Thermosonication process for optimal functional properties in carrot juice containing orange peel and pulp extracts

Thermosonication process for optimal functional properties in carrot juice containing orange peel and pulp extracts

Accepted Manuscript Thermosonication process for optimal functional properties in carrot juice containing orange peel and pulp extracts Oladipupo Q. A...

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Accepted Manuscript Thermosonication process for optimal functional properties in carrot juice containing orange peel and pulp extracts Oladipupo Q. Adiamo, Kashif Ghafoor, Fahad Al-Juhaimi, Elfadil E. Babiker, Isam A. Mohamed Ahmed PII: DOI: Reference:

S0308-8146(17)31729-6 https://doi.org/10.1016/j.foodchem.2017.10.090 FOCH 21913

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

30 April 2017 15 October 2017 16 October 2017

Please cite this article as: Adiamo, O.Q., Ghafoor, K., Al-Juhaimi, F., Babiker, E.E., Mohamed Ahmed, I.A., Thermosonication process for optimal functional properties in carrot juice containing orange peel and pulp extracts, Food Chemistry (2017), doi: https://doi.org/10.1016/j.foodchem.2017.10.090

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Thermosonication process for optimal functional properties in carrot juice containing orange peel and pulp extracts Oladipupo Q. Adiamo, Kashif Ghafoor*, Fahad Al-Juhaimi, Elfadil E. Babiker, Isam A. Mohamed Ahmed, Department of Food Science and Nutrition, College of Food and Agricultural Sciences, King Saud University, P.O. Box 2460 Riyadh, 11451, Saudi Arabia

*Correspondence author: Kashif Ghafoor Department of Food Science and Nutrition, College of Food and Agricultural Sciences, King Saud University, P.O. Box 2460 Riyadh, 11451, Saudi Arabia Email: [email protected] Phone number: +966-55-2174216

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Abstract Aqueous extracts of orange peel and pulp with high total phenolic contents (TPC) (25.94 and 11.38 mg GAE/g extracts, respectively) were employed in the formulation of functional carrot juice and functional juices were treated using thermosonication process. In accordance with Box-Behnken design, 17 runs with 3 variables and 3 levels was applied for the optimization of the carrot juice with peel (CJPL) and pulp (CJPP) extracts. Overlaid contour plots prediction showed that the optimal conditions for CJPL were 125 mL juice volume, 6.50 min ultrasound process time and 52.78˚C ultrasound process temperature for maximum TPC (30.25 mg GAE/100 mL) and DPPH scavenging activity (61.22%). Sample CJPP has maximum TPC (28.94 mg GAE/100 mL) and DPPH activity (55.87%) under optimal ultrasound process conditions of 125 mL juice volume, 5.04 min and 59.99˚C ultrasound process time and temperature, respectively. Optimization of thermosonication showed significant improvements in the quality of functional carrot juice.

Keywords: Carrot juice, orange by-product extracts, thermosonication, total phenolic content, response surface optimization

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1. Introduction Consumption of beverages, especially fruit and vegetable juices, is a convenient method of consuming large amounts of bioactive compounds and hence they can be used as vehicles to deliver health benefiting components (Carrillo, Fiszman, Lahteenmaki, & Varela, 2014). Carrot (Daucus carota L.) is cultiavted throughout the world and regarded as an important root vegetable of Umbelliferae family. Carrot and its products such as juices are widely known to be rich in phytonutrients particularly carotenoids, minerals and vitamins (Qin, Xu, & Zhang, 2005). However, carrots have lower phenolic compounds than some other fruits and vegetables such as apple, eggplant, orange etc. (Lutz, Hernandez, & Henriquez, 2015; Yahia, & Barrera, 2010). The positive contribution of phenolic compounds to human health in relation to their antioxidant, antimicrobial and antimutagenic activities have been reported (Mazzoni et al., 2016). Several sectors have increased their efforts for past decades to formulate functional foods using bioactive compounds from agricultural by-products such as fruits by-products (Jones, & Jew, 2007). Citrus fruits are one of the important horticultural crops which is also grown in Saudi Arabia with increasing yearly production of about 100000 in 2012 and 106292 tons in 2013 (FAO, 2014). Among the citrus fruits, orange is the most common and usually processed into juice or eaten as fresh. Considerable amounts of orange by-products may be produced during juice production which are prone to spoilage by microorganisms and hence can cause environmental problems (Omoba, Obafaye, Salawu, Boligon, & Athayde, 2015). Reports have shown that orange by-product extracts such as peel and pulp have high ascorbic acid, polyphenols and minerals and the antioxidant activity of orange by-products can be attributed to both ascorbic acid and phenolics (Barros, Ferreira, & Genovese, 2012). Orange peel is rich source of phenolic compounds (particularly quercitrin, rutin, and quercetin) and flavonoids. The bioactive compounds in orange peel carry good 2,2′-azino-bis(3-ethyl benzothiazoline-63

sulfonic acid) diammonium salt (ABTS) scavenging ability; ferric reducing antioxidant property (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability (Omoba et al. 2015). Hence, these orange by-products and their extracts have potential for use in functional food development due to their health promoting constituent (Barros et al., 2012). Quality of the functional food products is greatly influenced by the processing methods employed. Thermal pasteurization is the most widely used heat treatment for food preservation till date. It confers extended shelf-life and stability of fruit and vegetable juices; however, this preservation method may have negative effects on the nutritional and physicochemical properties like vitamins, carotenoids, polyphenols, pH and color. These negative effects include those on heat sensitive phenolic compounds and other nutrients (Santhirasegaram, Razali, & Somasundram, 2013). Phenolic compounds and other natural antioxidants such as anthocyanins are prone to thermal degradation in fruit juices and effective process modifications are required to preserve their structure and functionality (Mäkilä, Laaksonen, Kallio, & Yang, 2017). Due to scientific evidence and increased knowledge, consumers now demand for food not only having prolonged shelf-life but also providing health benefits. Consequently, researchers are working on innovative processing techniques which are least detrimental to bioactive compounds and nutritional value of fruit juices such as carrot juice. Several non-thermal processing techniques such as sonication, blanching (Jabbar et al. 2014), ultraviolet (UV) treatment (Riganakos, Karabagias, Gertzou & Stahl, 2017), pulse electric field and high hydrostatic pressure have been tested to extend the shelf-life and preserve nutritional quality of carrot juice (Davis, Moates, & Waldron, 2010). Recently ultrasound treatment combined with heat (thermosonication) has been employed in fruit juice processing as a better substitute for thermal treatment to retain most of juice health beneficial properties (Anaya-Esparza, Velazquez-Estrada, Roig, García-Galindo, 4

Sayago-Ayerdi, & Montalvo-Gonzalez, 2017). Martínez-Flores, Garnica-Romo, BermúdezAguirre, Pokhrel, and Barbosa-Cánovas (2015) reported significantly higher amounts of carotenoids, ascorbic acid and polyphenolics in thermosonicated carrot juice as compared to heat treated one. Similarly, an increase in total carotenoids, lycopene and lutein in carrot juice treated with ultrasound at 15˚C temperature, 20 kHz frequency and 70% amplitude has been reported by Jabbar et al. (2014). However, no studies have been reported on the effects of thermosonication on functional carrot juice developed using other fruits or orange byproducts. The objective of current work was, in addition to developing functional carrot juice using orange by-products (peel and pulp) extracts and thermosonication, to apply convenient statistical methods to optimize thermosonication process variables (temperature, time and juice volume) for carrot juice containing orange peel (CJPL) and pulp (CJPP) extracts for maximizing total phenolic contents and DPPH scavenging activity in juices using response surface methodology (RSM) and superimposed contour graphs. Furthermore, microbiological properties, carotenoid, total phenolic contents and antioxidant activities of the functional carrot juice produced at optimum thermosonication conditions were also compared with those pasteurized using thermal treatment.

2. Materials and methods 2.1. Materials, reagents, solvents and standards Fresh, ripened and locally grown Orlando oranges (Citrus sinensis) and carrots (Daucus carota L.) of good quality were procured from vegetable market in Riyadh, Kingdom of Saudi Arabia. Petroleum ether and methanol was purchased from Fisher Scientific Chemical Co. (Loughborough, UK). DPPH, Folin Ciocalteu reagent and β-carotene were from Sigma Chemical Co. (St. Louis, MO, USA). Anhydrous sodium carbonate was purchased from 5

Avon Chemical Limited (Cheshire, UK) and Gallic acid was acquired from Acros Organics Chemical Co. (New Jersey, USA). Nutrient agar and potato dextrose agar was obtained from Scharlab Chemical Co. (Barcelona, Spain). 2.2. Preparation of phenolic enriched extracts from orange peel and pulp The oranges were washed and peeled manually with knife. This was followed by manual extraction of the juice from the fruit. The orange by-products (peel and pulp) recovered were freeze-dried and powdered to pass through 24 mesh sieve. The phenolic extracts were prepared as described by Omoba et al. (2015) with slight modification by mixing orange byproducts with distilled water using magnetic stirrer (Ika-Combimag 22909, Friedberg/Hessen, Germany) at a ratio of 1g: 20 mL at temperature of 60 ºC for 30 min and the suspension was cooled to room temperature. The extracts were then filtered through Whatman No 1 filter paper followed by drying under reduced pressure at 40 ºC and stored at -20 ºC till further use. All chemicals used in this study were of analytical grade. 2.3. Determination of total phenolic contents (TPC) of extracts The TPC of the extracts were determined as described by Singleton and Rossi (1965) with slight modification. A 200 µL of the extracts (10 mg/mL) or standard solution (at varying concentrations) was mixed with 400 µL of Folin Ciocalteu reagents and 4.0 mL of distilled water and thoroughly mixed. The mixture was incubated for 10 min at 25 °C followed by addition of 20% Na2CO3 solution, mixed thoroughly and incubated for 2 hr. The spectrophotometer (PD-303UV, Apel, Saitama, Japan) was used to check the absorbance at 765 nm wavelength. Gallic acid solution of varying concentrations was used to obtain standard calibration curve while methanol was used as solvent and blank. The TPC was expressed as mg gallic acid equivalent per gram (GAE/g) of extracts from calibration curve (y = 4.4279x + 0.0364). 6

2.4. Functional carrot juice preparation The carrots were washed to remove adhering dirt and unwanted parts were trimmed off manually with knife followed by juice extraction using Santos Juicer (Al-Reziza Company, Jeddah, Saudi Arabia). Juice was kept in refrigerator at 4 ºC for cold settling and centrifuged at 4500 × g for 20 min. Dried extracts from orange peel and pulp were autoclaved at 121 °C for 15 min and then added to 100-150 mL of the juice, in quantities which carry 0.5 mg GAE of TPC by stirring on magnetic stirrer (Ika-Combimag 22909, Friedberg/Hessen, Germany) for 15 min. 2.5. Thermosonication processing of functional carrot juice The functional carrot juice was poured in 200 mL air tight sterilized media bottle and sonicated in an ultrasound bath (CPX2800H-E, Branson, USA) which was 230 cm wide × 140 cm long × 100 mm deep. The temperature and time of thermosonication treatment were varied and controlled from the panel. The juice was allowed to attain the desired temperature (40-60˚C) in the heated ultrasound water bath (set at the desired sonication temperature) by measuring the temperature at the geometric center of the bottle containing the juice using a digital probe thermocouple (Oakton, Eutech Instruments, China). Thereafter, sonication was performed at the desired temperature and time using a power of 110W and constant frequency of 40 KHz. After thermosonication treatment, the juice samples were immediately cooled in ice bath. 2.6. Experimental design The optimization involved three independent variables, i.e. time (5, 7.5 and 10 min), temperature (40, 50 and 60 ˚C) and juice volume (100, 125 and 150 mL). The dependent variables i.e. TPC and 1,1-diphenyl-2-picrylhydrazyl (DPPH) activity were based on a three

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factor experimental design using Design Expert (version 8.0, Stat Ease Inc., Minneapolis, USA). The design experiment was based on a series of preliminary experiment and a thermosonication processing time of 7.5 min, temperature 50 °C and juice volume of 150 mL were chosen as center points and the complete experimental design consisted of 17 experimental runs (Table 1). The results of triplicate measurement of responses (TPC and DPPH scavenging activity) were evaluated using regression methods and then the response surface analysis of the data was carried out to calculate optimum conditions. The prediction for optimum levels of variables was based on the equation below:

(1) where

is the predicted response, i.e. total phenolics and DPPH scavenging activity from

carrot juice containing peel (CJPL) and pulp (CJPP) extracts, and and

are the linear regression coefficients; and

,

and

is a constant. The

are quadratic terms and

, ,

are the interaction coefficients. The fitted polynomial equations were generated

to form surface and contour plots so as to visualize the relationship between the response and experimental levels of each factor. Moreover, the usage of overlaid contour plot of each response can interpret the optimum conditions directly. 2.7. Pasteurization of CJPP and CJPL Pasteurization of CJPL and CJPP in sterilized 200 mL media bottle was carried out by heating the juice in a shaking water bath (Janke & Kunkei HS—B 20D, Germany) at 75 ºC for 2 min according to the report of Khandpur and Gogate (2015) after slight modification. It took 7-9 min for the inner temperature of the juice to attain to 75 ºC. Samples were then held at 75 ºC for 2 min and immediately cooled in ice bath.

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Analysis was carried out on the thermosonicated CJPL and CJPP samples, processed at optimal conditions obtained from the overlaid contour plot, and the control (pasteurized samples). 2.8. Determination of total phenolic content and free radical scavenging activity of functional carrot juice The TPC of the juice was analyzed by the method of Singleton and Rossi (1965) as described earlier for orange by-products extracts after slight modification. The juice sample was diluted using distilled water (1:20). The TPC of the samples were expressed in milligram gallic acid equivalent per 100 mL of juice (mg GAE/100 mL) using gallic acid calibration curve equation (y = 4.4279x + 0.0364) obtained after preparing standard solution in the same way as for extract or juice sample. The free radical activity of the samples was determined using 1,1-diphenyl-2picrylhydrazyl (DPPH) (Lee, Mbwambo, Chung, Luyengi, Games, & Mehta, 1998). 1 mL solution of the juice extract at a concentration of 100 μL/mL methanol was mixed with 2 mL of 10 mg/L methanolic solution of DPPH. An equal amount of methanol and DPPH served as a control. The mixtures were shaken vigorously and allowed to stand at room temperature for 5 min and optical density (OD) was recorded at 517 nm. The percentage inhibition of the juice was calculated as follows:

2.9.

Microbial evaluation

The microbial loads of the juice were determined as described in APHA (2001). Pour plate method was employed to measure the total bacterial counts using nutrient agar and yeast and mold by potato dextrose agar. Incubation was done at 35 ˚C for 1 to 2 days for bacteria 9

and at 30 ˚C for 4 to 5 days for yeasts and molds. Three replicates from each treatment were tested and the results were expressed as log cfu/mL 2.10. Determination of total carotene (TC) content The TC of the sample was analyzed by modified method of Wallrauch (1984). Functional carrot juice (5 mL) was added to extraction solvent (petroleum ether: methanol 90:10, 50 mL) in a separating funnel. The mixture was vigorously mixed and allowed to stand for 5 min for solvent separation. The extraction process was repeated with 50 mL fresh extraction solvent. The solvents were pooled together and centrifuged at 3000 × g for 15 min. About 5 mL of the supernatant was mixed with 5 mL of petroleum ether in a test tube. A UV/visible spectrophotometer (Mod. 4050, Biochrom, Cambridge, UK) was used to measure the absorbance of the mixture at 450 nm. β-carotene solutions (1-20 µg/mL of petroleum ether) were used as standard and the results were expressed as mg β-carotene/L of juice. 2.11. Statistical analysis All determinations were carried out in triplicate and one-way analysis of variance (ANOVA) was employed to analyze the data using SPSS software (Version 23.0, IBM Corporation, New York, USA). Also means comparison was done using Duncan’s Multiple Range test at P < 0.05. The surface plots were constructed using Statistica 6.0 (Statsoft Inc., Okhlahoma, USA).

3. Results and discussion 3.1. Total phenolic content of orange peel and pulp extracts The results of total phenolic content of the orange by-products showed that higher values were obtained for peel (25.94 mg GAE/g extract) than pulp (11.38 mg GAE/g extract). 10

Similar observation has been reported by Gorinstein et al. (2001) that total phenolics contents of lemons, oranges, and grapefruit peels have 15% higher TPC than those in the peeled fruits. Furthermore, Al-Juhaimi (2014) reported that the TPC of peel from Orlando orange from Saudi Arabia was 31.2% higher than that of its pulp. The high TPC exhibited by the orange peel could probably be due to the presence of large amount of natural flavonoids and phenolic compounds in the peel as previously reported by Omoba et al. (2015). However, the result obtained disagrees with previous findings by Arora and Kaur (2013) where the TPC of orange pulp was higher than that of peel. The TPC of the peel extract obtained was comparable to that of peel extracts (21.11-30.18 mg GAE/g) of orange from Saudi Arabia. The differences in TPC could be due to the variety used, location and methods used for extraction. In the present study, the TPC of orange by-products obtained were higher than that of unripe (5.27 mg GAE/g extract) and ripe (9.40 mg GAE/g extract) sweet orange peel extract as reported by Omoba et al. (2015). However, the results were within the range of the TPC of Muscadine grape by-product (19.20-32.59 mg GAE/g extract) as reported by Pastrana-Bonilla, Akoh, Sellappan, and Krewer (2003). Therefore, the high amounts of phenolic compounds in the orange peel and pulp extract showed the importance of these orange by-products for use in functional foods due to their nutraceutical and health potential. 3.2. Modelling of the thermosonication process conditions from CJPL and CJPP Results of our preliminary study showed that thermal treatment (75 °C for 2 min) degraded the TPC of CJPL and CJPP by 12 and 8.59%, respectively. Similarly, Jabbar et al. (2015) reported 15.2% reduction in total phenolic content of carrot juice after thermal treatment at 80 °C for 1 min and this could be due to degradation of phenolic compounds in the juice at high temperature. Therefore, a combined sonication and heat (thermosonication) at temperatures lower than that of thermal treatment on TPC and DPPH scavenging activity was studied. Table 1, in addition to presenting the conditions of the complete design of 17 11

experiments, also shows the results of TPC and DPPH scavenging activity. Analysis of variance was used to analyze the data as shown in Table 2 together with the good fitness. The results showed good fitness with Equation (1), which were acceptable when p<0.05 with satisfactory R2 values. Three dimensional response surface plots were constructed using the full model to predict the relationships between independent and dependent variables. 3.3. Effect of process variables on total phenolic contents of CJPL and CJPP Analysis of multiple regressions was carried out on the experimental data and the significance was determined by evaluating the coefficients of the model (Table 2). The effect of ultrasound process time was more significant (p<0.0001) on TPC of CJPL than that of CJPP. Also the effect of juice volume was significant at p<0.05 and p<0.01 for CJPL and CJPP, respectively. The TPC of CJPP was significantly affected by the ultrasound temperature (p<0.05) while it showed non-significant effect on TPC of CJPL. The relationship, obtained using regression coefficient values, between the independent variables and the amounts of TPC of CJPL and CJPP (dependent variables or responses) are presented in Eq. (2) and (3), respectively. YTPC1 = 29.876 – 1.287X1 + 0.067X2 – 1.371X1X2 + 0.226X1X3 – 0.601X2X3 – 1.255X1X1 – 1.289X2X2 + 0.458X3X3……………………………………………………………………………………...Eq. 2) YTPC2 = 27.804 – 0.775X1 - 0.122X2 – 0.259X3 – 1.473X1X2 – 0.909X1X1 + 0.249X3X3………………………………………………………………………………………………………….Eq. (3) Where YTPC1 and YTPC2 are the total phenols (mg GAE/100 mL) in CJPL and CJPP, respectively, X1 is the ultrasound process time (min), X2 the ultrasound process temperature (°C) and X3 the juice volume (mL).

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Figure 1 shows the reference surface plot constructed in accordance with equations 2 and 3. The TPC of CJPL first increased gradually with increase in temperature and time as shown in Fig 1a. However, a sharp decline in the TPC was observed as time increased above 6.5 min at constant temperature (Fig 1a). The juice volume showed little effect on the TPC of CJPL and nearly reached a peak at the highest volume used (Fig. 1b). At constant volume there was a slow decline in the TPC as temperature increased more than mid-point used (Fig. 1c). The reference surface plots for TPC of CJPP are shown in Fig. 1(d, e and f). The total phenol content was mainly influenced by ultrasound process time. Fig. 2(d and e) showed that increase in the time of ultrasound processing first increased the TPC and later decreased as time reached 6 min. Slight decrease in the TPC occurred as juice volume increases as shown in Fig 2c but not much effect of ultrasound process temperature on TPC was observed with TPC having the maximum point at nearly the highest temperature used (Fig. 1f). The possible reason for the increase in phenolic contents as sonication time increases might be due to the cavitation which results in cell wall breakage of the extracts and juice constituents as result of sudden pressure change due to shear forces impacted by implosions of bubbles that may discharge the bound form of these phenolic compounds and eventually increase their availability in the juice (Jabbar et al. 2015). Increment in phenolic contents of apple juice (Abid et al., 2013) and mango juice (Santhirasegaram et al., 2013) have also been previously reported with increasing sonication time. However, as sonication time continues for longer period, there may be degradation of some phenolic compounds in the extracts and the juice, resulting in the decrease in the amount of TPC in the juice. Similar observations have been reported by Jabbar et al. (2015) where total phenols in carrot juice decreased as sonication time increased from 5 to 10 min at 40 and 60 ˚C. Furthermore, Ghafoor and Choi (2009) found that increase in the time and temperature of ultrasonication have significant effect on the total phenolic and antioxidant properties of grape peel. The efficiency of 13

ultrasonication could be explained by the fact that sonication simultaneously enhanced the hydration and fragmentation process while facilitating mass transfer of solutes to the liquid (Toma, Vinatoru, Paniwnyk, & Masom, 2001). 3.4. Effect of process variables on DPPH of CJPL and CJPP The result of DPPH scavenging activity of CJPL and CJPP at different temperature and time of ultrasound processing and juice volume is shown in Table 1. Multiple regression analysis was performed on the data and the coefficient of model was used to determine the level of significance. The thermosonication process conditions have significant effect on the DPPH scavenging activity of CJPL and CJPP. The ultrasound process time has significant effect at p<0.05 and p<0.0001 on the DPPH scavenging activity of CJPL and CJPP, respectively. A significant (p<0.05) effect was also observed on the DPPH scavenging activity of CJPL and CJPP with the former being highly significant. The DPPH scavenging activity of the samples was affected by the linear and quadratic terms of the independent variables. Equations 4 and 5 showed the relationship between the independent variables and the radical scavenging activity of CJPL and CJPP. YDPPH1 = 62.279 – 2.375X1 – 6.015X2 + 3.811X3 – 15.683X1X2 – 13.068X1X3 – 14.073X1X1 + 2.316X2X2 – 7.166X3X3……………………………………………………………………………………Eq. 4) YDPPH2 = 48.916 – 5.37X1 – 3.072X2 – 0.777X3 – 8.218X1X2 – 6.017X1X3 + 2.282X2X3 – 5.149X2X2 – 4.213X3X3……………………………………………………………………………………….Eq. 5) Where YDPPH1 and YDPPH2 represent the total phenols (mg GAE/100 mL) in CJPL and CJPP, respectively. The equations were based on the data of regression coefficients as shown in Table 2.

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In the reference plot as seen in Fig. 2, it could be observed that there is a sharp rise in DPPH of CJPL with increase in time at constant temperature and volume (Fig. 2a, b). As time increased nearly above the mid-point set, the DPPH decreased gradually. It can be depicted from Fig 2b that at constant time, the DPPH increased rapidly with increase in juice volume with the highest DPPH noticed very close to the largest volume of juice used. Temperature increase resulted in slight decrease in the DPPH of the juice (Fig. 2c). The first increase in antiradical activity might be attributed to the increase in phenolic compounds in the functional carrot juice caused by cavitation generated during sonication which enhanced the extraction and availability of these compounds from the extracts (Jabbar et al., 2015). The higher the phenolic compounds, more will be the antiradical activity. Therefore, the decrease in antiradical activity as sonication increased at higher temperature may be due to the lower amounts of correlated TPC obtained during that period. The decrease in antioxidant activity with increase in treatment temperature of thermosonicated watermelon juice has also been observed by Rawson et al. (2011). Studies show that the increase in temperature may favor extraction and availability of the phenolic compounds by improving the solute solubility and the diffusion coefficient; however, a too high temperature can also result in denaturation of phenolic compounds, which justifies the selection of the best temperature limits and process optimization (Spigno, Tramelli, De-Faveri, 2007; Yilmaz and Toledo, 2004). Fig. 2(d, e, f) shows the response surface plots for antiradical activity of CJPP. A linear decrease in DPPH with increase in time of ultrasound processing was noticed at constant temperature and volume (Fig. 2d, e). However slight increase in antiradical activity occurred as temperature rises with peak value at the maximum ultrasound temperature used (Fig. 2d, f). Formation of hydroxyl radical caused by bubble implosion during sonication to the aromatic ring of phenolic compounds may also results in the improvement of antioxidant 15

activity in the juice. The addition of second hydroxyl group in the ortho- or para- position of phenolic compounds may also result in antioxidant capacity increase (Ashokkumar et al. 2008). At constant temperature and time, the antiradical activity first decreased with lowest value occurring at nearly 125 mL and later increased gradually (Fig. 2e, f). 3.5. Optimum thermosonication process conditions for the maximum TPC and DPPH The estimated levels of optimum ultrasonic process conditions for maximum response of total phenolic contents and DPPH antiradical activity of CJPL and CJPP were determined. The predicted thermosonication process conditions were 6.48 and 5.12 min ultrasound process time, 48.73 and 59.95 °C ultrasound process temperature and 149.98 and 100.99 mL juice volume for the estimated maximum TPC (30.37 and 29.41 mg GAE/100 mL) for CJPL (R2 0.995, adjusted-R2 0.989 and p<0.0001) and CJPP (R2 0.995, adjusted-R2 0.988 and p<0.0001), respectively. Also estimated maximum DPPH for CJPL (66.89%, R2 0.992, adjusted-R2 0.982 and p<0.0001) and CJPP (60.58%, R2 0.993, adjusted-R2 0.985 and p<0.0001) was predicted at thermosonication process conditions of 5.24 and 5.00 min ultrasound process time, 57.14 and 60.0 °C ultrasound process temperature and 149.98 and 146.57 mL juice volume, respectively. These values showed that the model had adequately represented the real relation between the parameters chosen. Although we have obtained the optimum thermosonication conditions for each response individually, however, in order to predict optimal conditions for two responses simultaneously, the simplest strategy to adopt is visual inspection. The surface can be overlapped to find the experimental region that can satisfy the two responses studied (Bezerra, Santelli, Oliveira, Villar, & Escaleira, 2008). A superimposed contour plot for TPC and DPPH antiradical activity is shown in Fig. 3. By analyzing the effects of thermosonication process conditions on the TPC and antiradical activity, we found that the

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responses changed substantially with ultrasound process time and temperature. However, the juice volume showed minor effects, therefore the superimposed contour plot was fixed at the mid-value (125 mL) of juice volume used. The optimal thermosonication process conditions of 125 mL juice volume, 6.50 min ultrasound process time and 52.78 °C ultrasound process temperature are predicted in Fig. 3a, which can result in maximal TPC (30.25 mg GAE/100 mL) and DPPH (61.22%) of CJPL. The TPC (28.94 mg GAE/100 mL) and antiradical activity (55.87%) have maximum values under optimal thermosonication process conditions of 125 mL juice volume, 5.04 min and 59.99 °C ultrasound process time and temperature for CJPP (Fig. 3b). These total phenolic contents were higher than those reported by Jabbar et al. (2015) for control and thermosonicated carrot juice (without bioactive enriched natural extract). 3.6. Microbiological characteristics, total phenolic content, DPPH scavenging activity and total carotenoid content of thermosonicated and pasteurized CJPP and CJPL As shown in Table 3, no microbial growth was observed in thermosonicated CJPP (125 mL, 60°C, 5 min) and CJPL (125 mL, 52°C, 6.5 min) as well as. However, thermosonicated CJPL has total bacterial count of 1.85 log cfu/mL and yeast and mold count of 1.70 log cfu/mL. The differences in microbial loads could be due to low optimal thermosonication temperature (52 °C) used in the preparation of CJPL as compared to that used for CJPP (60 °C). Similar observations have been reported in ultrasound treated carrot juice (Jabbar et al., 2015). In addition, greater inactivation of microorganisms in thermosonicated (60 °C) mango juice has been reported (Kiang, Bhat, Rosma, & Cheng, 2013). Ultrasound treatment together with heat has been found to raise the efficiency in inactivating microorganism compared to ultrasound treatment alone (Lee, Zhou, Liang, Feng, & Martin 2009). The inactivation of micro-organisms may be due to sonication altering the structure of microbial cell membrane coupled with the increase in microorganisms’ sensitivity to heat (Bermúdez-Aguirre & 17

Barbosa-Canovas, 2012). Therefore, production of functional carrot juice with reduced microbial loads could be achieved by thermosonication at the optimal process conditions employed. The result of the total phenolic content and DPPH scavenging activity, as presented in Table 3, revealed that thermosonication of CJPP and CJPL exhibited significantly higher values than their pasteurized samples with thermosonicated CJPL having the greatest value. The increase in phenolic compounds might be attributed to the cavitation which results in cell wall breakage of the added extracts and that of juice as a result of sudden pressure change due to shear forces impacted by implosions of bubbles that may discharge the bound form of these phenolic compounds and eventually increase their accessibility in the liquid. An increase of phenolics compounds, from 768.29 to 829.32 µg/g, ws reported in apple juice treated with increasing ultrasound (25 kHz) treatment time from 30 to 90 min (Abid et al., 2013). Santhirasegaram et al. (2013) observed significant increase in carotenoids (4–9%) and phenolic compounds (30–35%) in mango juice when ultrasonic treatment (60 min, 25 °C, 40 kHz & 130 W) was given for 15 to 30 min in comparison to thermal treatment (90 °C for 30 and 60 s). Process temperature, although it may increase extractability, is very important in terms of stability of bioactive compounds in fruit juices. Jabbar et al. (2015) and Rawson et al. (2011) reported reduction in total phenolics in carrot juice and water melon, respectively, with increase in process temperature. The thermal degradation of phenolic compounds was also reported in conventionally pasteurized black currant juice (Mäkilä et al., 2017). The bioactive compounds may undergo isomerization at increased temperatures during conventional thermal pasteurization (Jabbar et al., 2015). The improved stability of phenolic compounds and increased antioxidant activity of thermosonicated (52-58˚C) carrot juice was previously reported (Martínez-Flores et al., 2015). Therefore, the application of thermosonication at the optimal process conditions to carrot juice containing orange peel and 18

pulp extract enhanced their TPC and DPPH scavenging activity as compared to conventional method, thereby improving the juice health beneficial properties. Phenolic compounds, carotenoids and other bioactive compounds are natural antioxidants that play an important biological function by scavenging free radicals, which are detrimental to human health and cause oxidative stress. Carotenoids are important natural pigments which not only give a characteristic color to carrots but also contribute to the total antioxidant activity (Yahia, & Barrera, 2010). The total carotenoid (TC) content of the samples ranged between 7.21-7.66 mg β carotene/L juice (Table 3). However, processing treatment showed non-significant effects on the TC content of samples CJPP and CJPL. The results obtained contradict the earlier findings reported by Jabbar et al. (2015) and Martínez-Flores et al. (2015) where the TC of thermosonicated carrot juice was significantly higher when processed at 20 and 24 kHz frequency using a probe sonicator directly into juice, respectively. According to Abid et al. (2014) sonication may increase total carotenoids by causing mechanical disruption of cell wall thereby enhancing free carotenoids. However, in the present study, although the water bath sonication frequency was 40 kHz but juice samples were sealed in a glass bottle which might have resulted in reduced free carotenoids release from cellular structure, different from the effects of same on phenolics. Hence optimization of more thermosonication variables, such as frequency, for other responses such as carotenoids, minerals and vitamins can result in further improvement of functional carrot juice quality. Based on findings being reported here, a study of other quality attributes of CJPL and CJPP formulated using increasing concentration of phenolics from respective orange by-product extracts and treated using optimal thermosonication conditions, showed that thermosonicated CJPL and CJPP, besides having improved functional quality, also showed better storage shelf life, sensory quality, titratable acidity, pH, browning index and viscosity (Adiamo, Ghafoor, AL-Juhaimi, Muhamed Ahmed, & Babiker, 2017). The 19

thermosonication process can also effect vitamins such as vitamin C and mostly a retention or slight reduction of its contents have been reported in various fruit and vegetable juice (Anaya-Esparza et al., 2017). Martínez-Flores et al. (2015) reported a 100% retention of vitamin C in thermosonicated carrot juice during 20-day storage. Furthermore, improvement in bioactive compounds, microbial stability and other physico-chemical properties were also reported in sonicated, blanched (Jabbar et al., 2014) and thermosonicated (Martínez-Flores et al., 2015; Jabbar et al., 2015) carrot juice. Hence, the optimization of thermosonication process for various quality attributes can be effectively utilized to improve quality and health benefits of functional carrot juice.

4. Conclusion This study showed that the inclusion of orange peel and pulp phenolic enriched extracts to carrot juice enhanced its phenolic contents and antioxidant activity. The thermosonication process variables such as time and temperature were observed to have main effects on the total phenolic content and DPPH scavenging activity of the functional carrot juice. Higher amounts of phenolics and antiradical activity were obtained in juice containing peel than that with pulp at their optimized thermosonication process conditions using overlaid contour plot. Thermosonication of carrot juice with orange peel extract at optimal conditions resulted in no detectable microbial growth. Furthermore, thermosonication improved the phenolic contents and antiradical activity of the functional carrot juice as compared to control (thermally pasteurized) juice samples. Thermosonication and process optimization techniques for various quality attributes of functional carrot juice can be effectively utilized for maximizing health benefits of functional foods.

20

Acknowledgement The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research [the Research Group no. RG-1435-049]. Conflict of interest: Authors declare no conflict of interest.

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26

Figure Captions:

Fig. 1. Response surface plot of the TPC (mg GAE/100 mL) of CJPL and CJPP as (a, d): a function of ultrasonic temperature and time; (b, e): a function of ultrasonic volume and time; (c, f): a function of ultrasonic volume and temperature. TPC: total phenolic content; CJPL: Carrot juice with orange peel extract; CJPP: Carrot juice with orange pulp extract Fig. 2. Response surface plot of the DPPH scavenging activity (% inhibition) of CJPL and CJPP as (a, d): a function of ultrasonic temperature and time; (b, e): a function of ultrasonic volume and time; (c, f): a function of ultrasonic volume and temperature. CJPL: Carrot juice with orange peel extract; CJPP: Carrot juice with orange pulp extract Fig. 3. The overlaid contour plots of TPC and DPPH scavenging activity of ultrasound process conditions of a) CJPL and b) CJPP as effected by ultrasound process time (X1) and temperature (X2) at fixed juice volume (125 mL). TPC: total phenolic content; CJPL: Carrot juice with orange peel extract; CJPP: Carrot juice with orange pulp extract

27

a

d

28

b

e

29

f

c

CJPL

CJPP

Fig. 1. Response surface plot of the TPC (mg GAE/100 ml) of CJPL and CJPP as (a, d): a function of ultrasonic temperature and time; (b, e): a function of ultrasonic volume and time; (c, f): a function of ultrasonic volume and temperature. CJPL: Carrot juice with peel; CJPP: Carrot juice with pulp

30

a

d

31

b

e

32

f

c

CJPL

CJPP

Fig. 2. Response surface plot of the DPPH activity (% inhibition) of CJPL and CJPP as (a, d): a function of ultrasonic temperature and time; (b, e): a function of ultrasonic volume and time; (c, f): a function of ultrasonic volume and temperature. CJPL: Carrot juice with peel; CJPP: Carrot juice with pulp

33

Design-Expert® Software Factor Coding: Actual Overlay Plot

a

Overlay Plot 70

DPPH: 24.2526

X1 = A: Time X2 = B: Temperature Actual Factor C: Volume = 125

B: Temperature (degree Celsius)

TPC DPPH Design Points

DPPH: 69.2814 TPC: 24.6244 65

60

TPC: 30.2633 DPPH: 61.2212 X1 6.49607 X2 52.784

55

50

5 5

6

7

8

9

10

A: Time (min)

b

Design-Expert® Software Factor Coding: Actual Overlay Plot

Overlay Plot 68

TPC DPPH Design Points

Actual Factor C: Volume = 125

B: Temperature (degree Celsius)

X1 = A: Time X2 = B: Temperature

DPPH: 27.3698

63

TPC: 28.9448 DPPH: 55.8692 X1 5.04001 TPC: 29.0163 X2 59.9948

DPPH: 63.4728

58

53

5

48 3.5

4.5

5.5

6.5

7.5

8.5

A: Time (min)

Fig. 3. The overlaid contour plots of TPC and DPPH activity of ultrasound process conditions of a) CJPL and b) CJPP as effected by ultrasound process time (X1) and temperature (X2) at fixed juice volume (125 ml). CJPL: Carrot juice with peel; CJPP: Carrot juice with pulp

34

Table 1 Box-Behnken experimental design of process variables for optimization of phenolic compounds and antiradical scavenging activity for thermosonicated CJPL and CJPP. Sonication condition

CJPL

CJPP

Time

Temp.

Vol

TPC

DPPH

TPC

DPPH

Runs

(min)

(ºC)

(ml)

(mg GAE/100ml)

(% inhibition)

(mg GAE/100ml)

(% inhibition)

1

10

50

150

27.81±1.09

30.67±2.16

26.27±0.98

40.99±2.88

2

10

40

125

27.48±1.02

69.28±5.43

27.70±0.69

48.26±11.66

3

5

40

125

27.30±1.04

45.43±5.33

26.34±1.05

44.36±14.19

4

7.5

60

100

29.95±0.31

50.41±1.63

28.21±1.89

41.36±9.43

5

7.5

50

125

29.86±1.73

61.58±1.44

27.79±0.22

48.78±6.94

6

7.5

60

150

28.52±2.09

54.05±4.96

27.66±0.71

42.19±6.40

7

7.5

50

125

29.76±0.96

62.92±3.50

27.79±1.23

47.46±11.43

8

5

50

150

29.94±0.31

58.80±4.92

27.71±1.55

63.47±2.31

9

7.5

40

150

29.34±0.31

68.66±8.91

28.19±1.22

43.12±1.87

10

7.5

50

125

30.01±0.29

62.30±2.48

27.86±1.08

49.90±2.30

11

10

50

100

27.77±1.53

49.42±5.28

26.51±1.72

55.45±10.19

12

7.5

40

100

28.3±0.35

56.59±1.98

28.60±0.79

48.80±7.94

13

7.5

50

125

29.94±3.09

60.87±5.78

27.77±2.49

48.52±3.15

14

5

50

100

30.80±1.80

25.27±3.54

28.10±0.34

53.86±16.66

15

5

60

125

29.93±0.98

63.13±5.42

29.02±1.83

56.71±3.13

16

7.5

50

125

29.82±1.51

63.73±4.89

27.81±1.79

49.92±4.03

17

10

60

125

24.62±0.16

24.25±2.50

24.48±0.87

27.37±10.73

CJPL: Carrot juice with orange peel extract; CJPP: Carrot juice with orange pulp extract; TPC: Total phenolic content; DPPH: 1,1-diphenyl-2-picrylhydrazyl

35

Table 2 Regression coefficients of predicted quadratic polynomial models for TPC and DPPH of sample CJPL and CJPP CJPL

CJPP

Coefficient

TPC1

DPPH

TPC

DPPH

β0

29.876a

62.279a

27.804a

48.916a

β1

-1.287a

-2.375b

-0.775a

-5.37a

β2

0.067d

-6.015a

-0.122b

-3.072a

β3

-0.161b

3.811c

-0.259c

-0.777b

β12

-1.371a

-15.683a

-1.473a

-8.218a

β13

0.226b

-13.068a

0.037d

-6.017a

β23

-0.601c

-2.105d

0.086d

-2.282c

β11

-1.255a

-14.073a

-0.909a

0.316d

β22

-1.289a

2.316b

-0.010d

-5.149a

β33

0.458c

-7.166c

0.249c

4.213a

Linear

Interaction

Quadratic

‘a’

means p<0.0001, ‘b’ means p<0.05, ‘c’ means p<0.01, ‘d’ means p>0.05

CJPL: Carrot juice with orange peel extract; CJPP: Carrot juice with orange pulp extract; TPC: Total phenolic content; DPPH: 1,1-diphenyl-2-picrylhydrazyl

Table 3 36

Microbiological characteristics, total phenolic content, DPPH scavenging activity and total carotenoid content of thermosonicated CJPP (125 mL, 60°C, 5 min) and CJPL (125 mL, 52°C, 6.5 min) as well as pasteurized (75 ºC, 2 min) CJPP and CJPL Analysis TBC

Treatment Thermosonicated Pasteurized

CJPP ND ND

CJPL 1.85 ± 0.02 ND

YMC

Thermosonicated Pasteurized

ND ND

1.70 ± 0.10 ND

TPC

Thermosonicated Pasteurized

29.13 ± 0.73a 26.58 ± 0.12b

32.54 ± 0.15a 28.58 ± 0.16b

DPPH

Thermosonicated Pasteurized

59.02 ± 0.31a 56.83 ± 0.78b

60.56 ± 0.12a 57.92 ± 0.23b

TC

Thermosonicated Pasteurized

7.60 ± 0.04a 7.66 ± 0.05a

7.21 ± 0.12a 7.44 ± 0.19a

Values are means of triplicate samples (±SE). Means not sharing a common superscript(s) in a column for each analysis are significantly different at P ≤ 0.05 as assessed by Duncan's Multiple Range Test. CJPL: Carrot juice with orange peel extract; CJPP: Carrot juice with orange pulp extract; TBC: Total bacteria count (log cfu/ml); YMC: Yeast and Mold count (log cfu/ml); TPC: Total phenolic content (mg GAE/100 ml juice); DPPH: 1,1diphenyl-2-picrylhydrazyl (% inhibition); TC: Total carotenoid (mg β carotene/L juice); ND: Not detectable

37

Highlights -

Extract of orange peel has higher total phenolic content (TPC) than pulp extract.

-

Temperature and time are important for thermosonication of functional carrot juice

-

Juice with orange peel extract has highest TPC and DPPH at 59.99°C and 5.04 min

-

Juice with orange pulp extract gave maximum response values at 52.78°C and 6.50 min

-

Thermosonication enhanced the TPC and DPPH of the juices compared to pasteurization

38