Application of Frozen Plasma-Activated Water to Sanitize and Precool Fresh Produce during Postharvest Handling Process

doi:10.21203/rs.3.rs-2327529/v1

Converting plasma-activated water (PAW) to frozen PAW (FPAW) imparts additional advantages since it can simultaneously conduct washing, precooling, and decontaminating when incorporated with water. This study investigates the efficacy of FPAW undergoes pH manipulation on decontaminating E. coli and S. aureus inoculated on okra and strawberries surfaces. The effects of dilution factor (3X and 5X dilution), type of PAW (fresh and FPAW), and immersion time (0, 10, 15, and 20 min) on PAW efficacy in combination with the surface properties of okra and strawberries are investigated. Although the reduction achieved by this study was generally lower than the treatment applied to planktonic E. coli and S. aureus, the efficacy of FPAW was not entirely eliminated by the dilution and the surface roughness of the treated product. The reduction of E. coli and S. aureus can be achieved to 2.5 log CFU/g and 1.0 log CFU/g, indicate that pH-manipulated-FPAW has the potential to be applied in the postharvest treatment of fresh produce. In addition, the combination of all treatment factors did not significantly affect the physical quality of the product; in fact, the precooling effects of frozen PAW prevent the chilling injury on okra stored at 4.5 ± 0.5°C for 10 days.

Industrial Relevance Conducting precooling with frozen PAW successfully diminishes the problem of chilling injuries and pathogenic bacteria contamination on fresh produce. Converting PAW into the frozen shape and accompanied by pH manipulation overcome the delays issues of the loss of PAW reactivity. The FPAW can be stored longer before it is applied in the precooling and decontamination steps during postharvest treatment. This study shows that FPAW is a potential postharvest treatment agent for fresh produce, and it does not affect the physical quality of the treated fresh produce significantly.

frozen PAW

pre-cooling

dilution

bacteria decontamination

fresh produce

Horticultural products are essential for they are sources of various components the human body requires to maintain its functions and health. It has been suggested that consuming more fruits and vegetables reduces the risk for many diet-related chronic diseases, such as obesity, type II diabetes, coronary heart disease, and some cancers (Deng et al., 2020). Encompassing fruits, vegetables, and ornamentals, horticultural products meet the aesthetical needs due to their attractive, colorful, and beautiful shapes. In many Asian countries, fruits and flowers are common materials in their culture. Therefore, the existence and availability of horticultural products throughout the season are crucial mainly because they are produced in specific regions and periods. In this case, postharvest handling and distribution are critical in fulfilling people's demand for fruit and vegetable products.

Generally, horticultural products are characterized by notable perishability and short shelf-life attributes. It is so because most of them contain high moisture content and various nutritional components, fragile epidermal tissue, a high respiration rate, and high transpiration coefficient, which are determined by the thickness of the cuticle and epicuticular wax layer, cracks in the latter, and the presence of stomata and lenticels (Veraverbeke et al., 2003). Hence, various postharvest technologies are developed to anticipate the effects caused by those properties. Review journals and books covering the technology are available widely and provide ready-to-use techniques (Duan et al., 2020; Elansari et al., 2019; Garrido et al., 2015). However, implementing the suggested technologies required the horticultural products handling to invest in some facilities to achieve the goals because cooling and cold storage require enough starting capital, running costs, and a reliable electricity supply (Makule et al., 2022). Introducing nonthermal plasma and plasma-activated water in the horticulture sector brings a new alternative for the handler in treating their product. The reactivity of nonthermal plasma can reduce various microorganisms harboring fresh products such as goji (Cong et al., 2022), blueberries (Lacombe et al., 2017), strawberries (Sarangapani et al., 2020), tomatoes (Ali et al., 2021), mushroom (Xu et al., 2016), mung bean sprouts (Xiang, Liu, et al., 2019), kale (Shah et al., 2019), etc. The continuous and increasing demands for fresh produce of fruits and vegetables put many challenges for suppliers (Dimoso et al., 2020). Extending the fresh product shelf-life so that the product's availability can be prolonged and widened depends on the harvesting stage and the following postharvest treatments. The harvesting stage is responsible for acquiring the best physiological qualities of the product, while postharvest treatments are responsible for maintaining the qualities as long as possible. Consequently, additional capital investment should be made by the handler first.

The impact of precooling on fresh produce showed a remarkable performance in prolonging or maintaining postharvest product qualities. A work conducted by Wijewardane & Guleria (2013) on apples reported that conducting precooling preserved firmness and titratable acidity. It slowed down the physiological weight loss of apples better than the untreated ones. In addition, combining the precooling treatment with another, such as coating, synergically benefited the apple. Similar results are reported by some works (Gariépy et al., 1991; Kongwong et al., 2019; Yan et al., 2018). Furthermore, a longer shelf-life and safety are essential attributes that fresh products should possess because they are frequently eaten fresh. Being free from pathogenic microorganisms and pesticides is a crucial goal for many fresh-produce handlers demanded to attain. Various substances have been applied to reduce the population of microorganisms and pesticide residues, including chemical, essential oils, and nonthermal plasma treatments (Bourke et al., 2018; Chizoba Ekezie et al., 2017; Goodburn & Wallace, 2013).

Based on the benefits of precooling and PAW ability, this study proposed a new approach to utilizing PAW in the postharvest processing stage for fresh produce: converting the PAW into the FPAW. Converting PAW into a frozen shape enhances its capability for some reasons. First, FPAW can be mixed with washing water for fresh produce to create cooling effects. Second, the reactive species of the PAW exert the deactivation effect on bacteria by which the product's safety can be increased. The last, FPAW can be stored for a longer time and utilized in a more flexible time. Based on the previous work we have conducted.

We have successfully developed FPAW, whose shelf life is longer, and its bactericidal feature is high. PAW can be stored for up to 10 days with a significant efficacy on planktonic E. coli and S. aureus, making the delay issue possible to be anticipated. Even though the effectiveness of FPAW on planktonic E. coli and S. aureus is high, it is necessary to investigate further the efficacy of FPAW we developed on the real object. Therefore, this article reports the influences of surface properties of fresh produce, dilution factor, and 3-day-delays of that FPAW on okra and strawberries. These two fresh products were taken to represent the fruit and vegetable model.

Sample preparation

Okra pods are collected from the traditional market in the morning. Okras are then sorted to collect the pod in good condition: green and fresh, no black/brown on its surface or ridges, and no wide wound at its pedicle base. Samples are grouped into two samples: microbial analysis samples consisting of 12 okra pods for each treatment and quality analysis samples. The samples are prepared as follows: Firstly, the pedicle of the pod is cut with an alcohol-sterilized knife to flatten the pod base. Okras are then washed in RO water to clean dirt and dried under air flown over the pods by a fan until no liquid water appears on the surface. Inverting the pod periodically is performed to dry the okras evenly. After that, samples for the microbial analysis group are moved to a laminar flow hood to be sterilized under UV light for 15 minutes/surface. To expose the okra to UV evenly, the pods are rolled to direct the surface facing to the UV light source. The okra pods are ready to be inoculated with bacteria solution. Meanwhile, the other group is submitted to the quality analysis treatment procedures.

Strawberries are bought from the strawberry wholesaler in the afternoon. Strawberries are stored in cold storage and retrieved when sold. The strawberries are sorted by the seller according to their size before storage; it is labeled as no. 2, 16–18 g in weight, and 2.5-3.0 cm in diameter. The fruits are then transported to the laboratory (about 1 hour) and stored in cold storage overnight. On the analysis day, the calyx of the fruit is cut with an alcohol-sterilized scissor before the fruits are washed in RO water. Then the strawberries are dried under blown air over the fruits with a fan. UV light sterilization was carried out in a laminar flow hood for 15 minutes on each surface as per okra. After that, the strawberries are ready to be inoculated with a solution of E. coli or S. aureus.

At the end of the preparation steps, the inoculation step is commenced: for okra, sterilized okras are laid on the sterile mesh in the laminar flow hood. A 100 µL of bacteria solution is pipetted onto the Okra surface. It is important to note that the inoculation should cover only two areas of the okra segment surface, with the farthest drip located ± 2 cm from the pod tip (Fig. 1). This is done so because the okra pod floats when immersed, thus is needed to put a cover/lid to push all inoculated surfaces immersed in water. Twelve okra pods are prepared, divided into four experimental units consisting of 3 pods for each unit: control, 10 min, 15 min, and 20 min experimental units. After the inoculation step, okras are left in the hood for 3 hours before plasma treatment.

For strawberries, sterilized strawberries are put on the sterile mesh and stand on their pedicle base. Then, a 100 µL bacteria solution is pipetted onto the strawberry's surface carefully to avoid the solution drop slipping from the strawberry surface, making the solution's volume less than 100 µL. The number of strawberries prepared is the same as the okra: 12 fruits, divided into four experimental units, three fruits for each unit. After the inoculation steps, strawberries are left for 3 hours in the hood to allow the bacteria to attach to the surface of the fruit.

2.2 FPAW preparation and treatment procedures

Two types of PAW are prepared before the contaminated okra and strawberries are treated, (1) fresh PAW, prepared by activating the DI water with corona plasma discharge for 30 minutes. At the end of the activation, the fresh PAW is diluted by mixing the fresh PAW and DI water with a ratio of 1 : 2 (g/g) to prepare 3X-diluted PAW and ratio 1 of : 4 (g/g) to prepare 5X-diluted PAW. The diluted PAW is then poured into three 250-mL-beaker glasses labelled with 10, 15, and 20 min immersion times. (2) FPAWpH7$\downarrow$3.5 is prepared by activating DI water with the same corona discharge three days before the treatment. After the activation PAW obtained is alkalinized with NaOH solution to increase its pH to 7 before storing at – 20°C for three days in plastic bottles to make FPAW. After the storage, the FPAWpH7$\downarrow$3.5 is crashed, weighed, and mixed with DI water. The mixture is stirred with an alcohol-sterilized glass rod to quicken the thawing process. After FPAW is dissolved, acidification with acetic acid is performed to restore the pH of the FPAW to 3.5, and the immersion step is commenced immediately. This step is called restoration treatment, which aims to restore the state of the PAW to the initial state (after the activation process). As in the previous step, the PAW is poured into the three 250 mL beaker glasses. Throughout the paper, FPAW will be used instead of frozen PAW, which means the FPAWpH7$\downarrow$3.5. Fresh PAW will be used consistently in this paper to differentiate it from FPAW.

The plasma treatment of okra is carried out as follows: three okra pods are immersed in fresh PAW or FPAW mixture for 10, 15, and 20 minutes. Stir the mixture every 5 minutes with okra by holding the pod tail altogether to expose the okra evenly. Point all the tails of the pods upward, then put a petri dish as a lid so that the surface of the okra is submerged instead of floating. Similarly, the plasma treatment of strawberries is conducted as follows: three sterilized strawberries are immersed in fresh PAW or FPAW mixture for 10, 15, and 20 minutes. Stir the strawberries gently with a sterile glass rod every 5 minutes to expose the strawberries evenly.

Microbiological Analysis

At the end of the exposure treatment, samples are lifted and put into a sterile plastic bag, followed by pouring 30 ml of PBS 1X into the bag. For the okra, the recovery step is done using a reciprocal shaker machine for 30 min. Meanwhile, for strawberries, the recovery steps are done using a stomacher machine set up to run for 3 min at 260 rpm. Because bacteria recovered from the okra is dispersed in PBS, a 100 µL solution from each unit can be pipetted directly into the tube for serial dilution. Whereas the recovered bacteria from strawberries are blended with strawberries pulp, thus it needs an additional step: aspirate the bacteria solution out of the pulp and then pour it into a 1.5 mL Eppendorf tube. Avoid sucking the solid pulp. After samples have been collected in the Eppendorf tube, the samples are centrifuged at 2000 rpm for 3 min. Pipetted 100 ${\mu }$L of the supernatant into the tube filled up with 9.9 ml DI water and carried out the serial dilution according to the standard procedures. A 100 ${\mu }$L each of the diluted bacterial solution from the last four tubes is pipetted into a Petri plate followed by pouring 20–25 mL sterile PCA (at 45–46°C) into the plate as per the pour-plate technique. Incubate the bacteria for at least 24 hours at 35°C when all plates are cold. Count the colonies growing on the media on the following day and report all colonies, which range from 25–250 per plate.

Physical Properties Analysis

As mentioned in section 2.1, the samples for quality analysis are chosen from the same sources. Okra and strawberry samples are washed with RO water two times to remove the dirt and then dried under flowing air. Ten okra and strawberries are prepared for weight loss and color analysis, while 20 samples are prepared for texture analysis. An equal number of the sample are used for control and treated analysis. Plasma treatment is carried out by immersing the samples in cold water (control group) or water-PAW admixture for 20 minutes. The procedures for making the mixture are presented in section 2.2. The type of FPAW applied in this study is based on the result of the same authors’ previous work. After the treatment, samples are dried under blown air to ensure no liquid water is on the surface of the samples. Finally, samples are put into a disposable plastic lunch pack with five holes of 5 mm diameter made at its cover and then stored in cold storage at 4.5 ± 0.5°C. Each time analysis is conducted, weight loss and color samples are retrieved and returned to cold storage until the end of the storage study. Five okra pods or strawberries are retrieved on the day of texture analysis without returning the sample for all tests conducted are destructive analyses.

Weight Loss

Tens pods and fruits are prepared for this analysis. Each sample is labeled with a number to ensure that the weight loss value is obtained from the respective sample, thus, the average value of the weight loss is derived from the weight changes from each sample. The weight loss is expressed as mass difference value in proportion to the initial fresh mass (%):

$$\varDelta w=\frac{{m}_{tn}-{m}_{0}}{{m}_{0}} x 100\%$$

1

Where: m_tn is the weight at observation n^th, m₀ is the initial weight of the treated product (g).

Color Parameters

The color component of fresh produce is one of the critical clues for the consumer in buying because a good color is perceived as good quality inside out. Generally, the color possessed at which produce is harvested is taken as the reference for color quality determination, especially non-climacteric produce. Thus, any changes experienced by the fresh produce determine the quality of postharvest treatment exerted. In this research, PAW treatment risks the fresh produce experiencing oxidative stress, leading to the degradation of color pigments such as chlorophyll or carotene. Therefore, the total color difference ($\varDelta$E), hue$^\circ$, Chroma, and Browning Index are measured to analyze the PAW effects on the product's color. The formula for those parameters is as follows (Kanwal et al., 2020):

$$\varDelta E=\sqrt{{{(L}_{2}-{L}_{1})}^{2}+{({a}_{2}^{*}-{a}_{1}^{*})}^{2}+{({b}_{2}^{*}-{b}_{1}^{*})}^{2}}$$

2

hue$^\circ =180^\circ +{(tan}^{-1}\frac{{b}^{}}{{a}^{}})$ ; for okra and, (3)

hue$^\circ ={tan}^{-1}\frac{{b}^{}}{{a}^{}}$ ; for strawberry (4)

Chroma = $\sqrt{{a}^{2}+{b}^{2}}$ (5)

Browning index (BI):

$$BI=\left[100\left(x-0.31\right)\right]/0.17$$

6

Where $x=\frac{{a}_{f}+1.75{L}_{f}}{(5.645{L}_{f}+{a}_{f}-3.012{b}_{f})}$ (7)

A pictorial illustration of those color parameters are presented in Fig. 2. According to equations 2–7, the product's L*, a*, and b* values are required. L* represents the lightness, a* denotes the greenness and redness, and b* represents the blueness and the yellowness of the product. The L* a* b* values of okra and strawberry are measured with a color meter (Nippon Denshoku Color Meter ZE2000, Nippon Denshoku Kogyo Co., LTD). The color value of okra pods and strawberry fruits are measured two times at two different spots randomly determined.

Texture

To measure the texture parameters of the okra, the setting test is as follows, distance: 10 mm, test speed: 1 mm/s, probe type: Warner-Bratzler shear or cutting. Two parameters measured on okra are firmness and snapping force. The firmness represents the highest force needed to cut the okra pod with the probe. The cutting point is located approximately 10 mm from the edge of the segment surface. The snapping force measures the freshness of the pod by snapping the thumb on the lower tip of the okra (Rai & Balasubramanian, 2009). The snapping force is measured with the same setting as the firmness test mentioned earlier, except the test point is located 10 mm from the pod tail’s tip (Fig. 3).

For strawberries, the setting test is distance: 10 mm, speed: 1 mm/s, and probe P5 diameter: 5 mm. The tested strawberry is laid such that the fruit stands firmly on the base of the testing frame. The puncture test is performed at the surface facing the probe tips. Two different positions of the puncture test are applied for each sample. Texture measured on TA.XT plus (Stable Micro Systems, UK).

Effect of FPAW on E. coli and S. aureus population inoculated on okra

Bacteria inoculated on the biological matrix (such as on a surface of fresh produce) interact with the matrix and, in such a way, give protection to the targeted bacteria making the treatment directed to kill bacteria less effective (Ma et al., 2015). Physically, okra's surface is hairy and not smooth, which provides a suitable surface for bacteria to attach and thrive. From the previous work, we found that exposing planktonic E. coli and S. aureus to fresh PAW reduces the population by 4.43 and 3.76 log CFU/mL, respectively. However, after storing the fresh PAW at a low temperature (-20°C) for three days, its efficacy decrease significantly. In contrast, FPAW showed remarkable efficacy in killing E. coli and S. aureus, whereas as high as 4.35 log CFU/mL of E. coli and 3.24 log CFU/mL of S. aureus reduction were registered by the treatment. The results proved that pH manipulation is effective in preserving and restoring the bactericidal properties of FPAW.

Deactivating planktonic bacteria with PAW is more effective than the bacteria attached to other media or dispersed in a solution containing organic substances or other impurities (Gutiérrez-Martín et al., 2011; Jo et al., 2018; Xiang, Kang et al., 2019). Thus, inoculating bacteria on okra’s surface and diluting the PAW concentration reduces the ability of fresh PAW and FPAW to deactivate E. coli and S. aureus significantly. Figure 4 depicts that diluting fresh PAW and FPAW reduces its respective efficacy on both bacteria. The highest reduction achieved by 3X and 5X dilution of fresh PAW for E. coli are 2.65 and 1.89 log CFU/mL and for S. aureus are 1.59 and 1.04 log CFU/mL, respectively. In addition, the highest reduction by 3X and 5X dilution of FPAW for E. coli are 2.93 and 1.90 log CFU/mL and for S. aureus are 0.97 and 0.47 log CFU/mL, respectively. The results depend on the reactive species concentration exposing the bacteria; the lower the dilution, the higher the reduction is. These results also demonstrate that the efficacy of FPAW is more sensitive to diluting effects. To our knowledge, no study intended to investigate dilution's influence on the efficacy of PAW. It is not surprising because PAW is mainly applied as a liquid-treating solution. Berardinelli et al. (2016) reported no significant reduction of E. coli inoculated on fresh-cut celery treated with plasma for 20 min or 60 min, even though there was a 1.7 log CFU/ml reduction attained when planktonic E. coli is submitted to plasma treatment for 20 min. This obviously proved that the media on which bacteria attach significantly affects the efficacy of plasma treatment, intertwined with other factors such as bacteria strain, treatment time, etc.

Table 1

The population of *E. coli* inoculated on okra (log CFU/mL) exposed to different types of PAW admixtures
	Immersion time (min)
Admixture type	Control	10	15	20
fresh PAW3X	10.18 ± 0.13^a	7.69 ± 0.06^b	7.63 ± 0.32^b	7.53 ± 0.16^b
fresh PAW5X	9.30 ± 0.10^a	7.47 ± 0.11^b	7.46 ± 0.28^b	7.24 ± 0.07^b
FPAW3X	10.36 ± 0.19^a	8.14 ± 0.05^b	7.77 ± 0.11^c	7.43 ± 0.13^c
FPAW5X	8.79 ± 0.19^a	7.94 ± 0.36^b	8.20 ± 0.35^b	8.01 ± 0.06^b

Note: population surviving E. coli after being immersed in different solutions.

Fresh PAW3X is a mixture of PAW and DI water (1 : 2).

Fresh PAW5X is a mixture of PAW and DI water (1 : 4).

FPAW is fresh PAW stored for 3 days before application and its pH is adjusted before storage.

FPAW3X is a mixture of FPAWpH7↓3.5 and DI water (1 : 2).

FPAW5X is a mixture of FPAWpH7↓3.5 and DI water (1 : 4).

Value (mean of three experiments ± SD) followed by a different lowercase letter is significantly different among treatments at the same admixture type at P < 0.05.

Table 2

Population of *S. aureus* inoculated in Okra (log CFU/mL) exposed to different types of PAW admixtures
	Immersion time (min)
Admixture type	Control	10	15	20
fresh PAW3X	10.14 ± 0.15^a	8.67 ± 0.11^b	8.56 ± 0.28^b	8.77 ± 0.17^b
fresh PAW5X	10.14 ± 0.19^a	9.06 ± 0.09^b	9.20 ± 0.27^b	9.00 ± 0.11^b
FPAW3X	10.23 ± 0.02^a	9.27 ± 0.09^b	9.26 ± 0.28^b	9.19 ± 0.10^b
FPAW5X	10.04 ± 0.09^a	9.71 ± 0.12^a	9.57 ± 0.06^a	9.59 ± 0.08^b

Note: population are surviving S. aureus after being immersed in different solutions.

See Table 1 note.

Effect of FPAW on E. coli and S. aureus population inoculated on strawberries

Similar to the results mentioned in the previous section, the reduction of E. coli and S. aureus are affected by the dilution, type of PAW, and type of bacteria strain. The dilution factor slightly decreases the efficacy of PAW on bacteria, but the difference is not statistically significant. In this case, however, the type of PAW (fresh and FPAW) does not demonstrate sensitivity to dilution compared to the okra case. For instance, a 2.77 log reduction of E. coli is attained by fresh 3X-diluted PAW, while the 5X-diluted fresh PAW makes a 2.67 log reduction at the same immersion time (20 min). Meanwhile, the decrease caused by the 3X-diluted and 5X-diluted FPAW is 2.02 and 1.88 log reduction, respectively. Hence, two groups of the reduction graph formed, demonstrating opposite trends in Fig. 4. The diluted-fresh PAW seems more effective in killing E. coli, while diluted FPAW is more effective in S. aureus.

Unlike the dilution factor and type of PAW, the immersion time showed unpredictable trends. As presented in Fig. 4, the trend is inconsistent; some treatments show significant differences due to the increment of immersion time, while others show insignificant population reduction. However, the results convincingly demonstrated that FPAW maintains its efficacy on E. coli and S. aureus under the influence of the weakening factors such as dilution and rough surface of the product.

Table 3

Population of *E. coli* inoculated in strawberry (log CFU/mL) exposed to different types of PAW admixtures
	Immersion time (min)
Admixture type	Control	10	15	20
fresh PAW3X	10.03 ± 0.06^a	6.95 ± 0.58^b	6.02 ± 0.07^c	7.62 ± 0.64^b
fresh PAW5X	10.03 ± 0.06^a	7.06 ± 0.02^b	7.29 ± 0.23^c	7.36 ± 0.15^bc
FPAW3X	9.71 ± 0.16^a	7.57 ± 0.08^b	7.39 ± 0.06^b	7.70 ± 0.43^bc
FPAW5X	10.61 ± 0.24^a	7.66 ± 0.35^b	7.90 ± 0.21^b	7.73 ± 0.15^b

Note: population are surviving S. aureus after being immersed in different types of solutions

Fresh PAW3X is a mixture of PAW and DI water (1 : 2)

Fresh PAW5X is a mixture of PAW and DI water (1 : 4)

frozen FPAWpH7↓3.5-3X is a mixture of frozen FPAWpH7↓3.5 and DI water (1 : 2)

frozen FPAWpH7↓3.5-5X is a mixture of frozen FPAWpH7↓3.5 and DI water (1 : 4)

Value (mean of three experiments ± SD) followed by a different lowercase letter is significantly different among treatments at the same admixture type at P < 0.05

Table 4

Population of *S. aureus* inoculated in strawberry (log CFU/mL) exposed to different types of PAW admixtures
	Immersion time (min)
Admixture type	Control	10	15	20
fresh PAW3X	9.89 ± 0.03^a	9.21 ± 0.19^b	8.81 ± 0.67^b	9.00 ± 0.26^bc
fresh PAW5X	10.19 ± 0.09^a	9.59 ± 0.07^b	9.47 ± 0.04^c	9.39 ± 0.16^b
FPAW3X	9.88 ± 0.10^a	8.21 ± 0.10^b	8.55 ± 0.29^c	8.18 ± 0.10^b
FPAW5X	9.85 ± 0.12^a	8.42 ± 0.28^b	8.53 ± 0.65^b	8.30 ± 0.16^b

Note: population are surviving S. aureus after being immersed in different types of solutions

Fresh PAW3X is a mixture of PAW and DI water (1 : 2)

Fresh PAW5X is a mixture of PAW and DI water (1 : 4)

frozen FPAWpH7↓3.5-3X is a mixture of frozen FPAWpH7↓3.5 and DI water (1 : 2)

frozen FPAWpH7↓3.5-5X is a mixture of frozen FPAWpH7↓3.5 and DI water (1 : 4)

Value (mean of three experiments ± SD) followed by a different lowercase letter is significantly different among treatments at the same admixture type at P < 0.05

Deactivating the bacteria inoculated on the biological surface is commonly hindered by the interaction between the reactive species and the surfaces, hence, it reduces the efficacy of PAW. Many works reported the excellent results of PAW efficiency in reducing the number of microorganisms inoculated in fruits, vegetables, or spices; unfortunately, only a few of those respective works compared the efficacy of PAW on planktonic bacteria and attached bacteria.

This current study found that the efficacy of fresh PAW and FPAW is strongly affected by the dilution, properties of the treated product, and the type of bacteria. Figure 4 depicts that increasing the dilution factor decreases the reduction of E. coli and S. aureus by about half, which is predictable. E. coli constantly demonstrates a higher susceptibility to PAW than S. aureus in any state, planktonic or attached. The cell wall differences between those bacteria seem to respond differently to PAW (Joshi et al., 2011). We can conclude that the bactericidal of PAW on E. coli and S. aureus can be maintained by converting PAW into FPAW treated with pH manipulation. Even though dilution, rough surface, and delays impose weakening effects on PAW simultaneously, those factors do not entirely eliminate PAW ability. In addition, the bacteria reductions attained by 3X dilution are relatively similar to other authors who treat various products in killing bacteria (Alwi & Ali, 2014;Min et al., 2017). The reduction attained is in the range of 1–3 log CFU.

Effects Of Fpaw Treatment On The Physical Properties Of Okra

Weight loss

Table 5 shows that during 4-day storage, FPAW treatment enhances the weight loss of the okra. However, its effects become insignificant on day six afterward. This pattern may indicate that okra can recover from the damage caused by the PAW treatment given for 20 min or that okra is not susceptible to reactive species of FPAW. Similar results were reported by Finger et al. (2008) that the weight loss of okra stored at low temperatures (5 and 10°C) reached > 10% after ten days of storage. Unwrapped Okra registered this result, while the wrapped okra maintained its weight loss lower than 5%. Weight loss above 5% is considered high enough to make the product unsalable (Kader & Saltveit, 2002). This means that okra can only be stored at 4.5 ± 0.5°C for less than six days for all treatments. However, the appearance of okra during storage seems well preserved in this study. This fact is supported by the results presented in Tables 6 and 7 where all color parameters and texture changes insignificantly during the storage.

Table 5

Weight loss of okra stored for 10 days
Treatment	Weight loss (%) on n day of storage
Treatment	0	2	4	6	8	10
Control	0.00 ± 0.00	1.93 ± 0.45^a	4.53 ± 0.97^b	7.64 ± 1.27^c	9.53 ± 1.33^d	11.5 ± 1.27^e
FPAW	0.00 ± 0.00	3.10 ± 0.62^a	5.45 ± 0.78^b	8.14 ± 1.00^c	10.2 ± 1.25^d	11.9 ± 1.41^e

The different superscript letters following the value represent the significant difference (P < 0.05)

All values are expressed with mean ± SD (n = 10)

Color Parameters

There is no significant effect caused by PAW treatment on okra color. All samples seem to experience a change in their color because of the physiological process. The prolonged storage period changes the color slightly on the control, but the color change range of FPAW samples is stagnant in the 4–5. This means that exposing okra to FPAW helps okra maintain its color from the browning process or chilling injury commonly experienced by the okra stored at low-temperature (Huang et al., 2012; Phornvillay et al., 2019). A study conducted by Phornvillay et al. (2019) found that stored okra at 4°C triggers chilling injury symptoms of pitting, translucency, and browning on the pericarp of the pods right from day four onwards. Based on the color parameters observed for ten days, there is no chilling injury symptoms appear on both okra samples (Table 6). According to the method of this study, both control and FPAW samples experience pre-cooling before storing at low temperatures. The results corroborated that pre-cooling on fresh produce helps remove the field heat and reduces the temperature gap between the product and the environment (storage), preventing them from chilling injury.

Additional observation in this study proved that retrieving okra on day 8th to room temperature for 2 days does not promote chilling injury; a contrast results according to Phornvillay et al. (2019). The color parameters of okra subjected to chilling injury evaluation are presented in Table 7. The table shows that chroma, hue angle, and browning index seem not to change significantly, making it easy to conclude that the treatments, in general, prevent okra undergoes measurable damage. Browning on fresh products is highly related to the loss of cell wall integrity due to lipid peroxidation. It is proved that plasma treatment triggers lipid peroxidation that could lead to the deterioration of the product (Jiang et al., 2004; Marangoni et al., 1996). In contrast, the current work reveals that the efficacy of FPAW in deactivating bacteria does not compromise the okra quality severely. The only parameter that looks different due to the treatment effects is the total color difference, in which the FPAW tends to cause a higher color change. The statistical analysis proved that the total color difference of okra is significantly affected. However, the changes are hardly recognized by the eye or captured by the camera. Radical species, especially OH^•, are involved in lipid peroxidation. However, according to Khlyustova et al. (2019) and Zhou et al. (2018) we can assume that hydroxyl radicals react to form hydrogen peroxide before it is dissolved in water. As a result, water contains reactive species beneficial in deactivating bacteria with less damaging effects on okra color.

Table 6

Color parameters of okra stored for 10 days at 4.5 ± 0.5°C
Storage day	Control			FPAW3X
Storage day	chroma	hue°	$\varDelta E$^#	chroma	hue°	$\varDelta E$
Initial*	29.14	178.44	0.00	29.14	178.44	0.00
day 0**	29.21	178.44	3.14	29.92	178.44	3.77
day 2	30.58	178.44	3.13	31.28	178.44	4.06
day 4	30.83	178.44	3.45	30.98	178.44	3.81
day 6	30.81	178.44	4.21	30.87	178.44	4.16
day 8	30.60	178.44	3.36	31.76	178.44	4.75
day 10	30.11	178.44	3.34	32.42	178.44	5.26

* The color parameter was obtained from the sample without any treatment.

** The color parameter obtained from the sample after treatment (ice : water) is carried out.

^# Total color difference.

Statistical analysis shows that there is no significant difference between the treatments.

Table 7

Color parameters of okra retrieved on the 8th day after storage
Color parameter	Monitoring day	Control	FPAW3X
$\varDelta E$	0	0.00 ± 0.000	0.00 ± 0.00
	1	1.43 ± 0.869	2.26 ± 0.46
	2	1.60 ± 0.157	3.73 ± 0.85
Hue°	0	178.44 ± 0.00	178.44 ± 0.00
	1	178.44 ± 0.00	178.44 ± 0.00
	2	178.44 ± 0.00	178.44 ± 0.00
Chroma	0	31.93 ± 0.78	31.53 ± 1.23
	1	31.74 ± 1.85	30.74 ± 3.03
	2	31.20 ± 3.19	30.10 ± 2.74
Browning index	0	59.74 ± 3.77	61.05 ± 5.41
	1	62.92 ± 3.57	57.93 ± 11.05
	2	59.95 ± 7.82	55.64 ± 7.72

Both treatments prevent the change in the color parameter of okras which are stored at 4.5 ± 0.5 and subsequently move to room temperature for 2 days on the 8th day. The statistical analysis shows that all color parameters and browning index influenced by the treatment are not significantly different.

Hardness

The maximum forces required to tear up the okra pod are presented in (Table 8). There is no significant increase in maximum forces required during 6 days of storage of FPAW-treated okra. However, a significant increase and decrease were registered on days 8th and 10th at which by the end of the storage study, the maximum force is lower approximately 3 N than that of the 0 days. A similar pattern is demonstrated by the control which shows an earlier increase and decrease of maximum forces. At the end of the storage, control okra hardness is lower than the FPAW, however, its hardness on day 0 and 10 differ around 3.8 N. The hardness of okra treated by FPAW is lower than the control with a narrower range as well. Both treatments show a similar pattern; starting with modest hardness, increasing to the maximum on day 6th, then followed by the decrease of the hardness to a lower point than the initial value as shown in Fig. 7. The results seem to be in accordance with(Rai & Balasubramanian, 2009) who found that the toughness changes of okra pod are opposite to its moisture loss. This study also found that the higher moisture loss is demonstrated by the okra treated with FPAW (Table 5).

Table 8

The forces are measured at 10 mm from the okra pod’s stem base
Treatment	Hardness (N) of okra on n days of storage
Treatment	0	2	4	6	8	10
Control	35.5 ± 0.99^a	37.6 ± 1.74^a	41.1 ± 4.65^b	41.1 ± 2.67^b	36.3 ± 3.36^a	31.7 ± 1.58^c
FPAW3X	37.5 ± 0.77^a	37.4 ± 1.68^a	37.9 ± 4.94^a	39.5 ± 4.01^a	33.0 ± 2.65^b	33.9 ± 4.47^ab
The forces are expressed in mean ± SD (n = 5)

Snapping Force

Snapping force is a common method used to evaluate the texture of brittle materials such as biscuits or chocolate bars by which the peak force is required. The snapping technique is commonly used by the consumer to subjectively determine the freshness of the okra pod. The snapping technique is done by griping the okra pod with 4 fingers and then thumb bending the tail of the okra pod until it breaks the pod at a particular part of the pod (approximately 1-1.5 cm from the tail). This study applied the snapping force required to determine the freshness of the okra treated with FPAW. The snapping forces of okra during the storage study are presented in Table 9. The pattern of snapping forces is similar to that of the hardness indicated by the lower forces required for FPAW-treated okra than the control. In contrast, the forces required tend to decrease along with the storage time and increase at the end of the storage. This phenomenon suggests that the moisture content significantly influences the snapping and hardness of the okra. On the one hand, moisture loss increases the hardness of okra. On the other hand, it decreases the snapping force. However, prolonging the moisture loss deforms the okra’s tail properties which increases the dryness of the tail, and increases the tail hardness. This is indicated by the drying process is started from the tail’s tip.Rai & Balasubramanian (2009) reported that the snapping force stored in packaged remains constant during storage while the unpackaged okra shows a decreasing trend. The reports suggested that moisture loss reduced the toughness (force required to snap the pod okra). Another possibility causing this phenomenon is that the hardening of okra is strongly related to the increase of cellulose content during the maturation process (Ren et al., 2021), which is probably decelerated by the treatment. However, the author emphasized that during the maturation process of okra, the cellulose content does not increase linearly, thus the average of the hardness and snapping force does not increase or decrease linearly as well.

Table 9

Snapping forces of okra stored at 4.5 ± 0.5°C
Treatment	Snapping forces* (N) on n day of storage
Treatment	0	2	4	6	8	10
Control	22.48 ± 2.24^a	18.58 ± 2.05^a	18.08 ± 1.48^b	18.71 ± 2.61^b	22.11 ± 3.14^a	23.16 ± 2.44^b
FPAW3X	17.95 ± 0.93^a	17.36 ± 1.05^a	17.61 ± 1.71^a	16.23 ± 2.37^a	16.78 ± 1.97^b	19.03 ± 2.49^a

*Snapping forces are measured at 10 mm from the okra pod’s tail.

All values are expressed as mean ± SD (n = 5)

Effects Of Fpaw Treatment On The Physical Characteristic Of Strawberries

Weight loss

The weight loss of strawberries treated by FPAW is presented in Table 10. In general, FPAW treatment does not influence the loss of weight for up to 4 days, after which the weight loss rate increases by almost double. It seems that low temperature delays the expression of the oxidative stress effects caused by the treatment. In addition, these results are similar to Kalt et al. (1993) who counted that the weight loss of strawberries stored at 5°C was about 4.89% in 8 days.

Strawberry fruits respire at a moderate rate at 4°C according to (Kader & Saltveit, 2002) while (Baka et al., 1999) reported that the strawberries respire at 15–20 ml CO₂/kg.h. According to the respiration equation C₆H₁₂O₆ + 6O₂ + 38ADP + 38 Pi→ 6CO2 + 6H₂O + 38 ATP + 686 kcal (Kader & Saltveit, 2002), it suggests that each mole of oxygen consumed will release one mole of water vapor and carbon dioxide which are both released into the environment. This means that the weight of strawberries decreases continuously accompanying water content loss. Low temperature decreases respiration (Saltveit, 2019), decelerating water loss and carbon dioxide from strawberry fruit. However, evaporation due to high water vapor pressure differences will occur rapidly without good humidity management.

Table 10

Weight loss of strawberry stored for 10 days at 4.5 ± 0.5°C
Treatment	Weight loss (%) on the n day of storage
Treatment	0	2	4	6	8	10
Control	0 ± 0.00	0.98 ± 0.37	1.52 ± 0.41	2.02 ± 0.57	1.99 ± 0.48	2.37 ± 0.54
FPAW3X	0 ± 0.00	0.91 ± 0.23	1.76 ± 0.37	2.58 ± 0.57	3.49 ± 0.70	4.27 ± 0.78
Values are expressed with mean ± SD (n = 10)

Statistical analysis shows that there is no significant difference between the treatments at P < 0.05.

Texture

Applying PAW instead of gaseous plasma benefits strawberries in terms of preserving their physical qualities. The loss of cell wall integrity and the increase of cell permeability are highly related to lipid peroxidation triggered by radical species generated in the gaseous plasma (Stoica et al., 2014). According to(Zhao et al., 2020) and(Kostya (ken) Ostrikov et al., 2020) hydrogen peroxide is produced when two hydroxyl radicals react •OH + •OH → H₂O₂. (8) and then pass through the gas-water interface and dissolve into the water. Form this point of view, the concentration of •OH in PAW is considered minuscule (Zhou et al., 2018), thus, the damage of strawberries tissue due to lipid peroxidation is considered insignificant as well (Verlackt et al., 2018). In addition, strawberry fruit is proved to be one of the best berries that demonstrate high antioxidant capacity compared to thornless blackberries (Rubus sp.), blueberries (Vaccinium spp.), cranberries (Vaccinium macrocarpon Aiton), raspberries (Rubus idaeus L. and Rubus occidentalis L.) according to (Wang & Jiao, 2000). The results obtained in this study support this concept. Table 11 shows that there is no significant impact of FPAW treatment on strawberries’ hardness during 9 days of storage (P < 0.05).

•OH + •OH → H₂O₂. (8)

Table 11

Hardness (N) of strawberries stored for 9 days at 4.5 ± 0.5°C
Treatment	Hardness (N) on n day of storage
Treatment	0	3	6	9
Control	3.59 ± 0.38^a	3.68 ± 0.72^a	4.22 ± 0.66^a	3.54 ± 0.58^a
FPAW3X	3.59 ± 0.38^a	3.82 ± 0.99^a	4.28 ± 1.07^a	4.15 ± 0.91^a

Statistical analysis shows that there is no significant difference between the treatment on strawberries hardness (P < 0.05) and there are no significant effects of storage day on strawberries hardness (P < 0.05)

The values followed by the same superscript letter for each treatment are not significantly different at (P < 0.05)

Color Parameters

Red color of strawberries is mainly dependent on the content of two anthocyanidin pigments namely glycosides namely pelargonidin 3-glucoside (Pg 3-gl) and cyanidin 3-glucoside (cy 3-gl) which vary among strawberries cultivars (Aaby et al., 2012). Roughly about 88% of the proportion belongs to Pg 3-gl (da Silva et al., 2007; Kalt et al., 1993). When strawberries are harvested at the fully red stage, there are no significant changes in anthocyanin content during low-temperature storage. However, the decline in Pg 3-gl occurs when strawberries are stored at 20 or 30°C. According to this, the changes in color parameters, especially the total color difference, are relatively constant during the storage due to, first, the low temperature used in storing the strawberries and, second, due to the samples obtained from the seller being fully red (Table 12).

Likewise, the polyphenol oxidase (PPO) causing browning effects on treated strawberries’ surface seems inactivated by the treatment or PPO which presents in the chloroplast thylakoid membrane (Teribia et al., 2021), undisturbed due to undamaging treatment to strawberries cell tissue as it is indicated by the relatively constant browning indexes. In addition, from our viewpoint, treating the fresh product with PAW is less damaging than direct gaseous plasma, as we mentioned in section 3.3.2. However, the research conducted by (Misra et al., 2015), who applied direct gaseous plasma on whole strawberries for 1 and 5 min, found no significant changes in anthocyanin content as well as the effects of PPO. Therefore, it is very likely that neither the FPAW nor fresh PAW treatment significantly affects the color parameters of whole strawberries at a certain level of treatment (Table 12).

Table 12

Color parameters of strawberry stored for 10 days
Treatment	Parameter	day of storage
Treatment	Parameter	0	2	4	6	8	10
Control	∆C	0.00 ± 0.00	3.69 ± 1.23	4.86 ± 1.25	5.12 ± 1.94	5.57 ± 2.08	5.50 ± 1.71
	Chroma	54.50 ± 1.69	57.44 ± 1.93	57.98 ± 2.18	58.15 ± 2.53	58.88 ± 2.88	59.17 ± 1.84
	Hue°	39.10 ± 1.95	39.90 ± 1.84	40.68 ± 2.09	40.11 ± 2.21	40.63 ± 1.93	40.81 ± 1.69
	BI	176.02 ± 1.69	200.23 ± 1.93	204.44 ± 2.18	208.10 ± 2.53	205.44 ± 2.88	204.38 ± 1.84
FPAW3X	∆C	0.00 ± 0.00	4.30 ± 1.21	4.24 ± 1.51	4.27 ± 1.64	4.65 ± 1.46	3.46 ± 2.28
	Chroma	52.95 ± 2.27	55.32 ± 1.87	54.96 ± 1.32	56.09 ± 1.86	55.36 ± 1.41	55.19 ± 1.54
	Hue°	38.12 ± 1.93	39.09 ± 1.76	38.55 ± 2.08	38.83 ± 1.66	38.46 ± 1.94	38.35 ± 1.20
	BI	169.38 ± 2.27	199.51 ± 1.87	198.37 ± 1.32	196.82 ± 1.86	196.15 ± 1.41	199.52 ± 1.54

Various factors influence the efficacy of PAW as a decontaminant agent for fresh produce. Some of them abate the reactivity of PAW, such as the roughness of produce surfaces, initial concentrations of the reactive species, the resistance of the bacteria treated, and even the delay of implementation. In a real environment where the PAW is being applied, the encounter is inevitable; thus, the results obtained from laboratory activities should be tried and evaluated for their retained efficacy. The efficiency of PAW in reducing the E. coli and S. aureus population has been proven to be profoundly high in the previous study, without the interference of delays, biological matrix, and dilution. This study included as many as lowering factors that seem encountered by the FPAW. Consequently, the lower ability of FPAW to reduce E. coli and S. aureus is demonstrated. The lower reduction of E. coli and S. aureus is caused by the intertwined effects of dilution, product surface, and the types of PAW incorporated in the treatment. However, the results convincingly demonstrate that the efficacy of FPAW in killing E. coli and S. aureus is preserved. Instead of injuring the product, the FPAW treatment in this study is able to prevent the chilling injury symptoms on okra and suppress the browning on okra and strawberries. The pre-cooling effect of incorporating FPAW in the treatment benefits the okra and strawberries in abating the chilling injury susceptibility.

Author contribution Gede Arda: Writing – original draft, Investigation, Data analysis. Chuan-Liang Hsu: Conceptualization, Writing-review, Supervision.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work in this paper.

Funding Declaration

This project is financially supported by the Ministry of Science and Technology, Taiwan, under Project No. MOST 110-2221-E-029-007.

Acknowledgments

The authors thank Dr. Li-Hsien Chen in the Department of Food Science at Tunghai University for advice on experimental design.

Data Availability Statement

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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No competing interests reported.

Application of Frozen Plasma-Activated Water to Sanitize and Precool Fresh Produce during Postharvest Handling Process

Status:

Version 1

Abstract

Figures

Introduction

Materials And Method

Sample preparation

Microbiological Analysis

Physical Properties Analysis

Weight Loss

Color Parameters

hue\(^\circ =180^\circ +{(tan}^{-1}\frac{{b}^{}}{{a}^{}})\) ; for okra and, (3)

hue\(^\circ ={tan}^{-1}\frac{{b}^{}}{{a}^{}}\) ; for strawberry (4)

Chroma = \(\sqrt{{a}^{2}+{b}^{2}}\) (5)

Texture

Results And Discussion

Effects Of Fpaw Treatment On The Physical Properties Of Okra

Weight loss

Color Parameters

Hardness

Snapping Force

Effects Of Fpaw Treatment On The Physical Characteristic Of Strawberries

Weight loss

Texture

•OH + •OH → H₂O₂. (8)

Color Parameters

Conclusions

Declarations

References

Additional Declarations

Status:

Version 1

Color parameter	Monitoring day	Control	FPAW3X
\(\varDelta E\)	0	0.00 ± 0.000	0.00 ± 0.00
	1	1.43 ± 0.869	2.26 ± 0.46
	2	1.60 ± 0.157	3.73 ± 0.85
Hue°	0	178.44 ± 0.00	178.44 ± 0.00
	1	178.44 ± 0.00	178.44 ± 0.00
	2	178.44 ± 0.00	178.44 ± 0.00
Chroma	0	31.93 ± 0.78	31.53 ± 1.23
	1	31.74 ± 1.85	30.74 ± 3.03
	2	31.20 ± 3.19	30.10 ± 2.74
Browning index	0	59.74 ± 3.77	61.05 ± 5.41
	1	62.92 ± 3.57	57.93 ± 11.05
	2	59.95 ± 7.82	55.64 ± 7.72

Application of Frozen Plasma-Activated Water to Sanitize and Precool Fresh Produce during Postharvest Handling Process

Status:

Version 1

Abstract

Figures

Introduction

Materials And Method

Sample preparation

Microbiological Analysis

Physical Properties Analysis

Weight Loss

Color Parameters

hue\(^\circ =180^\circ +{(tan}^{-1}\frac{{b}^{*}}{{a}^{*}})\) ; for okra and, (3)

hue\(^\circ ={tan}^{-1}\frac{{b}^{*}}{{a}^{*}}\) ; for strawberry (4)

Chroma = \(\sqrt{{a}^{2}+{b}^{2}}\) (5)

Texture

Results And Discussion

Effects Of Fpaw Treatment On The Physical Properties Of Okra

Weight loss

Color Parameters

Hardness

Snapping Force

Effects Of Fpaw Treatment On The Physical Characteristic Of Strawberries

Weight loss

Texture

•OH + •OH → H2O2. (8)

Color Parameters

Conclusions

Declarations

References

Additional Declarations

Status:

Version 1

hue\(^\circ =180^\circ +{(tan}^{-1}\frac{{b}^{}}{{a}^{}})\) ; for okra and, (3)

hue\(^\circ ={tan}^{-1}\frac{{b}^{}}{{a}^{}}\) ; for strawberry (4)

•OH + •OH → H₂O₂. (8)