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Optimization of time-electric field combination for PPO inactivation in sugarcane juice by ohmic heating and its shelf life assessment
LWT - Food Science and Technology 71 (2016) 329e338
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Optimization of time-electric field combination for PPO inactivation in sugarcane juice by ohmic heating and its shelf life assessment Juhi Saxena, Hilal Ahmad Makroo, Brijesh Srivastava* Department of Food Engineering & Technology, School of Engineering, Tezpur University, Assam, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 January 2016 Received in revised form 8 April 2016 Accepted 11 April 2016 Available online 13 April 2016
The effect of ohmic heat (OH) treatment was measured on polyphenol oxidase (PPO) activity in sugarcane juice under different electric field strengths (24, 32 and 48 V/cm) and holding times (0.25, 0.50, 0.75, 1.0, 1.25 min) at a temperature of 80 ± 2 C, optimized by conventional thermal (CT) treatment. The optimum temperature of PPO inactivation for CT-treatment was determined in a parallel study. The processing condition of 32 V/cm and 1 min holding time was found optimum and was analyzed for titrable acidity (TA), reducing sugars (RS), ascorbic acid (AA) and microbial load for 10 and 30 days at room and refrigeration temperatures respectively. During refrigerated storage, TA and RS remained significantly (p < 0.01) unchanged and the AA degradation was more pronounced at room temperature. Both treatments resulted in significant microbial reductions but growth resurfaced after 5th and 25 th day of room and refrigeration storage respectively. No yeast and mold growth was witnessed after OH-treatment. Overall, the OH-treatment was found to inhibit PPO enzyme activity in a shorter processing time than CT, while maintaining the potential quality attributes of the juice. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Sugarcane juice PPO activity Ohmic heating Optimization Leuconostoc mesenteroides
1. Introduction Numerous investigations have been carried out to devise a treatment that can increase the shelf life of sugarcane juice to allow its entry into the mainstream juice processing industries. Thermal processing is the most common method studied for sugarcane juice preservation (Chauhan, Singh, Tyagi, & Balyan, 2007; Mao, Yong, & Fei, 2007; Sangeeta, Hathan, & Khatkar, 2013). Although thermal treatment is very effective for microbial and enzyme inactivation, the use of high temperatures (80e90OC) is known to cause offflavor and discoloration in the processed product (Wang & Sastry, 2002). In the past decade, novel techniques like high pressure processing, pulsed electric field, microwave treatment, ohmic heating etc. have been studied for their application on various food products (Barba, Calabretti, d’Amore, Piccinelli, & Rastrelli, 2008; Castro, Macedo, Teixeira, & Vicente, 2004; Icier, 2005; Ohshima, Tamura, & Sato, 2007; Terefe, Yang, Knoerzer, Buckow, & Versteeg, 2010). Ohmic heating (OH) has gained wide popularity as an alternative thermal treatment as it causes volumetric heating of the sample
* Corresponding author. Department of Food Engineering & Technology, School of Engineering, Tezpur University, Assam 784028, India. E-mail address: [email protected] (B. Srivastava). http://dx.doi.org/10.1016/j.lwt.2016.04.015 0023-6438/© 2016 Elsevier Ltd. All rights reserved.
which leads to consistent and rapid heat generation especially in liquid foods. The rate of heat generation in OH is a function of electric field strength applied across the food material (Ramaswamy, Marcotte, Sastry, & Abdelrahim, 2014). Due to short processing times, OH causes minimum discoloration and maintains the nutritive value of the food (Leizerson & Shimoni, 2005; Wang & Sastry, 2002). This feature makes it one of the most desirable treatments particularly for sugarcane juice; as it contains sensitive flavor components that are easily destroyed at longer treatment times. Despite the problems of electrochemical degradation associated, OH has been successfully studied for its use in preheating, blanching, and extraction (Lakkakula, Lima, & Walker, 2004; Leizerson & Shimoni, 2005; Lima & Sastry, 1999). The major focus of most studies has been on commercial products like strawberry pulp, grape juice etc (Castro et al., 2004; Icier, Yildiz, & Baysal, 2008). Sugarcane juice is a proven health promoting food which is primarily known to fight cancer and prevent kidney problems (Singh, Gaikwad, & More, 2014). However, PPO browning and microbial spoilage have limited the storage of fresh juice to just a few hours. Over the years, sugarcane juice has been investigated for its shelf life by blending it with curd, lime juice and other preservatives (Khare, Lal, Singh, & Singh, 2012; Sneh, Chaturvedi, Kuna,
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Table 1 Processing times for different combinations of CT and OH-treatments. Treatment type
EFS
Holding temp. ( C)
Heating time (min)
Holding time (min)
Total processing time (min)
CT
e e
60 ± 1 70 ± 1
3.80 4.90
5,10,15,20 5,10,15,20
8.8, 13.8, 13.8, 18.8, 23.8 9.9, 14.9, 19.9, 24.9
e
80 ± 1
6.00
5,10,15,20
11.0, 16.0, 21.0, 26.0
e 24 32 48
90 ± 1
7.00
5,10,15,20
12.0, 17.0, 22.0, 27.0
80 ± 2 80 ± 2 80 ± 2
3.25 1.50 0.45
0.25, 0.5, 0.75, 1.0, 1.25 0.25, 0.5, 0.75, 1.0, 1.25 0.25, 0.5, 0.75, 1.0, 1.25
3.5, 3.75, 4.0, 4.25, 4.5 1.75, 2.0, 2.25, 2.5, 2.75 0.7, 0.95, 1.2, 1.45, 1.7
OH
& Dhanlakshmi, 2012), however, limited literature is available on the application of a novel technique such as OH to increase the shelf life of the product. Although the rate of heating under OH and conventional thermal (CT) are different, the present study was conducted to explore the option of an alternative treatment for sugarcane juice processing. Therefore, the objectives of this investigation were to determine the influence of OH on PPO inactivation in sugarcane juice under a wide range of electric field strengths and compare the observations with those of the CT treatment. Shelf-life studies were also conducted for the optimized treatments and quality attributes like titrable acidity (TA), ascorbic acid (AA), reducing sugars (RS) and microbial count was studied. 2. Materials and methods 2.1. Sample preparation Fully mature sugarcane stems of ‘Pharma’ variety were procured from the farms in Sonitpur, Assam, India. The stems were peeled, manually cut and crushed in ‘Usha’ Food Processor (make: FP2663, India) to yield the juice. The juice was subsequently filtered through four-folds of muslincloth to obtain a clear filtrate which was used throughout the study. Fresh juice was extracted prior to each treatment.
2.3. OH- treatment The OH-treatment was applied in a lab scale ohmic heater whose setup (Fig. 1) includes one movable and one stationary stainless steel electrode (grade 316) of 1.5 mm thickness and 25 mm diameter enclosed in a hollow cylindrical casing of Teflon with an outer and inner diameter of 50 mm and 25.5 mm respectively. An opening of 5 mm diameter was made on the surface, at a distance of 50 mm from each electrode for the feed and a tefloncoated thermocouple was inserted to measure the temperature at the centre of the cylinder. A power supply of 240 V and 50 Hz frequency was used to carry out the experiments. The OH-treatment was employed at three different electric field strengths (24, 32 and 48 V/cm) for five holding times (0.25, 0.50, 0.75, 1.0, 1.25 min). The electric field strengths were maintained by adjusting the distance between the two electrodes. Thus, the volumetric capacity of the equipment varied from 50 ml at 24 V/cm, 35 ml at 32 V/cm and 25 ml at 48 V/ cm. A digital temperature controller-cum-indicator was used to maintain the temperature of the juice at 80 ± 2 C (optimized temperature of CT-treatment, discussed in section 3.3). The heat-up time to reach 80 ± 2 C at different electric field strengths is shown in Table 1. The samples were held at 80 ± 2 C for the specified holding times. After both the treatments, the samples were immediately cooled and analyzed for their residual PPO activity.
2.2. CT-treatment The CT-treatment was carried out to establish effective comparison between the OH-& CT-treatment results. A volume of 50 ml of juice samples was heated at sixteen different processing combinations of temperature (60, 70, 80 and 90 C) and holding time (5, 10, 15 and 20 min) in a lab scale water bath (BW-20G,JEIOTech, Korea). The heating, holding and total processing times are shown in Table 1.
2.4. Polyphenol oxidase (PPO) enzyme assay The assay of the enzyme was carried out as described by Ozoglu and Bayindirli (2002). One ml of 0.2 mol/L Catechol solution was added to mixture of 0.5 ml of sugarcane juice and 2 ml of phosphate buffer (pH 6.5). The absorbance was measured at 420 nm at every 1 min interval. The enzyme activity was estimated from the linear
Fig. 1. Schematic Diagram of Ohmic heating set up.
J. Saxena et al. / LWT - Food Science and Technology 71 (2016) 329e338 Table 2 Physico-chemical properties of fresh sugarcane juice.
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Table 3a Solutions for CT-treatment.
Physico-chemical parameters
Units
Analysis
Water content Total Solids Acidity TSS pH Ascorbic acid Reducing Sugars PPO activity
g/100 mL g/100 mL g/100 mL B e mg/100 mL g/100 mL Units/mL
81.05 18.95 0.16 19.6 4.42 4.44 0.66 69.40
± ± ± ± ± ± ± ±
0.30 0.42 0.02 0.10 0.07 0.13 0.02 0.24
Number
Temperature
Time
EA
Desirability
1 2 3
80 90 90
10 10 5
4.0 4 8.0
1.00 1.00 0.111
Selected
Table 3b Solutions for OH-treatment. Number
Electric field strength
Time
EA
Desirability
1 2 3
32 48 48
1 1 1.25
7 7 4.5
1.00 1.00 1.00
Selected
portion of the curve of absorbance v/s time. One unit of PPO activity was defined as 0.001A420/min. The activity of the samples was expressed as % Residual PPO Activity (RA) as given in Eq. (1):
% RA ¼
Current Enzyme Activity 100: Initial Enzyme Activity
(1)
2.5. Optimization of process variables Both CT- and OH-treatments were optimized taking timetemperature and time-electric field strength as the independent variables, respectively. Optimization of the variable levels was achieved by desirable minimization of the % RA along the fitted two factor interaction (2FI) model by numerical optimization procedure of the design expert software 8.1.0. The statistical criteria like R2value, standard deviation, F-value and p-value were used to determine the goodness of fit. 2.6. Data validation The optimized process variables for both CT- and OH-treatments were validated for the % RA and the effect was also measured on the quality attributes viz. TA(%), RS(%) and AA(%) as per sections 2.7.1 and 2.7.2. 2.7. Shelf life studies The juice treated at optimized conditions of CT- and OHtreatments was stored in pre-sterilized glass vials of 10 ml each at room (25 ± 2) C and refrigeration (6 ± 1) C temperatures. A parallel shelf life study was conducted for untreated juice at both storage temperatures during which %RA,RS,TA,AA and total plate count, yeast and mold count, Leuconostoc mesenteroides count and E. coli counts were investigated at a regular interval of 5 days. The properties of the fresh juice were also determined. Fig. 2. Residual PPO activity (% RA) during (a) CT at 60 (-), 70 (C), 80 (:), 90 (;) C and (b) OH-treatment at 24 (-), 32 (C) and 48 (:) V/cm.
% Titrable acidity ¼
2.7.1. TA and AA determination TA was assayed by manual titration (AOAC, 1995) and was calculated as percent titrable acidity by Eq. (2).
Titre NNaOH Vol:made up 64 100 : Vol: of sample taken for estimation Vol: of sample 1000
(2)
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Table 3c Validation of optimized CT- and OH-treatments. Sample
Quality attributes Residual PPO activity (%)
CT80/10 OH32/1 a
a
Titrable acidity (g/100 mL) a
a
0.23 ± 0.04 0.20 ± 0.04a
6.47 ± 0.25 10.07 ± 0.32a
Reducing sugars (g/100 mL) 1.94 ± 0.01 1.80 ± 0.01a
Ascorbic acid (mg/100 mL) 2.96 ± 0.07a 2.98 ± 0.30a
Means within a column with different letters are significantly different (P < 0.01).
AA was estimated by 2,6-dichlorophenol indophenol method (AOAC, 1995) and was calculated as
Ascorbic acid ¼ 200 ðml indophenol sol:Þ ðCVitamin =ml indophenol sol:Þ
Fig. 3. Variation in titrable acidity (
), ascorbic acid (
(3)
) and reducing sugars (
2.7.2. RS content determination The RS content were analysed by the method (Mao et al., 2007) using glucose as the standard reducing sugar and 3,5dinitrosalicylic acid as the developer. 2.7.3. Microbiological analysis The treated-juice samples were diluted in sterile 0.85 g/100 mL saline solution and the total plate counts were incubated in nutrient agar (Himedia) at 37 C for 24 h. The yeast and mold count were estimated by incubation in potato dextrose agar (Himedia) at
) of untreated juice during storage at (a) Room and (b) Refrigeration temperature.
J. Saxena et al. / LWT - Food Science and Technology 71 (2016) 329e338
Fig. 4. Variation in titrable acidity (
), ascorbic acid (
) and reducing sugars (
25 C for 5 days. L. mesenteroides were maintained on Mayeux, Sandine, & Elliker agar at 21 C for 4 days. Escherichia coli were enumerated on Eosin Methylene Blue agar (Himedia) at 37 C for 24 h. All the tests were conducted in triplicates and the results were calculated in terms of log CFU/ml. 2.8. Statistical analysis All the experiments were conducted in triplicates. The data obtained was subjected to two-way analysis of variance (ANOVA) with a significance level of 0.01 (a ¼ 0.01). Mean values with standard deviations were reported and student's t-test was used as the Post-hoc test.
333
) of (a) CT80/10, and (b) OH32/1 treated samples under room temperature storage.
properties summarized in Table 2. The initial activity of PPO was found to be 69.4 ± 0.24 units/100 ml; which was higher than 39.5 and 28 units as reported in sugarcane juice by Eissa, Shehata, Ramadan, and Ali (2010) and Mao et al. (2007) previously. The samples were immediately analyzed for RA (%) after CTtreatment and the highest was observed at 60 C for 5 min (Fig. 2(a)). The activity of the enzyme decreased significantly (p < 0.01) as the treatment temperature and processing time was increased. However, no significant difference (p < 0.01) was found in the % RA of the enzyme at 80 and 90 C suggesting the development of resistance of the enzyme to inactivation after prolonged exposure to high temperature (Terefe et al., 2010). 3.2. Effect of OH-treatment on % RA
3. Results and discussion 3.1. Effect of CT-treatment on % RA Fresh sugarcane juice was analyzed for its physico-chemical
The analysis of OH-treated juice samples revealed a significant increase (p < 0.01) in the %RA initially, at 24 V/cm and 32 V/cm electric field strength which gradually decreased as the treatment time was increased (Fig. 2(b)). This behavior of the enzyme when
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Fig. 5. Variation in titrable acidity (
), ascorbic acid (
) and reducing sugars (
exposed to low electric field strengths suggests possible biochemical reactions that might have occurred due to changes in the molecular spacing that accelerated the inter-chain reactions (Castro et al., 2004). Similar results have been reported by Ohshima et al. (2007) where activity of peroxidase enzyme, derived from western horseradish, after 30 s exposure to an electric field of 12 kV/cm increased to 120% while 75% of the residual activity was observed at 23 kV/cm electric field for the same time. Higher electric field strength of 48 V/cm resulted in a significant reduction % RA, however, higher holding times of 1 and 1.25 min were found to be relatively insignificant. 3.3. Optimization of process variables Both CT- and OH- experiments were run in triplicates and 2FImodel was selected for analysis. The ANOVA revealed that temperature and time (for CT-treatment) and electric field strength and time (for OH-treatment) as well as their interaction were significant at p < 0.01. The general model equation is presented as follows (Eq. (4)):
) of (a) CT80/10, and (b) OH32/1 treated samples under refrigerated storage.
Yijk ¼ m þ ai þ bj þ ðabÞij :
(4)
Where, ai, bj represent the “main effect” of the i-th and j-th levels of A and B, respectively, (ab)ij is the effect of the interaction in that combination of levels. The R2 value for CT- and OH-treatment model was observed to be 0.994 and 0.986 while the standard deviation was reported to be 1.31 and 1.20 respectively. A target of obtaining a %RA between 4.5 and 8.5 units was set during optimization as this range has been reported to cause no deteriorative changes during storage of sugarcane juice (Mao et al., 2007). Three solutions (Table 3(a) and (b)) were obtained for both CT- and OH-treatment model. The CTtreatment with the lowest treatment time and temperature as well as minimum % RA was selected. Therefore, CT-treatment of 80 C for 10 min (CT80/10) was chosen to be studied for its shelf life at room and refrigerated conditions. Such results for CT-treatment have also been reported by Sneh et al.,2012 while Chauhan et al. (2007), and Sangeeta et al. (2013) found 70 C for 10 min suitable for shelf life stability of 20 days and 90 days at refrigeration
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An excessive increase in TA more often than not indicates microbial spoilage. The TA of CT80/10 and OH32/1 were observed to be 0.23 ± 0.04 and 0.20 ± 0.04 g/100 mL. The increase relative to untreated juice may be attributed to certain biochemical processes that might have been accelerated by the treatments. Such results have also been reported by Rivas, Rodrigo, Martinez, BarbosaCanovas, and Rodrigo (2006). The efficacy of a treatment is largely determined by its ability to retain heat sensitive nutritional components like AA. A number of studies have reported a relatively higher degree of AA degradation by OH than CT owing to the corrosion of electrodes and electrolysis of the sample (Assiry, Sastry, & Samarnayake, 2003). However, in the present study the reduction in AA was similar for both the treatments (Table 3(c)). Assiry, Sastry, and Samarnayake (2006) reported that at the same power level the reaction rate increases with increasing field strength. In the same study, it was also found that low temperatures (40 C) cause higher degree of degradation than higher temperatures (80 C) as there is a substantial amount of dissolved oxygen and the release of molecular oxygen by electrolysis causes oxidation of AA. Therefore, a temperature of 80 ± 2 C and treatment time of 1 min during OH treatment account for similar rate of degradation of AA as that of CT-treated samples. These observations for different quality parameters suggest a possible large scale utilization of the processing techniques. 3.5. Shelf life studies
Fig. 6. Variation in (a) Residual PPO activity (% RA) [room (-), refrigeration (;)], and (b) Total plate count [room (-), refrigeration (C)] in untreated juice during storage.
temperatures respectively. The optimum temperature of 80 C was used as the set temperature for OH-treatment using electric field strengths and treatment time as variables. The optimized OH-treatment was observed to be at 32 V/cm at 1 min holding time. The results for both were validated in triplicate for % RA (Table 3(c)) and their effect was also studied on the quality attributes like TA, RS and AA (Table 3(c)).
3.4. Changes in quality attributes Since OH exerts both thermal and non-thermal effects, evaluation of quality parameters viz., TA, AA, RS will provide substantial information on applicability of OH as an alternative to CT treatment. Sugar inversion during CT-treatment results in higher RS content leading to poor keeping quality of juice. The RS content of CT80/10 and OH32/1 was found to be 1.94 ± 0.01 g/100 mL and 1.80 ± 0.01 g/100 mL respectively (Table 3(c)). Jaggery contains about 10e15 g/100 g of RS (Rao, Das, & Das, 2007). These values represent only 12e13% of RS content in jaggery granules and visual inspection showed no signs of deterioration. Organic acids are most likely a product of biochemical processes or microbial degradation.
3.5.1. TA The TA of untreated juice increased significantly (p < 0.01) attaining the maximum value at day 6 of room and refrigerated storage conditions (Fig. 3(a) and (b)). The increase was significantly higher in the samples stored at room temperature indicating the importance of temperature for microbial growth and proliferation (Britz & Tracey, 1990). The TA of the CT80/10 juice samples stored at room temperature increased significantly (p < 0.01) to 0.72 ± 0.03% while the value for OH32/1 samples increased only to 0.32 ± 0.02% on day 10 of storage. However, both the samples showed the development of bad odor by day 10 and therefore had to be discarded (Fig. 4 (a) and (b)). The CT80/10 and OH32/1 juice samples stored under refrigerated conditions kept well for 30 days with a maximum TA of 0.38 ± 0.03% and 0.23 ± 0.07% respectively (Fig. 5(a) and (b)). The % TA for both treatments was found to be statistically insignificant under refrigerated storage. 3.5.2. AA The AA content of the untreated juice samples degraded significantly over the 6-day storage under both storage conditions (Fig. 3(a) and (b)). This decrease in the organic acid can be attributed to factors such as oxygen, heat, light, storage temperatures and storage time (Huelin, 1953). The AA content reduced by about 33.3% immediately after treatment in CT80/10 as well as OH32/1. On day 10 of room temperature storage, the AA content in CT80/10 and OH32/1 reduced significantly (p < 0.01) by 82.4% and 99.0% respectively (Fig. 4(a) and (b)). Such results have also been reported by Moreira, Ponce, Valle, and Roura (2006) for their study on AA degradation in lettuce leaves where they stated the use of high processing temperature as a cause of rapid degradation. Meanwhile, a 77.5% and 78.8% reduction of AA content was observed on day 30 of refrigerated storage for CT80/10 and OH32/1 samples respectively (Fig. 5(a) and (b)). The rate of decrease was significantly higher for the OH32/1 samples than CT80/10 samples under room temperature whereas the trend of degradation was slow but significant during refrigerated storage period with no significant difference between the two treatments.
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Fig. 7. Variation in Total plate count (
), Yeast and molds (
) and L. mesenteroides (
) in (a) CT80/10, (b) OH32/1 under room temperature.
3.5.3. RS The RS content for untreated juice increased significantly (p < 0.01) under both storage conditions from day 0 to 6 (Fig. 3(a) and (b)). The difference in RS increase under both conditions suggests that the microbial growth was greatly reduced under refrigerated conditions. For CT80/10 and OH32/1 treated samples stored at room temperature (Fig. 4(a) and (b)), the difference in RS for both treatments was statistically insignificant; however, the increase was significant with respect to storage time. There was no significant difference in the RS of CT80/10 and OH32/1 till day 20 under refrigerated conditions of storage (Fig. 5(a) and (b)). On day 25 of refrigeration, the %RS increase in OH32/1 was significantly different than CT80/10 indicating bacterial growth.
Fig. 8. Variation in Total plate count ( ) and L. mesenteroides ( ) in CT80/10 and Total plate count ( ) and L. mesenteroides ( ) in OH32/1 under refrigerated storage.
3.5.4. %RA The RA of untreated juice stored at room temperature initially increased significantly to 2.2 times on day 2 before reducing to 0.57 times and 0.35times on day 4 and 6 respectively. On the other hand, the %RA showed a steady increase throughout the refrigerated storage conditions (Fig. 6(a)). The unexplained decrease in the
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activity under room temperature storage in the final days can be attributed to the increase in acidity of the juice on storage which resulted in PPO inactivation. The effect of acidity on PPO has been verified by Mao et al. (2007) in their study of maintenance of sugarcane juice quality by blanching and ascorbic acid. Refrigeration conditions ensured minimal microbial activity which had a positive influence on the residual PPO activity. The % RA for CT80/10 and OH32/1 juice samples was found to be 6.47 ± 0.25 and 10.07 ± 0.32 respectively on day 0 (treatment day) and no activity was witnessed during the entire storage period under both room and refrigerated conditions indicating the efficacy of both the treatments. 3.5.5. Microbiological studies The microbiology parameters were investigated to observe thermal as well as non-thermal effects of OH on micro-organisms and to allow effective comparison with the destruction caused by conventional thermal treatment. The mechanism of microbial destruction by heat is well known. Microbial inactivation by electric field has been reported to be majorly by electroporation (Tsong, 1991) but some researchers have also suggested the formation of microbicidal agents such as chlorine, hydrogen peroxide etc., due to electric discharge in liquid media, that alter the DNA and cytoplasmic activity of the cells (Hulsheger, Potel, & Niemann, 1981). 3.5.5.1. Total plate count. The microbial count in untreated juice was found to be 5.78 ± 1.12 logCFU/ml that increased to 12.60 ± 1.10 and 10.67 ± 1.30 logCFU/ml on day 6 of storage at room and refrigeration temperatures respectively, which was the final day of storage as the juice started to become a viscous-gel (Fig. 6(b)). A 3.2and4.2 fold reduction was observed in the microbial growth for CT80/10 and OH32/1 samples respectively. Higher degree of inactivation by OH-treatment was due to the combined effect of heat as well as electric current (Leizerson & Shimoni, 2005). The room temperature storage observed a 3.1 and 3.96 fold significant increase in the count during the storage period of 10 days for CT80/10 and OH32/1 respectively (Fig. 7(a) &(b)), while the increase was limited to 1.4 and 1.6 fold for CT80/10 and OH32/1, respectively, during 30 days of refrigerated storage (Fig. 8). It is noteworthy that the growth of aerobic microbes remained insignificant for both CT80/10 and OH32/1 samples even at the end of refrigerated storage. 3.5.5.2. Yeast & mold count. The untreated juice observed a significant (p < 0.01) increase in the yeast and mold count (Fig. 9(a)). The growth was completed inhibited at refrigeration temperature for CT80/10, but not at room temperature which led to the spoilage of the CT80/10 within 10 days of storage (Fig. 7(a)). Yeast and mold were not detected in OH32/1 treated samples stored under both the conditions during the period of storage. Based on the results obtained, OH32/1 treatment may be regarded as more effective than CT80/10. Complete inactivation of yeast and mold in OH-treated orange juice have been reported by Leizerson and Shimoni (2005). 3.5.5.3. Leuconostoc mesenteroides. L. mesenteroides require sucrose and RS for growth and produce dextran which is an indicator of the deterioration of the sugarcane juice quality (Barker & Ajongwen, 1991). The initial count present in the untreated juice was 2.64 ± 0.13 log CFU/ml which rapidly increased by 3.5 and 3.2 times on day 6 of storage at room and refrigeration temperature respectively (Fig. 9(b)). For CT80/10 and OH32/1, immediate plating revealed no survivors of the L. mesenteroides population. However, when stored at room temperatures, their growth was witnessed in both the samples on day 5 which gradually increased on day 10 (Fig. 7(a)&(b)) with the formation of a viscous, jelly-like substance
Fig. 9. Variation in (a) Yeast and mold count, and (b) L. mesenteroides count for untreated juice under room (-) and refrigerated (C) conditions.
called dextran formed by the action of an extracellular enzyme, secreted by the Leuconostoc species, called dextransucrase on sucrose (Naessens, Cerdobbel, Soetart, & Vandamme, 2005). This increase in the L. mesenteroides count was also marked by the increase in the percentage of RS since the action of dextransucrase secreted by L. mesenteroides catalyzes the transfer of D-glucopyranosyl residues from sucrose to dextran with simultaneous release of a RS molecule (glucose or fructose) (Leathers, 2002). This verified that the species were viable and underwent multiplication in the first few days of storage at room temperature. At refrigeration conditions, no L. mesenteroides counts were observed until day 15 and 20 in CT80/10 and OH32/1 treated samples respectively (Fig. 8). However, the RS were found to increase in both CT80/10 and OH32/ 1 samples during the specified period but the increase was not significant (p < 0.01). The bacteria resurfaced on day 15 and 20 and the count increased to 2.05 ± 0.10 and 2.09 ± 0.15 logCFU/ml in CT80/10 and OH32/1 treated samples respectively. The increase in the count of L. mesenteroides from day 15 and day 20 can be accounted for the simultaneous increase in RS at day 15 and day 20 of T80/10 and OH32/1 respectively, under refrigerated conditions (Fig. 5(a),(b) and 8). Aronsson, Lindgren, Johansson, and Ronner (2001) reported that L. mesenteroides is one the most resistant
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microorganisms that observed no membrane damage to a PEF treatment of 35 kV/cm. Therefore, even though the bacteria lost its activity due to temporary shock, it wasn't completely destroyed since electric field strength of only 32 V/cm was applied. 4. Conclusion Optimization of the CT-treatment resulted in a residual PPO activity of 6.47 ± 0.25% in sugarcane juice at 80 C for 10 min. OH was able to achieve a similar residual PPO activity of 10.07 ± 0.32% when applied at 32 V/cm for a holding time of 1 min at 80 ± 2 C temperature. The degree of PPO inactivation achieved was sufficient to retard browning during storage and the parameters employed resulted in minimal changes RS, TA and AA. The OH treatment simultaneously achieved a considerable reduction in the microbial population in a processing time of 2.5 min whereas CTtreatment required a processing time of 26 (16 þ 10) min to achieve similar results. Also, complete inactivation of yeast and molds was possible only by the OH-treatment. Both the treatments resulted in a shelf life of 10 and 30 days at room and refrigeration temperatures respectively. During refrigerated storage, the quality attributes of OH- treated juice remained relatively stable in comparison to CT-treated juice. Therefore, time-efficiency of the OH renders the process applicable to food processing industries. However there is still scope for research in this area including use of improved packaging material, use of class-I preservatives like salt, lemon to aid further reduction in treatment time and prolong the shelf life of the product. Acknowledgment The authors would like to express their gratitude to the Department of FET, Central Workshop of Tezpur University. References AOAC. (1995). Official methods of analysis (16 ed.). Washington, DC: Association of Official Analytical Chemists. Aronsson, K., Lindgren, M., Johansson, B. R., & Ronner, U. (2001). Inactivation of microorganisms using pulsed electric fields: the influence of process parameters on Escherichia coli, Listeria innocua, Leuconostoc mesenteroides and Saccharomyces cerevisiae. Innovative Food Science and Emerging Technologies, 2(1), 41e54. Assiry, A., Sastry, A. K., & Samarnayake, C. (2003). Degradation kinetics of ascorbic acid during ohmic heating with stainless steel electrodes. Journal of Applied Electrochemistry, 33, 187e196. Assiry, A., Sastry, A. K., & Samarnayake, C. (2006). Influence of temperature, electrical conductivity, power and pH on ascorbic acid degradation kinetics during ohmic heating using stainless steel electrodes. Biochemistry, 68, 7e13. Barba, A. A., Calabretti, A., d'Amore, M., Piccinelli, A. L., & Rastrelli, L. (2008). Phenolic constituents levels in cv. Agria potato under microwave processing. LWT- Food Science and Technology, 41(10), 1919e1926. Barker, P. E., & Ajongwen, N. J. (1991). The production of enzyme dextransucrase using non-aerated fermentation techniques. Biotechnology and Bioengineering, 37(8), 703e707. Britz, T. J., & Tracey, R. P. (1990). The combination effect of pH, SO2, ethanol and temperature on the growth of Leuconostoc oenos. Journal of Applied
Biotechnology, 68(1), 23e31. Castro, I., Macedo, B., Teixeira, J. A., & Vicente, A. A. (2004). The effect of electric field on important food-processing enzymes: comparison of inactivation kinetics under conventional and ohmic heating. Journal of Food Science, 69(9), 696e701. Chauhan, O. P., Singh, D., Tyagi, S. M., & Balyan, D. K. (2007). Studies on preservation of sugarcane juice. International Journal of Food Properties, 5(1), 217e229. Eissa, H. A., Shehata, A. N., Ramadan, M. T., & Ali, H. S. (2010). Preservation of sugarcane juice by canning, effect of thermal and chemical pre-treatments on the enzymatic browning of sugarcane juice. Journal of American Science, 6(9). Huelin, F. E. (1953). Studies on the anaerobic decomposition of ascorbic acid. Food Research, 18, 633e639. Hulsheger, H., Potel, J., & Niemann, E. G. (1981). Killing of bacteria with electric pulses of high field strength. Radiation and Environmental Biophysics, 20, 53e65. Icier, F. (2005). Alternative heating method in food processing-Ohmic heating. Gida (Food), 30(2), 139e143. Icier, F., Yildiz, H., & Baysal, T. (2008). Polyphenoloxidase deactivation kinetics during ohmic heating of grape juice. Journal of Food Engineering, 85, 410e417. Khare, A., Lal, A., Singh, A., & Singh, A. (2012). Shelf lif enhancement of sugarcane juice. Croatian Journal of Food Technology, Biotechnology and Nutrition, 7(3), 179e183. Lakkakula, N. R., Lima, M., & Walker, T. (2004). Rice bran stabilization and rice bran oil extraction using ohmic heating. Bioresource technology, 92, 157e161. Leathers, T. D. (2002). Dextran. In E. J. Vandamme, S. De Baets, & A. Steinbuchel (Eds.), Polysaccharides I: Polysaccharides from prokaryotes: Vol. 5. Biopolymers (pp. 299e321). Weinheim: Wiley-VCH. Leizerson, S., & Shimoni, E. (2005). Effect of ultra-high temperature continuous ohmic heating treatment on fresh orange juice. Journal of Agriculture and Food Chemistry, 53, 3519e3524. Lima, M., & Sastry, S. K. (1999). The effects of ohmic frequency on hot-air drying rate and juice yield. Journal of Food Engineering, 41, 115e119. Mao, L. C., Yong, Q., & Fei, Q. (2007). Maintaining the quality of sugarcane juice with blanching and ascorbic acid. Food Chemistry, 104(2), 740e745. Moreira, M., Ponce, A., Valle, C., & Roura, S. (2006). Ascorbic acid retention< microbial growth and sensory acceptability of lettuce leaves subjected to mild heat shocks. Journal of Food Science, 71(2), 188e192. Naessens, M., Cerdobbel, A., Soetart, W., & Vandamme, E. J. (2005). Leuconostoc dextransucrase and dextran: production, properties and applications. Journal of Chemical Technology and Biotechnology, 80, 845e860. Ohshima, T., Tamura, T., & Sato, M. (2007). Influence of pulsed electric field on various enzyme activities. Journal of Electrostatistics, 65(3), 156e161. Ozoglu, H., & Bayindirli, A. (2002). Inhibition of enzymic browning in cloudy apple juice with antibrowning agents. Food Control, 13, 213e221. Ramaswamy, H. S., Marcotte, M., Sastry, S., & Abdelrahim, K. (2014). Ohmic heating in food processing. CRC press. International Standard Book Number: 13: 978-14200-7109-2. Rao, P. V. K. J., Das, M., & Das, S. K. (2007). Jaggery- a traditional indian sweetner. Indian Journal of Traditional Knowledge, 6(1), 95e102. Rivas, A., Rodrigo, D., Martinez, A., Barbosa-Canovas, G. V., & Rodrigo, M. (2006). Effect of PEF and heat pasteurization on the physicalechemical characteristics of blended orange and carrot juice. LWT- Food Science and Technology, 39, 1163e1170. Sangeeta, Hathan, B. S., & Khatkar, B. S. (2013). Studies on stability of sugarcane juice blended with anola juice at refrigerated and room temperature. International Journal of Agriculture and Food Science Technology, 4(10), 1027e1036. Singh, S., Gaikwad, K., & More, P. K. (2014). Spoilage of sugarcane Juice a problem in sugarcane industry- A review. International Journal of Agricultural Engineering, 7(1), 259e263. Sneh, S., Chaturvedi, A., Kuna, A., & Dhanlakshmi, K. (2012). Preservation of sugarcane juice using hurdle technology. Sugar Technology, 14(1), 26e39. Terefe, N. S., Yang, Y. H., Knoerzer, K., Buckow, R., & Versteeg, C. (2010). High pressure and thermal inactivation kinetics of polyphenol oxidase and peroxidase in strawberry puree. Innovative Food Science and Emerging Technologies, 11, 52e60. Tsong, T. Y. (1991). Electroporation of cell membranes. Biophysics Journal, 60, 297e306. Wang, W. C., & Sastry, S. K. (2002). Effects of moderate electrothermal treatments on juice yield from cellular tissue. Innovative Food Science and Emerging Technologies, 3, 371e377.
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Optimization of time-electric field combination for PPO inactivation in sugarcane juice by ohmic heating and its shelf life assessment Juhi Saxena, Hilal Ahmad Makroo, Brijesh Srivastava* Department of Food Engineering & Technology, School of Engineering, Tezpur University, Assam, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 January 2016 Received in revised form 8 April 2016 Accepted 11 April 2016 Available online 13 April 2016
The effect of ohmic heat (OH) treatment was measured on polyphenol oxidase (PPO) activity in sugarcane juice under different electric field strengths (24, 32 and 48 V/cm) and holding times (0.25, 0.50, 0.75, 1.0, 1.25 min) at a temperature of 80 ± 2 C, optimized by conventional thermal (CT) treatment. The optimum temperature of PPO inactivation for CT-treatment was determined in a parallel study. The processing condition of 32 V/cm and 1 min holding time was found optimum and was analyzed for titrable acidity (TA), reducing sugars (RS), ascorbic acid (AA) and microbial load for 10 and 30 days at room and refrigeration temperatures respectively. During refrigerated storage, TA and RS remained significantly (p < 0.01) unchanged and the AA degradation was more pronounced at room temperature. Both treatments resulted in significant microbial reductions but growth resurfaced after 5th and 25 th day of room and refrigeration storage respectively. No yeast and mold growth was witnessed after OH-treatment. Overall, the OH-treatment was found to inhibit PPO enzyme activity in a shorter processing time than CT, while maintaining the potential quality attributes of the juice. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Sugarcane juice PPO activity Ohmic heating Optimization Leuconostoc mesenteroides
1. Introduction Numerous investigations have been carried out to devise a treatment that can increase the shelf life of sugarcane juice to allow its entry into the mainstream juice processing industries. Thermal processing is the most common method studied for sugarcane juice preservation (Chauhan, Singh, Tyagi, & Balyan, 2007; Mao, Yong, & Fei, 2007; Sangeeta, Hathan, & Khatkar, 2013). Although thermal treatment is very effective for microbial and enzyme inactivation, the use of high temperatures (80e90OC) is known to cause offflavor and discoloration in the processed product (Wang & Sastry, 2002). In the past decade, novel techniques like high pressure processing, pulsed electric field, microwave treatment, ohmic heating etc. have been studied for their application on various food products (Barba, Calabretti, d’Amore, Piccinelli, & Rastrelli, 2008; Castro, Macedo, Teixeira, & Vicente, 2004; Icier, 2005; Ohshima, Tamura, & Sato, 2007; Terefe, Yang, Knoerzer, Buckow, & Versteeg, 2010). Ohmic heating (OH) has gained wide popularity as an alternative thermal treatment as it causes volumetric heating of the sample
* Corresponding author. Department of Food Engineering & Technology, School of Engineering, Tezpur University, Assam 784028, India. E-mail address: [email protected] (B. Srivastava). http://dx.doi.org/10.1016/j.lwt.2016.04.015 0023-6438/© 2016 Elsevier Ltd. All rights reserved.
which leads to consistent and rapid heat generation especially in liquid foods. The rate of heat generation in OH is a function of electric field strength applied across the food material (Ramaswamy, Marcotte, Sastry, & Abdelrahim, 2014). Due to short processing times, OH causes minimum discoloration and maintains the nutritive value of the food (Leizerson & Shimoni, 2005; Wang & Sastry, 2002). This feature makes it one of the most desirable treatments particularly for sugarcane juice; as it contains sensitive flavor components that are easily destroyed at longer treatment times. Despite the problems of electrochemical degradation associated, OH has been successfully studied for its use in preheating, blanching, and extraction (Lakkakula, Lima, & Walker, 2004; Leizerson & Shimoni, 2005; Lima & Sastry, 1999). The major focus of most studies has been on commercial products like strawberry pulp, grape juice etc (Castro et al., 2004; Icier, Yildiz, & Baysal, 2008). Sugarcane juice is a proven health promoting food which is primarily known to fight cancer and prevent kidney problems (Singh, Gaikwad, & More, 2014). However, PPO browning and microbial spoilage have limited the storage of fresh juice to just a few hours. Over the years, sugarcane juice has been investigated for its shelf life by blending it with curd, lime juice and other preservatives (Khare, Lal, Singh, & Singh, 2012; Sneh, Chaturvedi, Kuna,
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Table 1 Processing times for different combinations of CT and OH-treatments. Treatment type
EFS
Holding temp. ( C)
Heating time (min)
Holding time (min)
Total processing time (min)
CT
e e
60 ± 1 70 ± 1
3.80 4.90
5,10,15,20 5,10,15,20
8.8, 13.8, 13.8, 18.8, 23.8 9.9, 14.9, 19.9, 24.9
e
80 ± 1
6.00
5,10,15,20
11.0, 16.0, 21.0, 26.0
e 24 32 48
90 ± 1
7.00
5,10,15,20
12.0, 17.0, 22.0, 27.0
80 ± 2 80 ± 2 80 ± 2
3.25 1.50 0.45
0.25, 0.5, 0.75, 1.0, 1.25 0.25, 0.5, 0.75, 1.0, 1.25 0.25, 0.5, 0.75, 1.0, 1.25
3.5, 3.75, 4.0, 4.25, 4.5 1.75, 2.0, 2.25, 2.5, 2.75 0.7, 0.95, 1.2, 1.45, 1.7
OH
& Dhanlakshmi, 2012), however, limited literature is available on the application of a novel technique such as OH to increase the shelf life of the product. Although the rate of heating under OH and conventional thermal (CT) are different, the present study was conducted to explore the option of an alternative treatment for sugarcane juice processing. Therefore, the objectives of this investigation were to determine the influence of OH on PPO inactivation in sugarcane juice under a wide range of electric field strengths and compare the observations with those of the CT treatment. Shelf-life studies were also conducted for the optimized treatments and quality attributes like titrable acidity (TA), ascorbic acid (AA), reducing sugars (RS) and microbial count was studied. 2. Materials and methods 2.1. Sample preparation Fully mature sugarcane stems of ‘Pharma’ variety were procured from the farms in Sonitpur, Assam, India. The stems were peeled, manually cut and crushed in ‘Usha’ Food Processor (make: FP2663, India) to yield the juice. The juice was subsequently filtered through four-folds of muslincloth to obtain a clear filtrate which was used throughout the study. Fresh juice was extracted prior to each treatment.
2.3. OH- treatment The OH-treatment was applied in a lab scale ohmic heater whose setup (Fig. 1) includes one movable and one stationary stainless steel electrode (grade 316) of 1.5 mm thickness and 25 mm diameter enclosed in a hollow cylindrical casing of Teflon with an outer and inner diameter of 50 mm and 25.5 mm respectively. An opening of 5 mm diameter was made on the surface, at a distance of 50 mm from each electrode for the feed and a tefloncoated thermocouple was inserted to measure the temperature at the centre of the cylinder. A power supply of 240 V and 50 Hz frequency was used to carry out the experiments. The OH-treatment was employed at three different electric field strengths (24, 32 and 48 V/cm) for five holding times (0.25, 0.50, 0.75, 1.0, 1.25 min). The electric field strengths were maintained by adjusting the distance between the two electrodes. Thus, the volumetric capacity of the equipment varied from 50 ml at 24 V/cm, 35 ml at 32 V/cm and 25 ml at 48 V/ cm. A digital temperature controller-cum-indicator was used to maintain the temperature of the juice at 80 ± 2 C (optimized temperature of CT-treatment, discussed in section 3.3). The heat-up time to reach 80 ± 2 C at different electric field strengths is shown in Table 1. The samples were held at 80 ± 2 C for the specified holding times. After both the treatments, the samples were immediately cooled and analyzed for their residual PPO activity.
2.2. CT-treatment The CT-treatment was carried out to establish effective comparison between the OH-& CT-treatment results. A volume of 50 ml of juice samples was heated at sixteen different processing combinations of temperature (60, 70, 80 and 90 C) and holding time (5, 10, 15 and 20 min) in a lab scale water bath (BW-20G,JEIOTech, Korea). The heating, holding and total processing times are shown in Table 1.
2.4. Polyphenol oxidase (PPO) enzyme assay The assay of the enzyme was carried out as described by Ozoglu and Bayindirli (2002). One ml of 0.2 mol/L Catechol solution was added to mixture of 0.5 ml of sugarcane juice and 2 ml of phosphate buffer (pH 6.5). The absorbance was measured at 420 nm at every 1 min interval. The enzyme activity was estimated from the linear
Fig. 1. Schematic Diagram of Ohmic heating set up.
J. Saxena et al. / LWT - Food Science and Technology 71 (2016) 329e338 Table 2 Physico-chemical properties of fresh sugarcane juice.
331
Table 3a Solutions for CT-treatment.
Physico-chemical parameters
Units
Analysis
Water content Total Solids Acidity TSS pH Ascorbic acid Reducing Sugars PPO activity
g/100 mL g/100 mL g/100 mL B e mg/100 mL g/100 mL Units/mL
81.05 18.95 0.16 19.6 4.42 4.44 0.66 69.40
± ± ± ± ± ± ± ±
0.30 0.42 0.02 0.10 0.07 0.13 0.02 0.24
Number
Temperature
Time
EA
Desirability
1 2 3
80 90 90
10 10 5
4.0 4 8.0
1.00 1.00 0.111
Selected
Table 3b Solutions for OH-treatment. Number
Electric field strength
Time
EA
Desirability
1 2 3
32 48 48
1 1 1.25
7 7 4.5
1.00 1.00 1.00
Selected
portion of the curve of absorbance v/s time. One unit of PPO activity was defined as 0.001A420/min. The activity of the samples was expressed as % Residual PPO Activity (RA) as given in Eq. (1):
% RA ¼
Current Enzyme Activity 100: Initial Enzyme Activity
(1)
2.5. Optimization of process variables Both CT- and OH-treatments were optimized taking timetemperature and time-electric field strength as the independent variables, respectively. Optimization of the variable levels was achieved by desirable minimization of the % RA along the fitted two factor interaction (2FI) model by numerical optimization procedure of the design expert software 8.1.0. The statistical criteria like R2value, standard deviation, F-value and p-value were used to determine the goodness of fit. 2.6. Data validation The optimized process variables for both CT- and OH-treatments were validated for the % RA and the effect was also measured on the quality attributes viz. TA(%), RS(%) and AA(%) as per sections 2.7.1 and 2.7.2. 2.7. Shelf life studies The juice treated at optimized conditions of CT- and OHtreatments was stored in pre-sterilized glass vials of 10 ml each at room (25 ± 2) C and refrigeration (6 ± 1) C temperatures. A parallel shelf life study was conducted for untreated juice at both storage temperatures during which %RA,RS,TA,AA and total plate count, yeast and mold count, Leuconostoc mesenteroides count and E. coli counts were investigated at a regular interval of 5 days. The properties of the fresh juice were also determined. Fig. 2. Residual PPO activity (% RA) during (a) CT at 60 (-), 70 (C), 80 (:), 90 (;) C and (b) OH-treatment at 24 (-), 32 (C) and 48 (:) V/cm.
% Titrable acidity ¼
2.7.1. TA and AA determination TA was assayed by manual titration (AOAC, 1995) and was calculated as percent titrable acidity by Eq. (2).
Titre NNaOH Vol:made up 64 100 : Vol: of sample taken for estimation Vol: of sample 1000
(2)
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Table 3c Validation of optimized CT- and OH-treatments. Sample
Quality attributes Residual PPO activity (%)
CT80/10 OH32/1 a
a
Titrable acidity (g/100 mL) a
a
0.23 ± 0.04 0.20 ± 0.04a
6.47 ± 0.25 10.07 ± 0.32a
Reducing sugars (g/100 mL) 1.94 ± 0.01 1.80 ± 0.01a
Ascorbic acid (mg/100 mL) 2.96 ± 0.07a 2.98 ± 0.30a
Means within a column with different letters are significantly different (P < 0.01).
AA was estimated by 2,6-dichlorophenol indophenol method (AOAC, 1995) and was calculated as
Ascorbic acid ¼ 200 ðml indophenol sol:Þ ðCVitamin =ml indophenol sol:Þ
Fig. 3. Variation in titrable acidity (
), ascorbic acid (
(3)
) and reducing sugars (
2.7.2. RS content determination The RS content were analysed by the method (Mao et al., 2007) using glucose as the standard reducing sugar and 3,5dinitrosalicylic acid as the developer. 2.7.3. Microbiological analysis The treated-juice samples were diluted in sterile 0.85 g/100 mL saline solution and the total plate counts were incubated in nutrient agar (Himedia) at 37 C for 24 h. The yeast and mold count were estimated by incubation in potato dextrose agar (Himedia) at
) of untreated juice during storage at (a) Room and (b) Refrigeration temperature.
J. Saxena et al. / LWT - Food Science and Technology 71 (2016) 329e338
Fig. 4. Variation in titrable acidity (
), ascorbic acid (
) and reducing sugars (
25 C for 5 days. L. mesenteroides were maintained on Mayeux, Sandine, & Elliker agar at 21 C for 4 days. Escherichia coli were enumerated on Eosin Methylene Blue agar (Himedia) at 37 C for 24 h. All the tests were conducted in triplicates and the results were calculated in terms of log CFU/ml. 2.8. Statistical analysis All the experiments were conducted in triplicates. The data obtained was subjected to two-way analysis of variance (ANOVA) with a significance level of 0.01 (a ¼ 0.01). Mean values with standard deviations were reported and student's t-test was used as the Post-hoc test.
333
) of (a) CT80/10, and (b) OH32/1 treated samples under room temperature storage.
properties summarized in Table 2. The initial activity of PPO was found to be 69.4 ± 0.24 units/100 ml; which was higher than 39.5 and 28 units as reported in sugarcane juice by Eissa, Shehata, Ramadan, and Ali (2010) and Mao et al. (2007) previously. The samples were immediately analyzed for RA (%) after CTtreatment and the highest was observed at 60 C for 5 min (Fig. 2(a)). The activity of the enzyme decreased significantly (p < 0.01) as the treatment temperature and processing time was increased. However, no significant difference (p < 0.01) was found in the % RA of the enzyme at 80 and 90 C suggesting the development of resistance of the enzyme to inactivation after prolonged exposure to high temperature (Terefe et al., 2010). 3.2. Effect of OH-treatment on % RA
3. Results and discussion 3.1. Effect of CT-treatment on % RA Fresh sugarcane juice was analyzed for its physico-chemical
The analysis of OH-treated juice samples revealed a significant increase (p < 0.01) in the %RA initially, at 24 V/cm and 32 V/cm electric field strength which gradually decreased as the treatment time was increased (Fig. 2(b)). This behavior of the enzyme when
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Fig. 5. Variation in titrable acidity (
), ascorbic acid (
) and reducing sugars (
exposed to low electric field strengths suggests possible biochemical reactions that might have occurred due to changes in the molecular spacing that accelerated the inter-chain reactions (Castro et al., 2004). Similar results have been reported by Ohshima et al. (2007) where activity of peroxidase enzyme, derived from western horseradish, after 30 s exposure to an electric field of 12 kV/cm increased to 120% while 75% of the residual activity was observed at 23 kV/cm electric field for the same time. Higher electric field strength of 48 V/cm resulted in a significant reduction % RA, however, higher holding times of 1 and 1.25 min were found to be relatively insignificant. 3.3. Optimization of process variables Both CT- and OH- experiments were run in triplicates and 2FImodel was selected for analysis. The ANOVA revealed that temperature and time (for CT-treatment) and electric field strength and time (for OH-treatment) as well as their interaction were significant at p < 0.01. The general model equation is presented as follows (Eq. (4)):
) of (a) CT80/10, and (b) OH32/1 treated samples under refrigerated storage.
Yijk ¼ m þ ai þ bj þ ðabÞij :
(4)
Where, ai, bj represent the “main effect” of the i-th and j-th levels of A and B, respectively, (ab)ij is the effect of the interaction in that combination of levels. The R2 value for CT- and OH-treatment model was observed to be 0.994 and 0.986 while the standard deviation was reported to be 1.31 and 1.20 respectively. A target of obtaining a %RA between 4.5 and 8.5 units was set during optimization as this range has been reported to cause no deteriorative changes during storage of sugarcane juice (Mao et al., 2007). Three solutions (Table 3(a) and (b)) were obtained for both CT- and OH-treatment model. The CTtreatment with the lowest treatment time and temperature as well as minimum % RA was selected. Therefore, CT-treatment of 80 C for 10 min (CT80/10) was chosen to be studied for its shelf life at room and refrigerated conditions. Such results for CT-treatment have also been reported by Sneh et al.,2012 while Chauhan et al. (2007), and Sangeeta et al. (2013) found 70 C for 10 min suitable for shelf life stability of 20 days and 90 days at refrigeration
J. Saxena et al. / LWT - Food Science and Technology 71 (2016) 329e338
335
An excessive increase in TA more often than not indicates microbial spoilage. The TA of CT80/10 and OH32/1 were observed to be 0.23 ± 0.04 and 0.20 ± 0.04 g/100 mL. The increase relative to untreated juice may be attributed to certain biochemical processes that might have been accelerated by the treatments. Such results have also been reported by Rivas, Rodrigo, Martinez, BarbosaCanovas, and Rodrigo (2006). The efficacy of a treatment is largely determined by its ability to retain heat sensitive nutritional components like AA. A number of studies have reported a relatively higher degree of AA degradation by OH than CT owing to the corrosion of electrodes and electrolysis of the sample (Assiry, Sastry, & Samarnayake, 2003). However, in the present study the reduction in AA was similar for both the treatments (Table 3(c)). Assiry, Sastry, and Samarnayake (2006) reported that at the same power level the reaction rate increases with increasing field strength. In the same study, it was also found that low temperatures (40 C) cause higher degree of degradation than higher temperatures (80 C) as there is a substantial amount of dissolved oxygen and the release of molecular oxygen by electrolysis causes oxidation of AA. Therefore, a temperature of 80 ± 2 C and treatment time of 1 min during OH treatment account for similar rate of degradation of AA as that of CT-treated samples. These observations for different quality parameters suggest a possible large scale utilization of the processing techniques. 3.5. Shelf life studies
Fig. 6. Variation in (a) Residual PPO activity (% RA) [room (-), refrigeration (;)], and (b) Total plate count [room (-), refrigeration (C)] in untreated juice during storage.
temperatures respectively. The optimum temperature of 80 C was used as the set temperature for OH-treatment using electric field strengths and treatment time as variables. The optimized OH-treatment was observed to be at 32 V/cm at 1 min holding time. The results for both were validated in triplicate for % RA (Table 3(c)) and their effect was also studied on the quality attributes like TA, RS and AA (Table 3(c)).
3.4. Changes in quality attributes Since OH exerts both thermal and non-thermal effects, evaluation of quality parameters viz., TA, AA, RS will provide substantial information on applicability of OH as an alternative to CT treatment. Sugar inversion during CT-treatment results in higher RS content leading to poor keeping quality of juice. The RS content of CT80/10 and OH32/1 was found to be 1.94 ± 0.01 g/100 mL and 1.80 ± 0.01 g/100 mL respectively (Table 3(c)). Jaggery contains about 10e15 g/100 g of RS (Rao, Das, & Das, 2007). These values represent only 12e13% of RS content in jaggery granules and visual inspection showed no signs of deterioration. Organic acids are most likely a product of biochemical processes or microbial degradation.
3.5.1. TA The TA of untreated juice increased significantly (p < 0.01) attaining the maximum value at day 6 of room and refrigerated storage conditions (Fig. 3(a) and (b)). The increase was significantly higher in the samples stored at room temperature indicating the importance of temperature for microbial growth and proliferation (Britz & Tracey, 1990). The TA of the CT80/10 juice samples stored at room temperature increased significantly (p < 0.01) to 0.72 ± 0.03% while the value for OH32/1 samples increased only to 0.32 ± 0.02% on day 10 of storage. However, both the samples showed the development of bad odor by day 10 and therefore had to be discarded (Fig. 4 (a) and (b)). The CT80/10 and OH32/1 juice samples stored under refrigerated conditions kept well for 30 days with a maximum TA of 0.38 ± 0.03% and 0.23 ± 0.07% respectively (Fig. 5(a) and (b)). The % TA for both treatments was found to be statistically insignificant under refrigerated storage. 3.5.2. AA The AA content of the untreated juice samples degraded significantly over the 6-day storage under both storage conditions (Fig. 3(a) and (b)). This decrease in the organic acid can be attributed to factors such as oxygen, heat, light, storage temperatures and storage time (Huelin, 1953). The AA content reduced by about 33.3% immediately after treatment in CT80/10 as well as OH32/1. On day 10 of room temperature storage, the AA content in CT80/10 and OH32/1 reduced significantly (p < 0.01) by 82.4% and 99.0% respectively (Fig. 4(a) and (b)). Such results have also been reported by Moreira, Ponce, Valle, and Roura (2006) for their study on AA degradation in lettuce leaves where they stated the use of high processing temperature as a cause of rapid degradation. Meanwhile, a 77.5% and 78.8% reduction of AA content was observed on day 30 of refrigerated storage for CT80/10 and OH32/1 samples respectively (Fig. 5(a) and (b)). The rate of decrease was significantly higher for the OH32/1 samples than CT80/10 samples under room temperature whereas the trend of degradation was slow but significant during refrigerated storage period with no significant difference between the two treatments.
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Fig. 7. Variation in Total plate count (
), Yeast and molds (
) and L. mesenteroides (
) in (a) CT80/10, (b) OH32/1 under room temperature.
3.5.3. RS The RS content for untreated juice increased significantly (p < 0.01) under both storage conditions from day 0 to 6 (Fig. 3(a) and (b)). The difference in RS increase under both conditions suggests that the microbial growth was greatly reduced under refrigerated conditions. For CT80/10 and OH32/1 treated samples stored at room temperature (Fig. 4(a) and (b)), the difference in RS for both treatments was statistically insignificant; however, the increase was significant with respect to storage time. There was no significant difference in the RS of CT80/10 and OH32/1 till day 20 under refrigerated conditions of storage (Fig. 5(a) and (b)). On day 25 of refrigeration, the %RS increase in OH32/1 was significantly different than CT80/10 indicating bacterial growth.
Fig. 8. Variation in Total plate count ( ) and L. mesenteroides ( ) in CT80/10 and Total plate count ( ) and L. mesenteroides ( ) in OH32/1 under refrigerated storage.
3.5.4. %RA The RA of untreated juice stored at room temperature initially increased significantly to 2.2 times on day 2 before reducing to 0.57 times and 0.35times on day 4 and 6 respectively. On the other hand, the %RA showed a steady increase throughout the refrigerated storage conditions (Fig. 6(a)). The unexplained decrease in the
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activity under room temperature storage in the final days can be attributed to the increase in acidity of the juice on storage which resulted in PPO inactivation. The effect of acidity on PPO has been verified by Mao et al. (2007) in their study of maintenance of sugarcane juice quality by blanching and ascorbic acid. Refrigeration conditions ensured minimal microbial activity which had a positive influence on the residual PPO activity. The % RA for CT80/10 and OH32/1 juice samples was found to be 6.47 ± 0.25 and 10.07 ± 0.32 respectively on day 0 (treatment day) and no activity was witnessed during the entire storage period under both room and refrigerated conditions indicating the efficacy of both the treatments. 3.5.5. Microbiological studies The microbiology parameters were investigated to observe thermal as well as non-thermal effects of OH on micro-organisms and to allow effective comparison with the destruction caused by conventional thermal treatment. The mechanism of microbial destruction by heat is well known. Microbial inactivation by electric field has been reported to be majorly by electroporation (Tsong, 1991) but some researchers have also suggested the formation of microbicidal agents such as chlorine, hydrogen peroxide etc., due to electric discharge in liquid media, that alter the DNA and cytoplasmic activity of the cells (Hulsheger, Potel, & Niemann, 1981). 3.5.5.1. Total plate count. The microbial count in untreated juice was found to be 5.78 ± 1.12 logCFU/ml that increased to 12.60 ± 1.10 and 10.67 ± 1.30 logCFU/ml on day 6 of storage at room and refrigeration temperatures respectively, which was the final day of storage as the juice started to become a viscous-gel (Fig. 6(b)). A 3.2and4.2 fold reduction was observed in the microbial growth for CT80/10 and OH32/1 samples respectively. Higher degree of inactivation by OH-treatment was due to the combined effect of heat as well as electric current (Leizerson & Shimoni, 2005). The room temperature storage observed a 3.1 and 3.96 fold significant increase in the count during the storage period of 10 days for CT80/10 and OH32/1 respectively (Fig. 7(a) &(b)), while the increase was limited to 1.4 and 1.6 fold for CT80/10 and OH32/1, respectively, during 30 days of refrigerated storage (Fig. 8). It is noteworthy that the growth of aerobic microbes remained insignificant for both CT80/10 and OH32/1 samples even at the end of refrigerated storage. 3.5.5.2. Yeast & mold count. The untreated juice observed a significant (p < 0.01) increase in the yeast and mold count (Fig. 9(a)). The growth was completed inhibited at refrigeration temperature for CT80/10, but not at room temperature which led to the spoilage of the CT80/10 within 10 days of storage (Fig. 7(a)). Yeast and mold were not detected in OH32/1 treated samples stored under both the conditions during the period of storage. Based on the results obtained, OH32/1 treatment may be regarded as more effective than CT80/10. Complete inactivation of yeast and mold in OH-treated orange juice have been reported by Leizerson and Shimoni (2005). 3.5.5.3. Leuconostoc mesenteroides. L. mesenteroides require sucrose and RS for growth and produce dextran which is an indicator of the deterioration of the sugarcane juice quality (Barker & Ajongwen, 1991). The initial count present in the untreated juice was 2.64 ± 0.13 log CFU/ml which rapidly increased by 3.5 and 3.2 times on day 6 of storage at room and refrigeration temperature respectively (Fig. 9(b)). For CT80/10 and OH32/1, immediate plating revealed no survivors of the L. mesenteroides population. However, when stored at room temperatures, their growth was witnessed in both the samples on day 5 which gradually increased on day 10 (Fig. 7(a)&(b)) with the formation of a viscous, jelly-like substance
Fig. 9. Variation in (a) Yeast and mold count, and (b) L. mesenteroides count for untreated juice under room (-) and refrigerated (C) conditions.
called dextran formed by the action of an extracellular enzyme, secreted by the Leuconostoc species, called dextransucrase on sucrose (Naessens, Cerdobbel, Soetart, & Vandamme, 2005). This increase in the L. mesenteroides count was also marked by the increase in the percentage of RS since the action of dextransucrase secreted by L. mesenteroides catalyzes the transfer of D-glucopyranosyl residues from sucrose to dextran with simultaneous release of a RS molecule (glucose or fructose) (Leathers, 2002). This verified that the species were viable and underwent multiplication in the first few days of storage at room temperature. At refrigeration conditions, no L. mesenteroides counts were observed until day 15 and 20 in CT80/10 and OH32/1 treated samples respectively (Fig. 8). However, the RS were found to increase in both CT80/10 and OH32/ 1 samples during the specified period but the increase was not significant (p < 0.01). The bacteria resurfaced on day 15 and 20 and the count increased to 2.05 ± 0.10 and 2.09 ± 0.15 logCFU/ml in CT80/10 and OH32/1 treated samples respectively. The increase in the count of L. mesenteroides from day 15 and day 20 can be accounted for the simultaneous increase in RS at day 15 and day 20 of T80/10 and OH32/1 respectively, under refrigerated conditions (Fig. 5(a),(b) and 8). Aronsson, Lindgren, Johansson, and Ronner (2001) reported that L. mesenteroides is one the most resistant
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microorganisms that observed no membrane damage to a PEF treatment of 35 kV/cm. Therefore, even though the bacteria lost its activity due to temporary shock, it wasn't completely destroyed since electric field strength of only 32 V/cm was applied. 4. Conclusion Optimization of the CT-treatment resulted in a residual PPO activity of 6.47 ± 0.25% in sugarcane juice at 80 C for 10 min. OH was able to achieve a similar residual PPO activity of 10.07 ± 0.32% when applied at 32 V/cm for a holding time of 1 min at 80 ± 2 C temperature. The degree of PPO inactivation achieved was sufficient to retard browning during storage and the parameters employed resulted in minimal changes RS, TA and AA. The OH treatment simultaneously achieved a considerable reduction in the microbial population in a processing time of 2.5 min whereas CTtreatment required a processing time of 26 (16 þ 10) min to achieve similar results. Also, complete inactivation of yeast and molds was possible only by the OH-treatment. Both the treatments resulted in a shelf life of 10 and 30 days at room and refrigeration temperatures respectively. During refrigerated storage, the quality attributes of OH- treated juice remained relatively stable in comparison to CT-treated juice. Therefore, time-efficiency of the OH renders the process applicable to food processing industries. However there is still scope for research in this area including use of improved packaging material, use of class-I preservatives like salt, lemon to aid further reduction in treatment time and prolong the shelf life of the product. Acknowledgment The authors would like to express their gratitude to the Department of FET, Central Workshop of Tezpur University. References AOAC. (1995). Official methods of analysis (16 ed.). Washington, DC: Association of Official Analytical Chemists. Aronsson, K., Lindgren, M., Johansson, B. R., & Ronner, U. (2001). Inactivation of microorganisms using pulsed electric fields: the influence of process parameters on Escherichia coli, Listeria innocua, Leuconostoc mesenteroides and Saccharomyces cerevisiae. Innovative Food Science and Emerging Technologies, 2(1), 41e54. Assiry, A., Sastry, A. K., & Samarnayake, C. (2003). Degradation kinetics of ascorbic acid during ohmic heating with stainless steel electrodes. Journal of Applied Electrochemistry, 33, 187e196. Assiry, A., Sastry, A. K., & Samarnayake, C. (2006). Influence of temperature, electrical conductivity, power and pH on ascorbic acid degradation kinetics during ohmic heating using stainless steel electrodes. Biochemistry, 68, 7e13. Barba, A. A., Calabretti, A., d'Amore, M., Piccinelli, A. L., & Rastrelli, L. (2008). Phenolic constituents levels in cv. Agria potato under microwave processing. LWT- Food Science and Technology, 41(10), 1919e1926. Barker, P. E., & Ajongwen, N. J. (1991). The production of enzyme dextransucrase using non-aerated fermentation techniques. Biotechnology and Bioengineering, 37(8), 703e707. Britz, T. J., & Tracey, R. P. (1990). The combination effect of pH, SO2, ethanol and temperature on the growth of Leuconostoc oenos. Journal of Applied
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