Probing of ultrasonic assisted pasteurization (UAP) effects on physicochemical profile and storage stability of jambul (Syzygium cumini L.) squash

ABSTRACT The present study focused on evaluating the influence of ultrasonic-assisted pasteurization (UAP) on the quality and shelf stability of jambul squash. Squash was analyzed for physicochemical characteristics during a storage period of 4 months. There was a significant effect (p < .05) of storage period on the acidity, DPPH radical scavenging activity, and flavonoids of jambul squash with a significant increase in acidity and DPPH radical scavenging activity and decrease in flavonoids with the corresponding increases in sonication time and pasteurization temperature during storage. The microbial content of jambul squash during storage remained lower than untreated samples. Microstructure evaluation of jambul squash showed de-shaped middle lamella and cell wall after UAP treatment. Outcomes of the current study revealed that squash samples with increased sonication time and pasteurization temperature had less microbial content than untreated samples.


Introduction
Jambul (Syzygium cumini L.) is an important fruit from the family Myrtaceae. The well-known names of S. cumini are Jambul, Indian blackberry, Java plum, Jamun, etc. Ra Jamun and Kaatha varieties are commonly grown in Pakistan. [1] Jambul is rich in carbohydrates, such as glucose and fructose, and minerals like manganese, zinc, iron, calcium, sodium, and potassium. The major phytochemicals found in edible pulp are vitamin C, vitamin A, riboflavin, nicotinic acid, anthocyanins, and in seeds are glycoside jamboline; in bark and stem are tannins, phytosterols, and gallic acid . [2] The demand for jambul and its value-added products is rising due to the presence of plenty of natural antioxidants (i.e., anthocyanins) and therapeutic benefits, including anti-inflammatory, anti-diabetic, [2] anti-cancer, anti-microbial, gastrointestinal health-promoting properties. [3,4] Jambul fruit is widely available and beneficial; however, it is still an underutilized fruit that is not commercially processed in Pakistan. Jambul fruit is degraded because of microbial spoilage and deteriorative quality losses. The best strategy to minimize these losses and render jambul treated at 5, 10, and 15 min with pulse duration of 5s on and 5 s off and 20 kHz frequency. The depth of the probe was kept 5 cm in the juice samples. Then, the samples were pasteurized by placing them in a water bath for 30 min at 60, 70, and 80°C, respectively. Sample preparation and treatments were carried out in triplicate. Fresh untreated jambul squash was selected as a control (T 0 ). All experiments were performed in the dark. [19] Potassium-meta bisulfite (KMS) was used as a chemical preservative in treatment T 0 + to compare the squash with control and other treatments (sonication). The treatment plan presented in Table 1 was followed for further study.

Physico-chemical properties
Jambul squash was subjected to physicochemical analyses to assess the physical properties and chemical composition of squash. Total soluble solids in jambul squash were determined by hand refractometer (Atago Corp, Tokyo, Japan) as per AOAC Method No. 932.12. [20] pH of squash was measured with pH meter (Model: HI 2211, HANNA instruments) by following method No. 981.12 described in AOAC. [20] Acidity was determined by method No. 942.15 described in AOAC. [20] The acidity was calculated using the following formula: Acidity ð%Þ ¼ NaOH volume mL ð Þ x N NaOH ð Þ x acid meq:factor � 100 Titrated sample volume mL ð Þ Citric acid meq factor = 0.064

Total antioxidant activity (TAA)
To determine TAA, squash samples of all treatments were tested using the method described by . [21] Ascorbic acid was used for making standard calibration curves, and the results were expressed as microgram ascorbic acid equivalent (μg AAE)/mL sample. All determinations were performed in triplicates from triplicate experimental trials.

Total flavonoids contents (TFC)
Flavonoid contents were determined by the method described by. [19] Catechin (in ethanol) was used as a standard, and the results were expressed as μg of (+)-catechin equivalent (CE)/100 mL sample. All calculations were recorded in triplicates.

Total phenolic contents (TPC)
The TP contents were determined using Folin-Ciocalteu reagent with some modifications in the method, as explained by. [19] Gallic acid (in ethanol) was used as a standard, and the results of total phenolics were expressed as µg gallic acid equivalents (GAE)/ g samples. All determinations were carried out in triplicates.

DPPH radical scavenging activity
Squash treatments were assessed for their DPPH radical scavenging activity by [22] with minor changes. After diluting, 1 mL extract was taken, and 1 mL of DPPH (60 μmol in ethanol) solution was added.
The solution was then placed in the dark for 30 min, and then absorbance was measured at 517 nm using a spectrophotometer. A decrease in absorbance was calculated, and inhibition of radical scavenging activity (RSA) was calculated as μmol AAE/mL.

Microbiological analysis
Total plate counts (TPC) of all samples were carried out according to ISO 4833-1:2013. [23] A serially diluted sample (1 mL) was plated on plate count agar media (BioWorld, Ohio, Germany). The total aerobic plate counts (Log 10 CFU/g) were determined for 2 days at 30°C. For yeast & mold (Y & M) counts (Log 10 CFU/g), the AOAC method [20] was used in the same manner (after 4 days at 25°C) using potato dextrose agar (BioWorld, Ohio, Germany).

Microstructure evaluation of jambul squash after sonication
The microstructure evaluation of UAP treated fresh Jambul squash samples was taken to assess Jambul squash tissues' homogeneity using a compound microscope with permissible green channel (OPTIKA Microscope, 4083.B 3 , Optikam B 3 Digital Camera, Italy,) equipped with Image focus. A small drop of homogenized Jambul squash was placed on a glass slide and covered with a coverslip with no bubbles. The same method was adopted for the samples without UAP treatment. The prepared slides were examined under 40X magnification, and images were taken to evaluate the proper sonication time for a well-homogenized Jambul squash sample with excellent antioxidant potential.

Statistical analysis
The statistical analysis of data obtained from each parameter was conducted with the ANOVA technique at a significance level of p ≤ .05, and significant differences between mean values were determined by Tukey HSD pairwise comparison test. The statistical analyses were performed using Statistix 8.1 software (Analytical Software, Tallahassee, FL, USA).

UAP effects on total soluble solids ( 0 Brix)
An increase in the °Brix was observed within the treatment throughout the study. The highest ° Brix was found in JS 9 (53.76 ± 0.78°Brix), and the lowest 0 Brix was observed in JS 0-(49.08 ± 0.68°Brix) (Figure 1a). The impact of storage duration on °Brix of jambul squash illustrated that TSS gradually increased with the corresponding rise in storage period. At 120 days of storage, the highest °Brix was observed with the mean value of 53.07 ± 1.43°Brix, as shown in Figure 1a. Similar results were determined by [24] when they applied sonication on carrot grape blend juice and noticed that TSS increased from 12.7 to 13 °B with an increase in sonication time. As extraction efficiency improves, total sugars and soluble solids may be increased. Islam et al. [25] noticed that TSS increased with increasing storage duration, which could be attributed to polysaccharide breakdown into monosaccharides and oligosaccharides.

UAP effects on pH
The results of the effects of UAP on the pH of squashes are shown in Figure 1b. The maximum pH was observed in JS 9 (2.84 ± 0.03) and the lowest in JS 0-(3.31 ± 0.05) and JS 1 (3.32 ± 0.03).
The storage period delineated a momentous effect on the pH of jambul squash as it decreased from 3.30 ± 0.04 to 2.85 ± 0.04 (Figure 1b). Nadeem et al. [24] prepared sonicated carrot-grapes juice blend and observed a significant effect of sonication on the pH of blended juice. Abid et al. [26] observed similar results for pH in sonicated grapefruit juice. They observed nonsignificant variation (p > .05) in the pH of grapefruit juice upon sonication; however, slight increase in pH was observed on account of different phytochemicals entities like mineral elements or vitamins released from fruit tissues. Due to the destructive impact of sonication on the cell structure, it releases vitamins from their bound form, thus enhancing their concentration and increasing the pH.

UAP effects on titratable acidity
The more pronounced increasing trend in acidity (Figure 1c) was found in JS 0 (0.48 ± 0.01 to 0.85 ± 0.03%) followed by JS 1 to JS 5, and the lowest increasing trend was noticed in JS 6 to JS 9 (0.62 ± 0.05 to 0.77 ± 0.02%). The effect of the storage period on the acidity of jambul squash showed that the acidity gradually increased with the progression in storage time. [27] determined [28] *** similar results, who observed that apple juice's acidity significantly rose with corresponding increases in ultrasonic treatment time due to ultrasound-induced heat generation (in that study, the temperature was constant 25 ± 1°C). Another study by [26] revealed similar results in which apple juice was subjected to ultrasound treatment at 30, 60, and 90% amplitude level for 3 min and resulting samples were stored at 40°C for 30 days, and it was concluded that storage study increased the titratable acidity of apple juice from 0.23 ± 0.10 to 0.26 ± 0.02%.

UAP effects on total phenolic (TPC) and flavonoids contents (TFC)
TPC of jambul squash was observed in range of 55.20 ± 0.52 µg/mL gallic acid equivalent in JS 0 to 488.80 ± 10.62 µg/mL GAE in JS 6 at the start of the study ( Table 2). The TPC values of all the samples were decreased from 426.26 ± 5.42 to 77.80 ± 3.95 µg/mL GAE during storage of 120 days. The content of phenolic may be depleted due to the production of free radicals. [29] TFC of jambul squash was the minimum in JS 0 control samples (264.40 ± 2.59 µg CE/mL) and the maximum (469.00 ± 18.00 µg CE/mL) in JS 6 ( Table 3). TFC in all samples decreased during 4 months storage period. Nadeem et al. [24] reported a significant increase in total phenols and flavonoids content in a sonicated carrot-grapes juice blend. Abid et al. [30] determined the impact of sonication on the flavonoid content of apple juice, and it was observed that sonicated samples showed more improvement in TFC than non-sonicated sample control samples. These investigations recommended that increases in sonication time led to enhancement of flavonoids and bioactive compounds in the juice.

UAP effects on total antioxidant activity and DPPH-radical scavenging activity
The antioxidant values of all jambul squash samples ranged from 814.0 ± 45.0 to 1306.7 ± 56.9 µg/mL AAE at the start of the storage period ( Table 4). The highest antioxidants value was examined in JS 6, while the minimum value was observed in JS 0. A decreasing trend was observed during storage. [31] narrated similar results that antioxidant capacity and phenolic contents of grape fruit juice were increased with a corresponding increase in sonication time, i.e., 290 µg/g to 820 µg/g at 90 min. In both cases, treatment sonicated for 90 min in a bath-type sonicator was noticed more stability during 28 days of storage. The DPPH radical scavenging activity (RSA) of squash samples ranged from 3846.7 ± 20.2 to 4703.3 ± 58.6 µmol/mL AAE ( Table 5). The highest value was observed in JS 6 (4703.3 ± 58.6 µmol AAE/mL) followed by JS 5 (4553.3 ± 56.2 µmol/mL AAE), while the minimum value was recorded in JS 0-(3846.7 ± 20.2 µmol AAE/mL). [26] determined the impact of sonication on the DPPH-RSA of apple juice and concluded that sonicated samples showed significant improvement in DPPH-RSA than the non-sonicated control sample. It was further observed that sonication treatment enabled the accelerated release of antioxidants from the tissue matrices resulting in the scavenging of free radicals. These investigations recommended that an increase in sonication time enhances the flavonoids and bioactive compounds in juice. 310.74 ± 2.59 g-q 381.33 ± 22.50b-i 313.00 ± 11.14 g-q 219.67 ± 21.39q-w 143.01 ± 8.00 u-w

Impact of UAP on microbial content
Results for the microbiological status of jambul squash during the storage period showed a significant (p < .05) influence of temperature and treatment time on microbial count (Figure 2a,b). The outcomes of the current study revealed that squash samples subjected to increased sonication time and pasteurization temperature had less microbial content than untreated samples. The maximum total plate count was observed in JS 0-(2.05 ± 0.01 CFU/mL) at the start of the storage study and increased up to 2.68 ± 0.01 CFU/mL after 120 days, and the minimum total plate count was noticed in JS 9 (1.33 ± 0.04 CFU/mL) (Figure 2a). The maximum mold/yeast (Y&M) count was observed in JS 0-(1.86 ± 0.02 CFU/ mL), and the minimum Y&M count was observed in JS 9 (1.06 ± 0.07 CFU/mL) (Figure 2b). Nadeem et al. [24] reported a significant decrease in microbial count in sonicated carrot-grapes juice blend. Zou and Jiang [25] examined the impact of ultrasound on microbial load in carrot juice and examined a significant decrease in the total plate and mold counts in all samples that were sonicated for a time duration of 20, 40, and 60 min in comparison with non-sonicated juice samples. Abid et al. [26] claimed that the decrease in microbial content was due to increased biocides produced by sonication-induced cavitations. These cavitations increase pressure and generate free radicals, which inactivate bacteria.

UAP effects on microstructure
Microstructure evaluation of jambul squash showed de-shaped middle lamella and cell wall after UAP treatment. Figure 3a elucidates the microstructure evaluation of the control sample in which middle lamella and cell wall are seen intact; however, a progressive uniformity in microstructure profile on account of structural disintegration is seen in Figure 3(b-d), illuminating the effect of JS 1 , JS 6, and JS 9 respectively on the microstructure of jumbul squash. Moreover, a gradual improvement in the chemical and antioxidant profile was also observed in these treatments might be due to structural integration releasing bound compounds after sonication treatment. These results are in agreement with the findings of, [32] who earlier reported a similar cell de-shape (middle lamella & cell wall) effect in mangoes when observed under a compound microscope.

Conclusion
This study demonstrated the influence of ultrasonic-assisted pasteurization (UAP) on the functional properties of jambul squash during storage. The results showed a significant (p < .05) effect of UAP on total phenolic contents, flavonoid contents, DPPH radical scavenging activity, and antioxidant potential. Squash samples with the increased sonication time and pasteurization temperature had less microbial load than untreated samples. When heat and ultrasound were used together, the temperature during the process was considerably reduced compared to the conventional heating process, making it an ideal technique to preserve bioactive components and enhance nutritional profile. Sonication is an effective method that preserves fruit beverages quality by retaining a significant amount of bioactive compounds and reducing pathogenic microorganisms. Additionally, it contributes to the economy by requiring less energy. It could be concluded that UAP, as an environmentally friendly technique, can be effectively used to produce minimally processed foods, thereby preserving nutritional entities in foods and enhancing the dietetic perspective on foods with the least energy consumption and the highest costeffectiveness.

Disclosure statement
No potential conflict of interest was reported by the author(s).