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0INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
INFLUENCE OF THERMAL PASTEURIZATION ON POMEGRANATE AND SWEET ORANGE MIX JUICE BIOACTIVE COMPONENTS, COLOR, AND MICROBIOTA
1Anjali Ashokrao Bhoite, 2Shivaji Jagannath Sathe, 3Nilesh Nivruti Gaikwad
1Professor MITADTU 2Associate Professor, TC College 3Scientist, NRCP https://doi.org/10.5281/zenodo.13842070 Abstract. Present study deals with the effect of juice mixing (Pomegranate: Sweet orange 70:30) and various pasteurization temperature treatments (70 OC, 80 OC and 90 OC for 10 min) on bioactive components. At every applied temperature, thermal effect on Total Phenolic Compounds, (TPC) FRAP antioxidant content, anthocyanins, and ascorbic acid content, color, and the microbial load were analysed using standard methods. interaction factor (IF ) was determined by Gawlik-Dziki method. With a rise in temperature, an increase in TPC and antioxidant property , decrease in anthocyanin and ascorbic acid and decrease in all color parameters was reported in all juice samples. The Pearson correlation coefficient values of TPC and antioxidant activity for pomegranate juice and mix juice was found 0.77 and 0.95 respectively. The IFfactor study shows synergistic effect. The microbial load was found almost nil after pasteurization temperature 70 OC
Keywords: FRAP, TPC IF, Ascorbic acid, Anthocyanin, Pasteurization.
Introduction
Consumer demand for healthy food products has propelled the growth of the functional food industry and in turn the dynamic growth of the fruit juice industry. Fruit juices are now consumed as a part of daily breakfast to satisfy the daily requirement of minerals and vitamins, The main health benefits from juice consumption include, increased antioxidant capacity, reduced low-density lipoprotein oxidation, and improved cardiovascular and neurocognitive function, anti-inflammatory, anti-proliferative, anti-carcinogenic, and anti-microbial properties. The variant fruit juices are more preferred because of more nutritional content, and new organoleptic properties. This trend leads to cause the manufacturers developing fruit juices as functional beverages by enriching them with health beneficial components which include the addition of vitamins, minerals, probiotics. As per the global fruit and vegetable market report 2018-2025 the blending of juices is the natural way of value addition and formulations of functional juices are in high demand. The trend for natural and minimally mix/ blend juice consumption is more with few of the developed countries such as the United States, Canada, and Europe. Mixing different fruit juices without the addition of synthetic substances, which allows for attractive products with the content of bioactive substances is a more preferred processing method used to satisfy consumer demand. This could lead to more opportunities for fruits which are with significant aroma, taste but less health-promoting compounds. [22,42,36]. Fruit and vegetables are characterized by high amounts of phenolic compounds that offer a wide range of health benefits. The antioxidant property associated with fruits and vegetables is mainly attributed due to phenolics, they offer protection against diseases and oxidative damage. This leads to the interest of manufacturers towards high antioxidant activity enriched beverages and foods [9, 28]. The beverage compositions
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
of different fruits can have different antioxidant capacities which may be due to the different content and diversity of polyphenol compounds. The mixing of juices causes the interaction between polyphenol constitutes and there exist several kinds of interactions, either synergistic (at target sites of action) or antagonistic (inhibitory of action) [19]. Gawlik-Dziki (2012) method is popular to determine the extent of interaction to quote the safety and health properties of any formulated juice mix.
Considering the need for the formulation of quality juice mix juice, the present investigation deals with the study of bioactive component interaction of mix fruit juice of two medical fruits pomegranate and sweet orange. Pomegranate being rich in polyphenols offers significant antioxidant activity but lacks flavour, while sweet orange is of intense flavour but less with phenolics and high with ascorbic acid may offer mix juice with characteristically appealing aroma and high antioxidant mix juice product. This study was carried out to evaluate the pasteurization temperature effect on bioactive constituents and also for standardization of the pasteurization temperature for mix juice of pomegranate and sweet orange. Further, this study aimed to verify whether the interaction between pomegranate juice and sweet orange mix juice at various pasteurization temperatures is significant to quote its preference as functional mix juice.
Materials and Methods
Fruit collection and juice extraction
The fully matured pomegranate (Punica granatum L.) fruits of var. Bhagwa grown in Hastabahar (particular season to get good quality mature fruits) were harvested in month of March 2018 from the farm of ICAR-National Research Centre on Pomegranate Solapur, India. The sweet orange fruit(Citrus Sinensis ZJ(Mosambi) var.nucellar were harvested from farmer's field located in Aurangabad, India. Pomegranate and sweet orange fruits were transported on the same day to the laboratory and stored at 5°C until used for the experiment. Fruits were washed with chlorine water (200 ppm sodium hypochlorite) and cut into two equal halves. The juice was extracted in hydraulic press (Johnston make) from halved fruits. The juice was stored at 5oC for 24 hours for sedimentation and filtered using muslin cloth. All the chemicals required for the experiments were purchased from the Hi media.
Thermal treatment
The clarified pomegranate juice , sweet orange juice and mix juice ( 70:30) pomegranate: sweet orange were heat-treated (pasteurized) at 70,80, 90, °C ± 1°C for 5, and10 minutes. The juice is then packaged in glass bottles while still hot. The bottles were then allowed to cool down at ambient. The juice bottles were stored in a cold room (5oC) until used for analysis.
Bioactive compound assessment
Ascorbic acid in juices was determined using AOAC 2000 method based on the reduction of 2,6 di-chlorophenol indophenol by L-ascorbic acid[17]. Total phenolic compounds were determined by Folin-Ciocalteu (FC) colorimetric method at 765 nm as described by Singleton and Rossi (1965). The results were expressed as mg of Gallic acid equivalent (GAE) per liter of the sample. Total anthocyanin contents were estimated by a pH-differential method using two buffer systems, 0.025 M potassium chloride buffer at pH 1.0 and 0.4 M sodium acetate buffer at pH 4.5 using the spectrophotometric method at 510 and 700 nm wavelengths [41]. The antioxidant activity was determined by performing FRAP assay reported by (Benzie and Strain 1996 with slight modifications. All the juice samples (raw juices and juices treated at various temperature) were anlayzed to know the effect of temperature on these bioactive components.
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Microbiological analysis
Spread plate method was used to determine Colony-forming units (CFU). Decimal dilutions from untreated juice and thermally treated juice were made in sterile buffered peptone water and then 0.1 mL volumes of appropriate dilution were plated on sterile nutrient agar plate and plates were incubated at 37 oC for 24 hours and colonies were counted using colony counter max electronics, India.[1]
Colour measurement
Aliquots of 25 ml of pomegranate juice sample were transferred into a plastic beaker, covered and the color parameters were determined using a Lab Scan XE of hunter Lab, USA colorimeter [32] and expressed in dimensions of L*, a*, b*, C. The average values of triplicate readings were reported for each sample. Values of L*- indicate darkness and L*+ indicate lightness of sample color, while a*-indicates green color and a*+ indicates red color. The b*+ indicates a yellow color and b*- indicates blue color. The chroma (C) value is calculated as C = (a*2 + b*2)% and indicates the color intensity or saturation. Visual color appearance is determined by measuring Hue angle H° is a parameter is calculated as H° = tan-1 (b*/a*) (Solomon et al., 2006). The color index was calculated from (180 - H°)/(L* + C) [ 37,32].
Theoretical approach
The interaction factor (IF) factor was also determined according to the method described by [11,9]. IF which explains the mode of interaction: IF XA AM=AT where AM = measured the activity of a mixture of samples, and AT = theoretically calculated mixture activity (based on the dose-response of single components at various concentrations)
Statistical analysis
Results were presented as a mean ± standard deviation of three independent determinations. Experimental data were analysed for analysis of variance (ANOVA) and differences between means were assessed by Duncan's new multiple range test at the significance defined p< 0.001using SAS 9.3 software.
Results and discussion
Total Phenolic content
The phenolic content (TPC)of juices being a major important bioactive constituent is a determinant of the nutritional quality of juices, hence during the pasteurization along with the microbial destruction the reduction in phenolic compounds is also a major concern. To quote the effect of selected temperature ranges on total phenolic content three fruit juices treated at different temperatures and obtained resulted are presented in fig.1a(table 1), results indicates a significant increase in phenolic content in response to applied temperatures. An increase in phenolic content might be due to the heat-induced biochemical reactions which usually develop new phenolic compounds and also release bound phenolic compounds due to thermal effect [30]. The bound phenolic acids usually form bonds with structural carbohydrates and protein either through ester linkage with carboxylic groups or ether linkages with lignin through their hydroxyl groups in the aromatic ring or acetal bonds [21]. Application of heat may lead to breaking these bonds and in turn facilitates the release of phenolic compounds due to cell disruption and rupture of the food matrix. [18]. It is also due to the breakdown of polymeric phenolic compoundsand heat-induced molecular rearrangement of phenolic compounds [33,23].These results are in agreement with Ucan et al .,2016. However number of studies have reported the decrease in phenolic compounds because of the heat-labile phenolic destruction, hence the composition of the phenolic content of
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
juices is itself determining factor towards the thermal effect. [7]. Previous studies have revealed the increase in ellagitannin especially the increase in punicalagin after thermal treatment [13] could be the reason for the significant increase in phenolic content. Due to pasteurization, the amounts of extractable phenolic substances increased in all three juice samples, which might be due to the destructive effect of heat treatment on the cell membrane that membrane becomes pores which increases the mess transfer across the cell membrane. [3]. Ellagitannins being a noncolored phenolic not only contribute to the increase in phenolics but also in lowering the color quality of juices. ( lower L *and b*) value during each heat treatment.
Antioxidant activity
The antioxidant activity is mainly attributed due to the presence of phenolic compounds. In the present study, the antioxidant activity of pomegranate juice and sweet orange juice is conferred due to vitamin C and TPC. The values for total phenolic compounds and antioxidant activity and vitamin C for three different juices at various temperature is presented in fig 1b.(table 1).Larrauri et al have described the negative effect of thermal treatments on the antioxidant activity of fruit juices. In present study only sweet orange juice reported with negative correlation on antioxidant activity with an increase in temperature, this may be because of high vitamin C in sweet orange as major contributory in its antioxidant activity rather than phenolic content. The decrease in antioxidant activity concerning the loss of vitamin C of sweet orange was following first-order reaction kinetics. As per obtained results, the antioxidant activity of another two juices, pomegranate, and mix fruit juice is mainly associated with its TPC. The significant positive effect of temperature noticed on the antioxidant activity of this juice could, therefore, be correlated to the significant positive effect on its TPC. This study also highlights on the positive correlation between phenolic content and antioxidant activity. he findings of[10,25,39], they reported such positive correlations between TPC and antioxidant capacity in various fruits and vegetables.
Anthocyanin
All three juice samples, raw and pasteurized were analysed for anthocyanin content(ACN) and resulted were presented in fig.1C ( table 1)Anthocyanin content in juices is also most significant as they are contributory to the color of the juice and nutritional status. Temperature treatments cause more oxidation reactions, and cleavage of covalent bonds which causes the loss of anthocyanin especially studies have reported due to the formation of chalcone because of high temperature or loss of glycosyl moieties [24,27]. Similar results were obtained in the present study, which also highlights the correlation of anthocyanin degradation with loss of color. The stability of anthocyanin depends upon many factors besides heat, such as pH, storage temperature, the chemical structure of the anthocyanin compound, presence of UV light, oxygen, oxidative and hydrolytic enzymes, proteins and phenolic compounds that could have a protective effect, and the metallic ions that could enhance oxidation [14]. Heat causes the interaction of free radicals which in turn promotes the oxidation reaction also the presence of other organic acid or ascorbic acid leads to degradation of anthocyanin[29].
Ascorbic acid
The concentration of vitamin C ( ascorbic acid) orange juice and pomegranate juice as well is one of the most important attributes for the consumer. It is also considered an indicator of the nutritional quality of juices[6] and being a heat-sensitive bioactive compound, in this study it is important to detect the effect of thermal pasteurization on the ascorbic acid content. Therefore, the ascorbic acid content of raw and pasteurized juice samples was analysed and obtained resulted
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
were presented in fig.1d( table 1).The sweet orange was found to contain a high amount of ascorbic acid then pomegranate juice, mix juice ( pomegranate: sweet orange 70:30) therefore found to be enriched with ascorbic acid. The present study reported a decrease in ascorbic acid content with an increase in temperature in all three juices. Loss of ascorbic acid during long-term storage is also might be due to atmospheric oxygen[26]. It is also facilitated due to the effect of light, heat peroxides and enzymes such as ascorbate oxidase and peroxidase [8]. However, major factors such as storage temperature, method of processing and packaging materials affect the rate of ascorbic acid reduction [4].
Influence of thermal pasteurization on pomegranate and sweet orange mix components, color, and microbiota
Fig 1a
The values are mean of triplicates and vertical bars indicates SE. Means with different letters are significantly different from each other at P< 0.05according to Duncan's Multiple range test
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TEMPERATURE TREATMENTS FOR JUICE JUICE SAMPLES
Fig. 1b
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The values are mean of triplicates and vertical bars indicates SE. Means with different letters are significantly different from each other at P< 0.05according to Duncan's Multiple range test.
Anthocyanin content of juices at diffetent temperatures
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TEMPERATURE TREATMENTS FOR JUICE SAMPLE
Fig 1c
The values are mean of triplicates and vertical bars indicates SE. Means with different letters are significantly different from each other at P< 0.05according to Duncan's Multiple range test.
Ascorbic acid content of juices at different temperatures
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TEMPERATURE TREATMENTS FOR JUICE SAMPLES
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INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
The values are mean of triplicates and vertical bars indicates SE. Means with different letters are significantly different from each other at P< 0.05according to Duncan's Multiple range Test.
Figure 3.2. Studies on the effect of pasteurization temperature on bioactive constituents.1a) Effect of temperature on total phenolic content 1b) Effect of temperature on antioxidant content1c) Effect of temperature on anthocyanin content 1d) Effect of temperature on ascorbic acid content.
Colour analysis
The colour of fruit juice is an important determinant of the quality parameter which is governed by the presence of anthocyanin content of juices. The color parameters, hunter L, a* and b*were determined for all three juice samples (raw as well as thermally treated) and presented in table 2a. These parameters are the physical characteristics for indication of visual color.. L* parameter is an approximate measurement of luminosity. According to L* each colour can be considered as equivalent to a member of the greyscale that is between black and white [16]. The obtained results shows decrease in the L* value (i.e. the 'lightness' value) with rise in temperature in the case of all juice samples. The decrease in color is might be due to the loss of anthocyanin content which also indicates the formation of the coloured product. The reason for this change is not only non-enzymatic browning but also the unstable and suspended particles that cause partial precipitation in juice may lead to the depletion of L values. [12] .The parameter a* takes positive values for reddish colours and negative values for the greenish ones the positive a* value, which corresponds to red hue, was increased as the temperature of pasteurization increase in all juice samples. This indicates that the color becomes redder. On the contrary, b* takes positive values for yellowish colours and negative values for the bluish ones, positive b* value which is a measure of a yellow hue, not affected at different temperatures in case of pomegranate juice, while increases in sweet orange and mix juice. The overall decrease in all color parameters in all three juice samples at all applied pasteurization temperatures is as per the [31]. As presented in Table 2b, the hue angle of untreated juice and thermally treated juice was almost constant throughout the thermal treatment for pomegranate juice while it increases in sweet orange and mix fruit juice. C ho values of all thermally treated three different juice samples indicates, juice become less reddish and more yellow when the hue increases [7]. hue angle values indicate no effect of temperature on the color change in pomegranate juice while a more yellow character with sweet orange and mix fruit juice. Higher h values indicate yellowing of the sweet orange and mix fruit juice samples. The increase in ho values indicates anthocyanin degradation. However, Chroma (C*) is the quantitative attribute of colourfulness, is used to determine the degree of difference of a hue in comparison to grey colour with the same lightness. In the present investigation higher the chroma values indicate the higher colour intensity of all juice samples.
Hue angle (h*),is the attribute according to which colours have been categorised as reddish, greenish, etc., In the present study, a lower hue angle in sweet orange represents a more yellow character, while it was less in pomegranate and mix juice. The rise in temperature characteristically does not affect the yellow character.
The data represent the means ± SD of triplicate analyses from each trial. DMRT was used to determine the significance of differences as identified by different letters in the same column. Values of L*- indicate darkness and L*+ indicate lightness of sample color, while a*-indicates green color and a*+ indicates red color. The b*+ indicates a yellow color and b*- indicates blue color.
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Table 2 a) Effect of temperature treatments on the color quality of juice samples.
Type of juice Pomegranate Juice Sweet orange Juice Mix juice
L * a * b * L * a * b * L * a * b *
Raw juice 24.88+0. 0057 59.78+ 0.015 38.06+ 0.026 68.73+ 0.04 -2.4 23.71+ 0.020 36.43+ 0.098 43.21+ 0.14 28.39+ 0.11
70 23.16+0. 077 55.13+ 0.13 37.21+ 0.32 62.83+ 0.050 1.785 21.74+ 0.01 33.58+ 0.026 43.88+ 0.015 21.60+ 0.015
80 19.17+0. 020 53.44+ 0.015 37.64+ 0.020 63.15+ 0.080 1.265 22.65+ 0.01 31.59+ 0.017 42.86+ 0.032 20.71+ 0.026
90 16.13+0. 025 53.86+ 0.011 37.81+ 0.030 59.65+ 0.065 1.113 25.84+ 0.028 28.95+ 0.025 42.69+ 0.011 18.81+ 0.030
Table 2 b) Colour Quality parameters of all juice samples
Pomegranate uice Sweet lime juice Mix Juice
Temperature (oC) Croma value Hue angle Colour index Croma value Hue angle Colour index Croma value Hue angle Colour index
Raw juice 62.66 0.65 2.05 13.92 -1.40 2.19 46.96 0.40 2.15
70 64.36 0.64 1.84 21.78 -1.49 2.15 48.91 0.46 2.18
80 65.37 0.61 1.90 22.69 -1.52 2.11 50.74 0.49 2.18
90 73.34 0.64 1.80 25.84 1.56 2.16 59.19 0.72 2.03
Microbial inactivation
The effect of thermal processing on microbial inactivation is compared to all untreated juices and obtained results are presented in Table 3.Mild-temperature pasteurization (MTP) treatment 70 oC significant inactivation was found within 10 minutes, but only the high-temperature pasteurization (HTP) treatments 80 oC and 90 oC resulted in a nil microbial count within 10 minutes. Exposure to high temperatures (strong stresses) leads to lipid phase transitions and protein conformation changes which increases the cell membrane permeability eventually causing cell death. However, membrane fluidity changes may vary significantly, according to the extent of thermal stress [15]. Pomegranate juice is a low PH Juice, H >PH 4.5 requires stronger treatments to achieve the desired shelf life.
Table 3 Determination of interaction factor(IF) for various bioactive components of mix juice
at various temperatures.
Total phenolic content mg/L of GAE FRAP mg/ 100 ml of AAF Anthocyanin mg/100ml of cyanidin Ascorbic acid mg/100 ml
Heat treatm ent Theo retica l value Obs erve d valu e fold chan ge Theo retica l value Obs erve d valu e fold chan ge Theo retica l value Obs erve d valu e fold chan ge Theor etical value Obse rved value fold chan ge
Raw juice 912.9 0 134 0.66 0.68 14.72 18.5 0 0.80 17.35 18.5 0 0.94 47.95 35.83 1.34
70 oC 1119. 10 141 7.33 0.79 16.16 14.2 2 1.14 16.48 16.0 3 1.03 40.10 36.66 1.09
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80oC 1537. 50 164 2.33 0.94 13.29 19.1 2 0.70 12.97 16.4 2 0.79 37.99 30.83 1.23
90 0 C 1490. 40 220 1.33 0.68 16.09 20.5 1 0.78 17.22 17.8 8 0.96 38.58 30.43 1.27
A theoretical approach for the tested products
Table 3 shows interactions between bioactive compounds in pomegranate juice and sweet orange juice when they mix in 70:30 proportion and treated at various temperatures for 10 minutes. These interactions are determined by calculating the interaction factor (IF), which estimates the power of interaction. It is a simple way of defining the type of interactions between chemical compounds or extracts in processing. Their main advantage is testing between any number of parameters in the products[11].In all juices the IF value was lower than 1 and a synergistic interaction was found. The rise in temperature significantly affects the strength of interaction with all analysed parameters except ascorbic acid. According to Durak et al. (2015), the IF value of coffee and ginger (1:1 mixture) was 0.64 indicating a synergistic interaction. Furthermore, as per studies of[11] interaction factors of mixtures of vegetables revealed strongest synergistic interaction was reported for tomato and garlic mixture based in IF 0.11. while lowest synergistic interaction was in a tomato and onion mixture(IF = 0.80). In the present study, the lowest synergistic effect was reported concerning TPC, antioxidant activity and anthocyanin content. No interaction exists concerning ascorbic acid content at all applied temperatures in all three juice samples. The mixed juice of pomegranate and sweet orange ( 70:30) shoes more positive interaction with an increase in temperature while weak anthocyanin interaction (0.9 at 90 oC) exists with the anthocyanin as degradation of anthocyanin occurs at high temperature.
Conclusion
The present study recommends the use of 80 oC for 10-minute pasteurization treatment for healthy and safe mix juice. The obtained information may be used by the juice industry as a starting point for producers of natural attractive juice mixtures in India. The pomegranate and sweet orange fruit juice mix are also proven effective health-promoting juice. Present research boosted more research with different fruits regarding the formulation of mix juice and their phenolic interaction. This study also facilitates the application of sweet orange in the preparation of mix functional juice.
