Low-Pressure Plasma Treatment Increased the Quality and Characteristic Flavor of Lyophilized Lemon Slices

Low-pressure plasma (LPP) treatment was implemented as a nonthermal technology to preserve the organoleptic and nutritional qualities of lyophilized lemon slices. Efects of LPP treatment on the basic quality and favor characteristics of lyophilized lemon slices stored for 7d were evaluated. Fresh lyophilized lemon slices were prepared as a reference. Te total titratable acids and sugars were in the range of 1.00 ∼ 1.11mg/mL and 190.21 ∼ 197.37 mg/mL. Te total phenolic content and Vc gradually decreased during storage from 1.74 to 1.33mg/mL and 0.53 to 0.31mg/mL. LPP had minor efects on total sugars and total titrate acids but prevented the storage losses of vitamin C (Vc) and phenols. A total of 35 volatile organic compounds were identifed with C10 monoterpenoids being the major compounds. C6-C9 aldehydes corresponding to a green grassy aroma gradually decreased with increasing storage time. Te gallery plot confrmed the diferent compositions of volatile organic compounds in LPP-treated samples. LPP-treated lemon slices had a higher content of preferred aromatic substances (terpinen-4-ol, α -terpineol, α -terpinene, and limonene) with a citrus-like favor. Tese results demonstrated the positive efect of LPP treatment on retaining the favor characteristics of lemon slices.


Introduction
Lemons (Citrus limon) belong to the Citrus genus within the Rutaceae family.As a functional fruit, lemons are rich in limonene, vitamin C, and multiple trace elements such as calcium, magnesium, and sulfur [1].Lemon peels are rich in pectin and volatile organic compounds and possess antioxidization and antibacterial properties [2].Research has indicated that the small quantity of citral in lemons is responsible for the favor quality and distinctive aroma of lemon oil.Te lemon essential oil is extracted from the peel and has been proven to be benefcial in the alleviation of symptoms associated with anxiety [3].Furthermore, citric acid in lemons is efective in reducing the risk of recurrent stones [4].Currently, the most commonly used strategies for fruit processing in the fruit industry include juicing, pickling, and lyophilizing [5][6][7].Among these treatments, lyophilization is a way to completely preserve the nutritional value of fruits [5].In China, lyophilized lemon slices are popular amongst consumers due to their extensive use in the preparation of beverages.
Low-pressure plasma (LPP) treatment is an emerging nonthermal processing technique in the food industry, which can utilize the media surrounding food to produce photoelectrons, ions, free radicals, and other active substances [8].It has been shown that plasma technology can improve the functional value of food by increasing the concentration of bioactive compounds and reducing antinutritional components [9].Furthermore, cold plasma technology can inhibit the growth of microorganisms and improve the quality of food and has been a potential technology to be applied in the food industry [10,11].Research has demonstrated that industrial processing techniques, such as cutting, peeling, and slicing, can lead to microbial contamination by pathogens entering the internal tissues of the fruit [12].LPP can be an alternative for microorganism control that is more environmentally friendly and healthy than chemical methods such as the use of chlorine or SO 2 [13,14] and overcomes the defciencies of traditional physical sterilization such as heat (moist heat and dry heat), radiation (UV and microwave), and fltration [15].Heating is considered as the most commonly used method worldwide.However, thermal treatments are known to induce the caramelization reaction, which negatively infuences the appearance, taste, and nutrition of the fruit, resulting in a loss of quality [16].Nonthermal processing techniques have attracted an increasing attention in the food industry in order to maintain quality and nutritional value as much as possible.Tere are many studies focused on the inactivation of contaminating bacteria efect of low-pressure plasma; however, limited research eforts have been aimed at investigating the efect of LPP on the quality of fruit products [17][18][19].Te application of LPP in the food industry is still in an emerging stage.
Recently, gas chromatography-ion mobility spectrometry (GC-IMS) has been extensively applied in the separation and detection of volatile organic compounds [20].Compared with gas chromatography-mass spectrometer (GC-MS), GC-IMS technology is able to combine the virtues of both the strong separation ability of gas chromatography and higher sensitivity of ion migration spectrum and can quickly detect trace volatile organic compounds without any sample pretreatment, maximizing the loss of volatile substances during material pretreatment [21,22].In addition, the three-dimensional map of different volatile organic compounds obtained by GC-IMS technology can be transformed into the fngerprint of breeze favor, which is benefcial to recognize the diference in volatile organic compounds content in the samples more intuitively.
A particular focus has been put on nonthermal processing technologies, which are designed to eliminate the adverse efects of heat on food products.Cold plasma generates reactive oxygen species (ROS) in air mixtures, and the production of ozone not only has a highly antimicrobial efect but also causes strong oxidative stress on enzyme inactivation.LPP treatment will signifcantly reduce protease activity and, however, increase texture profle and color properties.In addition, it can be carried out at room temperature and atmospheric pressure, making it secure and energy-saving.Color, texture, and favor are important indicators of lemons.Since the relative humidity of the environment can weaken the LPP performance, it may be a suitable tool to maintain the quality of lyophilized lemon slices.Literature reported a controversial conclusion on the efects of cold plasma on polyphenols and favonoids, which may determine the favor of lemons.Herein, we investigated the efect of LPP on the basic quality parameters (titratable acids, total sugar, total phenol, vitamin C, and color index) and favor profle of lyophilized lemon slices.GC-IMS was used to evaluate the efects of LPP treatment on the volatile organic compounds in lyophilized lemon slices.

Materials and Methods
2.1.Materials.Lemons were purchased from a fruit market in Guangzhou (China), which belong to the Citrus genus within the Rutaceae family.Folinphenol, gallic acid, and acetic acid were obtained from Lvyin Biotechnology Co., Ltd.(Guizhou, China).Deionized water, acetone, hydrochloric acid, acetone, and phenolphthalein were purchased from Pengcai Fine Chemical Co., Ltd.(Langfang, China).

Sample Preparation.
Te fresh lemons (approx.60 g/per lemon) were cut into circular slices with a diameter of 5 cm and thickness of 0.5 cm after careful washing.Ten, the lemon slices were lyophilized for 48 h in a freeze-dryer.Next, the lyophilized lemon slices were divided into three groups: G1, G2, and G3.G1 are fresh lyophilized lemon slices without any treatment.G2 are lyophilized lemon slices stored at room temperature for 7 days.G3 are lyophilized lemon slices exposed to low-pressure plasma treatment and then stored at room temperature for 7 days.Two parallel circular aluminum electrodes with a 75 mm distance between them formed the plasma source (Figure 1).Samples were placed between the electrodes.Te low-pressure plasma treatment was conducted as described in our previous work [24].Briefy, the lyophilized lemon slices were treated at a discharge voltage of 40 kV root mean square (RMS) at 0.4 mbar for 1.5 min.All the treatments were carried out in triplicate.

Titratable Acid (TA) Content.
Approximately, 10 g of lyophilized lemon slices from each group were ground into powder and dissolved in 100 mL of distilled water.Two drops of 1% phenolphthalein indicator were added to the solution.A precalibrated 0.05 mol/L sodium hydroxide solution was titrated with the lemon solution (approximately 10 mL).Distilled water was also titrated and used as a blank control.Te TA of the sample was calculated according to the following formula, where the equivalent weight of malic acid is 67.04.

2
Journal of Food Biochemistry Titratable acid mg mL   � (ml base titrant) ×(N of base in mol/L) × equivalent weight of acid sample volume in ml . (1)

Total Phenol Content.
Te method used to extract the polyphenols from the lemon slices was previously described by El-Serafy and El-Sheshtawy [25] and with some modifcations.Based on the previous reports, ethanol is the best extracting solvent for determining the total phenol content [26].Terefore, 98% ethanol was used to extract the phenols from 10 g of powdered samples collected from each group (G1, G2, and G3).Approximately, 1.0 mL of the sample and 0.2 mL of Folin-Ciocalteu reagent were mixed with a magnetic stirrer in a beaker.After 2 min, 1.5 ml of 10% sodium carbonate solution and deionized water were added.Te mixtures were incubated at room temperature for 75 min.Te absorbance of the mixtures was detected with a spectrophotometer (Shenzhen Pukang Electronics Co., Ltd., Shenzhen, China) at 765 nm.Te total phenolic content of each sample was calculated after comparison with a gallic acid standard curve.

Total Sugar Content.
Te total sugar content was determined using the phenol sulfuric acid method [27] with a little modifcation.Approximately, 0.25 g of powdered samples from each group were mixed with 100 mL distilled water and 20 mL concentrated hydrochloric acid.After refux heating in a boiling water bath (100 °C) for 3 h, the mixture was analyzed using an enzyme marker analyzer (Shenzhen Pukang Electronics Co., Ltd., China).Te total sugar content was calculated by comparison with the standard curve.

Vitamin C (Vc) Content.
Te Vc content of the lyophilized lemon slices was determined using high-performance liquid chromatography (HPLC), according to the method described by Lafarga et al. [28] with some modifcations.
Approximately, 1 g of powdered samples from each group (G1, G2, and G3) were mixed with 6 mL distilled water and 6 mL ethanol (12%) in a beaker.After stirring for 30 s, the mixture was transferred to a 100 mL volumetric fask.Next, 5 mL of 2 mol/L acetic acids were added, and distilled water was used to fll the remaining volume of the fask.Ten, the prepared solution was transferred to an HPLC to measure the vitamin content on a reversed-phase Supelcosil TM LC18 (5 µm) stainless-steel column (250 × 4.6 mm i.d., Supelco, USA).An isocratic solvent system was used (0.1 mL/L of sulfuric acid, pH 2.5-2.6).Te fow rate was fxed at 1 mL/min, and the UV-Vis photodiode array detector was set at 254 nm.Te identifcation of vitamin C was performed by comparing the retention time and the obtained spectra to those previously obtained with a standard.

Color Index of Lyophilized Lemon Slices.
Te color index of the lyophilized lemon slices was measured using the colorimeter CR400 (Konica Minolta Co., Ltd., Japan).Te colorimeter was calibrated with a black and white standard plate prior to any sample measurement.Te color index (L * , a * , and b * ) was determined at three random surface areas of each sample.
2.8.Analysis of the Volatile Component.Te volatile component analysis of lyophilized lemon slices was conducted using an Agilent 490 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) and IMS instrument (Fla-vourSpec ® , Gesellschaft für Analytische Sensorsystem mbH, Dortmund, Germany).Headspace solid-phase microextraction (HS-SPME) was used to extract the volatile organic compounds from lyophilized lemon slices.Approximately, 0.5 g of ground lemon powder was placed into a 20 mL headspace bottle and incubated at 25 °C for 30 min.After that, 100 μL of the analyte was injected into the injector (85 °C) for analysis.GC-IMS conditions are FS-SE-54-CB-115MID column (15m × 0.53 mm), column temperature of 60 °C, and high purity N 2 as the carrier gas.Te fow program of N 2 was performed as follows: 2 mL/min for 2 min, increased to 10 mL/min within 8 min, to 100 mL/min within 10 min, and to 150 mL/min within 10 min.Te analytes were ionized in an ionization chamber by a 3H ionization source with a positive ion mode and then transferred to a 9.8 cm drift tube at a 500 V/cm constant voltage with a 150 ml/min nitrogen fow at 45 °C.
Te volatile compound contents were estimated using the internal standard quantitative method, which was expressed as an n-hydrocarbon (C 7 -C 30 ) equivalent according to the following equation (2) [29]: where W i represents volatile I; W z represents the mass of the prepositive hydrocarbon of volatile compound I; A i and A z represent the peak area of volatile compound I and the normal hydrocarbon before volatilization, respectively; f′ represents the correction factor of volatile compound I.
Te f′ value was assumed as 1 in this study.
2.9.Statistical Analysis.One-way analysis of variance (ANOVA) of the data was performed with Duncan's test using IBM SPSS Statistic software, and a least signifcant diference (LSD) with a confdence interval of 95% was applied to compare the means.All analyses were conducted in triplicate, and data are given as mean ± standard deviation (SD).

Physical and Chemical Properties
3.1.1.Total Titratable Acids, Total Sugars, Total Phenols, and Vc Content.Titratable acidity refects the quality index of food, as the organic acid content directly afects the favor, color, and storage stability of fruit.Te total titratable acids of G1, G2, and G3 samples were in the range of 1.00∼1.11mg/mL (as shown in Figure 2(a)).Te total sugar, which includes soluble monosaccharides and oligosaccharides in fruit, refects on the sensory quality, organizational morphology, and nutritional value of products.Meanwhile, total sugar content is a crucial factor that afects the choice of consumers, especially for diabetic consumers.As shown in Figure 2(b), no signifcant diferences (P > 0.05) were observed in the total sugar content from group G1 (197.37 mg/ mL), G2 (190.21 mg/mL), and G3 (196.76 mg/mL).It is well known that Vc is essential for the normal functioning of the human body.Recent research has reported that the healthpromoting efect of Vc might be associated with a low expression of proinfammatory cytokines in the spleen [30].In Figure 2(c), we found that Vc gradually decreased during storage, while the content of Vc in LPP-treated samples was 4% higher than that in G2.Te total phenolic content of fruit is a reference value in antioxidant research and plays an important role in the redevelopment of fruit products [31].It was found in Figure 2(d) that the total phenol content was 1.74 mg/mL in G1, 1.33 mg/mL in G2, and 1.68 mg/mL in G3.Vc and phenols are reported to be chemically unstable and easily decomposed when exposed to heat, light, and oxygen [32].Terefore, the phenol and Vc contents in the fresh samples (G1) were higher than that of samples stored for 7 d (G2).Also, the samples treated by LPP showed a higher content of phenol and Vc than that of G2 samples.Farias et al. reported that cold plasma reduced the PPO activity by 46% of apple cubes and 50% of apple juice [33].
Besides, the reactive species of plasma can cause the degradation of the cell membrane and enhance the release of phenolic compounds.Tere are contradictory reports about the efect of cold plasma on food antioxidants.Referring to Vc, cold plasma creates reactive species in the medium to oxidize Vc into deoxyascorbic acid.In addition, the exposure time is too short to cause the degradation of vitamin content.In this study, we proposed that LPP treatment can reduce the loss of antioxidants such as phenols and Vc that occur in lyophilized lemon slices during storage.

Color Index and Rehydration
Rate.Te appearance of the fruit is also a very important factor to evaluate the overall quality.In many cases, appearance and color play a significant role in the pricing and selling of fruit.Te L * .a* .b* value of lyophilized lemons was determined before and after rehydration using a chronometer, and the results are shown in Figure 3.According to colorimetric guidelines, diferent parameters can indicate the presence of diferent colors in samples.Te detailed guidelines for colorimetry are as follows: a higher a * value indicates that the color is closer to pure red, and a lower a * value indicates a color closer to pure green; a higher b * value indicates that the color is closer to pure yellow, while a lower b * value indicates that the color is closer to pure blue.As is well known, the storage conditions (temperature, time, light, oxygen, and moisture content) or the processing would afect the appearance of lemon slices.In this way, the diferences between the samples from the three groups were compared using the colorimetric guidelines.In terms of fresh G1 samples (as shown in Figure 3), no signifcant diference was observed in the L * value for the samples before (L * � 30.63) and after rehydration (L * � 34.63).However, a decrease in a * and an increase in b * were found, which was lower than that of samples after rehydration.It is interesting to note that the brightness (L value) was signifcantly enhanced after rehydration in fresh or LPP-treated lyophilized lemons after 7-d storage.In contrast, the yellowness (b * value) showed an opposite trend.Te lightness of lyophilized lemons may be explained as a result of the loss of water and the stability of pigments.In terms of LPP treatment, the penetration depths of plasma and the chemical reactions initiated by reactive species are probably the main reasons for changes in color and other physicochemical properties [34].In this study, LPP treatment was too short to generate a signifcant change in the color of lyophilized lemons.

GC-IMS Topographic Plot. GC-IMS technology is used
for the separation and detection of volatile organic compounds [20].A three-dimensional (retention time, migration time, and peak intensity) spectrum map of the organic matter by comparing the three groups of the lyophilized lemon slice is presented in Figure 4(a), and a 2D topographic top view plot of GC-IMS is shown in Figure 4(b).In Figure 4(b), each point represents one type of volatile organic compound.Te color represents the concentration of the substance, where white indicates a lower concentration and red indicates a higher concentration (the darker the color, the greater the concentration).Most of the signals were distributed in retention times of 100-950 s and a drift time of 1.0-1.75 with a high separation degree.Te retention times of some of the ions were between 500 s and 800 s, mainly because these compounds had a low polarity, and it is known that nonpolar compounds tend to retain longer on nonpolar columns than polar compounds [35].Te comparison spectrum showed the diference in the volatile compound content of lyophilized lemon slices more intuitively.Compared to the fresh samples (G1, left), the red plot in G2 (middle) and G3 (right) indicated higher volatile organic compounds, more concentrated in the range of 550-950 s.Te blue plot indicated a lower concentration of volatile organic compound than the fresh sample, more detected in the range of 100-200 s.It can be concluded that the concentration of small molecules decreased with storage time, while the concentration of larger molecules, such as terpenes, increased with storage time.Te distinct diferences observed from the 3D and 2D spectrum require an indepth statistical analysis.

Fingerprint Analysis of Volatile Organic Compound.
A total of 35 aroma compounds were detected by GC-IMS analysis in lemon slices, and the results are shown in Table 1.
Te results indicated that of the total aromatic compounds, olefns accounted for 77.43%, alcohols accounted for 14.69%, aldehydes accounted for 2.12%, ketones accounted for 5.4%, and lipids accounted for 0.36%.Tese results show that olefns (limonene accounted for 51.58% of the total) are the main aroma substances found in lemons, followed by ketones, alcohols, and aldehydes with lower proportions.Te detected volatile substances were lower than in previous studies of lemon slices subjected to diferent drying methods [32], probably due to the diferent processing or the diferent sources of lemon.As some individual compounds presented at diferent concentrations, several signals or spots were generated which represent the formation of corresponding dimers [36].Figure 4 was transformed into a gallery plot (Figure 5).In the fngerprint, each row represents all the signal peaks selected from a lyophilized lemon slice sample, while each column represents the signal peaks of the same volatile organic compounds in diferent lyophilized lemon samples.Te letters M and D following some substances represent the monomer or dimer of the same substance, respectively, and the numbers refer to the peaks of unidentifed components.As shown in Figure 5, the concentration of volatile organic compounds can be obtained using the color depth of the plot.Trough longitudinal comparison, the rule of the favor characteristics of diferent substances is more intuitive.In      Figure 5, the concentration of red squares detected in samples follows the order: G3 > G2 > G1.It was found that G3 had the highest concentration of 4-terpineol, α-terpineol, linalool, nonaldehyde, octyl aldehyde, α-terpinene, β-pinene, limonene, and α-pinene.In the previous report, aldehydes (nerolides and geranyl aldehyde) and esters (nerolides and geranyl acetate) are responsible for the aroma quality of lemons [37].Terefore, LPP showed no negative efect on the favor characteristics of lyophilized lemon slices.We also found that the concentration of compounds in the green squares decreased or even disappeared in G1 and G3, which included compounds such as 1-butanol, 1propanol, furfural, ethyl acetate, and 4-methyl-2-pentanone.
In terms of orange squares (unidentifed compound), there was also a small diference in the amount of the substance in the three groups of samples.Four terpenes including α-terpinene, limonene, β-pinene, and α-pinene were identifed in the lemon slices, accounting for 47%-52% of all volatile compounds.Tese C10 monoterpenoids accumulate in special oil glands of citrus fruit pericarps and are the main contributors to lemon favor and aroma properties [38].Te process of freezedrying facilitated the accumulation of these compounds by an increase of 9% and 7% in G2 and G3 compared with G1. α-Pinene and β-pinene might get degraded in the presence of ozone to give molozonide which will further disassociate to form formaldehyde [39].However, in this study, we did not observe a signifcant reduction of pinene, probably due to a short treatment time.Limonene might degrade in the presence of hydroxy ions to c-terpinene and α-terpinene, while c-terpinene is resistant to further degradation by LPP [40].In addition, six alcohols such as linalool, terpinen-4-ol, and α-terpineol, which contribute to the unique and desirable favors and aromas of lemon, were also identifed.Linalool and terpinen-4-ol are crucial volatile favor compounds with citrus fruits and rose aroma notes [41].Te GC-IMS results suggested that these characteristic alcohols were increased during the drying process and retained after LPP treatment.It has been reported that terpinen-4-ol imparted signifcant reduction during distillation and extraction because of its thermosensitive nature [42].In contrast, the increase of these compounds observed in this study was a result of the oxidative degradation of limonene exposed to reactive species induced by LPP, and they were maintained due to the low temperature during the whole processing.However, LPP treatments reduced the favor characteristics of fresh green orange peels corresponding to some C6-C9 micromolecule aldehydes such as hexanal, which are the key contributors of the fresh green orange peels with strong volatility and low threshold.In general, LPP increased the content of characteristic volatile compounds of lemon slices with a citrus-like favor.addition to efcient sterilization, LPP demonstrated a positive efect on the quality preservation of lyophilized lemon slices.Terefore, this study shows that LPP promises application in the food industry.

Figure 2 :
Figure 2: Comparison of physical and chemical properties of lyophilized lemon slices: (a) the total titratable acid content of lyophilized lemon slices in diferent treatment groups (G1: lyophilized lemon slice at 0 d; G2: lyophilized lemon slices at 7 d; G3: lyophilized lemon slices with LPP treatment at 7 d); (b) the total sugar content of lyophilized lemon slices in diferent treatment groups; (c) the vitamin C content of lyophilized lemon slices in diferent treatment groups; (d) the total phenol content of lyophilized lemon slice in diferent treatment groups.

Table 1 :
GC-IMS integration parameters of volatile organic compounds in the lyophilized lemon slice 1 .