A comparative study of high-pressure processing and thermal processing techniques on characteristics and microbial evaluation of orange juice

ABSTRACT This study investigates the impact of thermal and high-pressure processing on the quality indicator of orange juice and its storage stability. The pressure was applied at 400 MPa and 600 MPa for 3, 6, and 9 min to process the juice and evaluate the quality of orange juice during 60 days of storage with every 30-day interval. HPP conditions (400 MPa and 600 MPa for 3, 6, and 9 min) did not significantly change the ascorbic acid content of orange juice, in contrast to thermal processing where a crucial decrease in ascorbic content was observed. Ascorbic acid content increased non-significantly from 40.03 to 40.69 (mg/100 g) and 40.79 when applied for 3 and 6 min, respectively, while thermal processing reduced its content up to 33.45 (mg/100 g). Ascorbic acid at 600 MPa was observed at 41.19 (mg/100 g) when treated for 6 min. Carotenoid content was increased when HPP was from 8.6 to 9.14 (mg/100 ml) during 400 MPa for 9 min and 11.65 (mg/100 ml) for 6 min at 600 MPa compared to the corresponding untreated sample and thermally treated sample. The total soluble solids, pH, and acidity of the orange showed stability after HPP and during 60 days of storage. The natural microbiological load (yeast and mold, E-Coli log CFU/ml) was observed below 1 log reduction at 30 days of storage and 2 log reduction during days of storage. This study identified different pressure levels to produce nutritionally and microbiologically stable orange juice compared to thermally processed and untreated samples.


Introduction
The sweet orange fruit (Citrus sinensis (L.) Osbeck) accounts for roughly 50% of global citrus production.It is the most significant species within the Citrus genus and the most economically significant citrus fruit crop in the world [1] .Each year, Pakistan produces about 2.0 million metric tonnes of citrus fruit, predominantly kinnow. [2]This industry shares approximately 50% of the total fruit juice trade worldwide. [3]The most popular and well-liked product of orange fruit is its juice, which is liked worldwide.It is a great source of biologically active substances with oxidative qualities.It also has carotenoids, flavanones, and other metabolites in addition to vitamin C. Carotenoids are soluble in lipids that play a crucial role in maintaining good health in an individual. [1]Valencia orange juice is typically in demand by processors due to its significant amount of total soluble solids and color, and it is frequently blended with poor quality juices. [4]onsumer demand for safe and fresh foods has increased in the modern era while maintaining their nutritive aspects and calorie content.Conventional heat processing is the most often used technology for extending the shelf life and preserving fruit juices.Products' sensory and nutritional qualities might frequently suffer due to thermal processing. [5]Yet, interest in non-thermal technology has increased that are minimally and naturally processed. [2]Non-thermal technologies are methods of food preservation that work at normal and below temperatures, minimizing the unfavorable effects of heat on nutritional and quality characteristics. [6]A wide range of non-thermal processing technologies are now being developed.High-pressure processing (HPP) is one of these processing technologies. [7]ecause HPP may achieve pasteurization without heating (often at room temperature), it is a possible substitute for thermal pasteurization (TP).As a result, it is known to have less of an impact on the structure of macromolecules and taste components than TP. [6]he quality of orange juice after processing is a crucial topic for investigation.A number of deteriorative reactions occur in orange juice during processing and storage, lowering the product's quality.By using HPP, orange juice can have a longer shelf life than juice that was not processed while experiencing less product quality loss and retaining more of its fresh flavor. [8]Although multiple studies claimed that high-pressure processing of orange juice maintained its overall character and had a longer shelf life than juice that had not been treated, there were fewer studies that evaluate the use of constant pressures for different time intervals to check the nutritional stability and microbiologically safe orange use.The primary goals of the research were (i) to determine the best possible conditions of HPP with time to reduce the microbes up to non-detected limit during the storage period, (ii) to compare the non-thermal processing, i.e.HPP and conventional processing conditions in order to provide nutritionally stable orange juice. [9]

Procurement of raw material
The orange fruit (Valencia) was purchased from a hyperstore in Lahore, Pakistan.All the fruit were thoroughly washed and sorted out based on size and juice content.At the same time, all the chemicals used in the research were purchased from Sigma-Aldrich Chemical Co.(St.Louis, MO, USA) and Merck (Germany).

Extraction of juice
The orange juice content was pressed by using the conventional juice machine.All the juice extracted was then filtered using the muslin cloth to remove the seeds and peels of the orange.The orange juice was then stored at 4°C.

Thermal pasteurization
In a steam-jacketed kettle, the packed juice samples were heated to 85°C and kept for 45 s before being quickly cooled to 4°C in cold water.Brass stuffing boxes and T-type thermocouples from Ecklund-Harrison, Fort Myers, Florida, were used to measure the temperatures of the samples (Omega Engineering, Inc., Stamford, CT). Storage samples were placed in a walk-in cooler set to 4°C.The processed chilled samples were brought to the Pakistan Council of Scientific and Industrial Research (PCSIR), Lahore from the University of Veterinary and Animal Sciences (UVAS).All the samples were carried out under refrigerated conditions.

High-pressure processing
A 57 L capacity high hydrostatic pressurization unit (SHPP-57DZM-600, Shanshuihe Technology Co., Ltd., Taiyuan, China) was used to process the freshly squeezed orange juice at a temperature of 25°C.The pressure-transmitting medium was sterile water.Orange juice was poured into 200 mL plastic bottles before processing (PET).High pressure at 400 MPa and 600 MPa levels was applied for 3, 6, and 9 min, respectively.The pressure release time was less than 5 s, and the pressure rise rate was roughly 100 MPa/30 s.The time needed to increase and release the pressure was not included in the holding period in this investigation.All the samples were treated at 400 MPa and 600 MPa for 3, 6, and 9 min.

Storage conditions
High-pressure processing was done at the University of Veterinary and Animal Sciences (UVAS) and then transferred these treated samples to the Pakistan Council of Scientific and Industrial Research (PCSIR), Lahore.Orange juice bottles were treated with TP, HPP, and control treatments.They were stored in a refrigerator set to 4°C and tested for nutritional, microbial, and physicochemical analysis at 0, 30, and 60-day intervals during storage.Three replications of each treatment were included (individual bottles containing 250 mL juice).Approximately 24 h following treatment, all samples for the day 0 storage analysis were analyzed on the same day that the cargo was received.After an analysis of 0 days, control samples were removed.Additionally, sample bottles for HPP and TP treatments were kept at 4°C for 60 days for further analysis.

Ascorbic acid
In this study, the ascorbic acid of the sample was evaluated using the procedure described by AOAC, [10] with minor modifications.Iodine solution (0.005 mol/L) is used in this process to form iodide ions and dehydroascorbic acid by reacting with ascorbic acid as follows: In a short, 20 mL of sample was combined with 150 mL of water, 1 mL of starch solution, and 0.5 g/ 100 mL of water.Continuously agitate the solution, and Iodine is added until the endpoint is reached.The dark green color indicated the endpoint.The volume of iodine needed to reach the endpoint was used to compute the ascorbic acid concentration.The ascorbic acid concentration value was the mg ascorbic acid equivalent per 100 g of juice.

Total carotenoid content
The carotenoid value of orange juice was analyzed using the method represented by [11] with some modifications.In a short, 25 mL of the mixture was added to 80 mL of a 1:1 v/v hexane-acetone mixture in a separating funnel.Anhydrous sodium sulfate was added to the organic phase to absorb moisture after it had been collected.At room temperature, the absorbance was measured at 450 nm.Using β-carotene (2-10 μg/mL) mixed in hexane solution, a standard curve was created.

Total phenolic content
The methodology outlined by [12] was used to determine the total phenolic content (TPC).At room temperature, 2.5 mL of Folin-Ciocalteu reagent, diluted 10 times, was added to the samples (50 mL), followed by 2 mL of Na 2 CO 3 (7.5 g/100 mL).A spectrophotometer (HP 8452A, Agilent Technologies, Palo Alto, USA) measured the absorbance after the mixture was left in the dark for 30 min.Gallic acid (0.1-0.9 mg/mL) was used as the standard to generate a calibration curve, and the results were summarized in mg equivalent gallic acid per mL (mg GAE/ml).

Physicochemical analysis
A pH meter (Model FE20, Mettler Toledo, Schwerzenbach, Switzerland) was used to gauge the pH of the orange juice after being calibrated with buffer solutions.The total titratable acidity (TA), represented as g of citric acid per 100 mL, was calculated using titration.A digital refractometer (Pocket Pal-, Atago, Tokyo, Japan) was used to calculate the total soluble solids concentration in ° Brix. [13]

Microbial analysis
After processing (day 0) and storage, the identification of microflora of orange juice was carried out.The identification of microflora of orange juice was tested after 0 days and during storage.The materials were homogenized for 2 min after being diluted 1:10 in peptone water to enumerate total aerobic bacterial (TAB) (colony forming units cfu per ml) that are alive, and the suspensions were plated on PCA.The plates were incubated for 24 h at 30°C.The samples were also counted on DRBC agar to look for live yeast and mold.During 3-5 days, the plates were incubated at 25°C.A mechanical spiral plater (Eddy Jet, IUL Instruments, Barcelona, Spain) was mostly employed to achieve this.However, manual plating was also done at low dilutions.

Statistical analysis
The data were described as mean after analyzing each sample separately in triplicate using STATISTIX 8.1 at a 5% significance threshold to interpret the results using a two-way analysis of variance (ANOVA).

Ascorbic acid
Vitamin C is very crucial for human health and is composed of ascorbic and L-dehydroascorbic acid. [14]Its strong antioxidant capacity protects against free radicals and helps prevent the development of many degenerative diseases. [15]Since vitamin C is nutritionally significant and unstable, its presence ensures the existence of other nutrients and is used as a gauge for the nutritional value of processed foods.Citrus fruits are the primary source of vitamin C for consumers. [8]The mean values regarding the ascorbic acid content in the orange juice are presented in Table 1.The amount of ascorbic acid was measured in all orange juice samples.Because temperature adversely affects the ascorbic acid content, the thermally treated sample showed the lowest value of ascorbic acid content (38.44-33.45mg/100 ml).Ascorbic acid was observed 40.03 mg/100 ml in a fresh orange juice sample (control).Orange juice samples without any treatment were not analyzed further for storage study because they were untreated and unfit for human consumption during storage period.The increased concentration of ascorbic acid in high-pressure processed samples might be due to a higher extraction rate in juice due to high pressure.During storage, samples processed by thermal processing showed a significant decline in ascorbic acid content.In contrast to thermal processing, the sample treated with high-pressure processing showed better results.A non-significant reduction in ascorbic acid content was observed in HPP treated samples.A minimum reduction was observed in T6 ranged 41.19 to 39.87 mg/100 ml during 60 days of storage.
In samples subjected to high pressure, ascorbic acid is retained to the maximum value.This outcome was in line with a study by Lopez-Malo et al. [16] which discovered that a blend of orange, lemon, carrot, and water (8:1:4:7) maintained 99.6% of its vitamin C content after being subjected to a 500 MPa pressure for 5 min.Since ascorbic acid is extremely sensitive to the temperatures used during the thermal processing of juices, mild processing conditions result in higher ascorbic acid retention. [17,18]Furthermore, it has been observed that the degradation rate is slower in juice treated with HPP than in juice that has not been treated during storage, demonstrating that HPP can reduce the loss of ascorbic acid during juice storage. [19]

Carotenoid content
Table 2 displays the impact of pressure and time on orange juice's carotenoid concentration.Orange juice exhibited stability when processed under high pressure, but high temperatures reduced the carotenoid content because of the orange juice's adverse thermal reaction.Pressure treatment at 400 MPa for 3 min and 600 MPa significantly increased the carotenoid content from 6.7 mg/100 ml to 8.6 and 11.21 mg/100 ml.The extraction capacity of juice was boosted by applying pressure for a short period because carotenoid molecules have a complicated matrix and are not free.High pressure led to improved extraction, which led to an increase in carotenoid content.Every 100 MPa increase in pressure resulted in a 3 °C rise in temperature.The carotenoid content decreased due to a high exposure period to pressure, which was 600 MPa for 9 min.Orange juice underwent pressure treatment that considerably boosted the carotenoid concentration, while thermal treatment had a counter impact.The maximum value was achieved by applying pressure for 6 min at 600 MPa (11.65 mg/100 ml).A study was conducted to store these juices for 60 days to examine the carotenoid content's stability.The analysis is done at the 30 and 60-days points.Untreated fresh samples were discarded, while processed samples underwent analysis.The findings demonstrate that a 60-day storage period does not result in any appreciable loss.
An intriguing improvement in carotenoid extractability as a result of HPP treatment is reported. [20]arotenoids are mostly associated with macromolecules like proteins and membrane lipids in subcellular organelles (plastids), chloroplasts, and chromoplasts. [21]These findings concur with those of Pokhrel et al. [10] They reported the same outcomes with a carrot-orange juice mixture.No adverse effects of high-pressure processing of the carotenoid content on carrot-orange juice were discovered despite the carotenoid concentration increase.The findings of De Ancos et al. [7] likewise confirmed the findings.

Total phenolic content
Plants produce phenolic substances, which are found inside cells, during secondary metabolism.By increasing cell permeability with high pressure, phenolic substances may be retrieved or discharged. [10]In this study, the total phenolic content was measured.Statistically, there is no significant difference in phenolic content after high-pressure and thermal processing as phenolic compounds showed some stability to processing.Thermal processing reduces the phenolic content by up to 30% during 60 days of storage.Nevertheless, HPP showed better stability than thermal processing as HPP retains phenolic content.Due to the low pressurization levels, HPP did not notably impact the total phenolic substances (p > .05).The HPP level 600 MPa for 6 min showed maximum stability during processing and storage.Pressure applied at 600 MPa for 9 min showed some negative results due to increased temperature as the pressure applied time increased.The mean values regarding the total phenolic content in orange juice are presented in Table 3. High-pressure treatment for 9 min increased the cell disruption, increasing the total phenolic content of orange juice.These results are consistent with those reported by, [22] who found that after high-pressure treatment at 400 MPa for 15 min, both strawberry and blackberry puree had a non-significant increase in the total phenolic compound.There is little comparative research on HPP's impact on TPC in orange juice, but a few studies have found this influence in other fruit juices.In earlier investigations, it was discovered that different phenolic component concentration changes were observed following HPP processing.Thermal pasteurization reduces the total phenolic content as temperature negatively affects phenolic compounds but it reduces non-significantly.TP and HPP orange juices' TPC reduced after storage; however, this behavior was more pronounced in TP juices (p 0.05) after 60 days of storage.Similar results were found in an orange juice-milk beverage showing a significant increase at 100 MPa for 7 min but a non-significant decrease at 400MPa for 9 min. [23]According to certain research, TPC may even rise after processing when antioxidants are more easily removed. [24]Similar to this, a study using mulberry juice compared heat treatment (85°C, 15 min) with high-pressure processing (HHP) (500 MPa, 10 min), and discovered that HPP was able to preserve more TPC. [25]Varela-Santos [26] discovered that during 35 days of storage at 4°C, pomegranate juice treated with HHP showed a modest decline. [27]

Physicochemical analysis
The physicochemical characteristics of orange juice were observed after high-pressure processing and pasteurization and compared with untreated samples.Processing of orange juice non-significantly affects the pH of orange juice.High-pressure processing stabilizes the quality of orange juice as it has a non-significant effect on the pH, titratable acidity, and TSS of juice.The mean values regarding the physicochemical analysis in the orange juice were presented in Figures 1,2,3.HPP was found to have no appreciable impact on the product pH (p > .05),which is consistent with the study of Pokhrel et al. [10] There was a non-significant increase in the pH of orange juice during 60 days of storage after pasteurization, while high pressure processed samples showed a non-significant reduction in the pH of orange juice.Pressurization at 600 MPa for 6 min non-significantly affects the pH of the orange juice, hence retaining the juice quality after such a high-pressure level.The results were in line with the study of Pokhrel et al. [10] who reported that during storage, the pH of the control sample decreased by approximately 1 unit for the carrot orange juice blend with pH 5 and pH 6, and about 0.70 unit for the blend with pH 4. Similarly, no discernible impact of a 600 MPa high-pressure treatment lasting 10 min on pH was shown by Zhang et al. [28] for carrot juice.After initially being stable, the pH of processed samples started to fall substantially toward the end of storage, maintaining higher stability than untreated blends.
Juice quality and stability during the storage period of research was evaluated by the total soluble solids (TSS).TSS also showed the qualities of other parameters like amount of sugar and organic acids.Total soluble solid (TSS) is a parameter that allows us to evaluate the juice quality and stability while doing storage research since it indicates the entire quantity of sugar, organic acids, and other soluble molecules in a product.
Organic acids, produced by biochemical processes or fermentations by the development of certain spoilage microbes, contribute to the distinct flavors and palatability of orange juice.Acidity offers significant protection against the growth of hazardous microbes.Acidity alters the sweetness of sugar by helping to create flavor with a proper carbohydrate-to-acid ratio (Adeola and Aworh, 2010).HPP was found to have no appreciable impact on the titratable acidity of orange juice (p > .05).Thermal processing and high-pressure processing non-significantly affect the acidity of orange juice, hence retaining its fresh-like taste; however, storage affects the acidity of orange juice.High-pressure processing at 600 MPa for 6 min showed the best result and preserved better quality juice.W. Wu et al. [29] also represented a similar result.During storage, there were notable modifications, and the alterations mostly happened toward the conclusion of the storage period of HP treated and TP treated pineapple fruit juices.The results were also supported by the previous findings. [30,31]

Yeast and mold count
The mean values regarding the yeast and mold count in the orange juice are presented in Table 4.The microbiological quality of orange juice after high-pressure processing, thermal treatment, or no treatment was determined by monitoring the yeast and mold counts in orange juice.The mean initial  population of yeast and mold in a controlled sample of orange juice was 3.83 log CFU/ml.It was increased concerning time because of no treatment, and the sample was discarded because of high microbial load during further study.Thermal and pressure-processed juices show the very least count of yeast and mold at the initial stage.Post pasteurization contamination of the orange juice may have occurred with the bottle filler, adding to the microbial load in the thermally treated orange juice.Highpressure treatment at 400MPa for 3 min reduced the population levels up to 1.06 log CFU/ml but it was not detected when 400 MPa was applied for 6 and 9 min.Yeast and mold counts can be seen at 45 and 60 days of storage in samples treated with 400 MPa.The first pressurization at 600 MPa for 3, 6, and 9 min reduced the yeast and mold count up to 0.67, 0.64, and 0.62 log CFU/ml, respectively.The below detection limit made the juice highly acceptable for consumers.Only 1.15 log cfu/ml can be detected in the sample treated with 600 MPa for 6 min but it did not make juice unfit for consumption.HPP resulted in statistically significant (P-0.05)reductions in yeast and mold compared with untreated juice.The results were supported by the work of Sreedevi et al. [32] which investigated microbial load in navel and Valencia orange juice and reported significant yeast and mold reduction compared to non-treated samples and when treated with thermal techniques.Microbial safety was ensured in both HPP-treated and thermally-treated pineapple fruit. [33]

E-Coli count
The mean values regarding the E-coli count in the orange juice are presented in Table 5.The microbial quality of the orange was also determined by E-coli count.Thermal and high-pressure processing also reduce E-coli content significantly.The initial population of E-coli can be seen as 4.67 log CFU/ml without any treatment, but this value increased during storage and made juice unfit for further consumption.Thermal pasteurization reduced E-coli count up to 3.67 log CFU/ml and an increasing trend was observed in the samples.This increasing value of 6.49 log CFU/ml limits the life of orange juice compared to high-pressure processes where high pressure significantly reduced the value and retained juice completely fit for human consumption.Pressure applied 400 MPa for 3, 6, and 9 min showed a significant reduction in e-coli count compared to thermal treatment and untreated samples.When the pressure increased up to level 600 MPa, E-coli counts were not detected.E-coli could not be detected in orange samples at the initial stage of the study and could only be detected at a level of 1.07 log CFU/ml at 60 days of storage in samples treated with 600 MPa for 3 min.E-Coli was not detected in samples treated with 600 MPa for 6 and 9 min.A significant reduction is measured in orange juice samples compared to thermal treatment and to the samples that were not further processed.Pressure treatment significantly delayed the growth and recovery of the surviving microorganisms.Overall, the pressure level itself (400 or 600 MPa) had no significant effect (p > .05).Cao et al [34] also reported there processing at 600 MPa for up to 6 min could result in the complete inactivation of microbes when the initial microbial count was less than 4 logs CFU/ml in strawberry juice.Patterson et al. [35] also proposed similar findings in carrot juice.Untreated and pressure-treated carrot juices were evaluated for their microbiological quality.The total counts were lowered by about 4 log CFU/ml after highpressure treatment at 500 MPa and 600 MPa (1 min/20 C), and the survivors showed very little growth when stored at 4°C for up to 22 days.

Conclusion
Our findings showed that HPP at 600 MPa for 6 min could be used to make orange juice safely and with preserved quality and storage stability compared to commercially applied thermal processes.The quality of orange juice could be preserved by high-pressure processing while during thermal processing bioactive compounds like ascorbic acid, total phenolic compound, and carotenoid content are lost in maintaining the longevity of orange juice.This study identifies ideal high-pressure processing settings that can produce juice of superior quality and allow for a longer shelf life.It would help the present strategies for commercializing juice made from fruits and vegetables under high pressure under less strenuous circumstances.

Figure 1 .
Figure 1.Effect of treatments on pH of orange during 60 days of storage.

Figure 2 .
Figure 2. Effect of treatment on Total soluble solids (°Brix) of orange during 60 days of storage.

Figure 3 .
Figure 3.Effect of treatment on Titratable acidity of orange during 60 days of storage.

Table 1 .
Effect of treatment on the ascorbic acid content of orange during 60 days of storage.

Table 2 .
The effect of HPP on the carotenoid content of orange juice during 60 days of storage.

Table 3 .
Effect of treatment on total phenolic (mg GAE/100 ml) of orange during 60 days of storage.

Table 4 .
Effect of treatment on yeast and mold of orange juice with respect to storage.MPa for 3, 6, and 9 min, respectively.NT= Not Tested.Data represents mean ± SD Different letters shows significant difference while same letters shows results are non-significant.

Table 5 .
Effect of treatment on E-coli content of orange during 60 days of storage.Orange juice; OT1 = posturizes orange juice; OT2, OT3, OT4 represent orange juice treated at 400 MPa for 3, 6, and 9 min, respectively; OT5, OT6, OT7 represents orange juice treated at 600 MPa for 3, 6, and 9 min, respectively.NT = Not Tested.Data represent mean ± SD.Different letters show significant difference while same letters show results that are nonsignificant.