Quality evaluation of ozone-processed Kinnow (Citrus reticulata Blanco) juice at ambient temperature

ABSTRACT Non-thermal processing has been developed to increase the quality and storage life of fruit juices in response to consumer desire. Ozonation is a recently developed method that generates volatile oxygen atoms that can eliminate a broad spectrum of microbes and extend the shelf stability of food products. The present research was intended to investigate the ozone processing on the physicochemical characteristics, microbiological quality, sensory attributes, and antioxidant potential of Kinnow juice. In this study, Kinnow juice was processed to 150 mg/h of ozone gas at a constant concentration for 5, 10, and 15 min. The sample was then taken in a plastic container and stored at 35°C ± 2 for three months. The results revealed that ozone processing had a significant (p˂0.05) effect on the total phenolics (TP) contents, total flavonoids (TF) contents, total soluble solids (TSS), acidity, total antioxidant activity (TAA) and DDPH radical scavenging activity of Kinnow juice. In addition, the ozonation application also significantly (p˂0.05) decreased the population of the microbes when the ozone time was increased from 5–15 min. Among the treatments, it was observed that T3 had the highest TSS, acidity, TF contents, TAA, and DDPH radical scavenging activity values and the lowest mold and plate counts. Regarding sensory qualities, T0+ showed the highest sensory score during storage. Thus, it may be concluded from the findings of our study that ozone application improved the quality of Kinnow juice by increasing TSS, phytochemicals and decreasing the microbial population, thereby prolonging their shelf life.


Introduction
The nutritional benefits of minerals and vitamins of fruit juices are widely recognized.The fruit juices are also substantial sources of bioactive compounds such as vitamin C, carotenoids, and

Collection of fruit
Kinnow fruits were collected from a local farm and sorted to remove defective and infected fruit.The fruits were then rinsed with tap water to eliminate dirt and dust.

Chemical reagents
All analytical chemicals were acquired from Sigma-Aldrich (Gillingham, UK) and are readily available on the local market.

Extraction of Kinnow juice
The fruits were peeled before extracting the juice with an electrical juice extraction extractor (Model.No. KJ 1003 operating at 450W, frequency 60 Hz, and voltage 45 V).The clear juice was obtained by filtering the juice through a No. 18 filter sieve.Finally, the Kinnow juice was placed in a plastic container at ambient temperature (35°C ± 2) for three months.

Ozone processing of Kinnow juice
Ozone was done by subjecting 250 mL of Kinnow juice in a beaker to ozone gas at a fixed concentration of 150 g/ha via a pipe linked to a diffuser (Model No. TR-YCA, operating at 15 W of power, 50 Hz of frequency, and 220 V of voltage) that made bubbles and spread the gas evenly (Figure 1).The juice was exposed to ozone for 5 to 15 min.The samples were taken in sterilized bottles and stored at room temperature (35°C ± 2) for three months.

Chemical preservative
KMS (0.1%) was added to treatment T 0+ to compare the juice to the control and other treatments.The treatment plan is detailed in Table 1.

Physicochemical analyses
Total soluble solids were determined using a digital refractometer (Model AVl41984, ERMA, India).According to method 981.12 of the AOAC [23] the pH of Kinnow juice was determined using a pH meter (CPH-102, Contech, India).The acidity of the samples was measured using the standard titration method in line with method 942.15 of the AOAC. [23]The viscosity of Kinnow juice was determined using a ROTAVISC revolving viscometer with a No. L 2 spindle head.100 mL of each juice sample was taken in a beaker.Subsequently, the spindle was adjusted to a rotational speed of 1000 revolutions per min at a temperature of 28°C ± 1, following which the viscometer interpretation was recorded. [24]

Total phenolic (TP) contents
Total phenolic contents were measured using slightly modifying the Folin -Ciocalteu reagent technique. [25].5 mL prepared sample was taken in different test tubes.0.5 mL Folin solution (10%) was placed in the test tube and left for 6 minmin.Then, 1 mL of Na 2 CO 3 (20%) was taken to a test tube and stayed in the incubator at 30°C for 1 hour.Using a spectrophotometer (Du-8800d, UV-VIS double beam), the absorbance was measured at 760 nm.The findings were stated as mg equivalent gallic acid (GAE) per 100 mL of juice.

Total flavonoid (TF) contents
According to Iqbal et al., [14] the total flavonoids (TF) of Kinnow juice were calculated.250 µL sample was taken in different test tubes by preparing 1 mL diluted juice.Each sample made three replicates.Then, t 75 µL sodium nitrite was added and wait left for 6 min.150 µL aluminum chloride was taken in the above mixture and left for 5 min.Then, 500 µL NaOH was added, and further 9 mL distilled water was also added.The absorbance at 510 nm was then measured using a spectrophotometer (Du-8800d, UV-VIS double beam, Diepoldsau, Switzerland).The results were expressed in µg catechin equivalent (CE) per 100 mL of juice.

Antioxidant activity (TAA)
Kinnow juice samples were examined using the Prieto et al. [26] method to determine the antioxidant activity.The results were represented as µg ascorbic acid equivalent/100 mL juice and were calibrated using ascorbic acid as the standard.The evaluations were carried out in triplicate.

DDPH free radical scavenging activity
Kinnow juice samples were tested for DPPH free radical scavenging capacity using the prior method developed by Yi et al., [27] with slight changes.The following calculation was used to calculate the change in absorbance: (A 0 -A 1 /A 0 )×100= DPPH radical scavenging activity A 0 indicates the absorbance of the control, and A 1 indicatesthe absorbance of the extracts.Using Trolox as a standard, the identical procedure was used to assess DPPH radical scavenging activity, and the results were stated as µM equivalent of Trolox/mL juice.

Total plate count (TPC)
TPC determination was performed by FDA's standard method outlined in the Bacteriological Analytical Manual. [28]Using a sterilized pipette, 1 mL of the sample was transferred to a test tube, and 9 mL of saline solution was diluted.After adding diluents and shaking at a low speed for one to two min, the sample became completely clouded.9 mL of saline solution was used to follow the 10-fold serial dilution protocol.Each dilution was carefully stirred.In the petri dish, 12-15 ml of PCA (Plate Count Agar) was poured, sample dilutions were combined with media, and the media was solidified.The petri dishes were flipped over and incubated at 32°C for 48 hours.The total bacterial populations in the sample were calculated, multiplied by the reciprocal, and stated as log colony-forming units (CFU) per mL of juice.

Mold count (MC)
MC determination was performed by FDA's standard method outlined in the Bacteriological Analytical Manual. [28]One milliliter of juice was taken in a test tube.A solution exhibiting turbidity was obtained after 9 mL of saline solution and subsequent agitation for one to two min.Then, 1 mL of the 10 mL dilution was added to the 9 mL dilution previously prepared.All dilutions were thoroughly blended.Subsequently, 12-15 ml of PDA (Potato dextrose agar) was poured into a petri dish, the diluted sample was added, and the mixture was solidified.All petri dishes were incubated between 20 to 25°C.After 48 hours of incubation, the MC in each plate was measured and reported as log CFU/mL of juice.Each analysis was conducted in triplicate.

Sensory analyses
The purpose of assessing the product using the human senses is through sensory evaluation.Kinnow juice was evaluated in a sensory assessment laboratory for sensory attributes like texture, color, flavor, appearance, and overall acceptability at ambient temperature by a panel of semi-trained judges on a 9-point scale.The Hedonic Scale was described in detail in Land and Shepherd. [29]

Statistical analysis
The result was evaluated using statistics and the two-variance analysis method.Mean differences were assessed using Tukey's test with a 5% significance level. [30]

Impact of ozone treatments on TSS, pH, acidity, and viscosity
The ozone processing resulted in highly significant alterations in the TSS of Kinnow juice, but three months of storage led to a substantial reduction (Table 2).It was observed that the total soluble contents (TSS) were varied between treatments.The highest TSS were detected in T 3 (14.80°B),and the lowest values were in T 0-(13.77°B) at 0 day.Over time, TSS reduced from 14.80 to 4.10°B.After three months, the highest value of TSS in T 0+ (13.10°B) and the lowest in T 0-(4.10°B).The present findings of the study on TSS are consistent with prior research that stated similar patterns.Bulu and Koyuncu [31] and Aday et al. [32] also found the decreasing trends of TSS in ozonated pomegranate and strawberry fruits throughout the storage time, correlating with current results.The TSS is decreased by the breakdown of sugar into water, CO 2 , and ethyl alcohol.The raspberry samples undergoing ozone treatment (60 and 120 minmin) exhibited a higher soluble solid concentration than the control group.It may be attributed to a rise in the predominant carbohydrates, i.e. glucose and fructose, as a consequence of the results of ozone. [33]he findings revealed that the treatments and storage time significantly impacted the pH of Kinnow juice (Table 2).The pH was varied among the treatments.The pH of treated samples was increased during three months of storage.The highest pH value was noticed in T 0- (3.81) and the lowest in T 3 (3.60) at 0 day.Over time, pH increased from 3.60 to 3.99.After three months, the highest value of pH in T 0-(3.99) and the lowest in T 3 (3.81)were observed.Concurrent to the current findings, Alencar et al. [34] and Barboni et al. [35] demonstrated a rising tendency of pH in ozone-treated banana and kiwi fruit through storage.The same patterns were noticed across treatments (Fundo et al., 2018).The pH of both filtered and unfiltered ozone-processed watermelon juice decreased substantially (p˂0.05) over the duration of 5 to 25 min.The decrease in pH may be a result of the increase in malic acid in unfiltered and filtered ozone-processed watermelon juice after ozonation. [36]The rise in pH may be related to the inverse correlation between pH and acidity.The increase in pH may be the consequence of the conversion of acids to sugars or the chemical interaction between the organic components of the juice as a result of temperature and enzyme action. [37]he findings revealed that the treatments and storage time significantly impacted the acidity of Kinnow juice (Table 2).The acidity varied among the treatments.The acidity of treated samples was decreased during three months of storage.The highest acidity value was noticed in T 3 (0.173%), and the lowest acidity value in T 0-(0.126%) at 0 day.Over time, acidity decreased from 0.173 to 0.081%.After three months, the highest acidity value was in T 3 (0.109%) and the lowest in T 0-(0.081%).Ozone-treated watermelon and peach juices showed similar trends throughout treatments and storage durations. [24,36]During storage, the acidity of blends of Kinnow juice decreased.The invertase enzyme may be responsible for the degradation of acids into salt and sugar, hence decreasing acidity. [38]he findings revealed that the treatments and storage time significantly impacted the viscosity of Kinnow juice (Table 2).The viscosity was varied among the treatments.The viscosity of treated samples was reduced during three months of storage.The highest viscosity value was noticed in T 0+ (0.574 m.Pas), and the lowest was in T 3 (0.464 m.Pas) at 0 day.Over time, viscosity decreased from 0.574 to 0.143 m.Pas.After three months, the highest viscosity value was in T 0+ (0.218 m.Pas) and the lowest in T 3 (0.143 m.Pas).The findings are consistent with previous research, indicating a reduction in the viscosity of ozone-processed apple juice (1 to 4.8% w/w ozone) over a 12-min exposure period [39] Jaramillo-Sánchez et al. [24] found reductions in apparent viscosity of ozonized juices.Ozone exposure has been shown to reduce the molecular weight of dietary polysaccharides like pectins due to their potent oxidizing activity, which lowers viscosity. [40]

Impact of ozone treatment on total plate counts (TPC) and mold counts (MC)
The findings revealed that the treatments and storage time significantly impacted the TPC of Kinnow juice (Figure 2).The TPC were varied among the treatments.The TPC of treated samples were increased during three months of storage.The highest TPC value was noticed in T 0-(1.75CFU/mL) and the minimum TPC value in T 3 (1.59CFU/mL) at 0 day.After three months, the highest value of TPC was in T 0-(2.05CFU/mL) and the lowest in T 3 (1.90CFU/mL).The reduction of microbial populations in ozone-processed fresh-cut papaya fruits was noticed. [41]The ozone technique reduced the microbial population in soy milk by 0.19, 0.35, and 0.57 Logs at 1, 3, and 5 min of treatment. [42]he findings revealed that the treatments and storage time significantly impacted the MC of Kinnow juice (Figure 2).The MC was varied among the treatments.The MC of treated samples were increased during three months of storage.The highest MC value was perceived in T 0-(1.55CFU/ mL) and the minimum MC value in T 3 (1.68CFU/mL) at 0 day.After three months, the highest value of MC was in T 0-(1.72 CFU/mL) and the lowest in T 3 (1.91CFU/mL).The study conducted by Panigrahi et al. [22] stated that an increase in ozone processing time resulted in a significant decrease in MC in sugar cane juice.The mold and yeast count in filtered and non-filtered ozonated watermelon juice was reduced after 25 min.The findings demonstrated that the processing duration of ozone and the concentration of solids in watermelon juice affected the efficacy of ozone against yeast and mold. [36]0.5

Sensory analyses
The findings about sensory characteristics are presented in Figure 3.After three months of storage, the sensory evaluation score indicated that treatment T 0+ was highly acceptable, as determined by the sensory panel.The highest color score was observed in T 0+ (7.65), and the lowest was in T 3 (7.00) at 0 day (Figure 3a).During storage, the treatments exhibited a gradual decrease in color.During storage, the color score of Kinnow juice ranged from 7.65 to 2.75.The change in color might be due to changes in volatile compounds.Bhardwaj and Nandal [38] found that the color of Kinnow juice blend was significantly decreased during storage time at ambient and refrigerated temperatures with related present findings.Rajashri et al. [43] reported similar trends.The reduction of color may be attributed to the breakdown of pigment and copolymerization.
The findings revealed that the treatments and storage time significantly impacted the taste of Kinnow juice (Figure 3b).The highest taste score was observed in T 0+ (7.75), and the lowest was in T 3 (6.90) at 0 day.During storage, the treatments exhibited a gradual decrease in taste.During storage, the taste score of Kinnow juice ranged from 7.75 to 2.80.The loss of flavor in juice may be the result of considerable fermentation.Panigrahi et al. [22] found a slight change in the taste of ozone-processed sugar cane juice compared to the control, but non-significant results were observed.The taste of star fruit and sweet orange juices blended cordial was reduced with time. [44]The taste may be reduced due to the breakdown of ascorbic acid.Similar trends were observed in Kinnow banana RTS during storage at ambient and refrigerated temperatures.The taste of Kinnow banana RTS was reduced during storage. [45]he findings revealed that the treatments and storage time significantly impacted the texture of Kinnow juice (Figure 3c).The highest texture score was observed in T 0+ (7.90), and the lowest was in T 3 (6.85) at 0 day.During storage, the treatments exhibited a gradual decrease in texture.During storage, the texture score of Kinnow juice ranged from 7.90 to 2.75.Similar trends were shown in Kinnow banana RTS during storage at ambient and refrigerated temperatures.The texture of Kinnow banana RTS was reduced after three months. [45]he findings revealed that the treatments and storage time significantly impacted the overall acceptance of Kinnow juice (Figure 3d).The highest overall acceptance score was observed in T 0+ (7.75), and the lowest was in T 3 (7.0) at 0 day.During storage, the treatments exhibited a gradual decrease in overall acceptance.The overall acceptability of star fruit and sweet orange juice blend fruit cordial was reduced with time. [44]The reduction of overall acceptance might be attributed to the loss of appearance, uniformity, and flavor component of the juice.Similar trends were found in Kinnow banana RTS during storage at ambient and refrigerated temperatures.The overall acceptability of Kinnow banana RTS was reduced during storage by Kumar and Singh. [45]The reduction of overall acceptability of aloe juice complemented Kinnow nectar was also found by Shubhra et al. [46]

Impact of ozone treatment on phytochemicals (TP contents, TF contents), TAA and DPPH radical scavenging activity
The findings revealed that the treatments and storage time significantly impacted the TP content of Kinnow juice (Figure 4).The TP contents were varied among the treatments.The TP contents of treated samples were reduced during three months of storage.The highest TP contents were noticed in T 2 (489 mg GAE/mL), and the lowest were in T 0-(450.33mg GAE/mL) at 0 day.After three months, the highest values of TP contents were in T 2 (337 mg GAE/mL) and the lowest in T 0-(301 mg GAE/mL).A similar trend was observed by Shah et al.. [15] Alothman et al. [47] found that the total phenols increased substantially when pineapple and banana were exposed to ozone for up to 20 min.Ozone exposure may have altered the cell wall, which may have allowed some related phenolic chemicals to be released from the cell wall, leading to a rise in phenolic contents.Numerous free radicals are produced alongside the auto-decomposition of ozone.Various chemical reactions may also significantly contribute to the rise of these phenolic compounds.These reactions may involve a direct interface between ozone and the target molecule or its intermediate radicals.Shah et al. [48] observed that the total phenol of ozone -processed pummelo juice decreased during storage.It might be caused by the phenolic components performing their protective functions by scavenging ozone-generated free radicals, decreasing the polyphenol concentration of pummelo juice.
The findings revealed that the treatments and storage time significantly impacted the TF content of Kinnow juice (Figure 3).The TF contents varied among the treatments.The TF contents of treated samples were reduced during three months of storage.The highest TF contents were observed in T 3 (225.93mg CE/mL), and the lowest were in T 0-(165.43mg CE/mL) at 0 day.After three months, the highest value of TF contents was in T 3 (107.4mg CE/mL) and the lowest in T 0-(59.26mg CE/mL).During storage, the TF concentration in ozone-processed pineapple and tender coconut water were decreased. [43,47]The TF contents may be caused by the hyperresponsiveness of ozone, which prevents numerous enzymes responsible for the oxidation of TF content, such as polyphenol oxidase and peroxidase.
The findings revealed that the treatments and storage time significantly impacted the TAA of Kinnow juice (Figure 3).The TAA varied among the treatments.The TAA of treated samples was reduced during three months of storage.The highest TAA value was noticed in T 3 (386.42µg AAE/ mL) and the minimum TAA value in T 0-(302.81µg AAE/mL) at 0 day.The antioxidant capacity of freshly sliced kiwifruit was initially improved by ozone processing for 10 min, which may be associated with the proliferation of antioxidants. [49]Ozone processing raised AA, TP, and TF contents, thereby increasing TAA.Ozone lowered polyphenol oxidases, which caused enzymatic browning and increased TAA.After three months, the highest value of TAA was in T 3 (290.63µg AAE/mL) and the lowest in T 0-(71.71µg AAE/mL).After 10 to 20 min of exposure to ozone, the formation of lipoxygenase may contribute to an increase in TAA. [41,47]Al It was also observed that during the storage period, the TAA of ozone-processed raspberries decreased. [50]he findings revealed that the treatments and storage time significantly impacted the DDPH radical scavenging activity of Kinnow juice (Figure 3).The DDPH radical scavenging activity was varied among the treatments.The DDPH radical scavenging activity of treated samples was reduced during three months of storage.The highest DDPH activity value was noticed in T 3 (3744.2µM AAE/mL) and the minimum DDPH activity value in T 0-(3122.1 µM AAE/mL) at 0 day.Over time, DDPH activity reduced from 3744.2 to 2570.6 µM Trolox/mL.After three months, the highest value of DDPH activity was in T 3 (3171.2µM AAE/mL) and the lowest in T 0-(2570.6 µM AAE/mL).Increased DPPH activity after ozone exposure may have produced antioxidant enzymes like superoxide dismutase and phenylalanine ammonia-lyase, which remove harmful reactive oxygen species. [51]

Conclusion
The results of this study indicated that ozone processing resulted in Kinnow juice of superior quality, exhibiting exceptional stability in terms of both bioactive components and physicochemical attributes throughout the storage period.The ozone processing moderately increased the total phenols, flavonoids, acidity, DDPH activity, total soluble solid contents, and antioxidant activity of Kinnow juice.T 3 was the most effective treatment in increasing the bioactive components, physicochemical characteristics, and antioxidant capacity while lowering microbial loads within the permitted range.
Thus, ozone processing was a simple, cost-effective alternative to existing techniques for preserving product quality parameters and prolonging their shelf life.

Table 1 .
Treatment plan of the present study.