Sasikumar R*, Vivek K, Chakaravarthy S and Deka SC
Department of Agri-Business Management and Food Technology, North-Eastern Hill University, Meghalaya, India
Received Date: March 10, 2017; Accepted Date: April 06, 2017; Published Date: April 13, 2017
Citation: Sasikumar R, Vivek K, Chakaravarthy S, Deka SC (2017) Effect of Post-harvest Quality Parameters on Ultra-Sonication Treatment of Khoonphal (Haematocarpus validus) of Meghalaya, North-East India. J Food Process Technol 8:668. doi: 10.4172/2157-7110.1000668
Copyright: © 2017 Sasikumar R, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Freshly harvested khoonphal (Haematocarpus validus) were surface cleansed with ultrasonic treatment. The process variables i.e., ultrasonic amplitude, treatment time and temperature selected were optimized using response surface methodology (RSM) by three factor three level Box-Behnken design. Horn type ultra-sonicator with a power density of 460 W/cm2 with a constant frequency of 30 Hz was used for all the 17 experiments. Optimum independent variables selected by RSM were ultrasonic amplitude (100%), treatment time (5.10 min) and solvent temperature (25°C). The corresponding optimum values for dependent variables obtained were total plate count (2.94 log CFU/cm2), firmness (66.67 N) and respiration rate (42.32 N). Linear terms for all the dependent variables were found to be significant (p<0.05). Similarly, the interaction terms between ultrasonic amplitude and treatment time had showed significant negative effect on total plate count (p<0.001) and firmness (p<0.05). But significant positive effect was obtained for respiration rate (p<0.100). Therefore, from this study it was concluded that the ultra-sonication was found to be an effective technology in reducing surface microbial load. Hence, this may be used for extending the shelf-life and maintaining the quality of freshly harvested khoonphal, while RSM was proven to be an effective technique in controlling and optimizing the factors responsible for ultrasonic treatment.
Khoonphal; RSM; Ultrasonication; Total plate count; Firmness; Respiration rate
Khoonphal (Haematocarpus validus) is one of the rarest rather extinct fruits recoup after a period of 100 years in India. It added a recent disclosure of its habitats in Mawlakhieng village (Meghalaya, India) [1,2]. It is commonly known as Khoonphal/Raktaphal and is a rich source of iron, vitamin A, various alkaloids and functional phytochemicals. This fruit is famous for their traditional medicinal usages for treating jaundice, cancer, hypertension, arthritis, neurological problems, etc. [3-5]. Bioactive compounds present in this fruit neutralize free radical species generated as a part of biochemical reactions in our body system . Accessibility and availability of these bioactive compounds from dietary sources contribute significantly in traditional health system of tribal and rural population of developing world .
Haematocarpus validus is an evergreen perennial creeping woody climber capable of growing under extreme conditions, from very dry environments to highly acidic soils. The tiny, odorous, greenish white flowers unisex and dioecious produce fruit. The vines of the tree produce flower in middle of November-January. The fruits mature in April- May and the fruiting season is May to August . This fruit is highly perishable and has short shelf life i.e., 4-5 days at room temperature. It spoils rapidly during harvesting, storage and transportation due to surface bruising, senescence and surface microbial decay. The application of fungicides should be nullified due to the adverse effects on human health and environment . Therefore, there is an urgent need to develop a technology to maintain the quality and freshness of khoonphal for achieving the longer shelf life.
Ultrasound is an effective technology used in many industries including food [10,11]. It is a non-thermal technology which has wide spread applicability in heat-sensitive foods to retain sensory, functional and nutritional characteristics along with enhanced shelf life. Ultrasound is composed of sound waves with a frequency beyond the limit of human hearing. It is considered safe, environmental friendly and nontoxic. Among food industries fruits and vegetable industry has huge scope in using ultrasound to generate contamination-free products. Various authors have showed the effectiveness of ultrasonic cleaners in eliminating the contaminants and microorganisms present on objects, including sludge, mold, bacteria, fungi, worms and agrochemicals [9,12-14]. Other applications of ultrasound include degassing, inactivation of enzymes, crystallization, leaching, extraction, digestion, etc. . Low power (high frequency) ultrasound controls the food properties by monitoring the physicochemical properties and composition during storage and processing, which is also crucial for improving food quality and safety. This technology is relatively simple, cheap and energy saving hence it is applicable for improving the shelf life of post harvested fruits [9,12]. During ultra-sonication treatment various factors viz. frequency, solvent temperature, percentage of amplitude, treatment time, viscosity of the solvent, ultrasonic input and output power, etc. may affect the efficacy of the treatment . Controlling all these variables is difficult therefore; a robust and potent optimization tool is required for determining the effects of both individual operational factors and their interactions . Response surface methodology (RSM) is a simple and widely used technique in food engineering fields for optimizing food manufacturing operations and preservation techniques i.e., fresh cut lettuce, pear, kiwifruit, strawberry [9,12,17-19].
However, there has been no report published on effect and optimization of ultra-sonication on post-harvest khoonphal for maximizing the shelf life. Therefore, our aim of this study was to optimize the ultrasonic treatment and to study the sonication effect on total surface plate count, respiration rate, fruit firmness and some selected quality parameters. Independent variables considered for this study includes ultrasonic amplitude, treatment time and temperature.
The freshly ripened fruits (Khoonphal) were harvested and collected from the study area (West Garo Hills, Tura, Meghalaya, North-East India) in the month of May 2016. The good quality fruits were selected for the experimentation i.e., uniform size, absence of defects and visual wounds. Then the fruits were taken to the laboratory within 2 h from the time of harvest. The selected khoonphal had an initial total soluble solid (TSS) of 16 ± 1 °Brix and moisture content (M.C) of 85.00 ± 1.00% w.b. (wet basis). Khoonphal collected for experiments were shown in the Figure 1.
Optimization of ultrasonic treatment
Probe ultrasonicator (BBI-8535027, Sartorius Labsonic M, Germany) with a constant frequency of 30 kHz having a maximal output power density of 460 W/cm2 was used for treatment of khoonphal. A 3-mm titanium probe with maximum amplitude of 180 μm was used for experimental purpose. Experimental combination with different independent variables viz. amplitude, time and temperature was set according to design obtained from RSM – Box Benkhan model. The probe was immersed into solvent (distilled water) by 25 mm. While the Cut-off cycle time of 0.5 s was fixed for all the experiment to control the temperature of the solvent. 300 ml of solvent was used to treat 80- 90 gram fruits (ratio 1: 3.5). Ultrasonication treatment at maximum amplitude (100%) beyond 15 min would rupture the fruit tissues and severely loosens its firmness. After the treatment fruits were removed immediately and taken for further analyses. Untreated fruit samples are used as a control. All the experiments were conducted twice with two replicates of each treatment per experiment.
Response Surface Methodology (RSM) is considered as an effective optimization tool used to optimize the levels of independent variables. Screening designs were carried out to eliminate the minor variables which are not important during experiment. Therefore, independent variables with major effects on dependent variables (total plate count, respiration rate and firmness) were selected for optimization which includes ultrasonic amplitude, treatment time and temperature. Software design expert (version 7.00, Stat-Ease Inc., Minneapolis, MN) was used to construct model and analyse data. An efficient three-levelthree- factor, Box-Behnken design was employed with 17 experimental runs with four replicates at the centre point. The range and centre point values of the independent variables were given in the Table 1. Second order polynomial equation was used to express the dependent variables as a function of independent variables as follows:
|Ultrasonic amplitude (X1)||60||80||100|
|Treatment time (X2)||5||10||15|
Table 1: Independent variables and their level used for central composite design.
Where Y is the predicted variable/response, Xi and Xj are the independent variables. While β0 is the constant coefficient, βi, βij and βjj are the regression coefficients for the linear, interaction and quadratic, respectively. The Coefficients obtained were interpreted using the F test. Regression analysis, analysis of variance (ANOVA) was also performed to establish optimum conditions for ultra-sonication treatment for khoonphal. The surface plotting’s for the optimized results were shown in Figures 2-4.
Optimal conditions (ultrasonic amplitude: 100%, temperature: 25°C and treatment time: 5.10 min) obtained from RSM was used to compare with untreated fruits as a control sample. After ultra-sonication fruits were then immediately analysed for total plate count, firmness, respiration rate and vitamin C. There were three replicates of 80 ± 1 g of fruit each per treatment, and the same experimental combinations was conducted twice. Finally treated fruits were subjected to storage study after vacuum and normal packaging for 15 days (unpublished data).
Total plate count
Total plate count (TPC) was examined according to Pao et al.  with minor modification. 100 grams of ultra-sonicated sample was placed into sterilized bags consist of 1 litre of 0.1% (w/v) peptone solution. Then the sterilized sample bags were mixed thoroughly with the help of reciprocal shaker with 100 oscillations/ min at 6 ± 1°C for 3 h. After shaking, the wash solutions obtained were then taken immediately for enumeration of TPC. Appropriate dilutions (1:10) required for sampling (sample plating) were made with 0.1% (w/v) peptone solution. Each wash solution was then surface plated on plate count agar (PCA) and incubated for 48 h at 35°C. The results were expressed in colony forming unit per square centimeter (CFU/cm2).
Respiration rate was measured by sealing 100 ± 1 g fruits into 1 litre plastic container. The container used was fitted with an airtight rubber septum and held at 25 ± 1°C for 1 h. Respiration rate was measured in accordance with Vivek et al.  and the experiments were conducted thrice. The ultra-sonicated samples were then taken for measuring respiration rate using gas analyser (Checkmate 2, PBI, dansensor, Ringsted, Denmark). 3 ml head space gas (O2 and CO2) in container was taken by the gas analyser for respiration rate analysis. The results were then expressed in mg CO2 kg-1 h-1 fresh weight (FW).
Firmness and vitamin C
Firmness of the ultra-sonicated khoonphal was measured accordance with Vivek et al. . Texture analyzer (TA-HD plus, Stable Micro Systems, UK) was used to perform puncture test. The load cell was equipped with a 3 mm diameter stainless steel (SS) probe, at a constant speed of 3 mm s-1 to a depth of 0.20 mm. The peak puncture force was treated as firmness of khoonphal and is measured in netwton (N) [21,22]. While the vitamin C content was assayed by titration method (2,6-dichlorophenolindophenol titration method). The results of vitamin C were expressed as mg/100 g of FW .
All the experimental results obtained were statistically analysed by applying independent sample t-test using SPSS v16 for inspecting the significant differences in the mean values of dependent variables for both the control and ultra-sonicated samples. The mean absolute error (MAE) and root mean square error (RSME) were also calculated to find out the difference between predicted and experimental/observed values for describing the performance of the model. This also shows the deviation of predicted values to the experimental values. The formula used for calculating MAE and RSME were shown in Eqs. (1) and (2).
Where Rp is the predicted value; Rreal is the experimental/observed value; N is the number of points.
Mean values of all the selected dependent/response variables were shown in Table 2. Experimental data was used to obtain all the coefficients of second order polynomial equation for finding the significance of various coefficients of the model. The best fit of the experimental data to the regression model equation was finalised according to coefficients of multiple determinations (R2), adjusted coefficients of multiple determinations (Adj R2), mean average error (MAE), root mean square error (RSME), coefficient of variance (CV). Lack of fit for all the dependent variables were found to be insignificant, this indicates the error analysis obtained from RSM among centre points in the experimental combinations was minimum. The linear terms of the independent variables for all the dependent variables were found to be significant (p<0.05) which is shown in Table 3. Apart from this few quadratic and interaction terms also showed significant (p<0.05) effect and were shown in Table 3. Similar kind of results were shown by Cao et al.  and Vivek et al.  for kiwifruit and strawberry fruits, respectively. The number of experimental trails needed to assess and construct the model for multiple variables and their interactions was easily done by using RSM. Finally, the models constructed were statistically measured to describe the deviation in the data.
|Experimental no||X1||X2||X3||Total plate count (log CFU/cm2)||Respiration Rate (mg CO2 kg-1 h-1 FW)||Firmness (N)|
Table 2: Box behnkan design matrix and response values.
|Coefficients||Total plate count||Respiration||Firmness|
|Lack of fit||N.S||N.S||N.S|
* Significant at p<0.1.
** Significant at p<0.05.
*** Significant at p<0.001.
Table 3: Regression coefficient for the responses.
Total plate count: The overall model for the total plate count had showed significant difference at p<0.001 with F value 37.38. And the lack of fit for total plate count had showed non-significant difference. The independent variables ultrasonic amplitude and treatment time had showed the negative effect on total plate count. i.e., total plate count decreases with the increase in ultrasonic amplitude and treatment time. Both the independent variables had showed significant difference at p<0.001. The interaction terms between ultrasonic amplitude and treatment time had showed the significant negative effect on total plate count at p<0.001. The other interaction terms between ultrasonic amplitude and temperature had also showed the significant negative effect on total plate count at p<0.100. The quadratic terms for ultrasonic amplitude and treatment time had showed significant positive difference at p<0.100 and p<0.05 respectively. But the quadratic term for temperature had showed the significant negative effect on the total plate count at p<0.100. The coefficient of determination and adjusted coefficient of determination values (R2=0.98 and Adj R2=0.95) resulted high for TPC, which indicates the model fits extremely well. The RMSE and MAE values were also calculated (RMSE=1.76 and MAE=1.50), which tells the deviation in the experimental data. The maximum total plate count was observed at 60% ultrasonic amplitude, 5 min and 37.5°C and the minimum total plate count was observed at 100% ultrasonic amplitude, 10 min and 50°C. Similar kind of results were obtained for kiwifruit and strawberry [9,12]. This microbial reduction may be due to cavitation bubbles, localized temperature and pressure occur in solvent during ultrasonication .
Respiration rate: The overall model for the Respiration rate had showed significant difference at p<0.05 with F value 14.67. And the lack of fit for respiration rate had showed non-significant difference. Independent variables ultrasonic amplitude and treatment time had showed the positive effect on respiration rate. i.e., Respiration rate increases with the increase in ultrasonic amplitude and treatment time. Both the independent variables had showed significant difference at p<0.001. The interaction terms between ultrasonic amplitude and treatment time had showed the significant positive effect on respiration rate at p<0.100. The other interaction terms between treatment time and temperature had also showed the significant negative effect on respiration rate at p<0.05. The quadratic terms for ultrasonic amplitude and treatment time had showed significant positive difference at p<0.050 and p<0.100 respectively. The coefficient of determination and adjusted coefficient of determination values (R2=0.95 and Adj R2=0.88) resulted high for respiration rate, which indicates the model fits extremely well. The RMSE and MAE values were also calculated (RMSE=3.50 and MAE=2.20), which tells the deviation in the experimental data. The maximum respiration rate was observed at 100% ultrasonic amplitude, 15 min and 37.5°C and the minimum respiration rate was observed at 60% UA, 10 min and 25°C. Ultrasonication at higher power and longer treatment time for kiwifruits have showed higher respiration rates . This may be due to the due to the rupturing of fruit tissues and cell wall degradation [23,24]. Higher respiration rates indicate a more active metabolism and usually a faster deterioration rate . Heat treated mango fruits showed higher respiration rate .
Firmness: The overall model for the firmness had showed significant difference at p<0.05 with F value 6.71. And the lack of fit for firmness had showed non-significant difference. The Independent Variables ultrasonic amplitude and treatment time had showed the negative effect on firmness. i.e., Firmness decreases with increase in ultrasonic amplitude and treatment time. Both the independent variables had showed significant difference at p<0.001. The interaction terms between ultrasonic amplitude and treatment time had showed the significant negative effect on firmness at p<0.05 i.e., combined effect of ultrasonic amplitude and treatment time decreases the fruit firmness. Similar kind of results were reported for strawberry and kiwifruit [9,12]. The other interaction terms showed non-significant difference on fruit firmness. The quadratic terms for ultrasonic amplitude, treatment time and temperature had also showed non-significant difference on fruit firmness. The coefficient of determination and adjusted coefficient of determination values (R2=0.90 and Adj R2=0.76) resulted high for fruit firmness, which indicates the model fits well. The RMSE and MAE values were also calculated (RMSE=1.05 and MAE=1.20), which tells the less deviation in the experimental data. The maximum firmness was observed at 60% ultrasonic amplitude, 5 min and 37.5°C and the minimum firmness was observed at 100% ultrasonic amplitude, 15 min and 37.5°C. Various authors have reported that the cell wall degradation and tissue rupturing is mainly due to the disturbances in cell wall constituents i.e., polygalacturonase and pectin methylesterase [9,12,27,28].
Optimization of ultrasonic treatment conditions
The desirability function of 0.75 was obtained from numerical optimization using design of expert ‘7.0’. This approach is considered as an effective technique for the simultaneous determination of optimum settings of input variables that can determine optimum performance levels for one or more responses [29-31]. Importance of ‘5’ was given to total plate count, while importance level of ‘4’ and ‘3’ were given to firmness and respiration rate. Based on the relative contribution to final quality of product importance of ‘3’ was given to all the independent (ultrasound amplitude, treatment time and temperature) variables  were shown in Table 4. After giving all the preferences, computer program gives the optimum ultra-sonication conditions for the blood fruit. The optimum values of all the independent variables viz. ultrasound amplitude, treatment time and temperature of the solvent were shown in Table 5 [30,31].
|Goal||Lower limit||Upper limit||Lower weight||Upper weight||Importance|
|Total plate count||Minimize||2.77||3.02||1||1||5|
Table 4: Responses and limits of optimizer for optimization using numerical optimization in design expert.
|Ultrasonic amplitude (%)||100.00|
|Treatment time (min)||05.10|
|Total plate count (log CFU/cm2)||02.93|
|Respiration rate (mg CO2 kg-1 h-1 FW)||43.16|
Table 5: Optimized solution – response optimizer in design expert.
|Responses||Ultrasound treated sample (optimized conditions)||Un treated sample||p-value|
|Total plate count (log CFU/cm2)||2.9433||3.9767||0.00*|
|Respiration rate (mg CO2 kg-1 h-1 FW)||42.3167||33.2733||0.031*|
|Vitamin C (mg/100 g of FW)||70.0633||75.8267||0.416|
p-value corresponds to Student’s t-test to related samples (paired).
* Significant at p<0.05.
Table 6: Effect of ultrasonic treatment under the optimized conditions on responses.
Effect of ultrasonic treatment under optimum conditions on microbial population and quality of blood fruit
The optimum conditions obtained from the RSM [ultrasonic amplitude (%), temperature (25°C) and treatment time (5.10 min)] were considered for determining the microbial population on the surface of fruit. Other quality aspects like firmness and respiration rate for 80 to 100 grams of samples were also evaluated in order to verify and compare the effects of ultra-sonication with the control (Table 6). The mean values of total plate count, firmness, respiration rate and vitamin C for ultra-sonicated samples and control were shown in Table 6. Ultra-sonication significantly (p<0.05) inhibited the microbial growth on the surface by 1 log cycle. It also showed significant (p<0.05) difference for respiration and firmness when compared with the control (Table 6). Insignificant (p>0.05) difference was observed for vitamin C for both the ultra-sonicated and control samples. Similar kind of results was shown for kiwi fruits . The respiration rate for the ultra-sonicated samples were resulted higher (27%) compared to control samples. Total plate count, firmness and vitamin C for the ultra-sonicated samples were decreased (25.9%, 7.8%, 7.6%) compared to control samples. These results confirmed the validity and adequacy of the predicted models. Similar kind of results was shown by Cao et al.  where total bacterial count was decreased by 55.2%. Firmness of ultra-sonicated kiwi fruits were resulted 5.42% less compared to the NaOCl treated samples, while the TSS of the ultrasound treated samples were unaffected .
From this study it was concluded that the RSM was proven to be an effective technique in controlling and optimizing the factors responsible for ultrasonic treatment. The application of 100% ultrasonic amplitude for 5.10 min at 25°C was optimum conditions in terms of decreasing the surface microbial load by 1 log cycle and reducing decay by increasing the shelf life of the freshly harvested khoonphal. Ultrasonication treatment increased the respiration rate by 26.7% (33.27 to 42.32 mg CO2 kg-1 h-1 FW), decreases total plate count, firmness and vitamin C by 28% (3.98 to 2.94 log CFU/cm2), 7.8% (72.30 to 66.67 N) and 7.6% (75.83 to 70.06 mg/100 g of FW), respectively compared to control samples.
Authors would like to thank Prof. S.C. Deka, Department of Food Engineering and Technology, Tezpur University for his constant encouragement.