetables were investigated in this study. The vegetables were sweet mustard, bokchoy, spinach and lettuce obtained from a local market. Samples were stored at ±25 C and ±5 C. Analysis of nitrite, nitrate, and vitamin C was conducted in fresh samples, after storage for 24 and 48 hours. Nitrite was analyzed by spectrophotometry at 540 nm. Nitrate reduced into nitrite with Zn in acidic conditions and then analyzed as nitrite. Vitamin C was analyzed by titration with 2.6-dichlorophenolindophenol. During storage, nitrite and nitrate increased, while vitamin C decreased. Nitrite and nitrate content in fresh samples were 15.22 and 22.46 mg/kg (sweet mustard), 12.57 and 6.55 mg/kg (bokchoy), 20.26 and 90.60 mg/kg (spinach), 18.77 and 32.68 mg/kg (lettuce), respectively. Vitamin C content in fresh samples  was 101.15 mg/100g (mustard), 92.17 mg/100g (bokchoy), 88.95 mg/100g (spinach), 40.03 mg/100g (lettuce). After storage for 48 hours at ±25 C, nitrite and nitrate increased 44.97% and 53.19% (mustard), 46.18% and 62.59% (bokchoy), 43.86% and 16.48% (spinach), and 41.05% and 47.09% (lettuce), respectively. Vitamin C decreased 67.57% (mustard), 24.68% (bokchoy), 81.25% (spinach), and 79.74% (lettuce). Storage at ±5 C, showed that nitrite and nitrate increased 27.54% and 35.08% (mustard), 13.75% and 43.51% (bokchoy), 19.59% and 10.60% (spinach), 19.85% and 25.16% (lettuce), respectively. Vitamin C decreased 30.88% (mustard), 6.05% (bokchoy), 60.92% (spinach), and 74.94% (lettuce). During storage, nitrite and nitrate increased more significantly at ±25 C than  ±5 C while vitamin levels C decreased and were more effective at 25 C than 5 C.


Introduction
Vegetables are a major source of nitrite and nitrate intake from food. Nitrite and nitrate are also used as preservatives and coloring agents in processed meats [1][2][3][4][5] . Nitrate and nitrite contents in vegetables vary widely from 1 to 10.000 mg/kg, and this is affected by many factors, including environmental factors such as storage condition, processing procedure, temperature and agricultural practices [6][7][8][9] .
Nitrate can be reduced into nitrite by enzyme nitrate reductase and other reducing agents, including vitamin C, which is also contained in vegetables. Nitrite may react with alkylamines to form carcinogenic nitrosamines 10,11 . Therefore, the intake permitted (Acceptable Daily Intake = ADI) by the Food and Agriculture Organization of the United Nations/World Health Organization is 220 mg of nitrate and 8 mg of nitrite per day for adults weighing an average of 60 kg 12,13 . Previous studies reported that the longer the storage, the higher nitrite and nitrate contents. These effects are more influential at room temperature than at refrigeration. But the effect of temperature and storage condition on vitamin C have not yet been reported to the best of our knowledge. The aim of this study was to investigate the effect of storage condition on nitrite, nitrate and vitamin C contents in vegetables.

Samples
The vegetables analyzed in this study were sweet mustard (Brassica rapa chinensis), bokchoy (Brassica rapa L.), spinach (Amaranthus tricolor L), and lettuce (Lactuca sativa L). These vegetables were obtained from a local market in Medan, Indonesia. Samples were stored for 0, 24, until 48 hours at room temperature (±25°C) and in a refrigerator (±5°C).

Determination of absorbance curve of nitrite standard solution
In total, 4 ml of standard solution of nitrite (C=10.0 μg/ml) was transferred into 50 ml volumetric flask, added 2.5 ml sulfanilic acid solution and shaken. After 5 min, 2.5 ml NED reagent was added and made to volume with distilled water and homogenized (C=0.8 μg/ml). Absorbance was measured at wave length of 400-800 nm. Then, absorbance and wave length was plotted to construct absorbance curve. Wave length of maximum absorbance was determined from the absorbance curve 10 .
Absorbance stability of derivatized nitrite to determine working time In total, 4 ml of standard solution of nitrite (C=10.0 μg/ml) was transferred into volumetric flask of 50 ml, to which 2.5 ml of sulfanilic acid and stirred. After 5 min, 2.5 ml NED reagent was added and distilled water was added to make 50 ml. Absorbance was measured at wave-length of maximum absorbance obtained from absorbance curve (540 nm), and stability of absorbance was determined by observing absorbance at every minute for 1 hr. The absorbance was found to be relatively stable within 6 min in 7-12 min 10 .

Determination of calibration curve
Standard solution of nitrite (C=10.0 μg/ml) of different volume (0.5, 1, 2, 3, 4 dan 5 ml) were transferred into separated volumetric flasks of 25 ml, then 2.5 ml sulfanilic acid reagent added and stirred to homogenize. After 5 min, 2.5 ml NED reagent was added, then distilled water was added to make volume of 25 ml and homogenized. The series of concentration of prepared solutions were of 0.1 μg/ml, 0.2 μg/ml, 0.4 μg/ml, 0.8 μg/ml, 1.0 μg/ml. Absorbance of each solution was measured at wave-length of 540 nm within 7 min. Calibration curve was made by plotting absorbance versus concentration of each solution.
From the graph obtained, then linearity of regression equation and correlation coefficient were calculated (Y=aX+b) 10 .

Identification of nitrite and nitrate in vegetables
About 10 g ground sample, using blender, was transferred into a glass beaker. Distilled water was added to about 150 ml, heated in a waterbath (80°C) and shaken for 5 minute then cooled and filtered. The supernatant was transferred into a test tube, then 2.5 ml sulfanilic acid reagent was added and stirred. After 5 minutes, 2.5 ml reagent NED was added. Nitrite was identified using sulfanilic acid and NED solution, and the appearance of a violet color indicated the presence of nitrite. Nitrate was identified by adding several drops ferrous sulfate solution and then slowly adding a few drops of concentrated sulfuric acid. The formation of chocolate ring indicates the presence of nitrate 10 .

Quantification of nitrite and nitrate in vegetables
Nitrite. Determination of nitrite was carried out with procedure previously described 10 . Around ten (10) gram grounded sample transferred into 250 ml beaker glass to which hot distilled water (± 80ºC) was added about 150 ml. This mixture was homogenized by stirring and heated on waterbath for 15 minute while stirring. Allowed to cool and then transferred quantitatively into 250 ml volumetric flask, distilled water added to volume, then filtered. Ten (10 ml) of filtrate transferred into a volumetric flask of 50 ml, then 2.5 ml sulfanilic acid reagent was added and stirred. After 5 minutes, 2.5 ml reagent NED was added, then distilled water added to make 50 ml, and then homogenized. Absorbance was measured at wavelength of 540 nm after period of 7 to 12 minutes time.
Nitrate. In total, 10 g grounded sample transferred into 250 ml beaker glass to which hot distilled water (± 80ºC) was added to about 150 ml. This mixture was homogenized by stirring and heated on waterbath for 15 minute while stirring. Allowed to cool and then transferred quantitatively into 250 ml volumetric flask, distilled water added to volume, then filtered. 10 ml of filtrate transferred into a volumetric flask of 50 ml, then 0.1 g Zn powder and 1 ml HCl 1 N added and allowed to stand for 10 minutes to reduce nitrate to nitrite, then 2.5 ml sulfanilic acid reagent was added and stirred. After 5 minutes, 2.5 ml reagent NED was added, then distilled water added to make 50 ml, and then homogenized. Absorbance was measured at wavelength of 540 nm after period of 7 to 12 minutes time.
Nitrite concentration from reduction of nitrate into nitrite was calculated: Nitrite concentration from nitrate reduction = Total nitrite content -Initial nitrite concentration in samples Identification of vitamin C in vegetables 10 gram grounded sample using blender. About 0.5 ml of sample solution in a test tube was neutralized to a pH of 6-8 with NH 4 OH 1 N, three drops of 3% FeCl 3 was added -a purple color indicates the presence of vitamin C.
Analysis of vitamin C in vegetables 10 g grounded sample using blender was transferred into 100 ml volumetric flask, then acetic metaphosphoric acid 3% added to make 100 ml, then homogenized and filtered. Two (2) ml of filtrate was transferred into an erlenmeyer, and then added 5 ml of acetic metaphosphoric acid, then titrated with 2.6-dichlorophenol indophenol solution 0.025% until pink steadily 11 . The levels of vitamin C was calculated.

Nitrite and nitrate
It is found that samples contain nitrite indicated by the appearance of violet color to prove that all samples contained nitrite. The reaction with antipyrine in dilute hydrochloric resulted in the formation of green color to prove the present of nitrite. Nitrate in samples was identified using ferrous sulfate and concentrated sulfuric acid produced brown ring 10 .
Vitamin C Identification using FeCl 3 3% reagent generated violet color to prove that all samples contained vitamin C 11 .
The wave-length of maximum absorption of nitrite derivative The absorbance curve of the nitrite derivative solution (10 μg/ml) is presented in Figure 1. From Figure 1 it is shown that the maximum absorption was at 540 nm, which is similar to the value previously reported 2,7 , which was used to determine the analysis of nitrite and nitrate in samples.

Working time for measurement
Working time for nitrite and nitrate analysis was determined to know the period of time within which the absorbance of solution still remains stable. Absorbance of nitrite derivative with Griess reagent presented in Figure 2. Figure 2 shows that absorbance was stable within minute 7 to minute 12 then used in the analysis procedure 2,7 .

Influence of storage condition
The levels of nitrite, nitrate, and vitamin C during storage at ±25°C and ±5°C can be seen in Table 1 and Table 2.

Discussion
From Table 1 and Table 2 it can been seen that nitrite levels are generally relatively low in fresh vegetables compared to nitrate levels, except in bokchoy. This value is similar with those reported by researchers that nitrate is usually higher than nitrite 12 . The nitrate in plant also changes with age of the plant. The differences in nitrate content may be due to the fertilization, harvesting time, and storage time 8-12 .
The results indicate that storage temperature and time affect nitrite, nitrate, and vitamin C levels in vegetables. The longer the storage time the higher nitrite and nitrate levels and the lower vitamin C levels. These effects are more influential at room temperature than at refrigeration, as has previously been reported 3,12 .

62.32
Note: data is the mean of six replicates Table 1 and Table 2, suggest that the vitamin C and other antioxidant content in vegetables may reduce nitrate into nitrite, and then nitrite may react with amine compounds, especially secondary amines to form a carcinogenic nitrosamine. On the other hand, ascorbic acid available in fresh vegetables may prevent the formation of nitrosamine 8-12 .

Conclusion
Storage condition affects nitrite, nitrate and vitamin C content in vegetables. The higher the temperature and the longer the time of storage, the higher nitrite and nitrate levels,and the lower vitamin C levels. This effect is more influential at 25°C than at 5°C. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.