Influence of Cold Plasma Processing on the Stability of Phenolic Compounds of Araça-Boi (Eugenia stipitata) Juice

Abstract

Knowledge of the chemical changes caused during plasma treatment is essential to enhance food quality. In this work, the influence of two cold plasma technologies, dielectric barrier discharge plasma and glow discharge plasma, on the phenolic profile of araça-boi (Eugenia stipitata) juice was investigated and assessed by gas chromatography coupled to mass spectrometry. Eight phenolic compounds were identified in araça-boi, with cinnamic acid being the major phenolic compound of the fruit juice, followed by protocatechuic acid. The effects of excitation frequency and plasma flow rate were evaluated because these are the main operating conditions that can be set for plasma treatments. The phenolic profile slightly changed due to the reaction of the phenolics with the reactive plasma species produced during the treatment, with the highest increase in phenolic content observed in the dielectric barrier discharge plasma operating at 1000 Hz. Both plasma systems increased the bioavailability of phenolic compounds in the juice, which could be increased by up to 201% using the dielectric barrier discharge plasma. Plasma application increased the concentration of cinnamic, hydrocinnamic, benzoic, and p-coumaric acids. Overall, plasma treatment improved the bioavailability of the phenolic compounds and resulted in slight changes to the phenolic profile of araça-boi juice. Thus, the technology showed a positive effect on araça-boi juice. This work advanced our further understanding of the changes induced by cold plasma treatment on phenolic compounds and characterization of araça-boi (Eugenia stipatata).

1. Introduction

In contemporary times, there is a growing consumer demand for fresh food products that are not only tastier and healthier but also minimize the use of chemical preservatives and processing conditions. Among the popular choices for health-conscious consumers is fruit juice, recognized as a natural and healthy beverage. The increasing inclination towards juice consumption is driven by numerous studies highlighting the nutritional and health benefits associated with it. Fruit juices are considered rich in vitamins, minerals, and essential nutrients, contributing to their role as functional food products. The manufacturing process of fruit juice has currently become a focal point of attention for both consumers and researchers, emerging as a prominent topic in the industry.

Health-conscious consumers are actively seeking specific quality attributes in food products, including enhanced food safety, flavor, freshness, and nutritional value. The rising interest in clean-label foods, which assert their natural and additive-free qualities, is becoming increasingly prominent among consumers. Consequently, there is a growing demand within the food industry to adopt processing technologies capable of reducing additives while preserving food quality and natural flavors. This demand has led to the development of innovative non-thermal processing technologies, which must be adept at producing microbiologically stable products with extended shelf life coupled with elevated nutritional and sensory characteristics.

Cold plasma technology has been evaluated by numerous researchers for its potential application in the food processing industry due to its versatility and eco-friendly nature. Cold plasma, also known as non-thermal plasma, refers to a gas that is partially ionized, comprising electrons, ions, free radicals, and neutral particles. Its generation at temperatures near room temperature renders it well suited for treating heat-sensitive materials such as food [1,2].

The ionized particles generated in cold plasma can have various effects, such as promoting chemical reactions or modifying the surface properties of materials. Cold plasma has found successful applications across various sectors within the food industry, such as microbial inactivation [3,4,5,6], food property modification [7,8], surface modification and decontamination [9,10,11], reduction or removal of allergens [12], and sensory and nutritional improvements. The specific changes that can be induced depend on the types and concentrations of reactive plasma species that are generated, which also depend on the conditions of the cold plasma process, such as the gas composition, pressure, temperature, and plasma technology.

During the early exploration of cold plasma, there was an initial belief that applying plasma did not change the sensory and nutritional characteristics of food products, mainly because the processing took place at room temperature. As the investigations on cold plasma processes advanced, it became apparent that numerous chemical modifications were taking place, and many of these reactions had favorable effects on the food product quality [13].

Cold plasma has the capability to trigger diverse chemical reactions, inducing processes like ring opening, hydrolysis, isomerization, oxidation, scission, hydrogenation, and methyl group abstraction. Numerous studies have been undertaken to elucidate the interactions between reactive plasma species and various food compounds, with a particular focus on organic acids, sugars, oligosaccharides, aroma, and flavor compounds [13]. These studies showed that cold plasma processing changed the concentration of many foods’ chemical compounds, influencing aroma, taste, and nutritional quality changes. Utilizing atmospheric air as the working gas to produce non-equilibrium plasma discharges results in the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The primary active components among these are ozone (O₃), atomic oxygen (¹O₂), and hydroxyl radicals (OH). The OH radicals are generated through the direct dissociation of water molecules by electronic impact. These, along with several other minor reactive plasma species, interact with the food-bioactive compounds, partially reacting with them [1].

Although the understanding of cold plasma effects on food is improving, further research is necessary to comprehensively comprehend the impact of plasma processing on numerous other classes of compounds found in food, such as phenolic compounds. Studies with cold plasma technology have addressed phenolic compounds, but most refer only to total phenolic content. Few studies have evaluated the influence of the technology on the phenolic profile. Kungsuwan et al. [14] reported that the concentration of chlorogenic acid and caffeine reduced after plasma treatment of green coffee beans, while no significant change was observed in vanillic acid, vanillin, p-coumaric acid, and myricetin. The authors have not discussed what caused the reduction in phenolic compounds. Zhou et al. [15], working with blueberries, noticed that plasma treatment increased the concentration of chlorogenic acid, protocatechuic acid, and ferulic acid while decreasing the concentration of caffeic acid O-glucoside, cinnamic acid, and L-phenylalanine. Abouelenein et al. [16] reported for rocket-salad leaves increases in chlorogenic acid, ferulic acid, and p-coumaric acid and decreases in concentration for 4-hydroxybenzoic acid and 3,5-dicaffeoylquinic acid. As noted, the changes depend on the operating condition and the fruit matrix, since chlorogenic acid can either increase or decrease.

In this work, we advance the understanding of the chemical effects of cold plasma on fruit juices. Araça-boi (Eugenia stipatata) is an exotic Amazonian fruit that grows in a wide area of the western Amazon Forest comprising Brazil, Peru, Ecuador, and Colombia (Figure 1). The fruit belongs to the Myrtaceae family, which also includes guava (Psidium guajava L.). This fruit is large, round, or slightly oblong, with a smooth, thin, yellowish skin. The fruit weighs an average of 750 g and contains a thick pulp enclosing about 12 tiny seeds. The fruit is acidic, containing large amounts of cinnamic and malic acid, and has an intense flavor. The fruit also contains high amounts of carbohydrates (65 to 72% w/w) and proteins (8 to 10% w/w). Its pulp is creamy, with a custard-like texture. Araça-boi is consumed fresh or processed in the Amazon Forest into juice, jelly, jam, or other desserts. Araça-boi is a unique and flavorful tropical fruit showcasing the Amazon rainforest’s biodiversity. Its distinctive taste and nutritional benefits make it a valuable and interesting addition to the variety of fruits available in the region, making it worthwhile to study it [15].

This study evaluated the chemical changes induced by cold plasma treatment on the phenolic compounds profile of araça-boi (Eugenia stipatata). Two cold plasma technologies were evaluated: the dielectric barrier discharge plasma and the glow discharge plasma. The phenolic compounds were identified by gas chromatography coupled with mass spectrometry. The novelty of this work relies on further understanding the changes induced by cold plasma treatment on phenolic compounds, since, up to now, only a few works on other fruits have presented this effect on the phenolic profile composition. Furthermore, the work advances the characterization of araça-boi (Eugenia stipatata).

2. Materials and Methods

2.1. Materials

Araça-boi (Eugenia stipatata) was obtained directly from a producer from the Amazonian Forest (Manaus—AM). The fruit underwent peeling, and its pulp was extracted, subsequently being frozen (−4 °C) until processing. Prior to processing, the frozen pulp was thawed at room temperature (25 °C) and combined with distilled water (1:1 w/w) to produce the araça-boi juice.

2.2. Plasma Processing

Araça-boi juice was subjected to 6 different plasma processing using 2 cold plasma techniques: glow discharge plasma (GDP) and dielectric barrier discharge plasma (DBDP). The GDP assays were carried out at 3 air plasma flow rates (10, 20, and 30 mL/min) for 20 min at a fixed voltage (80 kV). The DBDP assays were carried out at 3 excitation frequencies (50, 500, and 1000 Hz) for 20 min at a fixed voltage (20 kV). A reference sample was stored without undergoing plasma treatment. The processing time was fixed at 20 min because the changes observed in other araça-boi food components, such as sugars, starch, amino acids, and aroma compounds, were maximal at this processing time [17]. All treatments were carried out in triplicate. The DBDP and GDP voltages were different due to equipment limitations.

The GDP processing was conducted using a Plasma Etch model PE-50 (Carson City, NV, USA). Cold plasma was generated in synthetic air (grade FID 4.0, purity 99.95%, White Martins, Brazil) using a 50 kHz radio-frequency source applying 80 kV through the electrodes. The gas flowing through the electrodes was directed into a processing chamber (190 mm × 220 mm × 90 mm) housing the samples. A vacuum pump maintained the processing chamber at 0.3 bar (Figure 2). For each trial, polypropylene tubes, each containing 40 mL of pulp, were placed in the treatment chamber and exposed to GDP treatment. The selection of the plasma flow rate and processing time was based on our group’s previous studies, indicating that these ranges significantly induce changes in fruit juices.

The DBDP processing was conducted with an Inergiae plasma generator (model PLS0130, Florianópolis, Brazil) connected to a DBDP system. This system comprised two aluminum electrodes 8 cm in diameter and two 5 mm-thick acrylic plates serving as dielectric barriers. A Petri dish made of acrylic, with a diameter of 55 mm and a height of 14 mm, containing 20 mL of araça-boi juice, was positioned within the 15 mm gap between the electrodes. The plasma discharge took place in an open space, with the sample not enclosed in any vessel, and atmospheric air served as the gas source at an ambient temperature of 25 °C (Figure 1). These specific operating conditions were selected based on prior research, which indicated that these frequencies and processing times induce noteworthy changes to the food-bioactive compounds.

The trials were conducted using 25 mL of araça-boi juice. For the DBDP, the juice was added to Petri dishes, which were placed directly between the electrodes of the plasma system. For the GDP, the juice was added to polypropylene tubes within the treatment chamber. Further information about the equipment is available in Farias et al. [18].

2.3. Extraction and Concentration of Phenolic Compounds

The phenolic compounds were extracted following the methodology described by Zuo et al. [19] with some adaptations. First, 10 mL of araça-boi juice was mixed with 20 mL of cold methanol (4 °C), 0.01 mL of HCl, and 0.10 g of ascorbic acid and refrigerated for 18 h at 4 °C. The mixture was centrifuged. The liquid phase was separated and stored (first aliquot). The solid phase was mixed with 10 mL of cold methanol, 0.005 mL of HCl, and 0.05 g of ascorbic acid and extracted for 3 h. The mixture was centrifuged, and the liquid phase was separated and stored together with the first aliquot. The liquid phase containing the phenolics was concentrated in a rotary concentrator (Tecnal model TE211, Piracicaba, Brazil) operating at 40 °C and 200 mmHg of vacuum to remove the methanol.

The phenolics of the concentrated liquid phase were extracted in a DSC-18 SPE tube. A solution of 0.01% HCl (5 mL) was passed through the SPE tube before eluting the phenolics with 3 mL of methanol. The methanol phase containing the phenolic compounds was dried to completion in a rotational vacuum concentrator (Christ model RVC 2-18, Osterode am Harz, Germany) operating at 40 °C and 200 mmHg of vacuum.

The dried phenolics were resuspended in 3 mL of distilled water and transferred to a 15 mL Falcon tube, to which 1 mL of a 1 M NaOH solution was added to perform an alkaline hydrolysis of the sample. The Falcon tubes were maintained under agitation at 200 rpm for 24 h in a thermoshaker (Kasvi model K80-200, São José dos Pinhas, Brazil). After the alkaline hydrolysis, the pH of the samples was adjusted to 1.25 using a 1 M HCl solution.

The phenolic compounds were extracted with ethyl acetate. The extraction was carried out by adding 1 mL of ethyl acetate to the Falcon tube, vigorously mixing the phases for 10 s using a vortex (Tecnal model TE211, Piracicaba, Brazil), waiting for 1 min, and collecting the ethyl acetate phase. This procedure was carried out 3 times, and the ethyl acetate phases were mixed. The ethyl acetate phase containing the phenolic compounds was dried to completion in a rotational vacuum concentrator (Christ model RVC 2-18, Osterode am Harz, Germany), operating at 40 °C and 200 mmHg of vacuum.

2.4. Chromatographic Analysis

The phenolic compounds were derivatized before the chromatographic analysis. A total of 30 μL of pyridine and 70 μL of BSTFA with 1% TMS were added to the Eppendorf containing the dried phenolics. The Eppendorfs were incubated at 600 rpm for 1.5 h in a thermoshaker (Kasvi model K80-200, São José dos Pinhas, Brazil) operating at 70 °C. After derivatization, 0.1 μL volumes of the samples were injected into the gas chromatograph [19].

Samples were analyzed in a gas chromatograph with a mass spectrometer (Thermos model ISQ). The injector and the interface temperatures were 250 °C, working in splitless mode. Chromatographic separations were performed using an Equity-1 column (30 m × 0.25 mm ID × 0.25 μm film). The temperature programming started at 80 °C for 4 min, increased to 80 °C at 2.5 °C·min⁻¹, increased to 100 °C at 5.0 °C·min⁻¹, holding at this temperature for 1 min, and then increased to 250 °C at 10.0 °C·min⁻¹, maintaining the final temperature for 1 min. The ion trap detector operated in the EI mode at 70 eV and 200 °C with a mass scan range of 50 to 400 m/z. The carrier gas was helium, set at a 1.0 mL·min⁻¹ flow rate. The mass spectra were compared with the NIST and Wiley mass spectra library.

2.5. Statistical Analysis

Statistical analysis was carried out using Statistica v.13 (Tbico Software). The experiments were conducted in triplicate, and both the mean and standard deviation were computed. A variance analysis was conducted, and the significance of differences between treatments was evaluated through Tukey’s multiple-sample comparison tests. Significance levels were examined at p ≤ 0.05.

3. Results

Eight phenolic compounds were identified in araça-boi: 2-aminobenzoxazole, benzoic acid, cinnamic acid, hydrocinnamic acid, oxanilic acid, p-coumaric acid, phenylacetic acid, and protocatechuic acid. Cinnamic acid was the major phenolic compound of the fruit juice, with 90.3% (w/w) of the fruit’s total phenolic content. Protocatechuic acid was the second-largest phenolic compound present in the juice (5.5% w/w). Other phenolic compounds were found in minor concentrations, such as benzoic acid, hydrocinnamic acid, p-coumaric acid, and phenylacetic acid. Two amino-phenolic acids, 2-aminobenzoxazole and oxanilic acid, were identified. The chemical structures of all phenolic compounds identified in araça-boi are presented in Figure 3.

In the available literature, the phenolic composition of araça-boi varies considerably. Guimarães et al. [20] reported that the major phenolic in araça-boi pulp was catechin, followed by ferulic acid, quercetin, chlorogenic acid, and rutin. Araújo et al. [21] reported the presence of caffeoyl tartaric acid, caffeoyl hexose, caffeoyl methylquinic acid, coumaroyl tartaric acid, fertaric acid, and gallic acid hexoside as the main phenolic compounds of araça-boi but did not present any quantification of these phenolics. Barros et al. [22] reported that the main phenolic compound found in araça-boi was protocatechuic acid, followed by p-coumaric acid, vanillic acid, vanillin, chlorogenic acid, ferulic acid, and cinnamic acid. No article has presented similar results; however, most of the compounds obtained herein are among the compounds reported by previous papers.

As previously mentioned, cinnamic acid was the phenolic compound with the highest concentration in araça-boi. Other fruits, such as Chilean strawberries (Fragaria chiloensis), physalis (Physalis peruviana), and guava (Psidium guajava), also present high concentrations of cinnamic acid in their glycosylated form [23,24].

Cold plasma treatment has induced some changes in the phenolic profile of araça-boi juice. These changes are presented in

Table 1. The phenolic compound profile of araça-boi juice (mass fractions) was treated on the dielectric barrier discharge plasma for 20 min. Different superscripts for each row indicate significant differences between the treatments.

Phenolic Compound	Control	Frequency
		50 Hz	500 Hz	1000 Hz
2-Aminobenzoxazole	0.71 ± 0.04 a	0.00 ± 0.00 b	0.00 ± 0.00 b	0.00 ± 0.00 b
Benzoic acid	0.84 ± 0.04 c	1.03 ± 0.05 b	1.58 ± 0.08 a	0.69 ± 0.03 d
Cinnamic acid	90.34 ± 1.81 b	96.70 ± 1.93 a	96.63 ± 1.93 a	95.40 ± 1.91 a
Hydrocinnamic acid	0.63 ± 0.03 c	1.61 ± 0.08 a	1.78 ± 0.09 a	1.05 ± 0.05 b
Oxanilic acid	0.47 ± 0.02 a	0.00 ± 0.00 b	0.00 ± 0.00 b	0.00 ± 0.00 b
p-Coumaric acid	0.00 ± 0.00 d	1.26 ± 0.06 b	1.97 ± 0.10 a	0.93 ± 0.05 c
Phenylacetic acid	0.29 ± 0.01 a	0.00 ± 0.00 b	0.00 ± 0.00 b	0.00 ± 0.00 b
Protocatechuic acid	5.49 ± 0.27 a	0.00 ± 0.00 c	0.00 ± 0.00 c	2.63 ± 0.13 b

and

Table 2. The phenolic compound profile of araça-boi juice was treated on the glow discharge plasma for 20 min. Different superscripts for each row indicate significant differences between the treatments.

Phenolic Compound	Control	Plasma Flow Rate
		10 mL/min	20 mL/min	30 mL/min
2-Aminobenzoxazole	0.71 ± 0.04 a	0.75 ± 0.04 a	0.29 ± 0.01 b	0.13 ± 0.01 c
Benzoic acid	0.84 ± 0.04 c	1.21 ± 0.06 a	0.97 ± 0.05 b	0.76 ± 0.04 c
Cinnamic acid	90.34 ± 1.81 c	96.38 ± 1.93 a	93.40 ± 1.87 bc	93.15 ± 1.86 bc
Hydrocinnamic acid	0.63 ± 0.03 c	0.00 ± 0.00 d	1.22 ± 0.06 b	2.20 ± 0.11 a
Oxanilic acid	0.47 ± 0.02 a	0.00 ± 0.00 c	0.00 ± 0.00 c	0.38 ± 0.02 b
p-Coumaric acid	0.00 ± 0.00 c	0.00 ± 0.00 c	0.91 ± 0.05 a	0.44 ± 0.02 b
Phenylacetic acid	0.29 ± 0.01 a	0.00 ± 0.00 b	0.00 ± 0.00 b	0.00 ± 0.00 b
Protocatechuic acid	5.49 ± 0.27 a	0.60 ± 0.03 c	3.48 ± 0.17 b	3.38 ± 0.17 b

for the treatment with the dielectric barrier discharge plasma and the glow discharge plasma.

After plasma treatment, cinnamic acid continued to be the major phenolic compound in araça-boi juice and increased its percentual content among the phenolics. Cinnamic acid rose from 90.3% to 95.4–96.7% when subjected to DBDP and 93.2–96.4% when subjected to GDP, indicating an increase of 7.0 and 6.7%, respectively, and a significant difference compared to control (untreated juice). The highest increase was attained in the DBDP-treated juice, and no significant difference was observed among the DBDP frequencies. For the GDP-treated juice, the highest content of cinnamic acid was observed in the treatment at a plasma flow rate of 10 mL/min, while at 20 and 30 mL/min, the increase in cinnamic acid was slightly lower.

Cinnamic acid and its derivates are highly abundant in fruits and vegetables, as observed in araça-boi. Numerous investigations have presented findings supporting the positive health outcomes associated with the daily intake of cinnamic acid and its derivatives across global populations [25]. Thus, the increase observed in the contents of cinnamic acid in araça-boi juice can be considered a positive effect of cold plasma processing.

The concentration of protocatechuic acid decreased significantly after plasma treatment. No protocatechuic acid was detected at 50 and 500 Hz in the DBDP, and a decrease of 52% was observed at 1000 Hz. This different decrease percentage is probably related to the type of reactive plasma species produced at these frequencies. At 50 and 500 Hz, DBDP presents a high concentration of hydrogen peroxide (Porto et al., 2023), which may induce oxygen and hydroxyl abstraction from the protocatechuic acid. The same decreasing tendency was observed in the juice treated by the GDP, where a significant decrease (89%) was observed at a plasma flow rate of 10 mL/min. A decrease in protocatechuic acid was also observed at plasma flow rates of 20 and 30 mL/min but at a lower amount (36 and 38, respectively).

The contents of benzoic acid tended to increase after cold plasma processing, except for the juice treated at 1000 Hz in the DBDP and at 30 mL/min in the GDP. The highest increases occurred at 500 Hz in the DBDP and 10 mL/min in the GDP. The increases in benzoic acid coincided with the decreases in the content of protocatechuic acid. It is probable that under these conditions, protocatechuic acid undergoes hydrogenation due to the oxygen abstraction forming benzoic acid, as presented in Figure 4. It should be noted that plasma acts as a catalyst. Reactive plasma species are highly reactive due to their nature. Normally, these species are formed as intermediates by catalysts, but when plasma is applied, it generates highly reactive species that directly react with many food compounds.

The concentration of hydrocinnamic acid considerably increased during the DBDP treatment, with an increase as high as 182% at 500 Hz. An increase in the hydrocinnamic acid content was also observed when the juice was treated by GDP at plasma flow rates of 20 and 30 mL/min, with increases of 93 and 249%, respectively. The increase in hydrocinnamic acid was associated with a decrease in the content of cinnamic acid, depicting that the formation of hydrocinnamic acid came from the hydrogenation of cinnamic acid, as presented in Figure 1. Structurally, hydroxycinnamic acids are hydroxy metabolites of cinnamic acid with a C6–C3 backbone. Hydrocinnamic acid contributes to the aroma and flavor of certain foods. It is used as a flavoring agent, providing a sweet, spicy, and slightly floral note. Its pleasant fragrance contributes to the acceptance of araça-boi juice. Hydrocinnamic acid, like many phenolic compounds, exhibits antioxidant properties, which can help combat oxidative stress in the body. Some studies suggest anti-inflammatory and antimicrobial effects, contributing to their potential therapeutic applications [26,27]. This phenolic acid can potentially inhibit the growth of certain microorganisms, extending the shelf life of food products [27]. Thus, the increase in hydrocinnamic acid can be considered a positive effect of plasma processing.

The plasma-treated araça-boi juice presented a small concentration of p-coumaric acid formed during the plasma treatment, since no p-coumaric acid was detected in the untreated juice. In the DBDP, the increase in p-coumaric acid occurred mainly under 50 and 500 Hz, conditions that generate the highest amounts of hydroxyl free radicals [28], which may be responsible for the hydrolysis of cinnamic acid into p-coumaric acid, following the reaction shown in Figure 1. The GDP treatment has formed p-coumaric acid, but only in the conditions employing 20 and 30 mL/min. The formation of this acid occurred in higher intensity in the DBDP than in the GDP treatments, showing that different plasma systems have different outcomes in the reactions induced in the juice phenolic content. Among the effects of p-coumaric acid are being a free radical scavenger, having antimicrobial activity, and having health benefits. p-Coumaric acid exhibits antioxidant properties, helping to neutralize free radicals in the body. This contributes to the potential reduction of oxidative stress and protection against cellular damage. Some studies suggest that p-coumaric acid may have anti-inflammatory effects [29,30]. Research indicates that p-coumaric acid may have cardioprotective effects, potentially contributing to cardiovascular health by influencing factors such as blood pressure and lipid metabolism [31]. Furthermore, studies suggest that p-coumaric acid may possess anticancer properties. It is being investigated for its potential role in preventing or inhibiting the growth of certain types of cancer cells [32,33]. Not only that, but p-coumaric acid contributes to the flavor and aroma of araça-boi. Due to all these factors, the increase in p-coumaric acid after plasma application can also be considered an advantage for the processed juice.

Phenylacetic acid and oxanilic acid were shown to be very susceptible to plasma treatment. No phenylacetic acid and oxanilic acid were detected in the plasma-treated juices. The exception was for the treatment at 30 mL/min in the GDP, where oxanilic acid remained in lower quantities. The aminophenolic compound 2-aminobenzoxazole was also shown to be very susceptible to plasma treatment. The DBDP-treated juice did not present any remaining 2-aminobenzoxazole, while its content was significantly reduced in the GDP-treated juice. The exception was for the juice treated in the GDP at a plasma flow rate of 10 mL/min, where no significant change was observed in the concentration of this compound. The reduction in the contents of oxanilic acid can be viewed as positive, since its excessive consumption can be harmful and has been associated with the formation of calcium oxalate kidney stones [34]. On the other hand, the significant decrease in 2-aminobenzoxazole is a disadvantage of the plasma process because this compound has been linked to antimicrobial, anti-inflammatory, and anticancer properties [35].

Plasma treatment tended to increase the total phenolic content of araça-boi juice, but this increase depended on the operating condition applied. The DBDP system increased the total phenolic content of the juice when operating at 50 and 1000 Hz, resulting in an increase of 83 and 201%, respectively, of total phenolics. No significant change was observed in the phenolic content when the DBDP was operated at 500 Hz. The increase attained in the GDP system was lower than in the DBDP system. The increase observed at 10, 20, and 30 mL/min of plasma flow rate was 8, 36, and 44%, respectively. The increase in total phenolic content is related to the higher bioavailability of phenolics in the juice. Reactive plasma species react with the surface of the cell membranes, breaking the bonds between the phenolic compounds and the structure of the membrane. This kind of reaction produces free phenolic compounds, which are easily detected by analytical methods and are directly correlated to their bioavailability in the juice.

Cold plasma affects the phenolic profile of fruits in different ways. Blueberries subjected to DBDP for 10 min resulted in an 11% decrease in cinnamic acid, showing a totally different trend from the trend reported herein for araça-boi juice [36]. Protocatechuic acid, on the other hand, tended to increase its concentration in blueberries and herein. The results for rocket-salad leaves followed a similar trend as reported herein, with an increase in the concentration of chlorogenic acid and p-coumaric acid after plasma treatment [16]. Although some authors have reported changes in phenolic compounds during plasma treatment, no reaction pathway is usually presented by these authors. The relationship between the changes in concentration and chemical reactions is rarely reported.

The results attained in this work are evidence that cold plasma technology is an option for the processing of araça-boi juice regarding the increase in biodisponibility and maintenance of its main phenolic compounds. These observations sum up to those of other reports on araça-boi showing the benefits regarding cold plasma processing on aroma compounds, volatiles, sugars, and other organic acids [18]. Furthermore, this study has contributed to other studies on plasma application to fruit juices, showing that nutritional quality improvement is much higher than the few drawbacks of the technology, such as the decrease in amino acid content [13,28]. This work has not performed a stability test of the phenolics on the juices because that was not the focus of this study. The reason behind this decision is that phenolics are known to be reasonably stable in food products, with little loss during storage, which has been proven by many studies that have addressed the stability of phenolic compounds during storage, especially evaluating total phenolic compounds (TPC).

Currently, cold plasma technology is under development and still needs approval from regional and national food and drug agencies for commercial application in food processing. Lab-scale systems have been developed by several research laboratories, and some pilot-scale systems have also been developed. Small industrial-scale systems are currently in use for non-food applications. Thus, cold plasma processing may come into future use in the industry due to its benefits and cost, which are similar to those of pulsed electrical field systems that are already in use in the food industry. For the street vendor, it would take longer, but in the long run, we could have plasma systems at prices a little bit higher than small ozone generators, since ozone is generated by plasma in this commercial equipment.

4. Conclusions

Eight phenolic compounds were identified in araça-boi, with cinnamic acid being the major phenolic compound of the fruit juice, followed by protocatechuic acid. Cold plasma technologies slightly changed the phenolic compound profile of araça-boi (Eugenia stipitata) juice. The total phenolic content in the juice tended to increase due to the release of phenolics bonded to cell membranes into the bulk of the juice, resulting in a higher bioavailability of phenolics. The increase in phenolic content depended on the operating condition applied. The highest increase in phenolic content was observed in the dielectric barrier discharge plasma operating at 1000 Hz.

The phenolic profile slightly changed after plasma treatment. However, cinnamic acid remained the major phenolic compound in the juice. An increase in concentration was observed for hydrocinnamic acid, benzoic acid, and p-coumaric acid. Amino-phenolic compounds, such as 2-aminobenzoxazole and oxanilic acid, were significantly reduced due to their reaction with the reactive plasma species formed during the treatment. Cinnamic acid content rose by 7.0%, showing a significant difference compared to control (untreated juice). The concentration of hydrocinnamic acid considerably increased during the DBDP and GDP treatment, with an increase as high as 182% at 500 Hz, and 249% at plasma flow rates of 30 mL/min.

Overall, plasma treatment improved the bioavailability of the phenolic compounds and resulted in slight changes to the phenolic profile of araça-boi juice. Thus, the technology showed a positive effect on araça-boi juice.