Influence of Glow Discharge Plasma Treatment on Cashew Apple Juice ’ s Aroma Profile and Volatile Compounds

. Cashew apple juice has a distinctive fruity aroma but contains an undesired balsamic/chemical note caused by the high concentration of styrene and other aromatic hydrocarbons. Cold plasma technology can induce chemical changes to fruit juices ’ volatile compounds, improving the aroma of fruit juices. This study is aimed at evaluating the chemical e ﬀ ects of cold plasma on the volatile compounds and aroma of cashew apple juice, which is characterized by a complex mixture of compounds. Glow discharge plasma was applied to cashew apple juice, varying the plasma ﬂ ow rate (10 to 30mL/min) and processing time (10 to 20min) at a constant voltage (80 kV). Plasma treatment induced several changes in the juice ’ s volatile compound composition, with a signi ﬁ cant decrease in fatty acids and fatty acid esters (92%) and an increase in aldehydes (50%), alcohols (86%), and short-chain esters (21%). The primary reaction observed during plasma treatment was the internal scission of fatty acid and fatty acid esters, which formed short-chain esters and aldehydes. Further hydrogenation of aldehydes produced alcohols. The chemical changes induced by plasma treatment intensi ﬁ ed cashew apple juice ’ s aroma by 28% while maintaining its aroma pro ﬁ le.


Introduction
Cashew apple (Anacardium occidentale) juice is widely consumed in some regions of Brazil, India, and Central Africa.The cashew apple is considered an exotic fruit in most of the world due to its short shelf life, making its distribution difficult to most markets.Cashew apple juice, therefore, is also considered an exotic juice.Its flavor is generally sweet with a combination of tartness and high astringency.Its flavor does not resemble any other commercial fruit.
The aroma of cashew apple juice has been described as fragrant, fruity, and tropical.The aroma profile of the juice can change slightly depending on the cashew variety (yellow, red, and orange), ripeness, and kind of processing method.Its primary aroma descriptor is fruity, correlated with its natural sweetness.Depending on the cultivar (variety), it may have floral, woody, earthy, and citrus notes [1].
From a chemical perspective, the aroma of cashew apple juice is a complex mixture of esters, fatty acids, aldehydes, terpenes, hydrocarbons, and aromatic hydrocarbons [1][2][3].The esters contribute to the primary fruity descriptor, while the other chemical compounds are responsible for the secondary notes.Cashew apple contains styrene in its composition, which gives it an aldehydic note that may be unpleasant for some persons [1].Reducing styrene concentration in cashew apple juice can improve its acceptance.
Cold plasma works through a process that involves ionized gas at relatively low temperatures, typically at or near room temperature.By definition, cold plasma refers to the fourth state of matter, consisting of partially ionized gas composed of free electrons, free radicals, charged ions, and neutral particles.Cold plasma is generated by subjecting a gas to an energy source, such as an electrical discharge, radio frequency, and microwave.This energy input causes the gas to lose electrons, forming positively charged ions and free electrons, which further react creating more complex free radicals.In contact with other compounds, cold plasma induces a series of chemical changes that can modify surfaces, kill microorganisms, induce enzymatic reactions, and change bioactive compounds [4,5].
Cold plasma can be utilized through various technological approaches, including glow discharge, dielectric barrier discharge, jet, arc, gliding arc, microwave-driven discharge, and radio frequency-driven discharge plasma [22].Each of these technologies has its own set of advantages and drawbacks.Compared to other plasma systems, the main advantage of glow discharge plasma is its generation of a gas plasma stream that is subsequently delivered into a spacious chamber for sample treatment.This approach offers the advantage of processing large sample volumes, yet it comes with the drawback of requiring low pressures (<0.5 bar) within the chamber.
The use of cold plasma technology on food property enhancement depends on a deep understanding of the chemical changes induced by plasma.In the past years, many studies have reported the chemical reactions and mechanisms that occur with sugars [23,24], oligosaccharides [25,26], furans, pyrazines, and pyridines [17,27], amino acids [28,29], esters and thioesters [30], terpenes, and sesquiterpenes [14,16] when subjected to plasma.However, due to the complexity of functional groups in foods, much work must be done to fully address this technology's capabilities.
Cold plasma processing of fruit juices showed high capability in modulating their aroma and mitigating off-flavors and undesirable aromas.Studies with orange juice showed that the undesired off-flavor caused by 4-terpineol was significantly decreased [14,15].Furthermore, 4-terpineol was converted into limonene, orange juice's most characteristic flavor compound.Studies with pineapple juice showed the capacity to change the ester compounds chemically.Such capability reduced the concentration of methyl hexanoate, which has a very pungent sweet flavor, improving the acceptance of the juice [30].The plasma treatment carried out with camu-camu juice, an exotic Amazonian fruit, showed the ability to modulate the juice's aroma, increasing or decreasing the odor activity value of several descriptors by changing the operating conditions of the treatment [16,31].
Cashew apple juice is an interesting case study because it has compounds from various classes of organic compounds, enabling us to understand the selectivity of reactive plasma species.This work intended to extend the knowledge on plasma treatment of fruit juices and its effects on aroma.Cashew apple juice was subjected to glow discharge plasma technology at three air flow rates (10 to 30 mL/min) and two processing times (10 and 20 min), and the volatile chem-ical profile of the juice aroma was identified by gas chromatography coupled to mass spectrometry.

Materials and Methods
2.1.Materials.Fruit Ltda (Caucaia, Brazil) provided the cashew apple pulp containing no additives.Cashew apple juice was produced by diluting the pulp with distilled water (1 : 1 v/v).
2.2.Plasma Processing.Cold plasma treatment was carried out in a glow discharge plasma system (Plasma Etch model PE-50, USA) fully described in [16].The assays were carried out at three synthetic air flow rates (10, 20, and 30 mL/min) and two processing times (10 and 20 min).These operating conditions were chosen based on prior experience with cold plasma technology applied to fruit juices [13,14,16].The operating conditions were also determined to fit a 2 3 facecentered experimental design with three levels for air flow rate and processing times (0, 10, and 20 min) with 0 min represented by the reference (unprocessed juice).The pressure inside the equipment was maintained at 0.4 bar.Glow discharge plasma should operate under a 0.2 to 0.5 bar pressure for better performance and higher generation of reactive species.The system was operated at 0.4 bar because it is the lowest pressure achieved by the system vacuum pump.
Cashew apple juice (40 mL) was placed inside polypropylene tubes and subjected to plasma treatment.The control group consisted of untreated cashew apple juice.All experiments were done in triplicate.
2.3.Plasma Characterization.The plasma's optical emission spectrum (OES) was attained using a fiber optic spectrometer (Ocean Insight model HR4Pro) associated with a 1 mm diameter optical fiber and a collimating lens.The spectra were measured at a wavelength range from 200 to 900 nm.The spectral resolution of the instrument was 0.06 nm.All analyses were done in triplicate.

Extraction and Chromatographic Analysis of the Volatile
Compounds.The volatile organic compounds of cashew apple juice were extracted using the solid-phase microextraction (SPME) technique.An aliquot of 10 mL of cashew apple juice and 1 g of sodium chloride was placed in a 20 mL vial, equilibrating at 40 °C for 20 min.The volatile compounds were extracted for 30 min using a DVB/CAR/ PDMS (50/30 μm) fiber placed in the headspace of the vial [32].
Samples were analyzed in a GC-MS (Thermos model ISQ).The samples were desorbed directly in the injector, set at 250 °C, working in splitless mode.The volatile was separated in an Equity-1 column (30 m × 0 25 mm ID × 0 25 μm film).The oven programming and chromatograph conditions followed the method described in Porto et al. [30].The mass spectra were compared with the NIST and Wiley mass spectral library.All analyses were done in triplicate.
2.5.Odor Profile.The volatile compounds were grouped according to their primary odor descriptors in the "The Good Scent Company" database [33].The odor activity 2 Journal of Food Processing and Preservation
The main volatile compounds observed in the cashew apple juice analyzed in this work are similar to those previously reported in the literature [1-3].Variations in the concentration of the compounds were due to differences in cultivar, year of collection, and maturity stage, which are part of the natural variation that occurs in the fruit composition profile.

Changes in Volatile Composition Profile Induced by
Plasma Treatment.Glow discharge plasma treatment has induced several chemical changes in the volatile composition profile of cashew apple juice.Table 2 presents the mass fraction of each compound before (control) and after plasma treatment at the tested operating conditions.Figure 1  The most significant net changes in mass fraction were observed for styrene, 3-methyl-1-butanol, 2,3-butadienol, ethyl 2-butenoate, ethyl 3-methylbutanoate, ethyl acetate, homomenthyl salicylate, methoxy-phenyl-oxime, methyl stearate, palmitelaidic acid, and palmitic acid.These were the compounds that most gained or lost mass during plasma treatment.
Among the chemical groups, plasma treatment increased the concentration of aldehyde (50%), esters (21%), linear hydrocarbons (37%), and sesquiterpenes (44%) and significantly decreased the concentration of fatty acids and fatty acid esters (92%).The changes in concentration of phenolic acids, alcohols, aromatic hydrocarbons, terpenes, and lactones depended on the operating conditions applied.Such behavior is explained by the different types and concentrations of reactive plasma species formed in each operating condition [16,30].
Mass balance analysis allowed us to identify chemical changes occurring during plasma treatment.The most significant differences were observed with fatty acids and fatty acid esters, which were reduced by 92% after plasma treatment.The changes affected all fatty acids and fatty acid esters, but the most significant reduction was observed for palmitic acid, palmitelaidic acid, myristic acid, methyl stearate, and stearic acid.
Fatty acids and fatty acid esters can undergo internal scission reactions, which can be induced by free radicals or enzymes [47].These reactions give rise to "green leaf volatiles," primarily small chain aldehydes, alcohols, and organic acids.The increase of all aldehydes and some alcohols accompanied the reduction in the fatty acids and fatty acid   be the source of the increase in hexadecanal through dehydration of the acid group and methyl group abstraction of the methyl and ethyl groups of the fatty acid esters.Palmitelaidic acid could also be the source of hexadecanal through dehydration of the acid group followed by the hydrogenation of the carbon double bond.Such reactions depend on the concentration of hydrogen radicals present in high amounts in air glow plasma (Figure 2).The optical emission spectra attained from the plasma chamber indicated the presence of N 2 + (427 nm), H α (486 nm), H β (656 nm), and atomic oxygen (777 and 845 nm) as the primary plasma reactive species.
The increase observed in 2-decenal and nonanal can be derived from the scission of ethyl oleate.Nonanal and octanal could be formed from the scission of stearic acid, methyl Ethyl (E)-2-butenoate stearate, and palmitelaidic acid.At the same time, the increase in hexanal concentration be linked to the internal scission of myristic acid, ethyl myristate, methyl myristate, and cis-5-dodecenoic acid.Zhou et al. [48], working with fresh-cut cantaloupes, also observed an increase in hexanal, nonanal, and other aldehydes after plasma application.However, they have not discussed the causes for the observed increase in concentration.The increase in hexanol, octanol, and nonanol can be attributed to the hydrogenation of hexanal, octanal, and nonanal produced by the scission of the fatty acids and fatty acid esters.Zhou et al. [48] observed an increase in several alcohols, such as heptanol, octanol, and nonanol, plasma application on fresh-cut cantaloupe, but have not discussed the causes for these chemical changes.
When ethyl and methyl esters undergo plasma-induced scission, they generate an aldehyde and a short-chain ester.This reaction generally occurs at the carbon double bond of unsaturated esters, but saturated esters may break near the central carbon when subjected to plasma.This reaction usually involves an oxygen free radical forming an epoxy group at the carbon double bond, further decomposing, forming an aldehyde and a short-chain ester [30].Such a reaction explains the increase in short-chain esters such as ethyl hexanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, and methyl octanoate.
The changes among the short-chain esters were also relevant.Extended treatment times in plasma (20 min) tended to produce shorter esters.Such phenomenon is explained by methyl group abstraction of the farthermost methyl group of the ester chain or a branched methyl group.These reactions have been detailed in pineapple juice subjected to plasma treatment [30].As observed previously with pineapple juice, ethyl esters showed to be more stable during plasma application than methyl esters and fatty acids.
The concentration of styrene in the plasma-treated juice fluctuated around the concentration of the control.Still, it was impossible to identify any major reaction that occurred or might have affected styrene.
Plasma treatment increased the OAV of most aroma descriptors in the cashew apple juice, intensifying its aroma (Table 3).The intensification of the natural aroma of cashew apple juice was between 24% (30 mL/min) and 28% (10 mL/ min).The intensification of aroma was higher at lower air plasma flow rates than at high flow rates.At higher flow rates, the higher concentration of reactive plasma species caused more chemical reactions that have affected compounds with low odor threshold, such as ethyl 3-methylbutanoate, ethyl 3-methylpentanoate, ethyl hexanoate, ethyl 2-hexenoate, and γ-dodecalactone, all of them having fruity aroma.
Despite the significant changes regarding the OAVs of each aroma descriptor between the untreated and plasmatreated, the aroma profile (Figure 2) did not present a significant shift regarding the contribution of each descriptor to the cashew apple juice aroma, except for the fatty descriptor that increased by 173%.Since the fatty descriptor is a minor descriptor of the cashew apple juice aroma, this increase does not compromise the aroma of the juice.Table 4 presents the normalized aroma profile of cashew apple juice.No significant changes were observed in the contribution of the fruity and aldehydic descriptors toward the juice aroma profile.The most important difference in the aroma profile was the considerable increase in the fatty notes, which became the fourth most noticeable aroma note in the plasma-treated juice.
Although several chemical changes were observed in the juice's volatile compounds, there were no significant changes in the chemical groups (Table 5).Esters and lactones, the main group that gives the fruity aroma, did not change sig-nificantly after plasma treatment, explaining why the fruity aroma continued to be predominant in cashew apple juice.The increase in the aldehyde mass fraction contributed to the increase of the OAVs of the aldehydic and fatty descriptors, which was mainly caused by the scission of fatty acids and fatty acid esters into 2-decenal (fatty), decanal (aldehydic), and nonanal (aldehydic).The OAV of the woody descriptor followed the changes in the mass fraction of the sesquiterpene group.
Styrene is the main contributor to the balsamic descriptor, which some consumers consider an undesired flavor or off-flavor of cashew apple juice.Glow discharge plasma could not reduce the contribution of this descriptor in the cashew apple juice's aroma profile.Plasma treatment could only slightly reduce the contents of styrene and dimethyl styrene in the juice, but not enough to significantly change the aroma profile.
10 Journal of Food Processing and Preservation Lower plasma flow rates increased the concentration of esters, especially short-chain esters with low odor due to the chemical changes discussed in Section 3.2.As the plasma flow rate increased, the concentration of short-chain esters with low odor thresholds slightly decreased due to methyl abstraction and isomerization, resulting in esters with higher odor thresholds.Furthermore, lipid scission reactions were more intense at higher plasma flow rates, increasing the concentration of aldehydes, which are less desired compounds.

Conclusions
This study evaluated the effects of glow discharge plasma on cashew apple juice, which contains a complex mixture of volatile compounds.Plasma treatment induced various changes in the composition of the juice's volatile compounds.The primary reaction observed during plasma treatment was the internal scission of fatty acids and fatty acid esters.Such a reaction decreased by 92% the content of fatty acid and fatty acid esters.The internal scission of fatty acids and fatty acid esters has formed several short-chain esters and aldehydes with low odor thresholds, contributing to the intensification of the odor activity value of cashew apple juice.The aldehyde and short-chain esters increased by 50% and 21%, respectively, increasing OAV by 28%.Hydrogenation of aldehydes resulted in the formation of alcohols, which increased by 86%.Plasma treatment has not significantly changed the concentration of styrene, toluene, and p-dimethylstyrene and could not reduce the concentration of these off-flavors.Glow discharge plasma increased the odor activity value of cashew apple juice, with the lowest flow rate (10 mL/min) resulting in the highest OAV.The lowest flow rate has also resulted in the highest fruity aroma and lowest balsamic and aldehydic notes, thus increasing desired notes and decreasing undesired ones.The increase in plasma flow rate induced undesired chemical changes, leading to a lower aroma quality.
shows a heat map indicating the changes in composition in a visual format.Blue indicates an increase in the compound concen-tration, and red indicates a decrease.Light colors indicate changes up to 50%, and bold colors indicate more than 50%.

FigureFigure 3 :
Figure Optical emission spectra of air produced inside the chamber of the glow discharge plasma.
significant statistical differences within each aroma descriptor (rows).

Table 1 :
Cashew apple juice volatile compounds with their residence time, Kovats index, odor threshold in water, and aroma descriptors.

Table 2 :
Mass fraction of volatile compounds cashew apple juice subjected to plasma treatment.

Table 3 :
Odor activity values (OAV) of the primary aroma descriptors of the untreated and plasma-treated cashew apple juice.Plasma treatment was carried out in glow discharge plasma for 20 min at plasma air flow rates of 10, 20, and 30 mL/min.

Table 4 :
Normalized odor activity values (OAV) of the primary aroma descriptors of the untreated and plasma-treated cashew apple juice.Plasma treatment was carried out in glow discharge plasma for 20 min at plasma air flow rates of 10, 20, and 30 mL/min.

Table 5 :
Mass fraction of the main chemical groups of the untreated and plasma-treated cashew apple juice.Plasma treatment was carried out in glow discharge plasma for 20 min at plasma air flow rates of 10, 20, and 30 mL/min.AromaControl 10 mL/min 20 mL/min 30 mL/min Esters 88 7 ± 2 7 a 88 9 ± 2 7 a 86 9 ± 2 6 a 88 0 ± 2 6 a