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Effect of plasma and heat treatments on orange juice quality

Efeito de plasma e tratamento térmico na qualidade do suco de laranja

Abstract

Heat treatment is used in the orange juice industry to neutralize the action of pathogenic microorganisms. However, it can reduce the nutritional value of the juice. Thus, our study assessed the cold plasma treatment as an alternative method against Escherichia coli and Candida albicans in 'Lima' orange juice. Both, plasma and heat treatments, reduced the amount of E. coli in the juice, inactivating 16.72 and 100%, respectively. Plasma did not inactivate C. albicans, but heat treatment inactivated 100%. Plasma and heat treatment increased Hue angle and luminosity (more yellowish juice). Plasma reduced vitamin C, carotenoids, and polyphenols content, while increased flavonoids. Heat treatment reduced the carotenoid content. However, neither heat nor plasma treatment altered the antioxidant activity. The plasma treatment reduced the intensity of color (chroma), the soluble solids content and the acidity ratio, total sugars, and the vitamin C content of juice compared to the heat-treated and control juices. Plasma-treated juice showed increased levels of yellow flavonoids, total phenolics and antioxidant activity until the 12th day of storage.

Keywords:
bioactive compounds; Candida albicans; Citrus sp.; Escherichia coli; non-heat treatment

Resumo

O tratamento térmico é utilizado na indústria de suco de laranja para neutralizar a ação de microrganismos patogênicos. No entanto, pode reduzir o valor nutricional do suco. Assim, o estudo avaliou o tratamento com plasma frio como método alternativo contra Escherichia coli e Candida albicans em suco de laranja 'Lima'. Ambos os tratamentos, plasma e calor, reduziram a quantidade de E. coli no suco, inativando 16.72 e 100%, respectivamente. O plasma não inativou C. albicans, mas o tratamento térmico inativou 100%. Plasma e tratamento térmico aumentaram o ângulo Hue e a luminosidade do suco (tom mais amarelo). O plasma reduziu o conteúdo de vitamina C, carotenóides e polifenóis, enquanto aumentou os flavonóides. O tratamento térmico reduziu o teor de carotenóides. No entanto, nem o calor nem o tratamento com plasma alteraram a atividade antioxidante. O tratamento com plasma reduziu a intensidade da cor (croma), o teor de sólidos solúveis e a razão de acidez, açúcares totais e o teor de vitamina C do suco em comparação com os sucos tratados termicamente e controle. O suco tratado com plasma apresentou maiores teores de flavonóides amarelos, fenólicos totais e atividade antioxidante até o 12º dia de armazenamento.

Palavras-chave:
compostos bioativos; Candida albicans; Citrus sp.; Escherichia coli; tratamento não-térmico

1. Introduction

Orange juice is widely consumed around the world, mostly because of its health benefits, such as high nutrition and pleasant aroma (Ni et al., 2020NI, M., GU, K., HASSAN, B., NING, D., ZHENG, Y., QI, Y. and XU, Y., 2020. Effect of oviposition by Bactrocera dorsalis on the antioxidant activity of orange juice. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 80, no. 3, pp. 641-647. http://dx.doi.org/10.1590/1519-6984.218661. PMid:31644657.
http://dx.doi.org/10.1590/1519-6984.2186...
). This juice is an important source of vitamin C, polyphenols, carotenoids, flavonoids, sugars, antioxidant properties and several bioactive compounds (Almeida et al., 2015ALMEIDA, F.D.L., CAVALCANTE, R.S., CULLEN, P.J., FRIAS, J.M., BOURKE, P., FERNANDES, F.A. and RODRIGUES, S., 2015. Effects of atmospheric cold plasma and ozone on prebiotic orange juice. Innovative Food Science & Emerging Technologies, vol. 32, pp. 127-135. http://dx.doi.org/10.1016/j.ifset.2015.09.001.
http://dx.doi.org/10.1016/j.ifset.2015.0...
; Liaquat et al., 2023LIAQUAT, M., ALI, I., AHMAD, S., MALIK, A.M., ASHRAF, H.M.Q., PARVEEN, N., TAREEN, M.J., SAEED, T., SHAH, S.H. and ZULFIQAR, B., 2023. Efficiency of exogenous zinc sulfate application reduced fruit drop and improved antioxidant activity of ‘Kinnow’mandarin fruit. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 83, p. e244593. http://dx.doi.org/10.1590/1519-6984.244593. PMid:34468512.
http://dx.doi.org/10.1590/1519-6984.2445...
; Morais et al., 2022MORAIS, R.A., TEIXEIRA, G.L., FERREIRA, S.R.S., CIFUENTES, A. and BLOCK, J.M., 2022. Nutritional composition and bioactive compounds of native Brazilian fruits of the Arecaceae family and its potential applications for health promotion. Nutrients, vol. 14, no. 19, p. 4009. http://dx.doi.org/10.3390/nu14194009. PMid:36235663.
http://dx.doi.org/10.3390/nu14194009...
). During the processing, the nutritional value as well as the microbiological security of the juice must be maintained in accordance with the standards, as it may also be used as a substrate for microorganisms to grow in, such as bacterial species Escherichia coli.

Generally, E. coli does not cause harm to human health, but some strains are very pathogenic and may bind to epithelial cells of the intestine and release toxins, causing gastric disorders, known as gastroenteritis (Ramadhianto et al., 2019RAMADHIANTO, A., NASUTION, J. and LUBIS, R., 2019. Bioaktivity test crude fruit of citrus lime (Citrus aurantifolia) on bacteria Escherichia coli in vitro. Budapest International Research in Exact Sciences Journal, vol. 1, no. 2, pp. 16-20. http://dx.doi.org/10.33258/birex.v1i2.219.
http://dx.doi.org/10.33258/birex.v1i2.21...
). The Brazilian Health Regulatory Agency (ANVISA) sets a limit of 10 colony-forming units(CFU) of fecal coliforms (group to which E. coli belongs) per milliliter of pasteurized juice (Brasil, 2001BRASIL. AGÊNCIA NACIONAL DE VIGILÂNCIA SANITÁRIA – ANVISA, 2001 [viewed 17 May 2020]. Resolução-RDC nº 12, de 2 de janeiro de 2001 [online]. Diário Oficial da República Federativa do Brasil, Brasília, 10 January. Section 1, pp. 01-10. Available from: https://bvsms.saude.gov.br/bvs/saudelegis/anvisa/2001/res0012_02_01_2001.html
https://bvsms.saude.gov.br/bvs/saudelegi...
). Candida albicans, a yeast-like fungus, is another important human pathogen in some cases, especially for people with weakened immune system. Under such circumstances, C. albicans causes severe diseases and it is hard to treat, which makes it of important concern (Macias-Paz et al., 2023MACIAS-PAZ, I.U., PÉREZ-HERNÁNDEZ, S., TAVERA-TAPIA, A., LUNA-ARIAS, J.P., GUERRA-CÁRDENAS, J.E. and REYNA-BELTRÁN, E., 2023. Candida albicans the main opportunistic pathogenic fungus in humans. Revista Argentina de Microbiologia, vol. 55, no. 2, pp. 189-198. http://dx.doi.org/10.1016/j.ram.2022.08.003. PMid:36411138.
http://dx.doi.org/10.1016/j.ram.2022.08....
).

The process of pasteurization is the most used treatment in the juice industry, consisting of the application of mild temperatures (under 100 °C) for a few seconds (Chiozzi et al., 2022CHIOZZI, V., AGRIOPOULOU, S. and VARZAKAS, T., 2022. Advances, applications, and comparison of thermal (pasteurization, sterilization, and aseptic packaging) against non-thermal (ultrasounds, UV radiation, ozonation, high hydrostatic pressure) technologies in food processing. Applied Sciences, vol. 12, no. 4, p. 2202. http://dx.doi.org/10.3390/app12042202.
http://dx.doi.org/10.3390/app12042202...
). Although this process presents high efficacy in the inactivation of pathogenic microorganisms and undesirable enzymes, nutrients loss and changes in the quality of the juice are also produced (Roobab et al., 2022ROOBAB, U., ABIDA, A., CHACHA, J.S., ATHAR, A., MADNI, G.M., RANJHA, M.M.A.N., RUSU, A.V., ZENG, X.A., AADIL, R.M. and TRIF, M., 2022. Applications of innovative non-thermal pulsed electric field technology in developing safer and healthier fruit juices. Molecules, vol. 27, no. 13, p. 4031. http://dx.doi.org/10.3390/molecules27134031. PMid:35807277.
http://dx.doi.org/10.3390/molecules27134...
). A study that applied pasteurization and thermosonication in order to assess the quality of tangor juice reported a reduction in ascorbic acid, carotenoids and antioxidant activity (Basumatary et al., 2022BASUMATARY, B., NAYAK, M., NAYAK, P.K. and KESAVAN, R.K., 2022. Assessment of quality changes of tangor fruit juice after pasteurization and thermosonication treatments. Journal of Food Process Engineering, vol. 45, no. 12, p. 14170. http://dx.doi.org/10.1111/jfpe.14170.
http://dx.doi.org/10.1111/jfpe.14170...
).

Due to these constrains, new technologies have been studied and developed to improve the quality of juice, and an increasing interest has been shown for the application of cold plasma. Plasma is formed by electric discharges produced by subjecting liquid or gas to a strong electromagnetic field (Starek et al., 2020STAREK, A., SAGAN, A., ANDREJKO, D., CHUDZIK, B., KOBUS, Z., KWIATKOWSKI, M., TEREBUN, P. and PAWŁAT, J., 2020. Possibility to extend the shelf life of NFC tomato juice using cold atmospheric pressure plasma. Scientific Reports, vol. 10, no. 1, p. 20959. http://dx.doi.org/10.1038/s41598-020-77977-0. PMid:33262535.
http://dx.doi.org/10.1038/s41598-020-779...
). It is comprised of electrons, ions, and both,energized and non-energized, neutral particles (Cherif et al., 2023CHERIF, M.M., ASSADI, I., KHEZAMI, L., HAMADI, N.B., ASSADI, A.A. and ELFALLEH, W., 2023. Review on recent applications of cold plasma for safe and sustainable food production: principles, implementation, and application limits. Applied Sciences, vol. 13, no. 4, p. 2381. http://dx.doi.org/10.3390/app13042381.
http://dx.doi.org/10.3390/app13042381...
). For such purpose, it is used a plasma in which the energy of the electrons is much higher than the other species. Since the plasma has a more chemical character and it is obtained at atmospheric pressure is known as cold atmospheric plasma (Ocaña de Jesús et al., 2022OCAÑA DE JESÚS, R.L., IBÁÑEZ, A.T.G., PUEBLA, I.R., DÍAZ, A.G., ROMERO, P.G.R., SÁNCHEZ, J.V., TORRES SEGUNDO, C. and VALENCIA, H.M., 2022. Microbiological study of the effect of a dielectric barrier discharge interaction on processed orange juices exposed to the environment. Food Science and Technology, vol. 42, p. e02622. http://dx.doi.org/10.1590/fst.02622.
http://dx.doi.org/10.1590/fst.02622...
).

Previous studies about plasma treatment have demonstrated interesting results for both inactivation of microorganisms and physicochemical characteristics indifferent food products, including juices. The treatment of orange juice inoculated with Staphylococcus aureus, E. coli, and C. albicans with dielectric barrier discharge (DBD) plasma for 12, 8 and 25 s, respectively, showed a 5-log reduction of the microorganisms (Shi et al., 2011SHI, X.M., ZHANG, G.J., WU, X.L., LI, Y.X., MA, Y. and SHAO, X.J., 2011. Effect of low-temperature plasma on microorganism inactivation and quality of freshly squeezed orange juice. IEEE Transactions on Plasma Science, vol. 39, no. 7, pp. 1591-1597. http://dx.doi.org/10.1109/TPS.2011.2142012.
http://dx.doi.org/10.1109/TPS.2011.21420...
). Almeida et al. (2017)ALMEIDA, F.D.L., GOMES, W.F., CAVALCANTE, R.S., TIWARI, B.K., CULLEN, P.J., FRIAS, J.M., BOURKE, P., FERNANDES, F.A.N. and RODRIGUES, S., 2017. Fructooligosaccharides integrity after atmospheric cold plasma and high-pressure processing of a functional orange juice. Food Research International, vol. 102, pp. 282-290. http://dx.doi.org/10.1016/j.foodres.2017.09.072. PMid:29195950.
http://dx.doi.org/10.1016/j.foodres.2017...
also reported that after plasma DBD treatment in prebiotic orange juice the oligosaccharides in the juice were minimally degraded, not affecting its quality, and color and antioxidant activity were preserved. The treatment of orange juice with dielectric barrier discharge plasmas for 2.5, 3.5, 5.0 and 6.0 min with electrical power of 40 W showed a reduction in Salmonella spp. colony forming units of 4.47 to 1.00 Log and from 4.00 to 1.00 Log for E. coli (Ocaña de Jesús et al., 2022OCAÑA DE JESÚS, R.L., IBÁÑEZ, A.T.G., PUEBLA, I.R., DÍAZ, A.G., ROMERO, P.G.R., SÁNCHEZ, J.V., TORRES SEGUNDO, C. and VALENCIA, H.M., 2022. Microbiological study of the effect of a dielectric barrier discharge interaction on processed orange juices exposed to the environment. Food Science and Technology, vol. 42, p. e02622. http://dx.doi.org/10.1590/fst.02622.
http://dx.doi.org/10.1590/fst.02622...
). Liao et al. (2018)LIAO, X., LI, J., MUHAMMAD, A.I., SUO, Y., CHEN, S., YE, X., LIU, D. and DING, T., 2018. Application of a dielectric barrier discharge atmospheric cold plasma (Dbd-Acp) for Eshcerichia Coli inactivation in apple juice. Journal of Food Science, vol. 83, pp. 401-408. http://dx.doi.org/10.1111/1750-3841.14045. PMid:29355961.
http://dx.doi.org/10.1111/1750-3841.1404...
also reported that, after treatment with plasma DBD in apple juice, it had a slight effect on the °Brix, pH, titratable acidity, color values, total phenolic content and antioxidant capacity of this product.

Thus, the present study aimed to assess the physicochemical quality and antioxidant potential of the whole orange juice (‘Lima’ variety) processed under the conventional thermal treatment and the cold plasma method.

2. Material and Methods

2.1. Microorganisms and inoculating suspensions

The strain ATCC 25922 of Escherichia coli was provided by the Laboratório de Bioprocessos from the Universidade Federal do Rio Grande do Norte – UFRN, and Candida albicans SC 5314 obtained from the Laboratório Integrado de Biomoléculas from the Universidade Federal do Ceará – UFC. The bacterial suspension of E. coli was prepared in Trypticase soy broth, and yeast suspension (C. albicans) in potato dextrose broth. After 24 h of incubation under constant stirring and temperature of 36±1 °C (Marconi incubator, model MA-420), bacterial colony-forming units were counted on violet-red bile agar (VRBA) for a concentration of 1.71 × 1010 CFUml-1, and C. albicans counted in PDA (3.05 × 108 CFU ml-1) (Tortora et al., 2012TORTORA, G.J., FUNKE, B.R. and CASE, C.L., 2012. Microbiologia. Porto Alegre: Artmed.).

2.2. Atmospheric plasma

We used an experimental prototype (unpublished data) developed by the research team of the Laboratório de Processamento de Materiais por Plasma of the Centro Integrado de Inovação Tecnológica do Semi-Árido from the Universidade Federal Rural do Semi-Árido – UFERSA. The device generates plasma DBD at a tension of 14 kV and 400 Hz.

2.3. Treatments

Oranges (‘Lima’ variety) bought from a local supermarket were sanitized with chlorinated water (100 ppm, 10 min) and squeezed using a kitchen processor to obtain the juice. Sterilization of the juice was performed in autoclave for 15 minutes at 121 °C. With the juice at room temperature, the inoculating suspensions were poured into the juice, separately, and the concentration of each microorganism was adjusted following the methodology described by Montenegro et al. (2002)MONTENEGRO, J., RUAN, R., MA, H. and CHEN, P., 2002. Inactivation of E. coli O157: H7 using a pulsed nonthermal plasma system. Journal of Food Science, vol. 67, no. 2, pp. 646-648. http://dx.doi.org/10.1111/j.1365-2621.2002.tb10653.x.
http://dx.doi.org/10.1111/j.1365-2621.20...
to 5.86 × 103 CFU ml-1 (E. coli) and 1.74 × 104 CFU ml-1 (C. albicans). Afterwards, treatments were applied: autoclaved, non-inoculated juice as an absolute control; inoculated juice (negative control); exposition of 5 ml of the inoculated juice to plasma DBD for 1 min in Petri dish (90 mm); and heat treatment for 1 min at 87 °C (water bath) of 50 ml in amber glass bottles (positive control).

Samples of each treatment were then plated on VRBA and potato dextrose agar (PDA) for assessment of colony-forming units of E. coli and C. albicans, respectively, and incubated on Biological Oxygen Demand (BOD) at 36±1 °C for 24 h (Siqueira, 1995SIQUEIRA, R.S., 1995. Handbook of food microbiology. Brasília: Embrapa. In Portuguese.).

A complete randomized experimental design was used, with five replicates. Experimental data were submitted to the non-parametric Kruskal-Wallis test and means compared with Dunn test (p ≤ 0.05) using the RStudio software (RStudio Team, 2020RSTUDIO TEAM, 2020 [viewed 17 May 2020]. RStudio: integrated development environment for R [online]. RStudio/PBC. Available from: https://posit.co/products/enterprise/connect/
https://posit.co/products/enterprise/con...
).

2.4. Physicochemical properties and bioactive compounds during storage

The experiment was conducted in a completely randomized design in a 3x5 split-plot scheme, with five replicates. The plot corresponded to treatments (control, plasma, heat treatment) and subplot to days of storage (0, 4, 8, 12, and 16 days).

The juice from orange fruits (‘Lima’ variety) underwent both plasma and heat treatments, separately, at the same conditions as described in the previous section. The orange juice was manually removed and deposited in sterilized beakers inside a laminar flow cabinet (Quimis). They were placed in amber glass bottles sterilized by autoclaving. Untreated natural juice was considered as the absolute control. Samples of each treatment and the control were evaluated for their quality attributes 12 h after the application of treatments (Time-0) and the others were stored in closed bottles at 2±1 °C and relative humidity of 53±3% (Continental TC41 refrigerator) for 16 days, with evaluations being performed every 4 days until the sixteenth day (Figure 1).

Figure 1
Experiment setup flowchart.

We performed analyses for the following traits: color, assessing luminosity (L), chroma (C) and hue angle (H), using a digital colorimeter (CR-400 Chroma Meter, Konica Minolta, Inc., Tokyo, Japan); pH, directly determined by a potentiometer (Tecnal, Piracicaba, SP, Brazil) with automatic temperature adjustment; soluble solids content (SSC), determined by direct refractometry of the juice sample in a digital refractometer with automatic temperature compensation (Palette, Atago, Tokyo, Japan) (Horwitz, 2002HORWITZ, W., 2002. Official methods of analysis of AOAC International. Gaithersburg: AOAC International, vol. 1.); and titratable acidity (TA), obtained by titration with 0.1 N NaOH using 1.0% phenolphthalein as color indicator (Zenebon et al., 2008ZENEBON, O., PASCUET, N.S. and TIGLEA, P., 2008. Métodos físico-químicos para análise de alimentos. São Paulo: Instituto Adolfo Lutz.); and soluble solids content/ titratable acidity ratio (SSC/TA).

Also, total soluble sugars content was determined by the Anthrone method and spectrophotometer reading at 620 nm (Yemn and Willis, 1954YEMN, E.W. and WILLIS, A.J., 1954. The estimation of carbohydrate in plant extracts by Antrone. The Biochemical Journal, vol. 57, pp. 504-514.); reducing sugars content, using the 3,5-dinitrosalicylic method and spectrophotometer reading at 540 nm (Miller, 1959MILLER, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, vol. 31, no. 3, pp. 426-428. http://dx.doi.org/10.1021/ac60147a030.
http://dx.doi.org/10.1021/ac60147a030...
); carotenoid content, following Higby (1962)HIGBY, W.K., 1962. A simplified method for determination of some aspects of the carotenoid distribution in natural and carotene‐fortified orange juice. Journal of Food Science, vol. 27, no. 1, pp. 42-49. http://dx.doi.org/10.1111/j.1365-2621.1962.tb00055.x.
http://dx.doi.org/10.1111/j.1365-2621.19...
methodology (spectrophotometer at 450 nm); yellow flavonoid content, following Francis (1982)FRANCIS, F.J., 1982. Analysis of anthocyanins. In: P. MARKAKIS, ed. Anthocyanins as food colors. New York: Academic Press, pp. 181-207. http://dx.doi.org/10.1016/B978-0-12-472550-8.50011-1.
http://dx.doi.org/10.1016/B978-0-12-4725...
methodology (spectrophotometer at 374 nm); total extractable polyphenol content, as described by Larrauri et al. (1997)LARRAURI, J.A., RUPÉREZ, P. and SAURA-CALIXTO, F., 1997. Effect of drying temperature on the stability of polyphenols and antioxidant activity of red grape pomace peels. Journal of Agricultural and Food Chemistry, vol. 45, no. 4, pp. 1390-1393. http://dx.doi.org/10.1021/jf960282f.
http://dx.doi.org/10.1021/jf960282f...
(spectrophotometer at 700 nm); vitamin C content, using Tillman’s solution (Strohecker and Henning, 1967STROHECKER, R. and HENNING, H.M., 1967. Análisis de vitaminas: métodos comprobados. Madrid: Paz Montalvo.); and antioxidant activity by capturing ABTS.+ radical (spectrophotometer at 734 nm) (Rufino et al., 2007RUFINO, M.D.S.M., ALVES, R.E., BRITO, E.S., MORAIS, S.M., SAMPAIO, C.D., PÉREZ-JIMENEZ, J. and SAURA-CALIXTO, F.D., 2007. Scientific methodology: determination of the total antioxidant activity in fruits by capturing the free radical ABTS+. Fortaleza: Embrapa Agroindústria Tropical. In Portuguese.).

Data were submitted to two-way analysis of variance and means were grouped by Tukey test (p ≤ 0.05) in R software (RStudio Team, 2020RSTUDIO TEAM, 2020 [viewed 17 May 2020]. RStudio: integrated development environment for R [online]. RStudio/PBC. Available from: https://posit.co/products/enterprise/connect/
https://posit.co/products/enterprise/con...
).

3. Results

3.1. Inactivation of microorganisms

A significant difference (p < 0.05) was observed for microbial count population of E. coli in the orange juice after plasma treatment, showing a reduced number of CFU ml-1 than the negative control, whereas both heat-treated and sterile samples showed to be free from both microorganisms (Table 1). According to the standards set by the ANVISA (Brasil, 2001BRASIL. AGÊNCIA NACIONAL DE VIGILÂNCIA SANITÁRIA – ANVISA, 2001 [viewed 17 May 2020]. Resolução-RDC nº 12, de 2 de janeiro de 2001 [online]. Diário Oficial da República Federativa do Brasil, Brasília, 10 January. Section 1, pp. 01-10. Available from: https://bvsms.saude.gov.br/bvs/saudelegis/anvisa/2001/res0012_02_01_2001.html
https://bvsms.saude.gov.br/bvs/saudelegi...
), only the juice treated with the conventional method (heat) would have been considered good for consumption. There was no significant difference between treatments in juice inoculated with C. albicans.

Table 1
CFU of E. coli ATCC 25922 and C. albicans ATCC SC 5314 in ‘Lima’ orange juices ubmitted to different treatments.

3.2. Physicochemical analysis and bioactive compounds of orange juice in storage

All treatments of juice caused changes in the luminosity values only in the first day of evaluation (the day of treatment), with no changes being reported in the following days of storage (Table 2, Figure 2).

Table 2
Summary of analysis of variance for luminosity (L), chroma (C), hue angle (°Hue), titratable acidity (TA), and pH of ‘Lima’ orange juice submitted to different treatments and stored under 2±1 °C for 16 days.
Figure 2
Color as luminosity (L), chroma (C) and hue angle (°Hue) of ‘Lima’ orange juice submitted to different treatments and stored under 2±1 °C for 16 days.

Plasma-treated juice samples showed a significant difference for chroma values only after 8 and 12 days of storage. The hue angle values of all treatments stayed near 100°, in the yellow color range, throughout the whole period of storage. The initial hue angle was higher in heat-treated juice but showed a slight reduction during the storage period, making the yellow color less intense (Figure 2). Samples treated with cold plasma showed the greatest values at the end, where their yellow was most vivid.

Only after 8 days of storage we observed some alterations in the titratable acidity and pH of the treated juices. After 16 days, plasma-tread samples showed lower pH, and consequently greater acidity than control and heat treatment (Figure 3). Similarly, differences in the SSC and SSC/TA ratio were observed only after 8 days of storage, with a reduction occurring in the juice treated with plasma (Table 3, Figure 4).

Figure 3
Titratable acidity and pH of ‘Lima’ orange juice submitted to different treatments and stored under 2±1 °C for 16 days.
Table 3
Summary of analysis of variance for soluble solids content (SSC), soluble solids content/titratable acidity ration (SSC/TA), total soluble sugars content (TSS), and reducing sugars content (RS) of ‘Lima’ orange juice submitted to different treatments and stored under 2±1 °C for 16 days.
Figure 4
Soluble solids content and soluble solids content/titratable acidity (SSC/TA) ratio of ‘Lima’ orange juice submitted to different treatments and stored under 2±1 °C for 16 days.

Total soluble sugars and reducing sugars contents were altered only after the 8th day of storage. However, after 16 days the plasma-treated juice showed similar content of total sugars to the absolute control. The juice under thermal treatment maintained the highest content of total sugars and reducing sugars (Figure 5).

Figure 5
Total soluble sugars and reducing sugars content of ‘Lima’ orange juice submitted to different treatments and stored under 2±1 °C for 16 days.

There were no significant differences in Vitamin C between factors treatment and storage. The values for the absolute control, plasma- and heat-treated juices were 35.09, 28.90, and 32.29 mg100g-1, respectively, with the plasma treatment showing the lowest values of all treatments. During the storage period, we observed a reduction in vitamin C from 42.68 in the day of processing to 27.53 mg100g-1 after 16 days of storage (Table 4, Figure 6).

Table 4
Summary of analysis of variance for Vitamin C (VitC), antioxidant activity (AA), carotenoids (Carot), total extractable polyphenols (TEP), and yellow flavonoids (Flav) of ‘Lima’ orange juice submitted to different treatments and stored under 2±1 °C for 16 days.
Figure 6
Vitamin C, antioxidant activity, carotenoids, total extractable polyphenols, and yellow flavonoids in ‘Lima’ orange juice submitted to different treatments and stored under 2±1 °C for 16 days.

There was a degradation of carotenoids in heat- and plasma-treated juices. No significant difference for carotenoid content after 4, 8, and 16 days of storage was seen. In general, both plasma and heat treatments caused the same level of loss in carotenoids (Figure 6).

A higher polyphenol content was observed in the absolute control and heat-treated juices at the day of processing and after 4 days of storage, in comparison to plasma treatment. After 8 and 12 days, the juice under plasma treatment showed an increase in polyphenols, significantly higher than the other treatments. A small reduction of polyphenols content was observed after 16 days of storage (Figure 6). Similar behavior as for the plasma effect was observed for the antioxidant activity after 16 days of storage. The juice under plasma treatment had a reduction in the antioxidant activity when compared to the absolute control. It was also possible to observe that in the day of the processing the antioxidant activity in heat- and plasma-treated juice was higher (3.72 and 3.37 µM Troloxg-1, respectively) (Figure 6).

4. Discussion

Cold plasma treatment significantly reduced the population of E. coli in the juice as compared to untreated juice (Table 1). Although plasma was less effective than heat treatment, our study opens perspectives for the use of this technique that has been tested against several microorganisms in many products. Klämpfl et al. (2012)KLÄMPFL, T.G., ISBARY, G., SHIMIZU, T., LI, Y.F., ZIMMERMANN, J.L., STOLZ, W., SCHLEGEL, J., MORFILL, G.E. and SCHMIDT, H.U., 2012. Cold atmospheric air plasma sterilization against spores and other microorganisms of clinical interest. Applied and Environmental Microbiology, vol. 78, no. 15, pp. 5077-5082. http://dx.doi.org/10.1128/AEM.00583-12. PMid:22582068.
http://dx.doi.org/10.1128/AEM.00583-12...
treated spores of C. albicans with plasma (10 kVpp, 1 kHz, under atmospheric pressure) in petri dishes for 30 s and observed about 4-log inactivation. Xiong et al. (2010)XIONG, Z., LU, X.P., FENG, A., PAN, Y. and OSTRIKOV, K., 2010. Highly effective fungal inactivation in He+O2 atmospheric-pressure nonequilibrium plasmas. Physics of Plasmas, vol. 17, no. 12, p. 123502. http://dx.doi.org/10.1063/1.3526678.
http://dx.doi.org/10.1063/1.3526678...
observed the effect of cold plasma at 8 kV, 9 kHz and helium gas at a rate of 2 L min-1 on C. albicans, showing a 99.9% of inactivation after 8 min in closed Petri dishes, while in open Petri dishes the inactivation was reduced to a few fractions, similarly to our results (also treated in open Petri dishes). The differences verified between the data obtained in this work and the works discussed here may be due to the differences between the technical specifications of the plasma devices used. The inactivation of fungi by treatment with cold plasma is given by the destruction and deformation of structures, and the degeneration and oxidation of proteins and DNA molecules in the cytoplasm (Xiong et al., 2010XIONG, Z., LU, X.P., FENG, A., PAN, Y. and OSTRIKOV, K., 2010. Highly effective fungal inactivation in He+O2 atmospheric-pressure nonequilibrium plasmas. Physics of Plasmas, vol. 17, no. 12, p. 123502. http://dx.doi.org/10.1063/1.3526678.
http://dx.doi.org/10.1063/1.3526678...
; Ye et al., 2012YE, S.Y., SONG, X.L., LIANG, J.L., ZHENG, S.H. and LIN, Y., 2012. Disinfection of airborne spores of Penicillium expansum in cold storage using continuous direct current corona discharge. Biosystems Engineering, vol. 113, no. 2, pp. 112-119. http://dx.doi.org/10.1016/j.biosystemseng.2012.06.013.
http://dx.doi.org/10.1016/j.biosystemsen...
; Kang et al., 2014KANG, M.H., HONG, Y.J., ATTRI, P., SIM, G.B., LEE, G.J., PANNGOM, K., KWON, G.C., CHOI, E.H., UHM, H.S. and PARK, G., 2014. Analysis of the antimicrobial effects of non thermal plasma on fungal spores in ionic solutions. Free Radical Biology & Medicine, vol. 72, pp. 191-199. http://dx.doi.org/10.1016/j.freeradbiomed.2014.04.023. PMid:24794411.
http://dx.doi.org/10.1016/j.freeradbiome...
; Lu et al., 2014LU, Q., LIU, D., SONG, Y., ZHOU, R. and NIU, J., 2014. Inactivation of the tomato pathogen Cladosporium fulvum by an atmospheric‐pressure cold plasma jet. Plasma Processes and Polymers, vol. 11, no. 11, pp. 1028-1036. http://dx.doi.org/10.1002/ppap.201400070.
http://dx.doi.org/10.1002/ppap.201400070...
).

Taking into consideration the large reduction of microbial population after plasma treatment in previous studies, the small effect in our study may have been caused by a combination of different factors such as the power our device compared to others in previous works, the input of gas flow into the system, distance between the sample and plasma, and amount of treated sample.

Regarding juice quality, the color of the juice was not significantly affected by the treatments used. Although some values of hue angle (H) and luminosity (L), color parameters, were significantly different during storage, it does not cause a difference in juice color that can be perceived by the consumer. Kovačević et al. (2016b)KOVAČEVIĆ, D.B., PUTNIK, P., DRAGOVIĆ-UZELAC, V., PEDISIĆ, S., JAMBRAK, A.R. and HERCEG, Z., 2016b. Effects of cold atmospheric gas phase plasma on anthocyanins and color in pomegranate juice. Food Chemistry, vol. 190, pp. 317-323. http://dx.doi.org/10.1016/j.foodchem.2015.05.099. PMid:26212976.
http://dx.doi.org/10.1016/j.foodchem.201...
also observed color change in pomegranate juice treated with plasma generated with argon gas (2.5 kV and 25 kHz). Such changes are influenced by the duration of the treatment, volume of the sample, and rate of gas flow into the system.

From eight days of storage, pH, titratable acidity (TA), soluble solids content (SSC), and SSC/TA ratio started to vary among treatments. At 16 days, the plasma-treated juice presented higher acidity and lower pH (Figure 3).Our results are consistent with Xiang et al. (2018)XIANG, Q., LIU, X., LI, J., LIU, S., ZHANG, H. and BAI, Y., 2018. Effects of dielectric barrier discharge plasma on the inactivation of Zygosaccharomyces rouxii and quality of apple juice. Food Chemistry, vol. 254, pp. 201-207. http://dx.doi.org/10.1016/j.foodchem.2018.02.008. PMid:29548443.
http://dx.doi.org/10.1016/j.foodchem.201...
, who observed a reduction of pH and an increase in acidity in apple juice treated with plasma DBD for 40 to 200 s at 90 W. However, SSC did not change over time. Differently, Shi et al. (2011)SHI, X.M., ZHANG, G.J., WU, X.L., LI, Y.X., MA, Y. and SHAO, X.J., 2011. Effect of low-temperature plasma on microorganism inactivation and quality of freshly squeezed orange juice. IEEE Transactions on Plasma Science, vol. 39, no. 7, pp. 1591-1597. http://dx.doi.org/10.1109/TPS.2011.2142012.
http://dx.doi.org/10.1109/TPS.2011.21420...
reported no alteration in pH and acidity in orange juice treated with plasma DBD (30 kV, 60 kHz) for 5 to 20 s.

Furthermore, total soluble sugars (TSS) and reducing sugars (RS) did not vary among treatments. From eight days of storage, the plasma-treated juice had lower TSS and RS, while the heat-treated juice had the highest contents (Figure 5). Almeida et al. (2015)ALMEIDA, F.D.L., CAVALCANTE, R.S., CULLEN, P.J., FRIAS, J.M., BOURKE, P., FERNANDES, F.A. and RODRIGUES, S., 2015. Effects of atmospheric cold plasma and ozone on prebiotic orange juice. Innovative Food Science & Emerging Technologies, vol. 32, pp. 127-135. http://dx.doi.org/10.1016/j.ifset.2015.09.001.
http://dx.doi.org/10.1016/j.ifset.2015.0...
have also observed the effect of plasma treatment DBD (70 kV, 50 Hz, under the presence of atmospheric air) on the levels of reducing sugars, where they reported reduction of reducing sugar fructose after 60 s of treatment. Such effect of the plasma after a small period under treatment was not observed in the present study, probably because the frequency and tension used (14 kV, 400 Hz) were different from the previous study.

The vitamin C degradation observed in the plasma-treated juice is probably associated with a greater exposition to the atmospheric air, since the juice in the open Petri dish formed a surface-layer of 9 cm diameter during the treatment. This may be due to the high concentration of oxygen in the air that degrades the vitamin C by activating oxidase enzymes (Shi et al., 2011SHI, X.M., ZHANG, G.J., WU, X.L., LI, Y.X., MA, Y. and SHAO, X.J., 2011. Effect of low-temperature plasma on microorganism inactivation and quality of freshly squeezed orange juice. IEEE Transactions on Plasma Science, vol. 39, no. 7, pp. 1591-1597. http://dx.doi.org/10.1109/TPS.2011.2142012.
http://dx.doi.org/10.1109/TPS.2011.21420...
). However, previous studies on the effect of plasma on vitamin C are very inconsistent. Shi et al. (2011)SHI, X.M., ZHANG, G.J., WU, X.L., LI, Y.X., MA, Y. and SHAO, X.J., 2011. Effect of low-temperature plasma on microorganism inactivation and quality of freshly squeezed orange juice. IEEE Transactions on Plasma Science, vol. 39, no. 7, pp. 1591-1597. http://dx.doi.org/10.1109/TPS.2011.2142012.
http://dx.doi.org/10.1109/TPS.2011.21420...
analyzed orange juice treated with plasma DBD (20 kV and 60 kHz) in open Petri dishes and no significant reduction in the vitamin C content was reported. However, Almeida et al. (2017)ALMEIDA, F.D.L., GOMES, W.F., CAVALCANTE, R.S., TIWARI, B.K., CULLEN, P.J., FRIAS, J.M., BOURKE, P., FERNANDES, F.A.N. and RODRIGUES, S., 2017. Fructooligosaccharides integrity after atmospheric cold plasma and high-pressure processing of a functional orange juice. Food Research International, vol. 102, pp. 282-290. http://dx.doi.org/10.1016/j.foodres.2017.09.072. PMid:29195950.
http://dx.doi.org/10.1016/j.foodres.2017...
, reported an increase in vitamin C when treating 20 ml of orange juice with plasma DBD (70 kV, 50 Hz, atmospheric air) for 1 min.

There was a degradation of carotenoids in heat- and plasma-treated juices. No significant difference for carotenoid content after 4, 8, and 16 days of storage was seen. In general, both plasma and heat treatments caused the same level of loss in carotenoids (Figure 6). Both plasma and heat treatments reduced carotenoids content in orange juice. The photooxidation of carotenoids is mainly influenced by oxygen and light (Dutra et al., 2012DUTRA, A.D.S., FURTADO, A.A.L., PACHECO, S. and OIANO NETO, J., 2012. Effect of thermal treatment on the carotenoid, phenolic compound and ascorbic acid concentrations, and the antioxidant capacity of murcott tangerine juice. Brazilian Journal of Food Technology, vol. 15, pp. 198-207. http://dx.doi.org/10.1590/S1981-67232012005000012.
http://dx.doi.org/10.1590/S1981-67232012...
), which were inevitable during the processes. Another possible factor related to the degradation of carotenoids is the heat (87 °C) used in one treatment. Dutra et al. (2012)DUTRA, A.D.S., FURTADO, A.A.L., PACHECO, S. and OIANO NETO, J., 2012. Effect of thermal treatment on the carotenoid, phenolic compound and ascorbic acid concentrations, and the antioxidant capacity of murcott tangerine juice. Brazilian Journal of Food Technology, vol. 15, pp. 198-207. http://dx.doi.org/10.1590/S1981-67232012005000012.
http://dx.doi.org/10.1590/S1981-67232012...
, when treated mandarin orange juice at 88 °C for 30 s (like what was used in this study) observed a reduction of approximately 11% in carotenoid content.

A higher polyphenol content was observed in the absolute control and heat-treated juices in the day of processing and after 4 days of storage, in comparison to plasma treatment. After 8 and 12 days, the juice under plasma treatment showed an increase in polyphenols, significantly higher than other treatments. A small reduction of polyphenol content was observed after 16 days of storage (Figure 6). Similar behavior as for the plasma effect was observed for the antioxidant activity after 16 days of storage. The juice under plasma treatment had a reduction in the antioxidant activity when compared to the absolute control. It was also possible to observe that in the day of the processing the antioxidant activity in heat- and plasma-treated juice was higher (3.72 and 3.37 µM Trolox g-1, respectively) (Figure 6). Almeida et al. (2015)ALMEIDA, F.D.L., CAVALCANTE, R.S., CULLEN, P.J., FRIAS, J.M., BOURKE, P., FERNANDES, F.A. and RODRIGUES, S., 2015. Effects of atmospheric cold plasma and ozone on prebiotic orange juice. Innovative Food Science & Emerging Technologies, vol. 32, pp. 127-135. http://dx.doi.org/10.1016/j.ifset.2015.09.001.
http://dx.doi.org/10.1016/j.ifset.2015.0...
treated 20 mL of orange juice supplemented with oligosaccharide with plasma DBD (70 kV, 50 Hz, atmospheric air) and observed a reduction in phenolic compounds after 15, 30, 45, and 60 s. Distinct results were reported by Herceg et al. (2016)HERCEG, Z., KOVAČEVIĆ, D.B., KLJUSURIĆ, J.G., JAMBRAK, A.R., ZORIĆ, Z. and DRAGOVIĆ-UZELAC, V., 2016. Gas phase plasma impact on phenolic compounds in pomegranate juice. Food Chemistry, vol. 190, pp. 665-672. http://dx.doi.org/10.1016/j.foodchem.2015.05.135. PMid:26213024.
http://dx.doi.org/10.1016/j.foodchem.201...
as an increase of phenolic compounds were seen in pasteurized (80 °C, 2 min) pomegranate juice and treated with plasma (argonium gas, 2.5 kW, 25 kHz). Such discrepancy in the polyphenol content found in the present work and by these other authors may be related to the difference of compounds found on different fruits (Kovačević et al., 2016aKOVAČEVIĆ, D.B., KLJUSURIĆ, J.G., PUTNIK, P. and VUKUŠIĆ, T., 2016a. Stability of polyphenols in chokeberry juice treated with gas phase plasma. Food Chemistry, vol. 212, pp. 323-331. http://dx.doi.org/10.1016/j.foodchem.2016.05.192. PMid:27374539.
http://dx.doi.org/10.1016/j.foodchem.201...
).

Our results showed that cold plasma treatment is promising for the inactivation of microorganisms in orange juice, besides maintaining better organoleptic properties as compared to heat treatment. However, further studies should focus on finding adequate treatment duration, volume of sample to be treated, rate of gas flow into the system to improve capacity of the treatment to improve the treatment's ability to inactivate microorganisms and to maintain beneficial compounds in juice.

Heat and plasma treatments reduce the Escherichia coli population in orange juice. Plasma treatment is not effective in inactivating Candida albicans, while heat treatment inactivates 100%. Plasma treatment reduces chroma (always being below the other treatments throughout the days of storage), soluble solids content/ titratable acidity ratio, total soluble sugars, and vitamin C content in orange juice, as compared to heat- and autoclaved-treat juices. This fact can end up harming the acceptance of the product by the consumer market, since it can change its flavor. However, plasma-treated orange juice shows increased levels of yellow flavonoids, total extractable polyphenols, and antioxidant activity (ABTS+) until the 12 days of storage as compared to heat- and autoclaved-treated juices.

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Publication Dates

  • Publication in this collection
    17 July 2023
  • Date of issue
    2023

History

  • Received
    07 Mar 2023
  • Accepted
    05 June 2023
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