Cangzhou Normal University, Cangzhou 061000, China

Abstract This paper studied and compared the effects of three preservation methods (normal temperature 20℃, fresh-keeping box cold storage 4℃, and fresh-keeping box+fresh-keeping film cold storage 4℃) on six kinds of vegetables. The results showed that the content of vitamin C in the six vegetables was higher in cold storage than in normal temperature storage; the nitrate content was lower in cold storage than in normal temperature storage. In both the normal temperature storage and cold storage, the vitamin C in vegetables declined with the increase of the storage time, while the nitrate content increased with the increase of the storage time. Thus, cold storage has better fresh-keeping effect for vegetables. In cold storage conditions, the vitamin C content was higher in vegetables packaged by fresh-keeping box, and the nitrate content was lower, followed by the fresh-keeping film+fresh-keeping box cold storage. In conclusion, the best preservation method for vegetable is fresh-keeping box cold storage.

Key words Preservation methods, Vegetable, VC content, Nitrate content, Ultraviolet spectrophotometry

1 Introduction

Vegetables are essential plant foods in people’s daily diet, and people can’t live without vegetables. Many kinds of nutrients and vitamins exist in vegetables, especially vitamin C is abundant in vegetables. Adequate vitamin C can strengthen the body’s immunity, enhance physical fitness, prevent cancer, and keep the skin smooth and tender. Some studies have shown that 90% of vitamin C in human diets comes from fruits and vegetables[1-2]. An important channel for human consumption of vitamin C is vegetables. However, vitamin C in vegetables is the most unstable. It will be destroyed during cooking, and may be decomposed from oxidation during the preservation of vegetables.

In recent years, different scholars have applied different methods in the determination of vitamin C, such as ultraviolet spectrophotometry, chromatography, titration, colorimetry and electrophoresis. Through summary of previous studies, it is known that UV spectrophotometry is the most common method because it is not so demanding on samples and operators, the instrument used is relatively simple to operate, the measurement results are accurate, and the measurement process is fast, thus reducing the oxidation of vitamin C, accordingly the data measured by ultraviolet spectrophotometry is relatively reliable.

According to the characteristics of the selected samples and their own capacities and the conditions of the laboratory, we used the UV spectrophotometry to determine the content of vitamin C in the six vegetables atλ=265 nm. According to the absorbance A, we checked the corresponding concentration on the standard curve, and then we calculated the vitamin C content in the sample using the formula.

With the continuous improvement in the quality of life of modern people, people pay more attention to the quality of life and to the nutrients in the diet. Vegetables occupy an important position in people’s daily diet, while the content of nitrate is an indicator for evaluating the quality of vegetables[3-6]. Besides, vegetables are particularly easy to accumulate nitrates. Therefore, research on changes in the content of nitrates in vegetables is extremely important. In previous studies, it was found that leafy vegetables and root vegetables are the most likely to accumulate nitrates, and 81.20% of the nitrate content in humans is derived from vegetables[7-9]. Nitrate itself is not very toxic to human body, but nitrate can be reduced to nitrite, especially in the human body, reduced to nitrite by the action of bacteria[10-12], nitrite is toxic and it can reduce the ability of blood to carry oxygen to the body, resulting in methemoglobinemia (MHb), which can lead to death in severe cases. Therefore, controlling the nitrate content in vegetables is one of the important channels to control the intake of nitrate in the human body.

At present, the methods for determining nitrate content mainly include ultraviolet spectrophotometry, spectroscopy, chromatography, and electrophoresis. Each method has its own advantages and disadvantages. According to the capacities and characteristics of selected samples and laboratory conditions, we decided to choose UV spectrophotometry. Through consulting the reference literature, we selected the saturated borax solution as the buffer solution to extract the nitrate ions in vegetables by heat extraction method, and determined the absorbance A according to the maximum absorption peak of nitrate ions atλ=220 nm, and checked the corresponding concentration on the working curve, and calculated the nitrate content in the samples. This method is accurate, reliable, simple and fast.

In this experiment, we selected six common vegetables as experimental materials: rape (BrassicacampestrisL.), cauliflower (BrassicaoleraceaL.), green pepper (CapsicumannuumL.), cucumber (CucumissativusL.), carrot (DaucuscarotaL.), and potato (SolanumtuberosumL.). We studied the effects of different preservation methods on the content of vitamin C and nitrate in vegetables, and obtained the best preservation method for fresh vegetables, in the hope of increasing the intake of vitamin C in vegetables and reduce the intake of nitrate.

2 Materials and methods

2.1 Experimental materialsRape (B.campestrisL.), cauliflower (B.oleraceaL.), green pepper (C.annuumL.), cucumber (C.sativusL.), carrot (D.carotaL.), and potato (S.tuberosumL.).

2.2 Medicines and reagentsMedicines: activated carbon, potassium ferrocyanide, potassium nitrate, zinc sulfate, borax.

Reagents: vitamin C standard solution, nitrate standard solution, chloroform, 1 mol/L hydrochloric acid, saturated borax, and distilled water.

Solution preparation: (i) Vitamin C standard solution: precisely weigh 0.1 g of vitamin C and place in distilled water, then transfer to a 100-mL volumetric flask, dilute to the desired volume, and shake up. Precisely pipette 10 mL of the prepared solution into a 100-mL volumetric flask, dilute to the desired volume, and shake up, then obtain 0.1 mg/mL of vitamin C standard solution. (ii) Nitrate standard stock solution: dry the potassium nitrate in an oven at 110℃, and precisely weigh 0.1631 g of dried potassium nitrate with an electronic balance, dissolve it in distilled water, then add with a few drops of chloroform to prevent corrosion, and then transfer to a 1 000-mL volumetric flask, dilute to the desired volume and shake up, to obtain the nitrate stock solution[13]. (iii) Saturated borax solution: precisely weigh 25 g of borax with an electronic balance, dissolve it with hot water, then pour it into a 500-mL volumetric flask, dilute it to the desired volume with hot water, shake up, place it in a glass bottle, prepare the solution just before the experiment. (iv) 1 mol/L hydrochloric acid solution: take 40 mL of concentrated hydrochloric acid in a measuring cylinder, pour into distilled water, then pipette 0.75 mL into distilled water, pour into a 500-mL volumetric flask, dilute to the desired volume, shake up, and place in a brown bottle for use. (v) Ferrous chloride potassium chloride solution: precisely weigh 75 g of ferrous chloride potassium chloride solution with an electronic balance, dissolve it with distilled water, then transfer a 500-mL volumetric flask, dilute it to the desired volume with distilled water, shake up, and place it in a glass bottle. (vi) Zinc sulfate solution: precisely weigh 150 g of zinc sulfate solution, dissolve it with distilled water, then pour it into a to 500-mL volumetric flask, dilute it to the desired volume with distilled, shake up, and place it in a glass bottle.

2.3 InstrumentsUltraviolet spectrophotometer (TU1810), constant temperature water bath, tissue tearor, electronic analytical balance, volumetric flask (50, 200, 500, 1 000 mL), 10 mL pipette, glass rod, measuring cylinder, beaker, conical flask, qualitative filter paper, gauze, funnel,etc.

2.4 Experiment method

2.4.1Experiment treatment. After the vegetables are bought, we selected the intact ones and divide them into three treatments: (i) store at room temperature, store the vegetables in the room at 20℃; (ii) put the vegetables in the fresh-keeping box, adjust the refrigerator to 4℃, and store them in the refrigerator; (iii) wrap the vegetables in plastic film and put them in the fresh-keeping box and store in the refrigerator at 4℃. Repeat each treatment three times, each repetition needs 20 g, and measure the contents of vitamin C and nitrate in the vegetables under each treatment after 0, 1, 2, 3 and 4 days, and make a record of the data in time.

2.4.2Plotting of standard curve. (i) Plot a standard curve of vitamin C: pipette the standard solution of 0.00, 1.00, 2.00, 4.00, 6.00, 8.00, 10.00, 12 mL of vitamin C into seven clean 50-mL volumetric flasks, and dilute with distilled water to the desired volume, and shake up. Then, obtain a series of standard solution with mass concentration of vitamin C of 0, 1, 2, 4, 6, 8, 10, 12 mg/L, respectively, take the distilled water as the reference, and atλ=265 nm, measure the absorbance using ultraviolet spectrophotometry[14-15], and plot the standard curve as shown in Fig.1. The regression equation isY=0.055X+0.070 andR2=0.998. (ii) Plot the nitrate standard curve: precisely pipette 0, 2, 4, 6, 8, 10, 12, 14 mL of the nitrate standard stock solution, respectively into eight 50-mL volumetric flasks, dilute to the desired volume and shake up. Then, obtain a series of standard solution with mass concentration of nitrate radical 0, 2, 4, 6, 8, 10, 12, 14 mg/L, respectively, take the distilled water as the reference, and atλ= 220 nm, measure the absorbance ultraviolet spectrophotometry[16], and plot the standard curve as shown in Fig.2. The regression equation isY=0.059X-0.036 andR2=0.999.

Fig.1 Standard curve of vitamin C

Fig.2 Standard curve of nitrate

2.4.3Extraction of solution. (i) Extraction of vitamin C. Firstly, select fresh, intact, and representative edible part, clean it, and use gauze to dry the surface. Secondly, weigh 20 g with an electronic balance, cut the samples into pieces, place it in a tissue tearor, and then add 50 mL of distilled water to squeeze the juice. Thirdly, pour the juice into a 100-mL measuring cylinder to obtain a volume of the extract, filter the extract with eight layers of gauze, and pour the filtrate into a 100-mL conical flask, to obtain the sample to be tested. (ii) Extraction of the nitrate[17-19]. Firstly, select fresh, intact, and representative edible part, clean it, and use gauze to dry the surface. Secondly, weigh 20 g with an electronic balance, cut the samples into pieces, place it in a tissue tearor, and then add 50 mL of distilled water to squeeze the juice. Thirdly, pour the juice into a 100-mL measuring cylinder, to obtain the volume of the extract. Fourthly, weigh 20 g of homogenate into a 200-mL conical flask, add 5 mL of saturated borax solution and 80 mL of 70-80℃, then put it into a water bath of 100℃ for 10 min, and stir with the glass rod when heating. Fifthly, ten min later, take it out, cool it, pour it into a 200-mL volumetric flask, add 5 mL of potassium ferrocyanide and 5 mL of zinc sulphate solution, 1 g of activated carbon, shake up when adding a reagent, dilute with distilled water to the desired volume, shake up, stand for 10 min, filter, and obtain the clear solution to be tested.

2.4.4Measurement and calculation of samples. (i) Measurement of vitamin C content: precisely pipette 1 mL of vitamin C to be tested in a 50-mL volumetric flask, dilute with the distilled water to the desired volume, shake up, measure the absorbance A atλ=265 nm, and calculate the content of vitamin C in the sample using the following formula: Formula: vitamin C (mg/100 g)=(C×Vtotal×Vtotal to be tested×100)/(V1×Wtotal×1 000)[20].

C: obtained on the basis of the standard curve (mg/L),V1: the volume (mL) of the sample solution absorbed when measuring the absorbance,Vtotal: the total volume of the sample absorbed (mL),Vtotal to be measured: the total volume of the sample to be tested (mL);Wtotal: vegetable weight (g).

(ii) Measurement of nitrate content: pipette 5 mL of clear filtrate into a 50-mL volumetric flask, add 1 mL of 1 mol/L hydrochloric acid, shake up, dilute with the distilled water to the desired volume, shake up, measure the absorbance atλ=220 nm on an ultraviolet spectrophotometer. Formula: Nitrate content (mg/kg)=(C×V1×V3) / (M×V2)[16, 21-22]

C: obtained from the standard curve (mg/L),V1: the volume of extract (mL),V2: volume of filtrate absorbed (mL),V3: volume of extract to be measured (mL),M: sample mass (g).

3 Results and analysis

3.1 Effects of different preservation methods on vitamin C in vegetablesFrom Table 1 and Fig.3, it can be known that whether it is stored at room temperature or in refrigerator, vitamin C in the six vegetables will be lost, but the degree of loss is different. After 4 d of storage under normal temperature conditions, the vitamin C of rape, cauliflower, green pepper, cucumber, carrot and potato dropped by 32.90, 5.37, 61.72, 17.57, 21.26, 22.66 mg/100 g, respectively; under fresh-keeping box cold storage condition, the vitamin C of rape, cauliflower, green pepper, cucumber, carrot and potato dropped by 21.13, 31.56, 45.79, 16.82, 17.14, 18.35 mg/100 g, respectively; under fresh-keeping box+fresh-keeping film cold storage condition, the vitamin C of rape, cauliflower, green pepper, cucumber, carrot and potato dropped by 23.16, 39.87, 47.68, 17.19, 20.62, 21.33 mg/100 g, respectively. These indicate that the loss of vitamin C in vegetables by cold storage is lower than that by normal temperature storage; in two kinds of cold storage, the loss of vitamin C in vegetables by fresh-keeping box cold storage is less than that by fresh-keeping box+fresh-keeping film cold storage. Therefore, the best preservation method for vegetables is fresh-keeping box cold storage. The small loss of vitamin C in the cold storage may be due to the limitation of the metabolism of vegetables in the low temperature environment. Low temperature reduces the oxidation of some enzymes, thereby reducing the loss of vitamin C. Therefore, in order to obtain more vitamin C from vegetables, it is recommended not to buy very much vegetable, but buy frequently, and after buying the vegetable, promptly store them in refrigerator.

Table 1 Content of vitamin C in vegetables under different preservation modes

VarietyTimedNormaltemperaturemg/100 gFresh-keepingbox cold storagemg/100 gFresh-keeping box+fresh keeping film coldstorage∥mg/100 gRape051.71--146.0149.6347.29240.1145.6543.57331.1637.3934.69418.8130.5828.55Cauliflower069.56--155.5966.9561.85242.7546.0843.25328.4042.3838.53419.1938.0029.69Green pepper0101.53--189.0297.4391.67281.3986.4185.73369.1078.4773.95439.8153.8555.74Cucumber029.61--120.6325.6122.97216.7818.8918.36315.1916.5515.89412.0412.7912.42Carrot038.83--133.9236.1235.68230.4632.0631.58322.6629.9928.1417.5721.6918.21Potato036.26--130.6336.0335.47223.0525.0923.87314.7428.2523.58413.6017.9114.96

Fig.3 Loss of the vitamin C in vegetables under different preservation methods

3.2 Effects of different preservation methods on nitrate content in vegetablesFrom Table 2 and Fig.4, it can be seen that the nitrate content of the six vegetables in the three preservation methods tends to increase with the extension of the preservation time. After 4 d of storage under normal temperature, the nitrate accumulation of rape, cauliflower, green pepper, cucumber, carrot and potato was 1 066.60, 519.43, 513.33, 577.84, 396.70, 400.95 mg/kg, respectively; under fresh-keeping box cold storage condition, the nitrate accumulation of rape, cauliflower, green pepper, cucumber, carrot and potato was 400.97, 199.00, 269.53, 228.17, 287.20, 273.17 mg/kg, respectively; under fresh-keeping box+fresh-keeping film cold storage condition, the nitrate accumulation of rape, cauliflower, green pepper, cucumber, carrot and potato was 426.00, 235.60, 356.56, 441.97, 303.42, 379.43 mg/kg, respectively. These indicate that the nitrate accumulation is the highest under normal temperature storage, followed by fresh-keeping box+fresh-keeping film cold storage, and the lowest is the fresh-keeping box cold storage. Therefore, the nitrate accumulation in vegetables is the lowest in cold storage condition, possibly because both nitrification and respiration have been inhibited in a low temperature environment, and the low temperature reduces the growth rate of nitrate. From Fig.4, it can be known that the nitrate accumulation in six kinds of vegetables is higher in normal temperature than in cold storage. Therefore, it is recommended to select cold storage method to keep freshness of vegetables, and it is better to use fresh-keeping box cold storage.

Table 2 Nitrate content in vegetables under different preservation methods

VarietyTimedNormaltemperaturemg/kgFresh-keepingbox cold storagemg/kgFresh-keeping box+fresh keeping filmcold storage∥mg/kgRape0495.07--11 017.90168.26534.2321 167.33541.50641.6331 544.13746.75875.3541 562.30896.67921.70

(To be continued)

(Continued)

VarietyTimedNormaltemperaturemg/kgFresh-keepingbox cold storagemg/kgFresh-keeping box+fresh keeping filmcold storage∥mg/kgCauliflower0155.07--1206.73186.93194.972308.87210.15279.233515.73321.73329.004675.13354.70391.30Green pepper0112.57--1133.00124.60129.932310.77152.67281.973375.93360.30371.254625.90382.10469.13Cucumber069.33--1145.6091.30122.102232.83136.95149.803261.30247.37252.974647.17297.50511.30 Carrot030.03--1186.93104.23117.902254.07139.30139.433263.57223.33228.604426.73317.23333.45Potato0139.70--1203.45161.80195.372251.10240.35241.533331.23304.97330.774540.65412.87519.13

Fig.4 Accumulation of the nitrate content in vegetables under different preservation methods

4 Conclusions and discussions

4.1 ConclusionsThe optimal preservation method is fresh-keeping box cold storage. By fresh-keeping box cold storage method, the loss of vitamin C is the least, and the nitrate accumulation is also the lowest. The effect of fresh-keeping box+fresh-keeping film cold storage is not better than simple fresh-keeping box cold storage, possibly because double layer of protection inhibits the respiration of vegetable cells, so that vegetables have to conduct anaerobic respiration, accordingly influencing the fresh-keeping effect. However, compared with the normal temperature storage, fresh-keeping box+fresh-keeping film cold storage is better.

4.2 DiscussionsThere is a certain difference between the data measured by UV spectrophotometry and the data measured by other methods, but the overall difference is not large, which is possibly associated with the characteristics of the vegetables, the planting area, fertilization and other factors. Some data have large difference and are not satisfactory, possibly because of the error that occurs during the operation. On the fourth day, when measuring the vitamin C content in green pepper, the vitamin C content in green pepper stored by fresh-keeping box was lower than that by the fresh-keeping box+fresh keeping film, possibly because vitamin C has been oxidized during the operation or during the storage; when measuring the nitrate content, there is difference in a group of data in the rape, the nitrate in the rape stored by fresh-keeping box dropped, possibly because part of the nitrate has been reduced to nitrite or a mistake has occurred during the extraction of the nitrate, or possibly because the extraction temperature is not sufficient. On the whole, the results of this experiment are reliable.