Good feasibility of ozone-microwave treatment as a sterilization technology to extend the edible life of candied fruit as a post-processed fresh fruit product

: In order to solve the problem of infestation of fresh fruit after making them 7 into candied fruit and the quality degradation caused by the infestation, a combined 8 ozone–microwave sterilization method was used to treat four candied fruit molds. 9 Response surface optimization analysis showed that when the ozone infusion time was 10 at 10 min and with 560 W microwave power for 20 s, the inhibition rates of the four 11 candied fruit molds S 1 , S 2 , X 1 , and H 1 were 100%, 94.28%, 100%, and 100%, 12 respectively. Moreover, according to the characterization of morphology, DNA 13 electrophoretic profiles, and nucleic acid/protein leakage test, the integrity of the mold 14 cell membrane was destroyed after the combined ozone–microwave treatment because 15 of the thermal and nonthermal effects of microwave and the strongly oxidizing 16 properties of ozone. The molds were dead because of the efflux of intracellular nucleic 17 acid and protein and the degradation of DNA, which influenced normal cellular 18 metabolism. Quality identification showed a better expected quality of candied fruit. 19


Introduction
Candied fruit is a dried product made from fresh fruit and vegetables which is processed with sugar or honey.Given the high osmotic pressure and low water activity due to the high sugar content, candied fruit can inhibit the growth of most microorganisms.However, some molds can still breed on the surface of candied fruit, thus causing a food-safety problem.Finding a fast, efficient, green sterilization technology is therefore necessary.
As an efficient sterilization technology, microwave sterilization has the advantages of rapid temperature rise, short time consumption, and broad-spectrum bacterial inhibition, making it widely used in the food industry (Hock, Chala, & Cheng, 2020).
According to references, upon microwave treatment of candied fruit of Shuanghua plum and bergamot, the total bacterial colonies were within the Chinese national standard (Kaixin, Chuyin, Jianhua, Shuxi, & Aimei, 2019).The dried apricots treated by microwave were still of high food quality and low microbiological count after 180 days of storage (Jiayuan, Wei, & Lihong, 2021).The wide use of ozone as a chemical sterilization method can damage the membrane structure of microorganisms with its strong oxidizing ability, which has been reported to have a significant sterilization effect on fresh-cut lettuce and peppers (Alexopoulos et al., 2013), drinking water (Watanabe et al., 2010), citrus (Boonkorn et al., 2012), oil palm bunches (Sukaribin & Khalid, 2009), and other foods.Moreover, ozone treatment causes no toxicity in fruit and vegetables.
A single sterilization technology that affects bacterial inhibition is still unsatisfactory (Yu et al., 2015).Combining different sterilization technologies with synergistic or interactive effects may reach the best effect and reduce the impact on food quality.Currently, ozone and ultrasound (Oliveira et al., 2018), ultraviolet radiation (Dev Kumar, Williams, Sumner, & Eifert, 2016); (Chen, Liu, Chen, Liu, & Duan, 2020), ultraviolet infrared-ultraviolet combination (Dev Kumar, Williams, Sumner, & Eifert, 2016); (Chen, Liu, Chen, Liu, & Duan, 2020), ozone and lowtemperature plasma (Mayookha et al., 2023), and other technologies combined with traditional sterilization method have been rapidly developed.These findings suggest that combining ozone and other sterilization technology is a reliable way of maintaining food quality.
In this research, the combination of ozone and microwave sterilization technology has been applied to candied fruit for mold sterilization.The best condition of the combined ozone-microwave treatment has been optimized and analyzed by response surface methodology.Moreover, the mechanism of ozone-microwave treatment was analyzed by in depth by morphology characterization, DNA electrophoresis profile, and nucleic acid/protein leakage from mold.Furthermore, the quality of candied fruit treated by ozone-microwave was analyzed to identify the feasibility of this combined sterilization.The results will help advance the theory of preservation of fresh fruit and vegetable post-processed products.

Mold culture and counting
Freshly cut candied fruit was left at room temperature for 4 months until the candied fruit was completely infested with mold.The mold strains were extracted from the candied fruit samples and isolated and purified according to a published method (Zeping, Yaohua, & Xianliang, 2022).Following the methods in the Chinese national standards for food safety (GB4789.92-2016 andGB4789.15-2016),we processed the candied fruit and performed the culture counts.Based on the ITS sequence analysis and phylogenetic tree results, the four molds were identified as Cladosporium velox, Penicillium citreonigrum, Penicillium sclerotiorum, and Alternaria tenuissima.The four molds were named S1, S2, X1, and H1, respectively.

Preparation of spore suspensions
The pure strain was cultured on a PDA medium and stored at 4 °C.Moreover, the dominant mold organism was transferred to a PDA medium and incubated at 28 °C for 5 days, and the spore suspension was prepared.We then took 1 mL of the spore suspension, incubated it at 28 °C for 5 days, and diluted the concentration to 10 5 -10 6 cfu/mL.

Combined ozone-microwave sterilization
The spore suspension (20 mL) mentioned above was transferred into PDA medium and then injected with ozone gas at 25 °C at 50% humidity supplied by an ozonegenerating device Shenzhen Feili Co.,Ltd.,Shenzhen,China).The ozone infusion times were 2, 5, and 10 min.
After the treatment above, the microwave generation device (G80F20CN2L-B8; Guangdong Granz Group Co., Ltd., Shunde, China) treated the mold suspension.The microwave power was set at 420, 490, and 560 W, and the microwave time was set at 10, 15, and 20 s.The inhibition rate was calculated by the dilution plate counting method.

Response surface methodology (RSM)
The effects of factors on mold inhibition rate were analyzed using RSM.Following the Box-Behnken design principle, a central composite design was set up to study the relationship between the inhibition rate of mold and the three controlled factors, namely, ozone infusion time (A), microwave time (B), and microwave power (C).The design was performed with the software SAS (SAS Institute Inc., NC, USA), and the effects of three factors on the inhibition rate were evaluated.

Morphological and ultrastructural observation
Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses were performed as described in previous studies (Du, Sun, Tian, Cheng, & Long, 2023;Gani et al., 2016;Songsamoe, Khunjan, & Matan, 2021).The mold cultured at 28 °C for 5 days in the PDA medium was prepared in a small piece (2 mm × 2 mm), sputtered with a layer of gold, and examined by SEM (Merlin-type, Carl Zeiss, Germany).The mold cultured at 28 °C for 5 days in the PDA medium was fixed with 2.5% glutaraldehyde, dehydrated with acetone, frozen, and stained with lead citrate solution and uranium peroxide solution.After slicing, the samples were observed by TEM (JEM-2100F; Nippon Electron Co., Ltd., Japan).

DNA electrophoresis profiles
Following the method in the reference (Flórez & Mayo, 2015), we measured the electrophoretic bands by using a Mini-SubCell GT Cell gel electrophoresis instrument (Bio-Rad Laboratories, Inc., USA) with 1% gel concentration at 120 V voltage for 20 min.Nucleic acid extraction was performed using an S-96 nucleic acid extractor (Bio-Rad Laboratories, Inc., USA).

Nucleic acid and protein leakage assay
We refer to the method of (Sun et al., 2018;Xiang et al., 2019).Ten milliliters of the mold suspension with different treatments were taken, centrifuged, and measured for absorbance at 260 and 280 nm by using a UV-visible spectrophotometer (UV2300; Jinghua Instruments Co., China) to determine the amounts of nucleic acid and protein leakages, respectively.The PDB medium was used as blank.

Quality identification
The molds analyzed in section 2.2 were cultured in the candied fruit again, and the quality of the candied fruit treated by combined ozone-microwave treatment was analyzed.

Total sugar content
According to (Deshavath, Mukherjee, Goud, Veeranki, & Sastri, 2020), candied fruit (1 g) was added to a liquid mixture of distilled water (3 mL) and 6 mol/L HCl (10 mL).The sample was heated in a water bath at 80 °C for 30 min and then cooled and filtered.The filtered liquor was neutralized with 6 mol/L NaOH, diluted with distilled water, and mixed with 1.5 mL DNS reagent, and the absorbance was measured at 540 nm.The total sugar content of the samples was calculated according to Eq. ( 1): where m1 is the glucose content (mg), n is the dilution times, and M is the dry weight of samples (mg).

Color evaluation
According to a reference (Wang et al., 2021), the candied fruit samples were selected, cut, and measured by a colorimeter (CR-410, Konica Minolta Holdings, Inc.).
The samples' a*, b*, and L* values were obtained.ΔE values were then calculated according to Eq. ( 2).

Sensory evaluation
According to (Kidoń & Grabowska, 2021), 8 adults (4 females and 4 males) were recruited from South China Agricultural University.The total sensory evaluation score for candied fruit samples was 100 points, consisting of color, aroma, tissue, and taste.
Furthermore, there was a 25-point category scale from 0 (none) to 25 (extremely).
Finally, the sensory scores were averaged.

Statistical analysis
Experiments were conducted in triplicate (n = 3), and the data were analyzed using SPSS 22.0 statistical software (SPSS, Inc., Chicago, IL) and SAS Software (SAS Institute Inc., NC, USA).The significance level was set at p < 0.05.

Effect of treatment conditions on the inhibition rate of mold
Factor levels according to the Box-Behnken design principle are shown in   The results were analyzed by ANOVA for Eq.(3), Eq. ( 4), Eq. ( 5), and Eq. ( 6) as listed in Table 3 and Table 4.The p values for all four models were less than 0.01, which indicated that the model differences were highly significant.The goodness of fit of the model was checked by regression coefficient (R 2 ).The R 2 values for S1, S2, X1, and H1   For the mold inhibition rate of X1, the ozone influx time was extraordinarily significant, and the microwave time was significant.There was an insignificant combined effect of the two factors from Fig. 1 (g) and Fig. 2 (g).Moreover, the effect of microwave power on the inhibition rate of X1 is significant.However, the combined effect of ozone infusion/microwave power was insignificant.From the analysis by the software, the optimal conditions were 8.81 min of ozone influx time, 559.55 W of microwave power, and 19.66 s of microwave time, which had the predicted mold inhibition rate of X1 at 100%.
For mold H1, as shown by Fig. 1 (j, k) and Fig. 2 (j, k), the combined effects of ozone influx time/microwave time and ozone influx time/microwave power were insignificant.In addition, there was a significant combined effect of microwave power/microwave time from Fig. 1 (l) and Fig. 2 (l).The optimal conditions were 9.33 min ozone infusion time, 531.48 W microwave power, and 18.59 s microwave time, of which the predicted mold inhibition rate was 100%.
A further experiment was conducted to verify the theoretically predicted result.At these reaction conditions (ozone influx time of 10 min, microwave power of 560 W, and microwave time of 20.00 s), the mold inhibition rates of S1, S2, X1, and H1 were 100%, 94.28%, 100%, and 100%, respectively, which are quite close to the predicted result, suggesting that the experimental and predicted values of mold inhibition rates were in good agreement.In the SEM images in Fig. 3, four mold spores showed smooth and intact surfaces without cracks.However, with ozone-microwave treatment, the surface of these molds was severely damaged.The spores of the S1 mold were not completely grown into shape, and some sporophores were broken.We observed fewer spores of S2 mold, which also had abnormal spore peduncles.Moreover, some spores of X1 mold were lost, and the bent sporophores became dried and rough.The morphology of H1's spores with treatment was similar to those of S1, S2, and X1.

Morphology of mold and its sporophores
The TEM images present the typical morphology of mold spores before treatment.
With ozone-microwave treatment, the cell wall of S1 was ruptured, the vesicles were ablated, and the nucleus disappeared.The cell walls of S2 and X1 both became thinner than before treatment, their organelles were destroyed, and the cytoplasm was solidified.
In addition, the vesicles of H1 disappeared in the cytoplasm, which had broken the plasma membrane and lost most organelles.
The morphological changes of these molds may be related to ozone and microwave in the combined treatment.First, ozone gas can destroy and deform cell walls.Although this may not kill microorganisms, it can impact their viability and reduce their growth and reproduction.Secondly, as an electromagnetic wave, the energy of microwaves can cause damage to DNA (Epelle et al., 2022).With microwave treatment, the mold was destroyed from the interior, damaging the nucleus and changing the permeability of the cell wall and membrane.Subsequently, the nucleic

DNA leakage
Fig. 4 DNA electrophoresis profile of molds with ozone-microwave treatment (M is the molecular weight marker, CK is the blank group, and 1 is the mold with ozone-microwave treatment).
As shown in Fig. 4, the DNA bands were intact, bright, and concentrated in the blank groups of each mold.However, with the ozone-microwave treatment, the DNA bands were seriously changed, some lanes disappeared, and the brightness decreased.
From these results, the mold's DNA degraded during ozone-microwave treatment.
Upon the molds' cell membrane disruption, DNA from the cell was in contact with the DNA degradation enzymes, which led to the degradation (Pinto et al., 2020).Intracellular nucleic acid and protein leakage are the key indicators of cell membrane breakage and permeability changes, which could reflect the mechanism of ozone-microwave treatment in sterilization (Gholinejad, Khadem Ansari, & Rasmi, 2019;Yi, Zhu, Fu, & Li, 2010).From Fig. 5, the leaking amount of nucleic acid and protein increased after the ozone-microwave treatment compared to the blank group; OD280 and OD260 for S1 increased by 2.64% and 2.50%, respectively.For S2, these increased by 1.21% and 6.71%.For X1, these increased by 3.48% and 7.78%.For H1, these increased by 0.48% and 3.86%.These results were consistent with those for morphology, in which the integrity of the cell membrane was disrupted and the cell contents leaked, resulting in the death of molds (Gani, Baba, Ahmad, Shah, Khan, Wani, Masoodi, & Gani, 2016).

Nucleic acid/protein leakage
As a nonthermal technology, ozone sterilization could be combined with other sterilization technology to enhance the effect.As one of the most reactive oxygen species, ozone could impact the viability and growth of microorganisms.However, its toxic effects should be considered (Epelle, Macfarlane, Cusack, Burns, Thissera, Mackay, Rateb, & Yaseen, 2022).Moreover, as another nonthermal technology, microwave treatment is widely applied in the food industry, which still risks deterioration of functional properties, destruction of active substances.(Almaiman et al., 2021).Given these limitations, this study has chosen the combined ozonemicrowave treatment for candied fruit sterilization.With the optimization, the molds in candied fruit were inhibited, and the candied fruit retained its quality.Values with different letters in the same column are significantly different at p < 0.05.

Physical and chemical indexes of candied fruit treated by ozone-microwave
From Table 5, the ozone-microwave treatment significantly affected the quality of the candied fruit, and all the physical and chemical indexes decreased.
Compared with the blank group, the water content of candied raisins with the treatment decreased by 3.76%, the water activity decreased by 0.062%, the total sugar content significantly decreased by 12.84%, the ΔE value decreased by 8.35, and the color was darkened.
The moisture content and water activity decreased by 9.42% and 0.082%, respectively, for the candied dried apricot.The total sugar content was vastly decreased because of the destroyed covalent bonds in polysaccharides (İlter et al., 2018).In addition, there was a minor change in color in candied dried apricot in the three types of candied fruit, for which the ΔE value was the smallest.
For the candied plums, the moisture content, total sugar content, and color difference were significantly affected by the ozone-microwave treatment, similar to the other two types of candied fruit.Moreover, the color difference changed significantly, and the color was not uniform, resulting in a decrease.Candied plums were the most affected by the combined treatment in terms of color of the three types of candied fruit.In Fig. 6 and Fig. 7, after ozone-microwave treatment, the candied raisins had less loss of texture and flavor, moderately sweet and sour flesh, and uneven color, which had a sensory score of 74.The dried apricots, with a sensory score of 80.4, became dark yellow after ozone-microwave treatment.The quality of the dried apricot was less damaged, and the original flavor and taste were retained, leading to a higher sensory score.Plums had a slightly saltier taste and a harder chewing sensation, which may be related to the lower moisture content after the combined treatment.With the reattachment of the molds S1, S2, X1, and H1, the candied fruit were stored for 20 days, and the physical and chemical indexes of the candied fruit were measured every 5 days.

Quality changes during storage
In Fig. 8 (a), during storage, the moisture content of the blank raisins decreased from 19.92% initially to 12.30%.The total sugar content of the untreated raisins decreased sharply from 37.92% to 28.49%.Compared to this, with combined treatment, the moisture content of the raisins decreased from 19.60% to 16.45%, and the total sugar content decreased by 6.57% in 20 days.Finally, there was no significant color difference for the first 10 days, decreasing significantly to that without pasteurization in the last 10 days.
In Fig. 8 (b), the dried apricots' moisture content, total sugar content, and color difference showed a decreasing trend.The total sugar content of the untreated dried apricots decreased from 29.63% to 25.18%.The total content of treated dried apricots decreased from 28.69% to 25.86%, which indicated that the ozone-microwave treatment could maintain the sugar content of candied fruit better.During the storage period, the color difference values of the untreated dried apricots in the blank group decreased significantly.However, the color difference values of dried apricots with ozone-microwave treatment leveled off after 10 days.In the 20 days of storage, no microorganisms were detected in the dried apricots treated with the ozone-microwave treatment.Putrefactive mold was detected in the dried apricots of the blank group.
In Fig. 8 (c), during storage, the moisture content of the untreated plum decreased from 35.75% to 29.23%.Moreover, the treated plum decreased from 34.14% to 31.09%.
Compared with raisins and dried apricots, the water loss of plums was higher due to their porous surface structure, which benefited transport of free water molecules.The total sugar content of plums decreased from 18.33% to 13.97% in the blank group and from 18.03% to 14.46% in the combined treated group.Moreover, they showed a decrease in color difference values.In the 20-day storage, no microorganisms were detected in the plums treated with ozone-microwave, while several strains of putrefactive mold were detected in the blank group without sterilization treatment.
The results in this part indicated that ozone-microwave treatment could reduce the deterioration rate of candied fruit and protect the candied fruit from mold during storage.

Conclusion
This study investigated the feasibility of ozone-microwave treatment for the sterilization of candied fruit.The optimal sterilization process parameters were analyzed by response surface methodology, which showed the best inhibition rate at 10 min of ozone influx time, 560 W of microwave power, and 20 s of microwave time.
The inhibition rates of the four candied fruit molds S1, S2, X1, and H1 were 100%, 94.28%, 100%, and 100%, respectively, close to the theoretical prediction.Moreover, the mechanism of ozone-microwave treatment had been clarified by morphological characterization and the cell's nucleic acid/protein leakage test, which disrupted the permeability of cell membranes, leaked the cell contents, and resulted in the death of molds during the processing.Furthermore, the quality analysis of candied fruit during storage indicated that the ozone-microwave treatment could protect the candied fruit from mold infestation and maintain its edibility.Ozone-microwave treatment can indeed prolong the shelf life of candied fruit by solving the critical problem of mold infestation.

Declaration of Interest Statement
were 0.9890, 0.9912, 0.9601, and 0.9890, respectively, indicating that the equations could reasonably predict the effects of ozone influx time, microwave time, and microwave power on mold inhibition rate.The experimental results were statistically significant.The order of the degree of influence on S1, S2, X1, and H1 inhibition rates based on the F values was judged as follows: A (ozone influx time) > C (microwave power) > B (microwave time), A > B > C, A > C > B, and A > C > B, respectively.

Fig. 1
Fig.1Response surface plots of the interaction between ozone influx time and microwave time (a, d, g, j), ozone influx time and microwave power (b, e, h, k), microwave time and microwave power (c, f, i, l), and inhibition rate on S1 (a, b, c), S2 (d, e, f), X1 (g, h, i), and H1 (j, k, l).

Fig. 2
Fig. 2 Contour plots of the interaction between ozone influx time and microwave time (a, d, g, j), ozone influx time and microwave power (b, e, h, k), microwave time and microwave power (c, f, i, l), and inhibition rate on S1 (a, b, c), S2 (d, e, f), X1 (g, h, i), H1 (j, k, l).

Fig. 3
Fig. 3 Morphological changes of molds with microwave-ozone treatment.
acids and proteins leaked, as demonstrated in the next section.Hence, ozonemicrowave treatment disrupted the permeability of the cell membranes, resulting in leakage of cell contents, which was confirmed by a reference (Glowacz, Colgan, & Rees, 2015).

Fig. 5
Fig. 5 Nucleic acid/protein leakage of molds with ozone-microwave treatment.

Fig. 7
Fig. 7 Sensory scores of three candied fruit with ozone-microwave treatment.

Fig. 8
Fig. 8 Changes in total sugar content, color difference, and moisture content of raisins (a), dried apricots (b), and plums (c) during storage.

Table 1 ,
and the experimental results are listed in Table2.Meanwhile, based on the experimental results in Table2, the model equations of the three factors A (ozone influx time), B

Table 3
Response surface ANOVA with S1 and S2 inhibition rates as the indicator.

Table 4
Response surface ANOVA with X1 and H1 inhibition rates as the indicator.

Table 5
Physical and chemical indexes of candied fruit samples.