1. Introduction
The carrot (
Daucus carota L.) is an important root vegetable with a pleasant taste, high nutritional value, and several benefits to human health due to its specific anti-anemic, anticancer, antioxidant, sedative, and healing characteristics [
1]. It contributes to the antioxidant potential due to the presence of significant amounts of bioactive compounds [
2]. Carrots are commonly eaten as fresh vegetables; however, due to their perishable nature, they are processed into a variety of products, including juices. During storage, undesirable browning reactions occur that ultimately cause discoloration of the product due to the condensation of phenolic compounds [
3]. Enzymes can be involved in the deterioration of food products that cause undesirable color, flavor, or nutritional changes [
4]. These enzymes include peroxidase (POD), pectin-methylesterase (PME), polyphenol-oxidases (PPO), and lipoxygenase (LOX). Microorganisms also cause deterioration of fruit and vegetable juices [
5]. At present, the shelf life of vegetable juices improved by the inactivation of the enzymes and microorganisms through heat treatments [
6]. However, these thermal treatments often cause adverse changes in the food products that account for color alterations, loss of nutrients, and off-flavor development [
7]. On the other hand, carrot juice is sensitive to the risk of microbial spoilage due to its low acidity, and it reported that natural micro-flora could play an important role in the deterioration of juice quality. Although high-temperature treatment can decrease the risk of enzymatic and microbial activities, it also causes the loss of heat-sensitive components [
8], particularly vitamin C, which is an essential vitamin for the proper functioning of the body, whereby deficiency can cause complications such as scurvy disease.
On the contrary, high-voltage cold plasma (HVCP) non-thermal treatment is a new kind of non-thermal sterilization technique [
9] that has more benefits and merits than traditional solvent extraction techniques, while also being more economical from an industry perspective. Because in the case, the equipment operation is simple; furthermore, the technique is non-thermal; it preserves heat-sensitive and water-soluble nutrients and reduces the solvent amount [
10]. The prospects of overcoming the existing conventional physical and chemical methods for sterilization make it a novel and economical technique to maintain food’s natural nourishment, composition, appearance, structure, and freshness [
11]. Furthermore, HVCP is quick and highly effective, with a low processing cost and low energy input; it was also found to be more efficient for decontamination [
12].
Therefore, high-voltage electric field cold plasma (HVCP) treatment can be beneficial, as it was found to stabilize wheat germ by inactivating lipase and lipoxygenase (LOX) enzymes, thereby extending the shelf life of wheat germs [
13]. Previous studies showed that HVCP could also inactivate POD in tomatoes [
14]. Therefore, the present work was designed with the aim of exploring the effect of HVCP on enzymes such as PPO, POD, PME, and LOX, as well as the levels of polyphenols, ascorbic acid, and coloring compounds (e.g., carotenoids, β-carotene, and lycopene), among other physicochemical properties, in carrot juice; microbial assays (e.g., total plate count, yeast and mold counts) were also carried out. Changes in the final product after treatment with HVCP should be considered for its practical implementation [
15]. Carrot juice color and flavor are important quality indicators that attract customers and fulfill customer demand [
16]. Some reports showed that HVCP did not affect color, particularly on lightness L* [
17]. HVCP-treated pomegranate juice also exhibited a reduction in a* and b* values [
18]. However, reductions in L* and b* values were also observed with a concomitant slight increase in a* value after the treatment of apple juice with an atmospheric pressure plasma jet (APPJ) [
17]. Thus, the effect on food quality differs significantly depending on the plasma source employed (e.g., APPJ, direct or indirect mode). The influence of the plasma used to sterilize large-scale liquid foods on the quality of fruit juices needs to be evaluated; therefore, it is also important to study the effect on color changes during HVCP treatment.
Moreover, the findings of this study are expected to be beneficial for the solution of quality and safety issues, to attain maximum consumer satisfaction. It is especially important, as consumers are now more conscious about health and diet, and their demand for fresh food brought about increased interest in the mild processing technologies used.
2. Material and Methods
2.1. Chemicals
FolinCiocalteu (FC) reagent was bought from Shanghai RyonBiological Technology Co., Ltd., China. Catechin and ascorbic acid were provided by Shanghai Aladdin Bio-chem Technology Co., Ltd. Methanol, hydrogen peroxide, petroleum ether, hydrochloric acid, and n-hexane were provided by Nanjing Chem. Reagent (Nanjing, China). All the other chemicals used in the experiment were of analytical grade.
2.2. Procurement of Raw Material, Blanching and Sample Preparation
Raw material (Carrots: Daucus carota cv Heitian-5) was bought from the local Suguo supermarket of Nanjing, China. For thermal treatment, good quality carrots (1 kg) were washed using tap water, peeled and then sliced manually to a thickness of approximately 2 cm, and diameter was 10 mm. The sliced carrots were blanched (thermal treatment) in hot water at 100 °C for 5 min. The approximate ratio between the weight of vegetables and water was kept 1:2. Blanched carrots were cooled to room temperature by dipping in cold water, and then these slices were pressed to make juice by using Hurom slow juicer (Model# HU7WN3L). The fresh carrot juice was selected as the control without any treatment. The fresh carrot juice was subjected to HVCP as a design in the experiment.
2.3. High Voltage Electric Field Cold Plasma Treatment (HVCP)
The dielectric barrier discharge (DBD) plasma system was used as a reaction cell. The apparatus was composed of an AC dielectric test set (BK-130, Phenix Technologies, Accident, MD, USA), high voltage wires, two circular aluminum electrodes (150 mm diameter), and dielectric barriers (2 mm polypropylene sheets). A high voltage power transformer (CTP-2000K) connected with the voltage regulator (Nanjing Suman Electronics Co., Ltd., Nanjing, China) was used to generate and provide power supply (0–80 kV) to upper and lower electrodes.
Approximately, 250 mL of carrot juice sample was taken for the treatment of HVCP. The samples were packed in pre-sterilized food grade bottles and were placed between two dielectric quartz plates with a gap of 30 mm and treated at 60, 70, and 80 kV for 3 and 4 min with three-time repetitions and a 30-second break interval in every treatment. Stable and uniform plasma was generated; the air was used as a working gas during the HVCP treatment. The observed rise in temperature was measured by a digital thermometer (2–5 °C) during the HVCP treatment. The schematic diagram of the HVCP treatment for carrot juice has been illustrated in
Figure 1.
2.4. Residual Enzyme Activity
2.4.1. Peroxidase Enzyme Residual Activity
The method of [
19] was used to determine POD residual activity in carrot juice. Absorbance was measured at 420 nm using a UV-VIS Spectrophotometer (Shimadzu Scientific Instruments Co., Ltd., Beijing, China). The POD residual activity was measured using Equation (1).
2.4.2. Polyphenol Oxidase Enzyme Residual Activity
The PPO residual activity in treated and untreated carrot juice samples was determined by the method of [
20]. Changes in absorbance were determined at 410 nm after every 60 s, starting from 0 s to 10 min using a UV-VIS Spectrophotometer. PPO residual activity was determined by using Equation (1). All measurements were determined in triplicate.
2.4.3. Pectin Methylesterase Enzyme Residual Activity
PME is measured as the amount of enzyme that releases 1 mmol of the carboxyl group in 60 s, as described by [
21]. A change in absorbance was determined at 620 nm after every 60 s, starting from 0 s to 10 min using a UV-VIS Spectrophotometer. PME residual activity was determined using Equation (1). All the measurements were determined in triplicate.
2.4.4. Determination of Lipoxygenase Residual Activity
Kim’s method was used to determine the lipoxygenase activity in carrot juice [
22]. Changes in absorbance were determined at 234 nm at room temperature using a UV-VIS Spectrophotometer. The oxidation of linoleic acid was calculated using Equation (1). All the measurements were determined in triplicate.
2.5. Determination of Coloring Compounds
2.5.1. Determination of Total Carotenoids
Total carotenoid was measured in all the treatments by a method previously reported in [
23] with minor modification. Briefly, the juice sample (25 mL) was poured into a separation funnel with an 80 mL volume of n-hexane/acetone and shaken well. We collected the organic phase after separation. We added 15 mL volume of n-hexane/acetone and shook well again and collected the organic phase. We repeated this step until the aqueous phase became colorless. The organic phase was dehydrated using anhydrous sodium sulfate, and standard β-carotene solutions of different concentrations were prepared for plotting a standard curve. A UV-VIS spectrophotometer was used to measure the absorbance at 450 nm to measure total carotenoids whereas; commercially available β-carotene was used as an external standard (2–10 µg/mL), and data were tested as µg of β-carotene equivalent per milliliter of sample.
2.5.2. Determination of Lycopene Contents
All the samples were treated, and the control was tested to determine the effect of HVCP on lycopene contents. A method explained by [
24] was used with slight modification, for the determination of lycopene contents of carrot juice. A juice sample (0.6 mL) was added to 5 mL of BHT in acetone (0.05:99.95, w/v), 5 mL of ethanol (95:5, v/v), and 10 mL of n-hexane of mixture solution. The mixture was then centrifuged for 15 min at 320 g. After shaking, 3 mL of distilled water was added to it. The vial was then agitated for 5 min and held for 2 min to allow the phase separation at room temperature. A UV-VIS spectrophotometer was used to measure the absorbance at 503 nm of the upper n-hexane layer. A pure n-hexane solution was used for the blanked reading. Equation (2) was used to measure lycopene content in carrot juice.
where MW is the stand for the molecular weight of lycopene, that is 536.9 g/mol, DF is the dilution factor, L is the path length in cm, and ε is the (172 000 L/mol cm) molar extinction coefficient as reported [
25] for lycopene. Lycopene content was determined as µg/mL of the sample. All the measurements were carried out in triplicate.
2.5.3. Determination of β-carotene and Lutein
Briefly, the juice sample (25 mL) was poured into a separation funnel with an equal volume of acetone three times and filtered by a No.1 Whatman filter paper. The filter cake was re-extracted with methanol, and the extract was vigorously mixed with an equal volume of petroleum ether. The upper petroleum ether layer was dehydrated using anhydrous sodium sulfate, and after filtration, it was concentrated using a rotary evaporator at 30 °C. Then, we added 10 mL acetonitrile–methanol–acetone solution (40:40:20, v/v) and kept it at 18 °C in the dark until further analysis. The samples were filtered through a syringe filter (0.45 μm, 13 mm Cat#AS-021345 N-Agela Technologies). Filtered samples were fractionated using Waters Auto Purification high-performance liquid chromatography (HPLC) (Cat. # 1501382671, X BridgeTM prep C18: 5 μm OBDTM, 150 mm × 19 mm, Waters, Ireland). The mobile phase was comprised of a mixture solution of acetonitrile-acetone-methanol (40:20:40, v/v), and the flow rate was adjusted to 0.80 mL/min. Commercially available β-carotene and lutein with different concentrations were used to draw a linear regression calibration curve.
2.6. Determination of Chlorogenic Acid
Kahle method with minor modifications was used to determined chlorogenic acid [
26]. The identification and quantification of chlorogenic acid were determined through HPLC (Waters 600 system). The juice sample was filtered (0.45 μm, 13 mm Cat. # AS 021345-N Agela Technologies), and samples were fractionated through the Waters Auto Purification HPLC column (X Bridge
TM prep C18: 5 μm OBD
TM, 150 mm × 19 mm, Waters, Ireland). The mobile phase consisted of a mixture solution of aqueous formic acid (A) with methanol (B) (0.1:99.9, v/v). We then injected 5 mL of filtrate sample into the column at a flow rate of 1 mL/minute. A linear biphasic gradient was used with 15–40% solvent B over 10 min, 40–50% over 15 min, 50–75% over 20 min, 75–90% over 25 min followed by a gradual column re-equilibration from 90–65% over 30 min, 65–40% over 35 min, 40–10% over 40 min. Samples were detected using a UV-VIS spectrophotometer at a wavelength of 320 nm, a commercially available chlorogenic acid was used as an external standard, and different concentrations of its solution (10–80 mg/mL) were used to draw a standard curve. Data were presented as mg of chlorogenic acid equivalent per milliliter of samples.
2.7. Determination of Sugar Content in Carrot Juice
Sugar contents were determined by [
27]. The identification and quantification of sugar contents were performed using a semi-preparatory high-performance liquid chromatography (HPLC) system (waters 600). The juice sample was filtered (0.45 μm, 13 mm Cat. # AS 021345-N Agela Technologies), and samples were fractionated through the Waters Auto Purification HPLC column (X Bridge
TM prep C18: 5 μm OBD
TM, 150 mm × 19 mm, Waters, Ireland). The mobile phase consisted of acetonitrile (75:25, v/v) with a 1 mL flow rate. A Cosmosil packed column of D-sugars (4.6×250 mm) was used. Commercially available sucrose, fructose, and glucose were used as external standards. Different concentrations of each solution standard were prepared. Sucrose, fructose, and glucose were used to draw a linear regression calibration curve. Data were analyzed as g of sucrose, fructose, and glucose equivalent per liter of sample.
2.8. Phytochemicals Analysis
2.8.1. Determination of Mineral Contents
American Public Health Association (1988) [
28] described method was followed to determine mineral contents. 1mL of the carrot juice sample was digested with 7 mL of nitric acid solution (65:35, v/v) in Teflon digestion (TFM) vessel. All the conditions for the detection of minerals are given below in
Table 1.
We added 1 mL of H
2O
2 to this mixture and heated at 200 °C in a microwave oven for 20 min with a one-minute rest after the first ten minutes for better acid digestion. The digested solution was transferred into a 50 mL volumetric flask and diluted up to the mark with pure water. The samples were tested using an inductively coupled plasma-optical emission spectrometer (OPTIMATM 2100 DV, Perkin Elmer Precisely, USA). Commercially available standards of each mineral were purchased and used as standards. All the measurements were conducted in triplicate. We followed the American Public Health Association (1988) described the method to determine mineral contents. 1mL of the carrot juice sample was digested with 7 mL of nitric acid solution (65:35, v/v) in a Teflon digestion vessel (TFM). All the conditions for the detection of minerals are given below in
Table 1.
2.8.2. Total Phenolic Content
Total phenolic content was determined following the method described by [
29]. Elution was monitored with a UV-VIS spectrophotometer at 760 nm, and commercially available gallic acid was used as the standard. Different concentration solutions of standard gallic acid were used to draw a standard curve, and the data were depicted as µg of gallic acid equivalent (GAE) per milliliter of sample.
2.8.3. Total Flavonoid Contents
The total flavonoid content was determined using the method described by [
29]. The absorbance was measured at 510 nm by a UV-VIS spectrophotometer. Results were expressed as µg of equivalent catechin per milliliter of carrot juice. All measurements were conducted in triplicate.
2.8.4. Total Tannin Contents
Tannin contents were determined by the method of [
30]. The absorbance of all samples was measured by a UV-VIS spectrophotometer at 720 nm using UV-VIS Spectrophotometer (Shimadzu Scientific Instruments Co., Ltd., Beijing, China). Catechin was used as the standard for analysis, and results were expressed as mg catechin per 100 mL of carrot juice.
2.8.5. Determination of Brix
Brix was measured by a hand refractometer (WYT-J 0–32% Chengdu HaochuanGuangdian company, China) at room temperature (25 ± 2 °C). All the measurements were taken in triplicate, and water was used to wash prism of hand refractometer.
2.8.6. Determination of pH in Carrot Juice
A digital pH meter was used to measure changes in pH in carrot juice samples. Different concentration buffer solutions (pH = 4.0 and 7.0) were used for the calibration of the pH meter. 15–20 mL of each treatment sample was poured into a beaker, and we measured the changes of pH with a pH meter. All measurements were conducted in triplicate.
2.8.7. Determination of Titratable Acidity
The titratable acidity of the carrot juice samples was measured by the standard method of the Association of Official Analytical Chemists [
31]. Briefly, a 15 mL carrot juice sample was taken in a beaker; the volume was completed to 100mL with distilled water. The mix was stirred while titrating against a standard solution of NaOH (4 g/L) to the endpoint (pH 8.2 ± 0.1). The titratable acidity was calculated as citric acid equivalents by Equation (3).
2.8.8. Determination of Color Changes in Carrot Juice
The color was measured using a colorimeter (CR-400, Konica Minolta, Osaka, Japan, with the CIELAB tristimulus parameters: L*, a*, and b*. The L* measures brightness/whiteness of color, a*describes the level of redness (+a*) or greenness (−a*), and b* used to describe the level of yellowness (+b*) and blueness (−b*). A standard white plate (L* = 97.95, a* = 0.89, and b* = 1.23) was used to calibrate the instrument. There are nine samples, and each piece was recorded three times.
2.8.9. Determination of Ascorbic Acid
The method described in [
29] was followed to determine the ascorbic acid content in carrot juice. The identification and quantification of ascorbic acid were made through HPLC (Waters 600 system). Filtered samples were separated through a TSKGEL OSD-100Z C18: (4.6 mm× 150 cm × 5 µm) column (Tosoh, Japan). Methanol (30% v/v) was used as a mobile phase at 1 mL/minute flow rate, and samples detection was conducted with UV-VIS spectrophotometer wavelength at 280 nm. Commercially available ascorbic acid, with different concentrations, was used to draw a linear regression calibration curve.
2.9. Microbiological Analysis
Microbiological counts were assessed by [
32], with slight changes. Serial dilution was performed to maintain the initial microbial load, and the pour-plating method was used to test total plate count (molten agar) and yeast and mold counts (potato dextrose agar). All samples were analyzed in triplicate, and the results were expressed as colony-forming units per gram based on fresh weight (log CFU g
−1).
2.10. Statistical Analysis
The mean and standard deviation values of the results were derived. A complete randomized design (CRD) with one-way Analysis of variance (ANOVA) was applied to calculate the level of significance (p < 0.05 the). Least significant difference (LSD) pair-wise comparison test was used to determine the level of significance between means. All the results were analyzed using the Statistical package for social science (SPSS), version 18.0 (SPSS, Chicago, IL, USA).
4. Conclusions
The findings of the recent work showed that HVCP processing has the potential for improving the quality and safety of carrot juice, and it may successfully be employed in beverage industries. Furthermore, the antimicrobial behavior of HVCP treatment has indicated that due to the presence of the reactive species and lethal interaction of hydrogen peroxide and acidified nitrites (NO− and NO2−), the treatment reduces microbial load and enhances the stability of carrot juice with better nutritional value. Due to this beneficial aspect, it is appropriate for industrial applications for heat-sensitive food items in which heat treatments lose nutrients. Moreover, HVCP treatment had a significant effect on the quality characteristics of carrot juice, such as color, acidity, and odor. Keeping all the above findings and also the certain benefits, i.e., simple and controllable equipment handling, and process operation compared to traditional processing and preservation techniques, we suggest that high voltage cold plasma may successfully be employed in the food industry.