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Effect of cold plasma on blueberry juice quality
Food Chemistry 290 (2019) 79–86
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Effect of cold plasma on blueberry juice quality a,1
Yanan Hou a b
b,1
, Ruixue Wang
a,⁎
, Zhilin Gan , Tao Shao
b,⁎
T a
a
, Xinxue Zhang , Mohe He , Aidong Sun
a
Department of Food Science and Engineering, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 10083, People’s Republic of China Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China
A R T I C LE I N FO
A B S T R A C T
Keywords: Cold plasma Blueberry juice Bacillus Anthocyanin Phenolics Vitamin C Antioxidant activity Color change
This work focusses on the effects of cold plasma, a novel, non-thermal technology, on the quality of blueberry juice, such as inactivation of Bacillus, anthocyanins, phenolics, vitamin C, antioxidant activity and color change. Oxygen concentration (0, 0.5% and 1%) of ionized gas was firstly confirmed to be the main factors for CP treatment, besides treatment time (2, 4 and 6 min). The increment of treatment time and oxygen concentration significantly promoted an increasing trend of death for Bacillus. Compared with thermal treatment, the content of phenolics was significantly increased by CP treatment, and also CP treatment could better keep the original color of blueberry juice. In terms of anthocyanin and vitamin C, relatively shorter exposure time to CP was more suggested. In antioxidant tests, increment of oxygen concentration resulted in the increasing trends of antioxidant activity in DPPH and ABTS assays.
1. Introduction Blueberry, a rich source of bioactive components including vitamin C, phenolics, anthocyanin and flavonoid, is one the most popular berries with high health value (Wang et al., 2017; Terefe, Delon, Buckow, & Versteeg, 2015). Previous reports have illustrated that blueberry could be recognized as one functional food with several activities, including antioxidant, anti-bacteria, anti-inflammatory, anti-allergic, anticancer and resistance to cardiovascular disease (Souza, Silva, Lima, Pio, & Queiroz, 2014; Folmer et al., 2014; Heinonen, 2010; Liu, Chen, Li, & Sun, 2016; Joshi, Howell, & D'Souza, 2016; Pertuzatti et al., 2014). Therefore, blueberry is considered as a fruit with a great processing potential. One of the most widespread blueberry products is the juice. Recently, blueberry juice is becoming more popular and attractive to consumers due to the high levels of antioxidants, such as anthocyanins and vitamin C, which is healthful to humans (Barba, Esteve, & Frigola, 2013). However, the blueberry juice with high fresh-like characteristics and long shelf-life is always a challenge. It is extremely important to preserve the above valuable compounds during processing in the highest possible extent. Traditionally, pasteurization is the most widely applied technique for inactivation of pathogenic microorganisms and enzymes during juice processing. However, the quality of juice, such as flavor, aroma, nutritional constituents, can be broken and degraded by thermal processing (Gómez, Welti-Chanes, & Alzamora, 2011). Consumers’ demand
for minimally processed juices has turned to non-thermal technologies. High pressure (Barba et al., 2013), ultrasound (Simunek, Jambrak, Dobrović, Herceg, & Vukušić, 2014), pulsed electric field (Chen et al., 2014), microwave heating (Elik, Yanık, Maskan, & Göğüş, 2016) are among the no-thermal technologies that have been applied to blueberry juice processing. Cold plasma (CP) is a novel, non-thermal technology, which is generated by excitation of process gas (carbon dioxide, argon, nitrogen, helioum, oxygen or air) with a strong electric field under ambient temperature (Niemira, 2012). During the processing, reactive chemical species, such as radicals, heat and UV light are excited, which vary according to the parameters of CP devices, such as voltage, frequency, flow rate and the type of gas that is used (Ramos, Miller, Brandão, Teixeira, & Silva, 2013). Several reports have demonstrated that CP could be efficiently applied in food industry, including pathogen decontamination (Lee, Kim, Chung, & Min, 2015), enzyme inactivation (Pankaj, Misra, & Cullen, 2013) and surface modification (Pankaj et al., 2014). The effect of CP on the quality of pomegranate and sour cherry juices have been studied previously (Herceg et al., 2016; Garofulić et al., 2015). The above literatures have demonstrated that treatment time was the main factor of CP on the quality of juice, and the CP was generated only by one pure gas, like nitrogen or argon. It is not clear if other parameters can affect it. Although Surowsky, Fröhling, Gottschalk, Schlüter, and Knorr (2014) have shown that CP acquired a better bactericidal effect after mixing oxygen to the ionized gas. Still
⁎
Corresponding authors. E-mail addresses: [email protected] (Z. Gan), [email protected] (T. Shao). 1 First authors: They had made equal contributions to this paper. https://doi.org/10.1016/j.foodchem.2019.03.123 Received 8 October 2018; Received in revised form 18 March 2019; Accepted 23 March 2019 Available online 25 March 2019 0308-8146/ © 2019 Published by Elsevier Ltd.
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little is known on the quality of fruit juices by using CP generated by a mixture of process gas. Additionally, no studies exist showing the effect of CP on the quality of blueberry juice which forms the focus of this work. Related to the current findings, the aim of this work is to evaluate the effect of CP treatment on the quality of blueberry juice, such as inactivation of Bacillus, anthocyanins, phenolics, vitamin C, antioxidant activity and color change. A quality based comparison of CP treatment of blueberry juice was done with thermal treatment (85 °C, 15 min). Also in this work, oxygen concentration (0, 0.5% and 1%) of ionized gas was firstly regarded as the main factor for CP treatment, besides treatment time (2, 4 and 6 min).
Table 1 Experimental design were under different conditions. Blueberry juices
Treatment time (min)
Oxygen concentration (%)
Temperature ( °C)
Untreated (UT) Heat treated (HT) A B C
0 15
– –
25 85
2, 4, 6 2, 4, 6 2, 4, 6
0 0.5 1
25 25 25
China) by applying an electrode. CP treatment system consisted of single-electrode atmospheric jet and computer-controlled mobile platform was designed by the Institute of Electrical Engineering of the Chinese Academy of Sciences. Such plasma produces a plasma jet extending out of the capillary tube to the length of about 2.4 cm to gas flow of 1.0 L/min. The spectra was collected by using a computercontrolled optical emission spectrometer (Andor, DH334T18U-03, Oxford Instruments plc, UK) together with an ICCD camera (Andor, SR500I, Oxford Instruments plc, UK). The fiber core has a diameter of 100 μm (19 in total) and is appropriate for measuring light in the wavelength range of 200 nm–1100 nm between ultraviolet and visible light. The emission spectra were gathered and stored at 11 kV for treatment for 4 min (Wang et al., 2017). For the juice treatment, cold plasma was running at a constant voltage of 11 kV and frequency of 1000 Hz, and varying at gas content (with oxygen concentration of 0, 0.5% and 1%) and treatment time (2, 4 and 6 min) according to the experimental design (Table 1). Distance from the plasma nozzle tip to the samples was fixed at 2.0 cm. Juice samples were placed in a tissue culture test plate consisted of 6 sample positions (Costar Electronic Material Co., Ltd) and each treatment was performed in triplicate. All the tests were conducted immediately at the end of the treatments.
2. Materials and methods 2.1. Chemicals and standards The blueberries were acquired from Organic Food Co., Ltd., Dandong City, Liaoning Province, China. Potassium chloride, Hydrochloric acid, Sodium acetate, Folin-Ciocalteu reagent, Sodium hydroxide, Ethanol, Ferrous sulphate (FeSO4), Ferric chloride (FeCl3), Salicylic acid, Hydrogen peroxide, Metaphosphoric acid, Glucose, gallic acid, 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), 6-hydroxyl-2,5,7,8-tetramethyl-2-carboxylic acid (Trolox), 2,4,6-tris(2-pyridyl)-S-triazine (TPTZ) were purchased from Sinopharm Chemical Reagent Co., Ltd. DPPH was provided by Shanghai Yuanye Biotechnology Co., Ltd. Hydrogen Peroxide assay kit was obtained from Nanjing Jiancheng Bioengineering Institute. All chemicals and reagents employed in the study were of analytical (AR) grade. 2.2. Preparation of blueberry juice The blueberries placed into distilled water at a ratio of 1:4, were squeezed by an organization stamp mill, and the slurry was centrifuged (4000 rpm for 10 min) at 4 °C. The supernatant was filtered by 4 layers of sterilized gauze to remove the coarse particles and impurities, and then the pH was adjusted to 3.3 to obtain the blueberry juice samples.
2.5. Determination of anthocyanin content Anthocyanin content was measured by pH-differential method (Giusti & Wrolstad, 2005). 1 mL sample was diluted with a buffer solution of pH = 4.5 and pH = 1.0 (7 mL), respectively, then mixed and rested for 1 h at room temperature. The respective absorbance values at 510 nm and 700 nm were measured by using an UV–visible spectrophotometer with deionized water as blanks. Then the following formula was used to calculate the total anthocyanin content:
2.3. Bacillus sp. strain Bacillus sp. was used throughout this experiment. The microorganism was maintained on LB nutrient agar under refrigeration with monthly transfers. Fresh culture was prepared by transferring a single colony of the nutrient agar into 100 mL of LB nutrient broth and incubated in a shaker (ZWY-200D, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd. Shanghai, China) at 200 rpm and 37 °C for 12 h. Then, the broth of the strain was harvested by centrifugation (TGL-20 M centrifuge, Kaida Scientific Instruments Co., Ltd., Changsha, Hunan, China) at 4000 rpm and 4 °C for 10 min, and washed twice by 0.85% sterile saline solution. The final bacteria cell density was adjusted to 109 CFU/mL. The cells were then diluted with sterile blueberry juice to obtain initial concentration of 107 CFU/mL. Liquid bacterial cultures with and without CP treatment were inverted on plates. Bacillus sp. was cultured in nutrient agar medium for 48 h at 37 °C, and cells were counted. The death rate lg S was indicates the bactericidal effect of CP treatment:
A = (A510 − A700)pH 1.0 − (A510 − A700)pH 4.5
Tacy = A × M × D F × 1000 / ε × 1 where A—the absorbance of the diluted sample; M—the molar mass of cyanidin-3-glucoside, 449.2 g/mol; DF—the dilution of the sample volume; ε—the cyanidin-3 Molar extinction coefficient of glucoside, 26,900 L/mol·cm; l —length of light path, 1 cm. 2.6. Determination of total phenolic content (TPC) The TPC was determined by Folin–Ciocalteu method according to Souza et al. (2014)’ protocol with a slight modification. The blueberry juice (1 mL) was mixed with 5 mL of Folin-Ciocalteu reagent (1 mol/L) and 2 mL of deionized water for 5 min in the dark. Then 2 mL of 10% sodium carbonate solution was added, and the mixture was stirred and kept at ambient temperature for 1 h in the dark. The absorbance was measured at 765 nm by using spectrophotometer. The TPC was calculated from the standard curve. A calibration curve was prepared based on the standard solution of gallic acid and the results of total phenols were expressed as µg of gallic acid equivalents (GAE) per mL of sample.
N lgS = lg ⎛ 0 ⎞ ⎝N⎠ where N0 and N are the numbers of microorganisms before and after cold plasma treatment (CFU/mL), respectively. 2.4. Cold plasma treatment The CP jet was generated by argon and oxygen with purities all above 99.99% (Beijing Huanyu Jinghui City Gas Technology Co., LTD, 80
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2.7. Determination of vitamin C
593 nm. The results were expressed as mM Fe(II)/mL juice.
The content of vitamin C in blueberry juice was measured according to modified protocols from literature (Zheng, 2006). Sample 1 determination: 0.1–0.5 mL sample was diluted to 10 mL with 2% metaphosphoric acid. The absorbance was measured at a wavelength of 262 nm. Sample 2 determination: 0.1–0.5 mL sample was homogenized with 0.3 mL 0.5 mol/L sodium hydroxide solution. The mixture was left standing for 40 min and then added 2% metaphosphoric acid to 10 mL. The absorbance was determined at a wavelength of 262 nm. The vitamin C content was calculated from the L-ascorbic acid standard curve, according to the difference value between the absorbance of the sample 1 and sample 2.
2.9. Colorimetric evaluation The variation of color for all experiments was measured as change in color before and after plasma treatment based on Bursać, Putnik et al. (2016)’s protocol. The data were determined by using a colorimeter (TCP2-A, Beijing Aoike Photoelectric Instrument Co., Ltd., Beijing, China) at CIE Standard Illuminant D65. The colorimetric variables (L*, a*, b*) were measured and total color difference (TCD), the saturation (C*) and the hue angle (H*) were calculated from:
TCD =
C∗ =
2.8. Determination of antioxidant activity
ΔL∗2 + Δa∗2 + Δb∗2
a∗2 + b∗2
(1) (2)
∗
b H ∗ = tan−1 ⎛ ∗ ⎞ ⎝a ⎠
2.8.1. DPPH free radical scavenging activity DPPH assay was carried out based on Souza et al. (2014)’ method. 10 mg of DPPH dissolved in 250 mL of ethanol was made into a DPPH solution with a final concentration of 0.1 mmol/L. 0.5 mL sample was mixed with 4 mL of DPPH solution and kept in the dark for 30 min. The absorbance of the mixture was measured at 517 nm. The DPPH radical scavenging rate was calculated as follows:
*
*
(3) *
where Δ L , Δ a and Δ b were calculated in reference to the untreated and treated blueberry juice.TCD shows the magnitude of the color change after cold plasma treatment. All values were record triplicate. 2.10. Experimental design and statistical analysis
A − A2 ⎤ × 100 DPPH radical scavenging activity(%) = ⎡1 − 1 ⎢ A0 ⎥ ⎦ ⎣
In this experiment, six-well plates were used as experimental treatment vessels, and 2 mL of samples were processed at each time. The experimental arrangement was as follows in Table 1. The samples of CP treatment were divided to three groups (A, B and C) according to different oxygen concentration. The data were indicated as means ± standard deviation. Significance levels were defined p < 0.05 and variables were analyzed using multivariate analysis of variance (MANOVA). All the analyses and graphs were performed by IBM SPSS Statistics 22.0 and Origin 2018.
where A1 refers to the absorbance of 0.5 mL of sample solution with 4 mL DPPH solution; A2 refers to the absorbance of 0.5 mL of sample solution with 4 mL of anhydrous ethanol; A0 refers to the absorbance 0.5 mL of deionized water with 4 mL DPPH. 2.8.2. Hydroxyl radical (%OH) scavenging ability Hydroxyl radical scavenging capacity was estimated by using Zou and Hou (2017)’s protocols. The sample (1 mL) were allowed to react with 10 mmol/L FeSO4 solution (1 mL), 10 mmol/L salicylic acid solution (dissolved in absolute ethanol, 1 mL), 6 mmol/L hydrogen peroxide solution (1 mL), then mixed at 37 °C for 1 h. Absorbance value of the mixture was measured at 510 nm. The hydroxyl radical scavenging rate was calculated as follows:
3. Results and discussion 3.1. Cold plasma spectroscopy Cold plasma discharge was identified by optical emission spectroscopy for identification of reactive species. Fig. 1 showed the peak intensity change of core excited state particles (%OH, O, Ar and N) from plasma with the change of oxygen concentration (Group A, B and C). The plasma jet emission spectra was shown in Fig. 1. It could be observed that pure Ar and oxygen-containing plasma jets had the same particle composition and radical emission peaks, which mainly contain molecules like N2 (290–440 nm), positive ions N2+ (391.4 nm), %OH (305–309 nm, 308. 84 nm), Argon (690–860 nm), O (777.02 nm) (Pankaj, Wan, Colonna, & Keener, 2017; Ramazzina et al., 2015), though the oxygen concentration was different among three groups. With the increase of oxygen concentration, the intensity of Ar, %OH, O and N increased and reached a maximum value at a concentration of 1% of oxygen concentration. Also a certain amount of reactive nitrogen species (RNS) and reactive oxygen species (ROS) were generated at the same time (Misra, Keener, Bourke, Mosnier, & Cullen, 2014; Liu et al., 2016).
A − A2 ⎤ × 100 OH radical scavenging activity (%) = ⎡1 − 1 ⎢ A0 ⎥ ⎦ ⎣ A1 refers to the absorbance of the sample; A2 refers to the absorbance of the mixed solution in which 1 mL the hydrogen peroxide solution is replaced by 1 mL of deionized water; A0 refers to the absorbance of the deionized water. 2.8.3. Determination of total antioxidant capacity The total antioxidant capacity was measured using the ABTS and FRAP methods according to the references (Chen et al., 2013; Caviasaiz, Muñiz, Ortega, & Busto, 2011). 2.45 mM potassium persulfate was added to the ABTS (7 mM in 20 mM sodium acetate buffer, pH 4.5) solution. Then the mixture was allowed to stand for 12 h at room temperature in the dark and stored for use. Before use, the solution was diluted with 20 mM sodium acetate buffer (pH 4.5) to an absorbance of 0.700 ± 0.001 at 734 nm to form the test reagent. Then 100 μL of the test sample was added into 3 mL of the reagent and mixed thoroughly. The mixture was incubated at room temperature for 0.5 h, and the absorbance at 734 nm was recorded. Trolox was used as reference standards, and the results were expressed as mM Trolox/mL juice. In FRAP assay, the reaction mixture was prepared by mixing 50 mL of 0.3 M sodium acetate buffer solution (pH 3.6), 5 mL of 10 mM TPTZ, 5 mL of 20 mM FeCl3, and 6 mL of water. Then 30 μL of the juice samples were added to the reaction mixture in a total volume of 1 mL and incubated at 37 °C for 30 min. The absorbance was tested at
3.2. Inactivation effect of CP on Bacillus sp. Fig. 2 shows the effects of treatment time and oxygen concentration on the inactivation of Bacillus. The processing times were 2, 4 and 6 min and the oxygen concentrations were 0, 0.5% and 1%. When the oxygen concentration was constant, the inactivation effect of CP on Bacillus increased with increasing treatment time. When the treatment time was constant, the inactivation effect of CP on Bacillus increased with increasing oxygen concentration. When the treatment time was 6 min and the oxygen concentration was 1%, the total number of Bacillus colonies 81
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Fig. 1. Optical emission spectroscopy for cold plasma jet and variations of intensity of different excited particles with the change of oxygen concentration.
declined by 7.2 log numbers. After analysis, the increase in treatment time and oxygen concentration significantly promoted death of Bacillus cells. Thus, the treatment time and oxygen concentration were the main factors in the bactericidal effect of CP. Similar results were acquired from some previous studies (Han et al., 2016; Pankaj et al., 2017). In the study of Han et al. (2016), the populations of E. coli and S. aureus were significantly decreased from 2 to 8 log cycles and 1.8 to 6.1 log cycles after treatment of CP for 1 and 5 min, respectively, which proved that inactivation effect of CP on Bacillus was related to treatment time, longer exposure time to CP, better inactivation effect for CP. Also Pankaj et al. (2017) found that the yeast inactivation was observed to be in linear relationship with the treatment time. 7.4 log10 CFU/mL inactivation of S. cerevisiae was achieved after CP treatment of 80 kV for 4 min. However, it has not been reported that oxygen concentration was related to the inactivation effect of CP on microorganism, which is firstly confirmed in this study. In terms of inactivation effect of CP on Bacillus, it could be attributed to the damaging effects of reactive gas species generated by CP and identified earlier by the emission spectra in
Fig. 2. Inactivation effect of cold plasma on Bacillus at different treatment time and oxygen concentration.
82
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showed that anthocyanin content in blueberry juice increased firstly and then decreased with the increase of oxygen concentration. Compared with Group A and C, Group B with an oxygen concentration of 0.5% exhibited a highest anthocyanin level at different treatment time. It is well known that anthocyanins are polyphenolic substances and natural pigments. However, it was susceptible to external factors like temperature, pH, oxygen, light and metal ions, and easy to degrade (Bursać,Gajdoš et al., 2016). In our study, obtained results showed that anthocyanin content decreased with the increase of treatment time by CP. The low stability of the anthocyanins in blueberry juice may be caused by oxidative degradation during exposures to plasma-generated reactive particles (Bursać, Putnik et al., 2016) and combined interaction of various reactive plasma species (Grzegorzewski, Ehlbeck, Schlüter, Kroh, & Rohn, 2011). Also the obtained results from this work showed that anthocyanin content went up firstly and then went down with the increase of oxygen concentration. As reported by Grzegorzewski et al. (2011), Ar ions generated by CP may cause the rupture of plant cells and therefore more anthocyanin is released. Based on the results of 3.1, increasing of oxygen concentration caused an increment of active particles, including Ar ions, which lead the increasing of anthocyanin content from Group A to B. Then degradation of anthocyanin from Group B to C was attributed to the greater effect of oxidation by more generated reactive oxygen species. Contrary to our work, Garofulić et al. (2015) found that CP treated sour cherry Marasca juice had the highest content of anthocyanins, compared to both pasteurized and untreated juice. They believed the possible explanation in the increase of anthocyanin was in the way of presence of undefined small-sized agglomerates or particles in the juice which would be dissociated by the plasma treatment. Inter and intramolecular association with other, especially polyglycosylated and polyacylated anthocyanins provide greater stability towards change of external factors. Differences in materials, CP equipments and composition of excited particles may lead the opposite conclusion between this work and ours. More detailed studies are required to understand the interaction mechanism between plasma reactive species and anthocyanin for further explanation. 3.3.2. Total phenolics content Fig. 3(b) showed the TPC in blueberry juice treated by CP, with oxygen concentration of 0, 0.5%, 1% during 2, 4 and 6 min. According to Fig. 3(b), the TPC increased with the prolonged processing time of CP treatment. The samples treated by CP exhibited a higher TPC than that of UT consistently except Group A in 2 min. The TPC in pasteurized blueberry juice was apparently decreased and lower than that of CP treatment and UT. During the same treatment time, increasing of TPC resulted in a significantly (p < 0.05) increment of oxygen concentration. By using an oxygen concentration of 1% during 6 min, a highest TPC (121.64 ± 0.45 μg/mL) was acquired, which was obviously (p < 0.05) higher than that of UT (113.32 ± 0.38 μg/mL) and HT (107.39 ± 0.51 μg/mL). Phenolics, as one kind of natural antioxidants, protect tissue cells from damage by reactive oxygen species and nitrogen species such as singlet oxygen, superoxide, peroxide radicals, hydroxyl radicals, and peroxynitrite. In some fruit, like blueberry, part of the phenolic compounds are bounded to cell membranes. So a certain level of energy is required to become free and available and increase its total amount in the samples (Rodríguez, Gomes, Rodrigues, & Fernandes, 2017). Recently, a few investigations have been focused on the changes of TPC when CP has been applied in food processing. Herceg et al. (2016) studied the effect of cold plasma application on pomegranate juice. When compared to untreated sample, CP treatment resulted in total phenolic content increasing 33.03%. A possible explanation for this effect has been attributed to breakdown of covalent bonds and cell membrane by chemically reactive species, charged particles, and UV photons created from CP, which promoted the release of phenol
Fig. 3. Effect of CP treatment on anthocyanin (a), TPC (b) and vitamin C (c) in blueberry juice. HT and UT refer to heat treatment and untreated, respectively.
Fig. 1. These active ingredients lead to the cell leakage by electroporation, lipid peroxidation, enzyme inactivation and DNA cleavage and finally result in bacteria death (Han et al., 2016; Pankaj et al., 2017). 3.3. Content of anthocyanin, total phenolics and vitamin C 3.3.1. Anthocyanin content Fig. 3(a) showed the relative amount (compared with the UT and HT samples) of anthocyanin in blueberry juice treated by means of CP, using oxygen concentration of 0, 0.5%, 1% during 2, 4 and 6 min. According to Fig. 3, the application of CP on blueberry juice promoted significantly reduction in the anthocyanin content with the increase of treatment time. After the treatment of 6 min, the decrement of 43.79, 35.12 and 37.49% of anthocyanin in Group A, B and C was observed, respectively, compared with the treatment of 2 min. The UT sample had the highest level of anthocyanin content, which was 15.16 ± 0.25 mg/L. After 2 min of treatment, the three groups of CP samples exhibited higher (p < 0.05) anthocyanin content than that of HT. However, when the treatment was extended to 4 and 6 min, HT sample showed a higher (p < 0.05) anthocyanin level than that of CP samples. Except treatment time, oxygen concentration was the other one factor related to the changes of anthocyanin content. The results 83
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Fig. 4. Effect of CP treatment on relative antioxidant activity in blueberry juice HT and UT referred to heat treatment and untreated, respectively. (1) DPPH radical; (2) %OH free radical; (3) ABTS; (4) FRAP. Table 2 Color parameters for different operating conditions during cold phase plasma treatment of blueberry juices. Sample UT HT A2 A4 A6 B2 B4 B6 C2 C4 C6
L* 33.26 30.26 33.69 34.17 34.72 33.41 34.86 35.04 33.65 34.92 35.11
a* ± ± ± ± ± ± ± ± ± ± ±
e
0.15 0.15f 0.14d 0.12c 0.16b 0.23de 0.19b 0.21a 0.23de 0.13a 0.11a
35.00 31.27 34.75 34.05 33.34 34.42 34.06 33.13 34.38 33.14 32.42
b* ± ± ± ± ± ± ± ± ± ± ±
a
0.08 0.26f 0.25ab 0.17c 0.14 cd 0.22b 0.26c 0.22d 0.12b 0.29d 0.14e
22.90 18.12 22.45 21.77 20.53 22.23 20.90 19.82 22.03 20.01 19.49
C* ± ± ± ± ± ± ± ± ± ± ±
a
0.13 0.14g 0.23b 0.21c 0.22d 0.19bc 0.20d 0.32f 0.11c 0.09e 0.12f
H* a
41.83 ± 0.11 36.2 ± 0.31j 41.34 ± 0.25b 40.44 ± 0.08e 39.15 ± 0.25 g 40.92 ± 0.21c 40.08 ± 0.1f 38.56 ± 0.3 h 40.81 ± 0.12d 38.66 ± 0.12 h 37.78 ± 0.21i
TCD e
1.3 ± 0 1.54 ± 0.04a 1.32 ± 0.03de 1.35 ± 0.02d 1.41 ± 0.03c 1.33 ± 0.01de 1.42 ± 0.03c 1.47 ± 0.05b 1.34 ± 0.01de 1.46 ± 0.01bc 1.46 ± 0.01bc
– 6.52 ± 0.29a 0.63 ± 0.17i 1.59 ± 0.3 g 3.22 ± 0.29d 1.16 ± 0.22 h 2.21 ± 0.28f 4.27 ± 0.26bc 1.1 ± 0.25 h 2.63 ± 0.11e 4.58 ± 0.17b
Results are expressed as mean ± standard error. Values represented with different letters are statistically different at p < 0.05.
Similar with the results obtained from 3.2.1, Vitamin C in blueberry juice was degraded with the increase of treatment time by CP treatment. After the treatment of 6 min, the decrement of 19.94, 57.14 and 52.33% for vitamin C in Group A, B and C was observed, respectively, compared with the treatment of 2 min. The UT sample had the highest level of vitamin C content, which was 4.03 ± 0.14 μg/mL. After 2 min of treatment, the three groups of CP samples exhibited higher (p < 0.05) vitamin C content than that of HT. When the treatment was extended to 4 and 6 min, HT sample showed a higher (p < 0.05) vitamin C level than that of group B and C, while, had a lower (p < 0.05) vitamin C level than that of group A. During the same treatment time, increasing of oxygen concentration resulted in a significantly (p < 0.05) decrement of vitamin C content. By using an oxygen concentration of 1% during 6 min, a lowest vitamin C content was acquired, which was obviously (p < 0.05) lower than that of UT (4.03 ± 0.14 μg/mL) and HT (2.49 ± 0.32 μg/mL). As reported by Rodríguez et al. (2017), when a N2 flow rate of 10 mL/min was used, an increment of 10.4 and 10.8% was observed after 5 and 10 min of treatment, respectively, but when the treatment was extended to 15 min, the sample exhibited a reduction of 4.5% in vitamin C. The authors explained that NO was one of the excited particles generated by CP, which could regulate the ascorbate–glutathione cycle and increased dehydroascorbate reductase activity.
compounds. A similar result has been found in Rodríguez et al. (2017)’s work. When CP was used, the TPC of the cashew apple juice exhibited an average increment of 108% although no significant differences among samples (using CP during 5, 10 and 15 min) were observed. However, the degradation of the total phenolics induced by CP has been found in recent literatures as well. In Pankaj et al. (2017)’s study, increasing the treatment time of the CP treatment resulted in a significant (p < 0.05) reduction in the total phenolics of the grape juices. The mechanism was that CP equipment used in this study with air generated many reactive oxygen species and ozone in the discharge. Phenolic compounds were particularly known to be susceptible for ozone attacks due to the degradation of aromatic rings in the structure (Stalter, Magdeburg, Wagner, & Oehlmann, 2011; Pérez, Torrades, Domènech, & Peral, 2010). Similar degradation of phenolic compounds after CP treatments were also reported in lettuce and orange juice (Almeida et al., 2015; Grzegorzewski et al., 2011). More detailed studies are required to understand the interaction mechanism between plasma reactive species and phenolics for further explanation. 3.3.3. Vitamin C content Fig. 3(c) showed the vitamin C in blueberry juice treated by CP, with oxygen concentration of 0, 0.5%, 1% during 2, 4 and 6 min. 84
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demonstrated to be another factor have influence on the antioxidant activity of juice.
Dehydroascorbic acid was an oxidative form of ascorbic acid, which could be converted to ascorbic acid by dehydroascorbic acid reductase (Sun et al., 2015; Shan, Zhou, & Liu, 2015). After 5 and 10 min of treatment, the rate of regeneration of ascorbic acid by the ascorbate–glutathione cycle was greater than the rate of decay of ascorbic acid through its reaction with other plasma generated reaction species, while, the rate of decay exceeded the rate of regeneration after 15 min. However, no increment of vitamin C was observed in this work. The stronger degradation of vitamin C could be attributed to reactive oxygen species.
3.5. Color stability The effect of CP on color of the blueberry juice was shown in Table 2. Generally, higher a* values indicate more reddish overall impression of the samples, whereas b* values evaluate blue (-b*) to yellow (+b*) tonality of the samples ((Bursać, Putnik et al., 2016)). In this work, upward trend of L* and H* value, and downward trend of a*, b* and C* value, regarding with the increasing of treatment time or oxygen concentration after CP treatment were found in Table 2. Compared with CP samples, HT showed the higher L* and H* value, and the lower a*, b* and C* value. Also among experimental samples, treatments of 2 min and 4 min were no visual difference in color for CP treatment vs UT, since color difference TCD = 1.5–3.0 reflects merely noticeable color difference. However, a range greater than TCD = 1.5–3.0 was observed in treatment time of 6 min and HT, reflecting noticeable color difference compared with untreated sample. Thus, CP treatment could better keep the original color of blueberry juice compared with HT, since HT sample had the highest TCD value. Several literatures reported the various results about the color changes after CP treatment. For example, the TCD values were 4.47 and 9.63 in fresh tomato and lettuce, respectively, after CP treatment time of 3 min (Bermudez-Aguirre, Wemlinger, Pedrow, Barbosa-Canovas, & Garcia-Perez, 2013). In another work (Pankaj et al., 2017), the TCD values of grape juice were 3.23, 4.22, 3.87 after CP treatment of 2, 3 and 4 min, respectively, which were all higher than that of thermal treatment (1.71). Thus, more studies are suggested to investigate the mechanism of color changes in different food system after CP treatment.
3.4. Antioxidant activity The antioxidant activity was determined by using different methods, including organic radical scavenging capacity (DPPH, %OH free radical and ABTS) and metal reduction ability (FRAP – ferric reduction antioxidant power), which could comprehensively and reliably represent the antioxidant activity of fruit juices (Mariadosocorrom et al., 2010). Fig. 4(1) (DPPH), (2) (%OH), (3) (ABTS) and (4) (FRAP) show the antioxidant activity of blueberry juice treated by CP. Different trends were observed when using the different assays denoting that the bioactive compounds that respond better to certain kind of assay are affected differently by CP application and exposure time. The antioxidant activity of the UT samples measured by DPPH, %OH free radical, ABTS and FRAP assays were of 93.03%, 58.25%, 4.06 mg Trolox/ g and 1.70 mM FeSO4/mL, respectively, which were all higher than those of HT samples. According to DPPH assay, it could be observed a significant effect of the treatment time, namely, longer treatments led to lower antioxidant activities. After the treatment of 6 min, the decrement of 14.10, 14.29 and 19.15% for antioxidant activity in Group A, B and C was observed, respectively, compared with the treatment of 2 min. Group C showed an equal (p > 0.05) antioxidant activity with UT, and higher (p < 0.05) than HT during 2 min. The behavior of the antioxidant activity resembles the behavior observed for vitamin C and anthocyanin, which may be the dominant factor determining the antioxidant activity at different time. Similar with the results of TPC content in 3.2.2, increasing of DPPH scavenging capacity resulted in a significantly (p < 0.05) increment of oxygen concentration during the same treatment time. On the contrary, higher oxygen concentration led to the lower (p < 0.05) antioxidant activities in %OH free radical scavenging assay after same time of treatment. It could be attributed to a certain amount of %OH free radical generated by CP. Based on the results of 3.1, more % OH was excited by CP with the increase of oxygen concentration. Also a significant effect of the treatment time was found in this assay, longer exposure to CP with lower antioxidant activities, which may due to the decrease in vitamin C and anthocyanin. Group A and B had greater %OH free radical scavenging capacity at all the experimental time, compared with that of UT and HT samples, which may relate to the increase of TPC. Similar trend with DPPH assay was observed in ABTS assay. The antioxidant activity was decreased with the increase of treatment time by CP treatment, while, except Group A in 4 and 6 min, the others’ were all significantly (p < 0.05) higher than that of UT and HT samples. Also increasing of oxygen concentration resulted in a significantly (p < 0.05) increment of antioxidant activity during the same treatment time. Also it could be observed a significant effect of the treatment time in FRAP assay, longer treatments led to lower antioxidant activities. However, different from the results of other three methods, no regular trends related to oxygen concentration was found in this assay. In a previous study (Pankaj et al., 2017), a decrease in the both DPPH free radical scavenging and antioxidant capacity was observed after CP treatment with increasing of treatment time, which is similar with this work. While, oxygen concentration of ionized gas was also
4. Conclusions Effects of cold plasma technology on the quality of blueberry juice have been presented in this study, including inactivation of Bacillus, contents of anthocyanin, total phenol, vitamin C, and antioxidant activity. Treatment time and oxygen concentration have been confirmed to be the main factors for CP treatment. The increment in treatment time and oxygen concentration significantly promoted death of Bacillus cells. CP treatment at 1% of oxygen concentration was resulted in 7.2 log reduction of Bacillus. In general, increasing of treatment time resulted in a significantly (p < 0.05) decrement of anthocyanin, vitamin C and antioxidant activity, and increment of TPC. Lower vitamin C content and %OH free radical scavenging capacity were induced by higher oxygen concentration, while, TPC, antioxidant activity in DPPH and ABTS assays were increased with the increase of oxygen concentration. Compared with HT, relatively shorter exposure time and lower oxygen concentration are better to keep the above components. In the tests of color change, blueberry juice treated by CP are more closer to UT juice, in comparison to HT juice, especially when the treatment is 2 or 4 min, CP juice reflects merely noticeable color difference with UT juice. In summary, the present study showed that cold plasma processing had more positive influence on quality of blueberry juice compared with thermal processing.
Declaration of interests None.
Acknowledgement This work was financially supported by the Fundamental Research Funds for the Central Universities (BLX201616). 85
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86
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Effect of cold plasma on blueberry juice quality a,1
Yanan Hou a b
b,1
, Ruixue Wang
a,⁎
, Zhilin Gan , Tao Shao
b,⁎
T a
a
, Xinxue Zhang , Mohe He , Aidong Sun
a
Department of Food Science and Engineering, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 10083, People’s Republic of China Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China
A R T I C LE I N FO
A B S T R A C T
Keywords: Cold plasma Blueberry juice Bacillus Anthocyanin Phenolics Vitamin C Antioxidant activity Color change
This work focusses on the effects of cold plasma, a novel, non-thermal technology, on the quality of blueberry juice, such as inactivation of Bacillus, anthocyanins, phenolics, vitamin C, antioxidant activity and color change. Oxygen concentration (0, 0.5% and 1%) of ionized gas was firstly confirmed to be the main factors for CP treatment, besides treatment time (2, 4 and 6 min). The increment of treatment time and oxygen concentration significantly promoted an increasing trend of death for Bacillus. Compared with thermal treatment, the content of phenolics was significantly increased by CP treatment, and also CP treatment could better keep the original color of blueberry juice. In terms of anthocyanin and vitamin C, relatively shorter exposure time to CP was more suggested. In antioxidant tests, increment of oxygen concentration resulted in the increasing trends of antioxidant activity in DPPH and ABTS assays.
1. Introduction Blueberry, a rich source of bioactive components including vitamin C, phenolics, anthocyanin and flavonoid, is one the most popular berries with high health value (Wang et al., 2017; Terefe, Delon, Buckow, & Versteeg, 2015). Previous reports have illustrated that blueberry could be recognized as one functional food with several activities, including antioxidant, anti-bacteria, anti-inflammatory, anti-allergic, anticancer and resistance to cardiovascular disease (Souza, Silva, Lima, Pio, & Queiroz, 2014; Folmer et al., 2014; Heinonen, 2010; Liu, Chen, Li, & Sun, 2016; Joshi, Howell, & D'Souza, 2016; Pertuzatti et al., 2014). Therefore, blueberry is considered as a fruit with a great processing potential. One of the most widespread blueberry products is the juice. Recently, blueberry juice is becoming more popular and attractive to consumers due to the high levels of antioxidants, such as anthocyanins and vitamin C, which is healthful to humans (Barba, Esteve, & Frigola, 2013). However, the blueberry juice with high fresh-like characteristics and long shelf-life is always a challenge. It is extremely important to preserve the above valuable compounds during processing in the highest possible extent. Traditionally, pasteurization is the most widely applied technique for inactivation of pathogenic microorganisms and enzymes during juice processing. However, the quality of juice, such as flavor, aroma, nutritional constituents, can be broken and degraded by thermal processing (Gómez, Welti-Chanes, & Alzamora, 2011). Consumers’ demand
for minimally processed juices has turned to non-thermal technologies. High pressure (Barba et al., 2013), ultrasound (Simunek, Jambrak, Dobrović, Herceg, & Vukušić, 2014), pulsed electric field (Chen et al., 2014), microwave heating (Elik, Yanık, Maskan, & Göğüş, 2016) are among the no-thermal technologies that have been applied to blueberry juice processing. Cold plasma (CP) is a novel, non-thermal technology, which is generated by excitation of process gas (carbon dioxide, argon, nitrogen, helioum, oxygen or air) with a strong electric field under ambient temperature (Niemira, 2012). During the processing, reactive chemical species, such as radicals, heat and UV light are excited, which vary according to the parameters of CP devices, such as voltage, frequency, flow rate and the type of gas that is used (Ramos, Miller, Brandão, Teixeira, & Silva, 2013). Several reports have demonstrated that CP could be efficiently applied in food industry, including pathogen decontamination (Lee, Kim, Chung, & Min, 2015), enzyme inactivation (Pankaj, Misra, & Cullen, 2013) and surface modification (Pankaj et al., 2014). The effect of CP on the quality of pomegranate and sour cherry juices have been studied previously (Herceg et al., 2016; Garofulić et al., 2015). The above literatures have demonstrated that treatment time was the main factor of CP on the quality of juice, and the CP was generated only by one pure gas, like nitrogen or argon. It is not clear if other parameters can affect it. Although Surowsky, Fröhling, Gottschalk, Schlüter, and Knorr (2014) have shown that CP acquired a better bactericidal effect after mixing oxygen to the ionized gas. Still
⁎
Corresponding authors. E-mail addresses: [email protected] (Z. Gan), [email protected] (T. Shao). 1 First authors: They had made equal contributions to this paper. https://doi.org/10.1016/j.foodchem.2019.03.123 Received 8 October 2018; Received in revised form 18 March 2019; Accepted 23 March 2019 Available online 25 March 2019 0308-8146/ © 2019 Published by Elsevier Ltd.
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little is known on the quality of fruit juices by using CP generated by a mixture of process gas. Additionally, no studies exist showing the effect of CP on the quality of blueberry juice which forms the focus of this work. Related to the current findings, the aim of this work is to evaluate the effect of CP treatment on the quality of blueberry juice, such as inactivation of Bacillus, anthocyanins, phenolics, vitamin C, antioxidant activity and color change. A quality based comparison of CP treatment of blueberry juice was done with thermal treatment (85 °C, 15 min). Also in this work, oxygen concentration (0, 0.5% and 1%) of ionized gas was firstly regarded as the main factor for CP treatment, besides treatment time (2, 4 and 6 min).
Table 1 Experimental design were under different conditions. Blueberry juices
Treatment time (min)
Oxygen concentration (%)
Temperature ( °C)
Untreated (UT) Heat treated (HT) A B C
0 15
– –
25 85
2, 4, 6 2, 4, 6 2, 4, 6
0 0.5 1
25 25 25
China) by applying an electrode. CP treatment system consisted of single-electrode atmospheric jet and computer-controlled mobile platform was designed by the Institute of Electrical Engineering of the Chinese Academy of Sciences. Such plasma produces a plasma jet extending out of the capillary tube to the length of about 2.4 cm to gas flow of 1.0 L/min. The spectra was collected by using a computercontrolled optical emission spectrometer (Andor, DH334T18U-03, Oxford Instruments plc, UK) together with an ICCD camera (Andor, SR500I, Oxford Instruments plc, UK). The fiber core has a diameter of 100 μm (19 in total) and is appropriate for measuring light in the wavelength range of 200 nm–1100 nm between ultraviolet and visible light. The emission spectra were gathered and stored at 11 kV for treatment for 4 min (Wang et al., 2017). For the juice treatment, cold plasma was running at a constant voltage of 11 kV and frequency of 1000 Hz, and varying at gas content (with oxygen concentration of 0, 0.5% and 1%) and treatment time (2, 4 and 6 min) according to the experimental design (Table 1). Distance from the plasma nozzle tip to the samples was fixed at 2.0 cm. Juice samples were placed in a tissue culture test plate consisted of 6 sample positions (Costar Electronic Material Co., Ltd) and each treatment was performed in triplicate. All the tests were conducted immediately at the end of the treatments.
2. Materials and methods 2.1. Chemicals and standards The blueberries were acquired from Organic Food Co., Ltd., Dandong City, Liaoning Province, China. Potassium chloride, Hydrochloric acid, Sodium acetate, Folin-Ciocalteu reagent, Sodium hydroxide, Ethanol, Ferrous sulphate (FeSO4), Ferric chloride (FeCl3), Salicylic acid, Hydrogen peroxide, Metaphosphoric acid, Glucose, gallic acid, 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), 6-hydroxyl-2,5,7,8-tetramethyl-2-carboxylic acid (Trolox), 2,4,6-tris(2-pyridyl)-S-triazine (TPTZ) were purchased from Sinopharm Chemical Reagent Co., Ltd. DPPH was provided by Shanghai Yuanye Biotechnology Co., Ltd. Hydrogen Peroxide assay kit was obtained from Nanjing Jiancheng Bioengineering Institute. All chemicals and reagents employed in the study were of analytical (AR) grade. 2.2. Preparation of blueberry juice The blueberries placed into distilled water at a ratio of 1:4, were squeezed by an organization stamp mill, and the slurry was centrifuged (4000 rpm for 10 min) at 4 °C. The supernatant was filtered by 4 layers of sterilized gauze to remove the coarse particles and impurities, and then the pH was adjusted to 3.3 to obtain the blueberry juice samples.
2.5. Determination of anthocyanin content Anthocyanin content was measured by pH-differential method (Giusti & Wrolstad, 2005). 1 mL sample was diluted with a buffer solution of pH = 4.5 and pH = 1.0 (7 mL), respectively, then mixed and rested for 1 h at room temperature. The respective absorbance values at 510 nm and 700 nm were measured by using an UV–visible spectrophotometer with deionized water as blanks. Then the following formula was used to calculate the total anthocyanin content:
2.3. Bacillus sp. strain Bacillus sp. was used throughout this experiment. The microorganism was maintained on LB nutrient agar under refrigeration with monthly transfers. Fresh culture was prepared by transferring a single colony of the nutrient agar into 100 mL of LB nutrient broth and incubated in a shaker (ZWY-200D, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd. Shanghai, China) at 200 rpm and 37 °C for 12 h. Then, the broth of the strain was harvested by centrifugation (TGL-20 M centrifuge, Kaida Scientific Instruments Co., Ltd., Changsha, Hunan, China) at 4000 rpm and 4 °C for 10 min, and washed twice by 0.85% sterile saline solution. The final bacteria cell density was adjusted to 109 CFU/mL. The cells were then diluted with sterile blueberry juice to obtain initial concentration of 107 CFU/mL. Liquid bacterial cultures with and without CP treatment were inverted on plates. Bacillus sp. was cultured in nutrient agar medium for 48 h at 37 °C, and cells were counted. The death rate lg S was indicates the bactericidal effect of CP treatment:
A = (A510 − A700)pH 1.0 − (A510 − A700)pH 4.5
Tacy = A × M × D F × 1000 / ε × 1 where A—the absorbance of the diluted sample; M—the molar mass of cyanidin-3-glucoside, 449.2 g/mol; DF—the dilution of the sample volume; ε—the cyanidin-3 Molar extinction coefficient of glucoside, 26,900 L/mol·cm; l —length of light path, 1 cm. 2.6. Determination of total phenolic content (TPC) The TPC was determined by Folin–Ciocalteu method according to Souza et al. (2014)’ protocol with a slight modification. The blueberry juice (1 mL) was mixed with 5 mL of Folin-Ciocalteu reagent (1 mol/L) and 2 mL of deionized water for 5 min in the dark. Then 2 mL of 10% sodium carbonate solution was added, and the mixture was stirred and kept at ambient temperature for 1 h in the dark. The absorbance was measured at 765 nm by using spectrophotometer. The TPC was calculated from the standard curve. A calibration curve was prepared based on the standard solution of gallic acid and the results of total phenols were expressed as µg of gallic acid equivalents (GAE) per mL of sample.
N lgS = lg ⎛ 0 ⎞ ⎝N⎠ where N0 and N are the numbers of microorganisms before and after cold plasma treatment (CFU/mL), respectively. 2.4. Cold plasma treatment The CP jet was generated by argon and oxygen with purities all above 99.99% (Beijing Huanyu Jinghui City Gas Technology Co., LTD, 80
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2.7. Determination of vitamin C
593 nm. The results were expressed as mM Fe(II)/mL juice.
The content of vitamin C in blueberry juice was measured according to modified protocols from literature (Zheng, 2006). Sample 1 determination: 0.1–0.5 mL sample was diluted to 10 mL with 2% metaphosphoric acid. The absorbance was measured at a wavelength of 262 nm. Sample 2 determination: 0.1–0.5 mL sample was homogenized with 0.3 mL 0.5 mol/L sodium hydroxide solution. The mixture was left standing for 40 min and then added 2% metaphosphoric acid to 10 mL. The absorbance was determined at a wavelength of 262 nm. The vitamin C content was calculated from the L-ascorbic acid standard curve, according to the difference value between the absorbance of the sample 1 and sample 2.
2.9. Colorimetric evaluation The variation of color for all experiments was measured as change in color before and after plasma treatment based on Bursać, Putnik et al. (2016)’s protocol. The data were determined by using a colorimeter (TCP2-A, Beijing Aoike Photoelectric Instrument Co., Ltd., Beijing, China) at CIE Standard Illuminant D65. The colorimetric variables (L*, a*, b*) were measured and total color difference (TCD), the saturation (C*) and the hue angle (H*) were calculated from:
TCD =
C∗ =
2.8. Determination of antioxidant activity
ΔL∗2 + Δa∗2 + Δb∗2
a∗2 + b∗2
(1) (2)
∗
b H ∗ = tan−1 ⎛ ∗ ⎞ ⎝a ⎠
2.8.1. DPPH free radical scavenging activity DPPH assay was carried out based on Souza et al. (2014)’ method. 10 mg of DPPH dissolved in 250 mL of ethanol was made into a DPPH solution with a final concentration of 0.1 mmol/L. 0.5 mL sample was mixed with 4 mL of DPPH solution and kept in the dark for 30 min. The absorbance of the mixture was measured at 517 nm. The DPPH radical scavenging rate was calculated as follows:
*
*
(3) *
where Δ L , Δ a and Δ b were calculated in reference to the untreated and treated blueberry juice.TCD shows the magnitude of the color change after cold plasma treatment. All values were record triplicate. 2.10. Experimental design and statistical analysis
A − A2 ⎤ × 100 DPPH radical scavenging activity(%) = ⎡1 − 1 ⎢ A0 ⎥ ⎦ ⎣
In this experiment, six-well plates were used as experimental treatment vessels, and 2 mL of samples were processed at each time. The experimental arrangement was as follows in Table 1. The samples of CP treatment were divided to three groups (A, B and C) according to different oxygen concentration. The data were indicated as means ± standard deviation. Significance levels were defined p < 0.05 and variables were analyzed using multivariate analysis of variance (MANOVA). All the analyses and graphs were performed by IBM SPSS Statistics 22.0 and Origin 2018.
where A1 refers to the absorbance of 0.5 mL of sample solution with 4 mL DPPH solution; A2 refers to the absorbance of 0.5 mL of sample solution with 4 mL of anhydrous ethanol; A0 refers to the absorbance 0.5 mL of deionized water with 4 mL DPPH. 2.8.2. Hydroxyl radical (%OH) scavenging ability Hydroxyl radical scavenging capacity was estimated by using Zou and Hou (2017)’s protocols. The sample (1 mL) were allowed to react with 10 mmol/L FeSO4 solution (1 mL), 10 mmol/L salicylic acid solution (dissolved in absolute ethanol, 1 mL), 6 mmol/L hydrogen peroxide solution (1 mL), then mixed at 37 °C for 1 h. Absorbance value of the mixture was measured at 510 nm. The hydroxyl radical scavenging rate was calculated as follows:
3. Results and discussion 3.1. Cold plasma spectroscopy Cold plasma discharge was identified by optical emission spectroscopy for identification of reactive species. Fig. 1 showed the peak intensity change of core excited state particles (%OH, O, Ar and N) from plasma with the change of oxygen concentration (Group A, B and C). The plasma jet emission spectra was shown in Fig. 1. It could be observed that pure Ar and oxygen-containing plasma jets had the same particle composition and radical emission peaks, which mainly contain molecules like N2 (290–440 nm), positive ions N2+ (391.4 nm), %OH (305–309 nm, 308. 84 nm), Argon (690–860 nm), O (777.02 nm) (Pankaj, Wan, Colonna, & Keener, 2017; Ramazzina et al., 2015), though the oxygen concentration was different among three groups. With the increase of oxygen concentration, the intensity of Ar, %OH, O and N increased and reached a maximum value at a concentration of 1% of oxygen concentration. Also a certain amount of reactive nitrogen species (RNS) and reactive oxygen species (ROS) were generated at the same time (Misra, Keener, Bourke, Mosnier, & Cullen, 2014; Liu et al., 2016).
A − A2 ⎤ × 100 OH radical scavenging activity (%) = ⎡1 − 1 ⎢ A0 ⎥ ⎦ ⎣ A1 refers to the absorbance of the sample; A2 refers to the absorbance of the mixed solution in which 1 mL the hydrogen peroxide solution is replaced by 1 mL of deionized water; A0 refers to the absorbance of the deionized water. 2.8.3. Determination of total antioxidant capacity The total antioxidant capacity was measured using the ABTS and FRAP methods according to the references (Chen et al., 2013; Caviasaiz, Muñiz, Ortega, & Busto, 2011). 2.45 mM potassium persulfate was added to the ABTS (7 mM in 20 mM sodium acetate buffer, pH 4.5) solution. Then the mixture was allowed to stand for 12 h at room temperature in the dark and stored for use. Before use, the solution was diluted with 20 mM sodium acetate buffer (pH 4.5) to an absorbance of 0.700 ± 0.001 at 734 nm to form the test reagent. Then 100 μL of the test sample was added into 3 mL of the reagent and mixed thoroughly. The mixture was incubated at room temperature for 0.5 h, and the absorbance at 734 nm was recorded. Trolox was used as reference standards, and the results were expressed as mM Trolox/mL juice. In FRAP assay, the reaction mixture was prepared by mixing 50 mL of 0.3 M sodium acetate buffer solution (pH 3.6), 5 mL of 10 mM TPTZ, 5 mL of 20 mM FeCl3, and 6 mL of water. Then 30 μL of the juice samples were added to the reaction mixture in a total volume of 1 mL and incubated at 37 °C for 30 min. The absorbance was tested at
3.2. Inactivation effect of CP on Bacillus sp. Fig. 2 shows the effects of treatment time and oxygen concentration on the inactivation of Bacillus. The processing times were 2, 4 and 6 min and the oxygen concentrations were 0, 0.5% and 1%. When the oxygen concentration was constant, the inactivation effect of CP on Bacillus increased with increasing treatment time. When the treatment time was constant, the inactivation effect of CP on Bacillus increased with increasing oxygen concentration. When the treatment time was 6 min and the oxygen concentration was 1%, the total number of Bacillus colonies 81
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Fig. 1. Optical emission spectroscopy for cold plasma jet and variations of intensity of different excited particles with the change of oxygen concentration.
declined by 7.2 log numbers. After analysis, the increase in treatment time and oxygen concentration significantly promoted death of Bacillus cells. Thus, the treatment time and oxygen concentration were the main factors in the bactericidal effect of CP. Similar results were acquired from some previous studies (Han et al., 2016; Pankaj et al., 2017). In the study of Han et al. (2016), the populations of E. coli and S. aureus were significantly decreased from 2 to 8 log cycles and 1.8 to 6.1 log cycles after treatment of CP for 1 and 5 min, respectively, which proved that inactivation effect of CP on Bacillus was related to treatment time, longer exposure time to CP, better inactivation effect for CP. Also Pankaj et al. (2017) found that the yeast inactivation was observed to be in linear relationship with the treatment time. 7.4 log10 CFU/mL inactivation of S. cerevisiae was achieved after CP treatment of 80 kV for 4 min. However, it has not been reported that oxygen concentration was related to the inactivation effect of CP on microorganism, which is firstly confirmed in this study. In terms of inactivation effect of CP on Bacillus, it could be attributed to the damaging effects of reactive gas species generated by CP and identified earlier by the emission spectra in
Fig. 2. Inactivation effect of cold plasma on Bacillus at different treatment time and oxygen concentration.
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showed that anthocyanin content in blueberry juice increased firstly and then decreased with the increase of oxygen concentration. Compared with Group A and C, Group B with an oxygen concentration of 0.5% exhibited a highest anthocyanin level at different treatment time. It is well known that anthocyanins are polyphenolic substances and natural pigments. However, it was susceptible to external factors like temperature, pH, oxygen, light and metal ions, and easy to degrade (Bursać,Gajdoš et al., 2016). In our study, obtained results showed that anthocyanin content decreased with the increase of treatment time by CP. The low stability of the anthocyanins in blueberry juice may be caused by oxidative degradation during exposures to plasma-generated reactive particles (Bursać, Putnik et al., 2016) and combined interaction of various reactive plasma species (Grzegorzewski, Ehlbeck, Schlüter, Kroh, & Rohn, 2011). Also the obtained results from this work showed that anthocyanin content went up firstly and then went down with the increase of oxygen concentration. As reported by Grzegorzewski et al. (2011), Ar ions generated by CP may cause the rupture of plant cells and therefore more anthocyanin is released. Based on the results of 3.1, increasing of oxygen concentration caused an increment of active particles, including Ar ions, which lead the increasing of anthocyanin content from Group A to B. Then degradation of anthocyanin from Group B to C was attributed to the greater effect of oxidation by more generated reactive oxygen species. Contrary to our work, Garofulić et al. (2015) found that CP treated sour cherry Marasca juice had the highest content of anthocyanins, compared to both pasteurized and untreated juice. They believed the possible explanation in the increase of anthocyanin was in the way of presence of undefined small-sized agglomerates or particles in the juice which would be dissociated by the plasma treatment. Inter and intramolecular association with other, especially polyglycosylated and polyacylated anthocyanins provide greater stability towards change of external factors. Differences in materials, CP equipments and composition of excited particles may lead the opposite conclusion between this work and ours. More detailed studies are required to understand the interaction mechanism between plasma reactive species and anthocyanin for further explanation. 3.3.2. Total phenolics content Fig. 3(b) showed the TPC in blueberry juice treated by CP, with oxygen concentration of 0, 0.5%, 1% during 2, 4 and 6 min. According to Fig. 3(b), the TPC increased with the prolonged processing time of CP treatment. The samples treated by CP exhibited a higher TPC than that of UT consistently except Group A in 2 min. The TPC in pasteurized blueberry juice was apparently decreased and lower than that of CP treatment and UT. During the same treatment time, increasing of TPC resulted in a significantly (p < 0.05) increment of oxygen concentration. By using an oxygen concentration of 1% during 6 min, a highest TPC (121.64 ± 0.45 μg/mL) was acquired, which was obviously (p < 0.05) higher than that of UT (113.32 ± 0.38 μg/mL) and HT (107.39 ± 0.51 μg/mL). Phenolics, as one kind of natural antioxidants, protect tissue cells from damage by reactive oxygen species and nitrogen species such as singlet oxygen, superoxide, peroxide radicals, hydroxyl radicals, and peroxynitrite. In some fruit, like blueberry, part of the phenolic compounds are bounded to cell membranes. So a certain level of energy is required to become free and available and increase its total amount in the samples (Rodríguez, Gomes, Rodrigues, & Fernandes, 2017). Recently, a few investigations have been focused on the changes of TPC when CP has been applied in food processing. Herceg et al. (2016) studied the effect of cold plasma application on pomegranate juice. When compared to untreated sample, CP treatment resulted in total phenolic content increasing 33.03%. A possible explanation for this effect has been attributed to breakdown of covalent bonds and cell membrane by chemically reactive species, charged particles, and UV photons created from CP, which promoted the release of phenol
Fig. 3. Effect of CP treatment on anthocyanin (a), TPC (b) and vitamin C (c) in blueberry juice. HT and UT refer to heat treatment and untreated, respectively.
Fig. 1. These active ingredients lead to the cell leakage by electroporation, lipid peroxidation, enzyme inactivation and DNA cleavage and finally result in bacteria death (Han et al., 2016; Pankaj et al., 2017). 3.3. Content of anthocyanin, total phenolics and vitamin C 3.3.1. Anthocyanin content Fig. 3(a) showed the relative amount (compared with the UT and HT samples) of anthocyanin in blueberry juice treated by means of CP, using oxygen concentration of 0, 0.5%, 1% during 2, 4 and 6 min. According to Fig. 3, the application of CP on blueberry juice promoted significantly reduction in the anthocyanin content with the increase of treatment time. After the treatment of 6 min, the decrement of 43.79, 35.12 and 37.49% of anthocyanin in Group A, B and C was observed, respectively, compared with the treatment of 2 min. The UT sample had the highest level of anthocyanin content, which was 15.16 ± 0.25 mg/L. After 2 min of treatment, the three groups of CP samples exhibited higher (p < 0.05) anthocyanin content than that of HT. However, when the treatment was extended to 4 and 6 min, HT sample showed a higher (p < 0.05) anthocyanin level than that of CP samples. Except treatment time, oxygen concentration was the other one factor related to the changes of anthocyanin content. The results 83
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Fig. 4. Effect of CP treatment on relative antioxidant activity in blueberry juice HT and UT referred to heat treatment and untreated, respectively. (1) DPPH radical; (2) %OH free radical; (3) ABTS; (4) FRAP. Table 2 Color parameters for different operating conditions during cold phase plasma treatment of blueberry juices. Sample UT HT A2 A4 A6 B2 B4 B6 C2 C4 C6
L* 33.26 30.26 33.69 34.17 34.72 33.41 34.86 35.04 33.65 34.92 35.11
a* ± ± ± ± ± ± ± ± ± ± ±
e
0.15 0.15f 0.14d 0.12c 0.16b 0.23de 0.19b 0.21a 0.23de 0.13a 0.11a
35.00 31.27 34.75 34.05 33.34 34.42 34.06 33.13 34.38 33.14 32.42
b* ± ± ± ± ± ± ± ± ± ± ±
a
0.08 0.26f 0.25ab 0.17c 0.14 cd 0.22b 0.26c 0.22d 0.12b 0.29d 0.14e
22.90 18.12 22.45 21.77 20.53 22.23 20.90 19.82 22.03 20.01 19.49
C* ± ± ± ± ± ± ± ± ± ± ±
a
0.13 0.14g 0.23b 0.21c 0.22d 0.19bc 0.20d 0.32f 0.11c 0.09e 0.12f
H* a
41.83 ± 0.11 36.2 ± 0.31j 41.34 ± 0.25b 40.44 ± 0.08e 39.15 ± 0.25 g 40.92 ± 0.21c 40.08 ± 0.1f 38.56 ± 0.3 h 40.81 ± 0.12d 38.66 ± 0.12 h 37.78 ± 0.21i
TCD e
1.3 ± 0 1.54 ± 0.04a 1.32 ± 0.03de 1.35 ± 0.02d 1.41 ± 0.03c 1.33 ± 0.01de 1.42 ± 0.03c 1.47 ± 0.05b 1.34 ± 0.01de 1.46 ± 0.01bc 1.46 ± 0.01bc
– 6.52 ± 0.29a 0.63 ± 0.17i 1.59 ± 0.3 g 3.22 ± 0.29d 1.16 ± 0.22 h 2.21 ± 0.28f 4.27 ± 0.26bc 1.1 ± 0.25 h 2.63 ± 0.11e 4.58 ± 0.17b
Results are expressed as mean ± standard error. Values represented with different letters are statistically different at p < 0.05.
Similar with the results obtained from 3.2.1, Vitamin C in blueberry juice was degraded with the increase of treatment time by CP treatment. After the treatment of 6 min, the decrement of 19.94, 57.14 and 52.33% for vitamin C in Group A, B and C was observed, respectively, compared with the treatment of 2 min. The UT sample had the highest level of vitamin C content, which was 4.03 ± 0.14 μg/mL. After 2 min of treatment, the three groups of CP samples exhibited higher (p < 0.05) vitamin C content than that of HT. When the treatment was extended to 4 and 6 min, HT sample showed a higher (p < 0.05) vitamin C level than that of group B and C, while, had a lower (p < 0.05) vitamin C level than that of group A. During the same treatment time, increasing of oxygen concentration resulted in a significantly (p < 0.05) decrement of vitamin C content. By using an oxygen concentration of 1% during 6 min, a lowest vitamin C content was acquired, which was obviously (p < 0.05) lower than that of UT (4.03 ± 0.14 μg/mL) and HT (2.49 ± 0.32 μg/mL). As reported by Rodríguez et al. (2017), when a N2 flow rate of 10 mL/min was used, an increment of 10.4 and 10.8% was observed after 5 and 10 min of treatment, respectively, but when the treatment was extended to 15 min, the sample exhibited a reduction of 4.5% in vitamin C. The authors explained that NO was one of the excited particles generated by CP, which could regulate the ascorbate–glutathione cycle and increased dehydroascorbate reductase activity.
compounds. A similar result has been found in Rodríguez et al. (2017)’s work. When CP was used, the TPC of the cashew apple juice exhibited an average increment of 108% although no significant differences among samples (using CP during 5, 10 and 15 min) were observed. However, the degradation of the total phenolics induced by CP has been found in recent literatures as well. In Pankaj et al. (2017)’s study, increasing the treatment time of the CP treatment resulted in a significant (p < 0.05) reduction in the total phenolics of the grape juices. The mechanism was that CP equipment used in this study with air generated many reactive oxygen species and ozone in the discharge. Phenolic compounds were particularly known to be susceptible for ozone attacks due to the degradation of aromatic rings in the structure (Stalter, Magdeburg, Wagner, & Oehlmann, 2011; Pérez, Torrades, Domènech, & Peral, 2010). Similar degradation of phenolic compounds after CP treatments were also reported in lettuce and orange juice (Almeida et al., 2015; Grzegorzewski et al., 2011). More detailed studies are required to understand the interaction mechanism between plasma reactive species and phenolics for further explanation. 3.3.3. Vitamin C content Fig. 3(c) showed the vitamin C in blueberry juice treated by CP, with oxygen concentration of 0, 0.5%, 1% during 2, 4 and 6 min. 84
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demonstrated to be another factor have influence on the antioxidant activity of juice.
Dehydroascorbic acid was an oxidative form of ascorbic acid, which could be converted to ascorbic acid by dehydroascorbic acid reductase (Sun et al., 2015; Shan, Zhou, & Liu, 2015). After 5 and 10 min of treatment, the rate of regeneration of ascorbic acid by the ascorbate–glutathione cycle was greater than the rate of decay of ascorbic acid through its reaction with other plasma generated reaction species, while, the rate of decay exceeded the rate of regeneration after 15 min. However, no increment of vitamin C was observed in this work. The stronger degradation of vitamin C could be attributed to reactive oxygen species.
3.5. Color stability The effect of CP on color of the blueberry juice was shown in Table 2. Generally, higher a* values indicate more reddish overall impression of the samples, whereas b* values evaluate blue (-b*) to yellow (+b*) tonality of the samples ((Bursać, Putnik et al., 2016)). In this work, upward trend of L* and H* value, and downward trend of a*, b* and C* value, regarding with the increasing of treatment time or oxygen concentration after CP treatment were found in Table 2. Compared with CP samples, HT showed the higher L* and H* value, and the lower a*, b* and C* value. Also among experimental samples, treatments of 2 min and 4 min were no visual difference in color for CP treatment vs UT, since color difference TCD = 1.5–3.0 reflects merely noticeable color difference. However, a range greater than TCD = 1.5–3.0 was observed in treatment time of 6 min and HT, reflecting noticeable color difference compared with untreated sample. Thus, CP treatment could better keep the original color of blueberry juice compared with HT, since HT sample had the highest TCD value. Several literatures reported the various results about the color changes after CP treatment. For example, the TCD values were 4.47 and 9.63 in fresh tomato and lettuce, respectively, after CP treatment time of 3 min (Bermudez-Aguirre, Wemlinger, Pedrow, Barbosa-Canovas, & Garcia-Perez, 2013). In another work (Pankaj et al., 2017), the TCD values of grape juice were 3.23, 4.22, 3.87 after CP treatment of 2, 3 and 4 min, respectively, which were all higher than that of thermal treatment (1.71). Thus, more studies are suggested to investigate the mechanism of color changes in different food system after CP treatment.
3.4. Antioxidant activity The antioxidant activity was determined by using different methods, including organic radical scavenging capacity (DPPH, %OH free radical and ABTS) and metal reduction ability (FRAP – ferric reduction antioxidant power), which could comprehensively and reliably represent the antioxidant activity of fruit juices (Mariadosocorrom et al., 2010). Fig. 4(1) (DPPH), (2) (%OH), (3) (ABTS) and (4) (FRAP) show the antioxidant activity of blueberry juice treated by CP. Different trends were observed when using the different assays denoting that the bioactive compounds that respond better to certain kind of assay are affected differently by CP application and exposure time. The antioxidant activity of the UT samples measured by DPPH, %OH free radical, ABTS and FRAP assays were of 93.03%, 58.25%, 4.06 mg Trolox/ g and 1.70 mM FeSO4/mL, respectively, which were all higher than those of HT samples. According to DPPH assay, it could be observed a significant effect of the treatment time, namely, longer treatments led to lower antioxidant activities. After the treatment of 6 min, the decrement of 14.10, 14.29 and 19.15% for antioxidant activity in Group A, B and C was observed, respectively, compared with the treatment of 2 min. Group C showed an equal (p > 0.05) antioxidant activity with UT, and higher (p < 0.05) than HT during 2 min. The behavior of the antioxidant activity resembles the behavior observed for vitamin C and anthocyanin, which may be the dominant factor determining the antioxidant activity at different time. Similar with the results of TPC content in 3.2.2, increasing of DPPH scavenging capacity resulted in a significantly (p < 0.05) increment of oxygen concentration during the same treatment time. On the contrary, higher oxygen concentration led to the lower (p < 0.05) antioxidant activities in %OH free radical scavenging assay after same time of treatment. It could be attributed to a certain amount of %OH free radical generated by CP. Based on the results of 3.1, more % OH was excited by CP with the increase of oxygen concentration. Also a significant effect of the treatment time was found in this assay, longer exposure to CP with lower antioxidant activities, which may due to the decrease in vitamin C and anthocyanin. Group A and B had greater %OH free radical scavenging capacity at all the experimental time, compared with that of UT and HT samples, which may relate to the increase of TPC. Similar trend with DPPH assay was observed in ABTS assay. The antioxidant activity was decreased with the increase of treatment time by CP treatment, while, except Group A in 4 and 6 min, the others’ were all significantly (p < 0.05) higher than that of UT and HT samples. Also increasing of oxygen concentration resulted in a significantly (p < 0.05) increment of antioxidant activity during the same treatment time. Also it could be observed a significant effect of the treatment time in FRAP assay, longer treatments led to lower antioxidant activities. However, different from the results of other three methods, no regular trends related to oxygen concentration was found in this assay. In a previous study (Pankaj et al., 2017), a decrease in the both DPPH free radical scavenging and antioxidant capacity was observed after CP treatment with increasing of treatment time, which is similar with this work. While, oxygen concentration of ionized gas was also
4. Conclusions Effects of cold plasma technology on the quality of blueberry juice have been presented in this study, including inactivation of Bacillus, contents of anthocyanin, total phenol, vitamin C, and antioxidant activity. Treatment time and oxygen concentration have been confirmed to be the main factors for CP treatment. The increment in treatment time and oxygen concentration significantly promoted death of Bacillus cells. CP treatment at 1% of oxygen concentration was resulted in 7.2 log reduction of Bacillus. In general, increasing of treatment time resulted in a significantly (p < 0.05) decrement of anthocyanin, vitamin C and antioxidant activity, and increment of TPC. Lower vitamin C content and %OH free radical scavenging capacity were induced by higher oxygen concentration, while, TPC, antioxidant activity in DPPH and ABTS assays were increased with the increase of oxygen concentration. Compared with HT, relatively shorter exposure time and lower oxygen concentration are better to keep the above components. In the tests of color change, blueberry juice treated by CP are more closer to UT juice, in comparison to HT juice, especially when the treatment is 2 or 4 min, CP juice reflects merely noticeable color difference with UT juice. In summary, the present study showed that cold plasma processing had more positive influence on quality of blueberry juice compared with thermal processing.
Declaration of interests None.
Acknowledgement This work was financially supported by the Fundamental Research Funds for the Central Universities (BLX201616). 85
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