GET THE APP

Cabbage Inhibits Nitrite Formation In Other Vegetable Juices During Storage
Journal of Nutrition & Food Sciences

Journal of Nutrition & Food Sciences
Open Access

ISSN: 2155-9600

Research Article - (2018) Volume 8, Issue 6

Cabbage Inhibits Nitrite Formation In Other Vegetable Juices During Storage

Jinming Huang1*, Cynthia Robinson1, Conner Callaway1, Samuel Pope1, Mackenzie Willis1, Nathan Probst1, Daniel B. Kim-Shapiro2, Trent Roberts1, Autumn Webb1 and Joshuah Hathcox1
1School of Mathematical and Natural Sciences, University of Arkansas at Monticello, Monticello, AR 71656, USA
2Department of Physics, Wake Forest University, Winston Salem, NC27109, USA
*Corresponding Author: Jinming Huang, Associate Professor of Chemistry, School of Mathematical and Natural Sciences, University of Arkansas at Monticello, Monticello, AR 71656, USA, Tel: 1-870-460-1866, Fax: 870-460-1316

Abstract

Although it is well known that green vegetables contain high content of nitrate, reported results regarding nitrite accumulation in fresh green vegetables are controversial. Recent studies suggest that nitrate from food has health benefits including reduce blood pressure and increase blood flow. Intake excess amount of nitrite is believed to cause increased risk of some cancers and methemoglobinemia in infants. In this study, we investigated the dynamics of nitrite and nitrate contents in spinach juice, iceberg lettuce juice, celery juice, green cabbage juice, and red cabbage juice. Nitrite concentration increased significantly in home-made spinach juice, iceberg lettuce juice, and celery juice after only two days of cold storage at 4°C; while nitrate concentrations in these vegetable juices decreased significantly with time during the storage, suggesting a conversion from nitrate to nitrite. However, no significant change in nitrite and nitrate concentrations observed in green cabbage juice and red cabbage juice during the storage. We further discovered that both green cabbage juice and red cabbage juice can completely inhibit the formation of nitrite in spinach juice, iceberg lettuce juice, and celery juice during cold storage at 4°C. Sodium tungstate, an inhibitor of nitrate reductase, was effective in inhibiting nitrite formation in celery juice during storage. The nitrite formation in these vegetable juices was much faster during storage at ambient temperature. The ability of cabbage to inhibit the formation of nitrite in other vegetable juice was also observed during storage at ambient temperature. However, if cabbage juice was boiled for five minutes prior to mixing with other vegetable juice then no inhibitory effect was observed. Cabbage had an inhibitory effect on nitrite formation when mixed with other vegetable juice. This effect was lost upon boiling, suggesting that cabbage may contain some compounds that can inhibit nitrate reductase that was decomposed by boiling.

Keywords: Nitrate; Nitrite; Cabbage; Vegetable juice; Inhibition; Chemiluminescence analysis

Introduction

The highest sources of nitrate in human diet are found in certain vegetables, such as spinach, lettuce, and celery. It was estimated that vegetables contribute about 80% of human dietary nitrate intake [1,2]. Although nitrate is not an essential nutrient for humans, recent studies have suggested that nitrate from food has health benefits [2,3]. It was reported that drinking nitrate rich beetroot juice could significantly reduce blood pressure [4] and increase regional blood flow in the brain of older adults [5].

Pure inorganic nitrate is an inert anion. However, nitrate in vegetables can be reduced to nitrite by some enzymes or bacteria during storage. The intake of excess amount of nitrite is believed to cause increased risk of some cancers and methemoglobinemia in infants [6,7]. There are many reports on the nitrate and nitrite contents in retail leave vegetables [2,8,9]. In respect to nitrate and nitrite contents change in leave vegetables during storage, there are inconsistent and controversial results; some reports indicated that nitrate levels dropped and nitrite levels increased dramatically in leave vegetables during storage in a few days at ambient temperature, but nitrate and nitrite levels did not change significantly over 7 days of storage in a refrigerator [10,11]; some researchers reported that the nitrite content in spinach increased about 27% after being frozen for 6 months at -25°C while no significant change in nitrate content [12]. However, other researchers reported that the nitrate content in canned vegetable- based infant foods increased 15% and 29% after 48 hours of refrigerated storage and room temperature storage respectively, while no significant change in nitrite content was observed [13]; it was also reported that both nitrate and nitrite contents in lettuce increased significantly during storage of 48 hours at refrigerated and room temperature [14]. Tamme et al. reported that the nitrite concentrations in raw home-made carrot juice, beetroot juice, and radish juice increased 1870, 275, and 518 fold respectively after 48 hours of storage at room temperature, while the nitrate concentrations in these juices decreased significantly [15]. The increase in nitrite concentration and decrease in nitrate concentration in these juices were much smaller after 48 hours of storage at refrigerated temperature. The study also showed no significant increase in nitrite concentration in raw home-made cabbage juice after 48 hours of storage at room temperature or refrigerated temperature [15].

We hypothesize that cabbage may contain some compounds that can resist the degradation of nitrate to nitrite. The aim of this study was to investigate if home-made cabbage juice can inhibit the formation of nitrite and the degradation of nitrate in other home-made vegetable juice during storage. If our hypothesis is correct it will provide useful information in food or vegetable juice storage procedure, since nitrate from vegetables has health benefit while nitrite has potential harmful effect to human, the formation of nitrite in vegetables during storage should be avoid.

Materials and Methods

All vegetables were purchased from a local supermarket. All chemicals were purchased from Sigma- Aldrich Chemical Company (St. Louis, MO, USA) unless otherwise indicated.

Sample preparation

For spinach (Spinacia oleracea), iceberg lettuce (Lactuca sativa), green cabbage (Brassica oleracea var. capitate), and red cabbage or purple cabbage (Brassica oleracea var. capitata f. rubra), all edible parts were used in preparing the juice; for celery (Apium graveolens), only edible stalks were used in preparing the juice. Eighty grams of any of the above fresh vegetables were weighed and washed with de-ionized water (DI H2O) and then homogenized in a blender with 200 ml DI H2O. The homogenized mixture was then filtered through cheesecloth to remove the solid residue and the juice volume was measured and recorded. All sample juices were immediately stored at different temperature conditions in dark before analysis.

Electrochemical measurement of nitrate

A nitrate ion selective electrode (OAKTON Instruments, Inc., Vernon Hills, IL) was used to determine the nitrate concentration in the vegetable samples using the standard addition method. Since very high concentrations of nitrate were present in all tested vegetables, sample juices were diluted 100 times with DI H2O before the determination. Briefly, 1.0 ml vegetable juice was added to 99.0 ml DI H2O while stirring and then the electric potential was measured. Next, a series of small volumes of standard solution of 0.1 M NaNO3 was added to the diluted juice and the electric potential was measured after each addition of standard nitrate solution. After a series of electric potential measurements, the nitrate concentration initially present in the diluted juice was calculated by fitting the series of electric potentials measured and the corresponding standard nitrate concentrations added with the Nernst equation: E = E0 –S*log (X + X0), where X0 is the nitrate concentration initially present in the diluted juice (μM), X is the standard nitrate concentration added to the diluted juice (μM), E0 is the reference electric potential which is a constant with a given electrode, E is the electric potential measured (mV), and S is the electrode slope (~ 59 mV at 25°C). The nitrate concentrations in vegetable juices or juice mixtures measured were then converted from μM to mg.l-1. The nitrate concentrations in the same type of vegetables or vegetable juices were reported from five different samples (Mean ± SD, n=5).

Griess assay of nitrite

A well-known and widely used method, the Griess assay, was used to determine the nitrite concentration. Briefly, 1.0 ml of a 1% sulfanilamide solution (in 5% H3PO4) was added to 1.0 ml of the vegetable juice that had been previously centrifuged and the solid residue was completely removed. This solution was incubated at room temperature in the dark for 10 min and then 1.0 ml of a 0.1% N-1- napthylethylenediamine (NED) solution (in DI H2O) was added and the mixture solution was again incubated at room temperature in the dark for another 10 min. The absorbance was then measured at 540 nm using a spectrophotometer (Shimadzu PharmaSpec UV-1700). The nitrite concentration (μM) was obtained by comparing the absorbance to a calibration curve. A calibration curve was constructed prior to the measurement of nitrite in the vegetable samples. A blank test using 1.0 ml DI H2O instead of 1.0 ml vegetable juice with everything else same was used to zero the spectrophotometer. A background test was done by adding 1.0 ml DI H2O instead of 1.0 ml of 0.1% NED solution with everything else same, the absorbance from this background was subtracted from absorbance of each vegetable samples. The nitrite concentrations in vegetable juices or juice mixtures measured were then converted from μM to mg.l-1. The nitrite concentrations in the same type of vegetables or vegetable juices were reported from five different samples (Mean ± SD, n=5).

Chemiluminescence measurement of nitrite

The chemiluminescence method is the most sensitive and commonly used method in measuring nitric oxide (NO) and its various derivatives including nitrite and nitrate. An aliquot (5 μl) of the vegetable juice was injected into the reaction vessel of a Sievers 280i Nitric Oxide Analyzer chemiluminescence detector. This apparatus directly detects NO and can be used for nitrite and nitrate detection under conditions where these species are converted to NO. For nitrite analysis, the reaction vessel contained 1% w/v KI in glacial acetic acid to reduce nitrite to NO. The concentration of nitrite was determined on the basis of a calibration curve [16]. The nitrite concentrations in the same type of vegetables or vegetable juices were reported from five different samples (Mean ± SD, n=5).

Nitrite and nitrate contents change during storage

For the cold storage experiments (4 ± 1°C) in vegetable juices, the nitrate and nitrite concentrations were determined every two days (48 hours) for up to eight days starting from day zero when the juices were prepared. For the ambient temperature storage experiment (25 ± 1°C) in vegetable juices, the nitrate and nitrite concentrations were determined every 24 hours for up to 72 hours (three days). For the cold storage experiment in whole fresh spinach, iceberg lettuce, celery, and cabbage, nitrate and nitrite concentrations were determined on the day when the vegetable were purchased (day zero) and day eight. For the ambient temperature storage experiment in whole fresh vegetables, nitrate and nitrite concentrations were measured every day over three days.

Cabbage juice inhibits the degradation of nitrate and the formation of nitrite in other vegetable juices

To investigate if cabbage juice can inhibit the degradation of nitrate and the formation of nitrite in other home-made vegetable juices (spinach, iceberg lettuce, or celery) during storage, 50 ml of cabbage juice was mixed with 50 ml of the other home-made vegetable juices (spinach, iceberg lettuce, or celery), in the volume ratio of 1:1 or 50% of cabbage juice. Nitrate and nitrite concentrations were then measured every 48 hours for up to eight days starting from day zero when the juices were prepared and mixed during cold storage in dark at 4°C. Each of these home-made vegetable juices (spinach, iceberg lettuce, or celery) were mixed with DI H2O instead of cabbage juice in the same volume ratio was set as a control sample. In a similar manner as was done for storage at 4°C, we studied if cabbage can inhibit the formation of nitrite in other home-made vegetable juices during storage at ambient temperature (25°C). In order to determine the minimum concentration of cabbage juice needed to inhibit the formation of nitrite and degradation of nitrate in other home-made vegetable juices, home-made green or red cabbage juices were mixed with home-made celery juice at volume ratios of 1:9, 1:5.7, 1:4, 1:3, and 1:1, or 10%, 15%, 20%, 25%, 50% of cabbage juice respectively; the nitrite concentrations were then measured every 48 hours for up to eight days during cold storage.

Experiments of sterilization and inhibitor

To study the mechanism of cabbage juice inhibiting the degradation of nitrate and the formation of nitrite in other home-made vegetable juice, cabbage juice was boiled at 100°C for five minutes before the incubation with other vegetable juice; the results were compared with those results from the incubation of other vegetable juice mixed with the cabbage juice without boiling. In inhibitor experiment, celery juice was mixed with 1mM sodium tungstate (from a stock solution of 100 mM in DI H2O), an inhibitor of nitrate reductase enzyme (NR), and stored in a refrigerator at 4°C; celery juice itself was set as a control and store under the same condition. Nitrate and nitrite concentrations were measured in the same manner as described above.

Statistical analysis

All results were obtained from five replications with different samples of the same type of vegetable. The data were analyzed using MS Excel 2010 software (Microsoft Corporation, Redmond, WA, USA). A t-test was used to reveal the statistical significance of differences in measured variables at different time points. Results with p<0.05 were considered to be statistically significant.

Results and Discussion

Nitrate contents in fresh spinach, iceberg lettuce, celery, green cabbage, and red cabbage on the day purchased were 2.65 ± 0.34 mg.g-1, 1.54 ± 0.23 mg.g-1, 2.14 ± 1.18 mg.g-1, 0.54 ± 0.28 mg.g-1, and 0.38 ± 0.29 mg.g-1 respectively. Previous reported nitrate contents in fresh spinach, iceberg lettuce, celery, and cabbage varied in a wide range of 0.029 ~ 8.00 mg.g-1, 0.079 ~ 4.38 mg.g-1, 0.046 ~ 2.67 mg.g-1, and 0.008 ~ 0.929 mg.g-1 respectively [2,12,17-25]. Nitrite contents were less than 0.1 mg.kg-1in all tested fresh vegetables. Previous reported nitrite contents in these vegetables were much lower than that of nitrate and varied in a wide range of 0.01- 200 mg.kg-1 [2,12,17,19,20,23-25].

No significant changes in the nitrate and nitrite contents were observed in all fresh vegetables tested during cold storage at 4°C over eight days (data not shown). However, nitrite concentrations increased significantly (p<0.05) in home-made spinach juice, iceberg lettuce juice, and celery juice after only two days of cold storage at 4°C. Table 1 shows that the nitrite concentrations in home-made spinach juice, iceberg lettuce juice, and celery juice increased more than 3860 fold (from less than 0.023 mg.l-1 to 88.79 mg.l-1), 1032 fold (from less than 0.023 mg.l-1 to 23.75 mg.l-1), and 1845 fold (from less than 0.023 mg.l-1 to 42.45 mg.l-1) respectively after eight days of cold storage at 4°C; while no significant change in nitrite concentration was observed in home-made green cabbage juice and red cabbage juice during the cold storage over eight days. Interestingly, we found that no significant change in nitrite concentration in mixed juice of green cabbage with spinach, iceberg lettuce, or celery (at the volume ratio of 1:1 or 50% cabbage juice) during eight days of cold storage; the same results were observed in red cabbage juice mixed with any of these vegetable juices during cold storage. However, if green cabbage juice or red cabbage juice was boiled for five minutes and brought to room temperature before mixing with these vegetable juices, the inhibitory effect on the formation of nitrite in these vegetable juices disappeared.

Vegetable juice Nitrite concentration (mg.l-1)a
Day 0 Day 2 Day 4 Day 6 Day 8
Spinach juice+ DI H2O (1:1)b <0.023 1.87d 33.37 49.79 88.79
Iceberg juice+ DI H2O (1:1)b <0.023 0.13d 1.83 5.87 23.75
Celery juice+ DI H2O (1:1)b <0.023 0.19d 1.38 10.05 42.45
Green cabbage juice + DI H2O (1:1)b <0.023 <0.023 <0.023 <0.023 <0.023
Red cabbage juice + DI H2O (1:1)b <0.023 <0.023 <0.023 <0.023 <0.023
Boiled spinach juicec <0.023 <0.023 <0.023 <0.023 0.37
Boiled iceberg juicec <0.023 <0.023 <0.023 <0.023 <0.023
Boiled celery juicec <0.023 <0.023 <0.023 <0.023 <0.023
Spinach juice + green cabbage juice (1:1) <0.023 <0.023 <0.023 <0.023 0.33
Iceberg juice + green cabbage juice (1:1) <0.023 <0.023 <0.023 <0.023 <0.023
Celery juice + green cabbage juice (1:1) <0.023 <0.023 <0.023 <0.023 <0.023
Celery juice + red cabbage juice (1:1) <0.023 <0.023 <0.023 <0.023 0.69
Spinach juice + boiled green cabbage juice (1:1) <0.023 0.63d 20.91 59.94 90.85
Iceberg juice + boiled green cabbage juice (1:1) <0.023 0.33d 2.26 7.26 25.89
Celery juice + boiled green cabbage juice (1:1) <0.023 0.15d 2.62 11.82 44.08
Celery juice + boiled red cabbage juice (1:1) <0.023 0.13d 2.37 12.83 45.52

Note: aNitrite concentrations were measured by the Griess assay, detection limit was 0.023 mg.l-1.
bVegetable juice mixed with DI H2O (volume ratio 1:1) was set as a control sample.
cVegetable juice was boiled for five minutes and cooled to room temperature before storage at 4°C was set as another control sample.
dNitrite concentration increased significantly in two days, p < 0.05.

Table 1: Changes of mean nitrite concentration in home-made vegetable juices and juice mixtures during storage at 4 ± 1°C (n=5).

That cabbage juice inhibits nitrite formation in other vegetable juices during cold storage at 4°C was also confirmed by the chemiluminescence method. Figure 1 shows very strong nitrite chemiluminescence signals in celery juice after six days of cold storage, while nitrite signals in the mixed juices of celery and green cabbage (at the volume ratio of 1:1 or 50% cabbage juice) were very weak indicating that green cabbage inhibits nitrite formation in celery juice. The very weak nitrite signals most likely came from cabbage itself as it contains a little nitrite. Figure 1 also shows that red cabbage can inhibit nitrite formation completely in celery juice. Similar to the results in Table 1 using the Griess assay, the chemiluminescence method confirmed that boiled green cabbage juice or red cabbage juice lost their ability in inhibiting nitrite formation in celery juice. This inhibitory effect on nitrite formation in other vegetable juices can be observed at concentrations of green or red cabbage juice as low as 10 ~ 15% (volume ratio of 1:5.7 to 1:9, Table 2).

nutrition-food-sciences-formation-nitrite

Figure 1: Cabbage inhibits the formation of nitrite in celery juice during storage at 4°C (at volume ratio of 1:1, or 50% cabbage juice). Nitrite concentrations in various vegetable juices or juice mixtures were measured by the chemiluminescence method on day 6 during storage. Each sample analysis was carried out in three injections.

Vegetable juice Nitrite concentration (mg.l-1)a
Day 0 Day 2 Day 4 Day 6 Day 8
Celery+Green Cabbage (1:1) <0.023 <0.023 <0.023 0.035 0.035
Celery+DI H2O (1:1)b <0.023 0.19c 1.38d 10.05d 42.45d
Celery+Green Cabbage (3:1) <0.023 <0.023 <0.023 0.036 0.03
Celery+DI H2O (3:1)b <0.023 0.033c 0. 44d 38.08d 89.71d
Celery+Green Cabbage (4:1) <0.023 <0.023 <0.023 0.043 0.33
Celery+DI H2O (4:1)b <0.023c 0.045c 0. 82d 44.61d 97.03d
Celery+Green Cabbage (5.7:1) <0.023 <0.023 0.026 0.052 0.41
Celery+DI H2O (5.7:1)b <0.023c 0.067c 0.89d 45.23d 99.35d
Celery+Green Cabbage (9:1) <0.023 <0.023 0.033 0.979 16.59
Celery+DI H2O (9:1)b <0.023c 0.12c 0.89d 48.36d 107.52d

Note: aNitrite concentrations were measured by the Griess assay, detection limit was 0.023 mg.l-1.
bCelery juice mixed with DI H2O at the same volume ratio as it mixed with green cabbage juice was set as the control sample.
cNitrite concentration in control celery juices increased significantly in two days, p<0.05.
dNitrite concentration in control celery juices was significantly higher than it in the mixed juice of celery with green cabbage on the same day, p<0.05.

Table 2: The effect of the concentration of green cabbage juice on inhibiting nitrite formation in celery juice during storage at 4 ± 1 °C (n=5)

Nitrate concentrations decreased 61.6% (from 343.1 mg.l-1 to 131.7 mg.l-1), 60.5% (from 203.5 mg.l-1 to 80.3 mg.l-1), and 34.1% (from 591.4 mg.l-1 to 390.0 mg.l-1) in home-made spinach juice, iceberg lettuce juice, and celery juice respectively after eight days of cold storage at 4°C (Table 3); while no significant change in nitrate concentration in green or red cabbage juice was observed during eight days of cold storage (Table 3). Green and red cabbage can completely inhibit the degradation of nitrate in home-made spinach juice, iceberg lettuce juice, and celery juice at a volume ratio of 1:1 (or 50% cabbage juice) during cold storage over eight days (Table 3). Again, if green cabbage juice or red cabbage juice was boiled for five minutes and brought to room temperature before mixing with these vegetable juices, no inhibitory effect on the degradation of nitrate in these vegetable juices was observed (Table 3).

Vegetable juice Nitrate concentration (mg.l-1)a
Day 0 Day 2 Day 4 Day 6 Day 8
Spinach juice+ DI H2O (1:1)b 343.1 338 281 208.9 131.7
Iceberg juice+ DI H2O (1:1)b 203.5 197.5 187.2 128.8 80.3
Celery juice+ DI H2O (1:1)b 591.4 568.3 525.5 468.5 390
Green cabbage juice + DI H2O (1:1)b 129.8 132.7 133.3 126.6 130
Red cabbage juice + DI H2O (1:1)b 86.7 85.6 84.9 84.3 84.6
Boiled spinach juicec 664.3 651.5 655.5 643.5 615.7
Boiled iceberg juicec 410.2 420.1 425.5 414.1 403.6
Boiled celery juicec 1265 1267 1199 1189 1156
Spinach juice + green cabbage juice (1:1) 488.4 490.7 491.5 488.9 470.6
Iceberg juice + green cabbage juice (1:1) 331.7 333.6 334. 9 336.9 335.7
Celery juice + green cabbage juice (1:1) 710.5 708.6 720.4 702.7 710.5
Celery juice + red cabbage juice (1:1) 668.4 667.8 666.7 659.1 655.3
Spinach juice + boiled green cabbage juice (1:1)d 495.3 479.9 436.9 326.1 250.4
Iceberg juice + boiled green cabbage juice (1:1)d 340.4 331.1 312.5 243.6 218.3
Celery juice + boiled green cabbage juice (1:1)d 733.2 719.6 677.7 567.3 390.1
Celery juice + boiled red cabbage juice (1:1)d 691 687.2 673.4 630.7 504.3

Note: aNitrate concentrations were measured by nitrate ion selective electrode (ISE).
bVegetable juice mixed with DI H2O (volume ratio 1:1) was set as a control sample.
cVegetable juice boiled for five minutes and cooled to room temperature before storing in a refrigerator was set as another control sample.
dVegetable juice mixed with the boiled cabbage juice (volume ratio 1:1) was set as another control sample.

Table 3: Changes of mean nitrate concentration in home-made vegetable juices and juice mixtures during storage at 4 ± 1°C (n=5).

The increases in nitrite concentrations in home-made iceberg lettuce juice and celery juice at ambient temperature (25°C) are much faster than that at 4°C. Nitrite concentrations increased more than 1215 fold (from less than 0.023 mg.l-1 to 27.98 mg.l-1) and 565 fold (from less than 0.023 mg.l-1 to 13.04 mg.l-1) in home-made iceberg lettuce juice and celery juice respectively after only one day (24 hours) storage at 25°C (Table 4). Again, no significant change in nitrite concentration was observed in green cabbage juice over three days of storage at 25°C. Furthermore, green cabbage juice can inhibit nitrite formation in home-made iceberg lettuce juice and celery juice over two days (at a volume ratio of 1:1) of storage at 25°C (Table 4).

Vegetable juice Nitrite concentration (mg.l-1)a
0 hr 24 hr 48 hr 72 hr
Iceberg juice + DI H2O (1:1)b <0.023 27.98d 61.55e 47.21
Celery juice + DI H2O (1:1)b <0.023 13.04d 26.52e 44.31
Green cabbage juice + DI H2O (1:1)b <0.023 <0.023 <0.023 0.55
Boiled iceberg juicec <0.023 <0.023 <0.023 2.3
Boiled celery juicec <0.023 <0.023 0.11 45.2
Iceberg juice + green cabbage (1:1) <0.023 <0.023 4.50e 10.3
Celery juice + green cabbage (1:1) <0.023 <0.023 3.33e 22.13

Note: aNitrite concentrations were measured by the Griess assay, detection limit was 0.023 mg.l-1
bVegetable juice mixed with DI H2O (volume ratio 1:1) was set as a control sample.
cVegetable juice boiled for five minutes and cooled to room temperature before storage was set as another control sample.
dNitrite concentration in iceberg juice and celery juice that mixed with DI H2O (volume ratio 1:1) increased significantly in 24 hours (p<0.05).
eNitrite concentrations in iceberg juice and celery juice that mixed with DI H2O were significantly higher than that in iceberg juice and celery juice that mixed with green cabbage juice after 48 hours of storage (p<0.05).

Table 4: Changes of mean nitrite concentrations in home-made vegetable juices and juice mixtures during storage at 25 ± 1°C (n=5).

It is well known that nitrate in plants can be reduced to nitrite by nitrate reductase (NR) [26-29]. Spinach and lettuce contain NR [30-33]. The above results suggest that NR initially present in spinach, iceberg lettuce, and celery may be responsible for the reduction of nitrate to nitrite. Our experimental results showed that the nitrate concentration decreased with time in spinach juice, iceberg lettuce juice, and celery juice (Table 3), while the nitrite concentration increased with time during storage in a refrigerator at 4°C (Table 1), suggesting a conversion from nitrate to nitrite. When enzymes were deactivated by boiling for five minutes, no changes in nitrate concentration or nitrite concentration were observed during the storage (Tables 1 and 3). Our experimental results from these boiled vegetable juices suggest that NR may be responsible for the reduction of nitrate to nitrite during the storage, since boiling for five minutes deactivated only the enzyme initially present in the vegetables. If exogenous bacteria such as bacteria from the air were responsible for the nitrate reduction, we should observe some nitrite formation in these boiled vegetable juices as bacteria can enter juices during the eight days of storage. The experimental results of tungstate, an inhibitor of NR, inhibiting nitrite formation in celery juice during storage at 4°C confirmed that NR was responsible for nitrite formation in celery juice during storage (Table 5). However, these results cannot exclude the role of bacteria that were initially present in these vegetables as they were deactivated as well by boiling. Recent report from Watanabe’s lab showed the evidence of microbial NR involved in the formation of nitrite in spinach leaves during storage [8].

Vegetable juice Nitrite concentration (mg.l-1)a
Day 0 Day 2 Day 4 Day 6 Day 8
Celery juice + DI H2O (1:1, v/v)b <0.023 0.19c 1.38 10.05 42.45
Celery juice + DI H2O (1:1, v/v) + 1mM Na2WO4 <0.023 <0.023 <0.023 <0.023 <0.023
Celery juice + green cabbage juice (1:1, v/v) <0.023 <0.023 <0.023 <0.023 <0.023

Note: aNitrite concentrations were measured by the Griess assay, detection limit was 0.023 mg.l-1.
bCelery juice mixed with DI H2O (volume ratio 1:1) was set as a control sample.
cNitrite concentration in celery juice mixed with DI H2O increased significantly in two days, p<0.05.

Table 5: The effect of nitrate reductase inhibitor (1 mM Na2WO4) on the formation of nitrite in home-made celery juices during storage at 4 ± 1°C (n=5).

Our experimental results showing no significant increase in nitrite concentration in cabbage juices during cold storage are similar to a report from Tamme’s lab except that their storage period was 48 h [15]. Our finding that no significant change in nitrate and nitrite contents in all fresh vegetables tested during cold storage at 4°C over eight days is consistent with the suggestion of nitrite accumulation merely occurs in intact fresh vegetables but only occurs in physically damaged vegetables as discussed by Phillips [34]. More interestingly, we discovered that cabbage (green or red) can inhibit the formation of nitrite and the degradation of nitrate in other vegetable juices during storage. This inhibitory effect can be observed at cabbage juice concentrations as low as 10 ~ 15%. The mechanism of cabbage juice inhibiting the formation of nitrite and degradation of nitrate in other vegetable juices during storage is not clear at present. It is possible that two components were involved in the inhibitory effect. It appears that a component contained in green cabbage lost its inhibitory effect upon boiling. On the other hand, another component contained in red cabbage retained some activity even after the treatment as Figure 1 shows. These results suggest that one component is heat sensitive and the other one in red cabbage is heat resistant. Cabbage contains large amount of glucosinolates which can be hydrolyzed to volatile sulfur compounds such as isothiocyanates [35]. These molecules are known as bioactive compounds that show multiple functions such as antimicrobial activity [36]. Red cabbage is rich in a number of bioactive compounds including anthocyanins which also exhibits multiple functions such as antioxidant activity, enzyme inhibition, and antimicrobial activity [37]. It is also possible that plant 14-3-3 proteins involved in the inactivation of NR [38,39]. We will further study the mechanism of cabbage juice inhibiting the formation of nitrite and the degradation of nitrate in other vegetable juices in the future. To the extent that the reduction of nitrate to nitrite during storage has potential harmful effects on health, our finding that cabbage juice inhibits the formation of nitrite in other vegetable juices may provide useful information for storage procedures.

Conclusion

Cabbage can completely inhibit the increasing in nitrite and decreasing in nitrate contents in other home-made vegetable juices (spinach, iceberg lettuce, and celery) during the cold storage at 4°C over eight days or ambient temperature storage over two days. The lowest concentration (volume ratio) that can inhibit nitrite formation in other home-made vegetable juices is about 10 ~ 15% of cabbage juice (or volume ratio 1:5.7 to 1:9). However, cabbage juice lost its inhibitory effect upon boiling for five minutes prior to mix with other home-made vegetable juices.

Acknowledgement

We thank Dr. Mary Stewart for her critical reading of the manuscript. We also thank Ms. Alexandra White for her technical assistance. This work was supported by Arkansas Space Grant Consortium [grant numbers 19096, 20078, 21053, 22073, 23022, 24020]; National Science Foundation and Arkansas Science & Technology Authority [Grant numbers 11-EPSCoR-0030, 12-EPS2-0026]; Arkansas ASSET Initiatives [grant numbers EPS-0701890, EPS1003970]; Arkansas Department of Higher Education [SURF grant]. This work was also partially supported by University of Arkansas at Monticello Faculty Development Fund.

References

  1. Lundberg JO, Carlstrom M, Larsen FJ, Weitzberg E (2011) Roles of dietary inorganic nitrate in cardiovascular health and disease. Cardiovasc Res 89: 525-532
  2. Hord NG, Tang YP, Bryan NS (2009) Food sources of nitrates and nitrites: the physiologic context for potential health benefits. Am J Clin Nutr 90: 1-10
  3. Milkowski A, Garg HK, Coughlin JR, Bryan NS (2010) Nutritional epidemiology in the context of nitric oxide biology: a risk–benefit evaluation for dietary nitrite and nitrate. Nitric Oxide 22: 110-119
  4. Webb AJ, Patel N, Loukogeorgakis S, Okorie M, Aboud Z, et al. (2008) Acute blood pressure lowering, vasoprotective and anti-platelet properties of dietary nitrate via bio-conversion to nitrite. Hypertension 51: 784-790
  5. Presley TD, Morgan AR, Bechtold E, Clodfelter W, Dove RW, et al. (2011) Acute effect of a high nitrate diet on brain perfusion in older adults. Nitric Oxide 24: 34-42
  6. Cassens R (1997) Residual nitrite in cured meat. Food Technol 51: 53-55
  7. Chan TYK (2011) Vegetable-borne nitrate and nitrite and the risk of methaemoglobinaemia. Toxicol Lett 200: 107-108
  8. Watanabe NS, Yamasaki H (2016) Dynamics of nitrite content in fresh spinach leaves: evidence for nitrite formation caused by microbial nitrate reductase activity. J Nutr Food Sci 7: 572
  9. Nuñez de González MT, Osburn WN, Hardin MD, Longnecker M, Garg HK, et al. (2015) A survey of nitrate and nitrite concentrations in conventional and organic-labeled raw vegetables at retail. J Food Sci 80: C942-C949
  10. Chung JC, Chou SS, Hwang DF (2004) Changes in nitrate and nitrite content of four vegetables during storage at refrigerated and ambient temperatures. Food Addit Contam 21: 317-322
  11. Lin JK, Yen JY (1980) Changes in the nitrate and nitrite contents of fresh vegetables during cultivation and postharvest storage. Food Cosmet Toxicol 18: 597-603
  12. Jaworska G (2005) Nitrates, nitrites, and oxalates in products of spinach and New Zealand spinach: effect of technological measures and storage time on the level of nitrate, nitrite, and oxalates in frozen and canned products of spinach and New Zealand spinach. Food Chem 93: 395-401
  13. Tamme T, Reinik M, Roasto M, Meremae K, Kiis A (2009) Impact of food processing and storage conditions on nitrate content in canned vegetable-based infant foods. J Food Prot 72: 1764-1768
  14. Silalahi J, Nasution AF, Ginting N, Silalahi YCE (2016) The effect of storage condition on nitrite and nitrate content in lettuce. Int J Pharm Tech Res 9: 422-427
  15. Tamme T, Reinik M, Pussa T, Roasto M, Meremae K, et al. (2010) Dynamics of nitrate and nitrite content during storage of home-made and small-scale industrially produced raw vegetable juices and their dietary intake. Food Addit Contam 27: 487-495
  16. Huang J, Hadimani SB, Rupon JW, Ballas SK, Kim-Shapiro DB, et al. (2002) Iron nitrosyl hemoglobin formation from the reactions of hemoglobin and hydroxyurea. Biochemistry 41: 2466-2474
  17. Siciliano J, Krulick S, Heisler EG, Schwartz JH, White JW (1975) Nitrate and nitrite content of some fresh and processed market vegetables. J Agric Food Chem 23: 461-464
  18. Santamaria P, Elia A, Serio F, Todaro E (1999) A survey of nitrate and oxalate content in fresh vegetables. J Sci Food Agric 79: 1882-1888
  19. Chou SS, Chung JC, Hwang DF (2003) A high performance liquid chromatography method for determining nitrate and nitrite levels in vegetables. J Food Drug Anal 11: 233-238
  20. Chung SY, Kim JS, Kim M, Hong MK, Lee JO, et al. (2003) Survey of nitrate and nitrite contents of vegetables grown in Korea. Food Addit Contam 20: 621-628
  21. De Martin S, Restani P (2003) Determination of nitrates by a novel ion chromatographic method: occurrence in leafy vegetables (organic and conventional) and exposure assessment for Italian consumers. Food Addit Contam 20: 787-792
  22. Sanchez CA, Crump KS, Krieger RI, Khandaker NR, Gibbs JP (2005) Perchlorate and nitrate in leafy vegetables of North America. Environ Sci Technol 39: 9391-9397
  23. Tamme T, Reinik M, Roasto M, Juhkam K, Tenno T, et al. (2006) Nitrates and nitrites in vegetables and vegetable-based products and their intakes by the Estonian population. Food Addit Contam 23: 355-361
  24. Czech A, Pawlik M, Rusinek E (2012) Contents of heavy metals, nitrates, and nitrites in cabbage. Pol J Environ Study 21: 321-329
  25. Afzali SF, Elahi R (2014) Measuring nitrate and nitrite concentrations in vegetables, fruits in Shiraz. J Appl Sci Environ Manage 18: 451-457
  26. Crawford NM (1995) Nitrate: nutrient and signal for plant growth. Plant Cell 7: 859-868
  27. Campbell WH (1999) Nitrate reductase structure, function and regulation: bridging the gap between biochemistry and physiology. Annu Rev Plant Physiol Plant Mol Biol 50: 277-303
  28. Suzuki S, Kataoka K, Yamaguchi K (2000) Metal coordination and mechanism of multicopper nitrite reductase. Acc Chem Res 33: 728-735
  29. Einsle O, Messerchmidt A, Huber R, Kroneck PM, Neese F (2002) Mechanism of the six-electron reduction of nitrite to ammonia by cytochrome c nitrite reductase. J Am Chem Soc 124: 11737-11745
  30. Ida S, Morita Y (1973) Purification and general properties of spinach leaf nitrite reductase. Plant Cell Physiol 14: 661-671
  31. Nakagawa H, Yonemura Y, Yamamoto H, Sato T, Ogura N, et al. (1985) Spinach nitrate reductase. Plant Physiol 77: 124-128
  32. Swamy U, Wang M, Tripathy JN, Kim SK, Hirasawa M, et al. (2005) Structure of spinach nitrite reductase: implications for multi-electron reactions by the iron-sulfur: siroheme cofactor. Biochemistry 44: 16054-16063
  33. Matraszek R (2008) Nitrate reductase activity of two leafy vegetables as affected by nickel and different nitrogen forms. Acta Physiol Plant 30: 361-370
  34. Phillips WEJ (1968) Changes in the nitrate and nitrite contents of fresh and processed spinach during storage. J Agric Food Chem 16: 88-91
  35. Rouzaud G, Young SA, Duncan AJ (2004) Hydrolysis of glucosinolates to isothiocyanates after ingestion of raw or microwaved cabbage by human volunteers. Cancer Epidemiol Biomarkers Prev 13: 125-131
  36. Tang L, Paonessa JD, Zhang Y, Ambrosone CB, McCann SE (2013) Total isothiocyanate yield from raw cruciferous vegetables commonly consumed in the United States. J Funct Foods 5: 1996-2001
  37. Wiczkowski W, Szawara-Nowak D, Topolska J (2013) Red cabbage anthocyanins: Profile, isolation, identification, and antioxidant activity. Food Res Int 51: 303-309
  38. Lambeck IC, Fischer-Schrader K, Niks D, Roeper J, Chi JC, et al. (2012) Molecular mechanism of 14-3-3 protein-mediated inhibition of plant nitrate reductase. J Biol Chem 287: 4562-4571
  39. Moorehead G, Douglas P, Morrice N, Scarabel M, Aitken A, et al. (1996) Phosphorylated nitrate reductase from spinach leaves is inhibited by 14-3-3 proteins and activated by fusicoccin. Curr Biol 6: 1104-1113
Citation: Huang J, Robinson C, Callaway C, Pope S, Willis M, et al. (2018) Cabbage Inhibits Nitrite Formation in other Vegetable Juices during Storage. J Nutr Food Sci 8: 742.

Copyright: © 2018 Huang J, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.