Effect of ultra-sonication on postharvest quality parameters and microbial load on Docynia indica

Scientia Horticulturae 225 (2017) 163–170

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Effect of ultra-sonication on postharvest quality parameters and microbial load on Docynia indica

MARK

⁎

Vivek Ka, , Sabyasachi Mishraa, Sasikumar Rb a b

Department of Food Process Engineering, National Institute of Technology, Rourkela, Odisha, 769008, India Department of Agri-Business Management & Food Technology, North Eastern Hill University, Tura Campus, Chandmari 794002, West Garo Hills, Meghalaya, India

A R T I C L E I N F O

A B S T R A C T

Keywords: Docynia indica RSM Ultrasonication Total platecount Firmness Respiration rate

Freshly harvested Docynia indica fruits were surface cleansed with ultrasonic treatment at different amplitudes. Partly matured fruits were selected for all the experimental studies. 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. Probe type ultra-sonicator with a power density of 600 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 (80%), treatment time (5.82 min) and solvent temperature (25 °C). The corresponding optimum values for all the dependent variables obtained were total plate count (2.82 log CFU/cm2), firmness (65.22 N) and respiration rate (43.30 mg CO2 kg−1 h−1 FW). The linear terms, some interaction and quadratic terms were found to be significant (p < 0.05). 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, ultra-sonication treatment extremely useful for extending the shelf-life (58%) and maintaining the quality of freshly harvested Docynia indica by one month at refrigerated temperature (7–8 °C). While RSM was proven to be an effective technique in controlling and optimizing the factors responsible for ultrasonic treatment.

1. Introduction Docynia indica (wall.) Decaisne is a wild edible underutilised fruit of India. It belongs to Rosaceae family and is native to the eastern Himalayas and is also distributed in some parts of China, Myanmar, Thailand and Vietnam. Although this fruit is popularly known as Assam apple but closely related to quince (Cydonia oblonga). This fruit is sour in taste and has high nutritive value. This fruit is used as a natural remedy for treatment of digestive problems, many infectious diseases and it also showed hypoglycemic and hypolipidemic effect (Shende et al., 2016). It has high concentration of various alkaloids and functional phytochemicals hence exhibit higher antioxidant activity in DPPH and FRAP assay (Sharma et al., 2015). Bioactive compounds present in this fruit neutralizes free radical species generated as a part of biochemical reactions in our body system (Najjaa et al., 2011; Singh et al., 2012; Rahim et al., 2015; Ahmad et al., 2015; Caprioli et al., 2016). Accessibility and availability of these bioactive compounds from dietary sources contribute significantly in traditional health system of tribal and rural population of developing world (Odeja et al., 2014; Wong et al., 2014). ⁎

Corresponding author. E-mail addresses: [email protected], [email protected] (V. K).

http://dx.doi.org/10.1016/j.scienta.2017.07.006 Received 27 April 2017; Received in revised form 26 June 2017; Accepted 3 July 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.

The Docynia indica tree is semi evergreen or deciduous and grows up to 3 m tall at open places, thickets, slopes and stream sides between the elevation of 2000–3000 meters Nepal, Sikkim, Meghalaya, Arunachal Pradesh and Bhutan states of India. It also widely grows in some parts of China and found less in some parts of Myanmar, Thailand and Vietnam. Leaves are elliptic or oblong-lanceolate, (3.5–8 × 1.5–2.3) cm, firmly papery, abaxially sparsely pubescent or sub glabrous, adaxially alabrous, lustrous, base broadly cuneate or sub rounded, margin shallowly crenate, rarely serrate or entire only at apex, apex acute or acuminate; pedicel short or nearly absent, pubescent. Docynia indica flowers develop in March to April and the fruits mature in September (Reshma et al., 2012). This fruit is perishable and has short shelf life 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 (Vivek et al., 2016a). Therefore, there is an urgent need to develop a technology to maintain the quality and freshness of post-harvest Docynia indica for achieving the longer shelf life.

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treat 120 ± 2 g fruits (ratio 1:5). Ultrasonication treatment at maximum amplitude (80%) beyond 15 min would severely affects the fruit tissues and loosens its firmness accordingly treatment time was restricted accordingly. Fruits were then taken immediately to the laboratory for analysis after the ultra-sonication treatment. Untreated fruit samples are used as a control. All the experiments were conducted in duplicate with two replicates of each treatment per experiment.

Ultrasound is an effective technology used in many industries including food (Knorr et al., 2004; Vilkhu et al., 2008). 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 (Alegria et al., 2009; Baumann et al., 2009; Cao et al., 2010; Vivek et al., 2016b). Other applications of ultrasound include degassing, inactivation of enzymes, crystallization, leaching, extraction, digestion, etc. (Jiao and Zuo 2009). 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 (Cao et al., 2010). 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 (Cao et al., 2010). 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 (Baş et al., 2007). 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 (Abreu et al., 2003; Beirão-da-Costa et al., 2006; Ölmez and Akbas 2009; Cao et al., 2010; Vivek et al., 2016a,b). However, there has been no report published on effect and optimization of ultra-sonication on postharvest Docynia indica for maximizing the shelf life. Therefore, our aim of this study is to investigate the effectiveness of ultra-sonication treatment on various quality parameters like total surface plate count, respiration rate, fruit firmness and to optimize the process variables for some selected quality parameters. The main process variables considered for this study includes ultrasonic amplitude, treatment time and temperature.

2.3. Experimental design Response Surface Methodology (RSM) is considered as an effective optimization tool used to optimize the levels of independent variables (Vivek et al., 2016a). 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. Design expert 8.00, Stat-Ease Inc., Minneapolis, MN was used to construct optimized model and analyse data. An efficient three-level-three-factor, Box-Behnken design was employed with 17 experimental runs with four replicates at the centre point (Bruns, 2007; Tomadoni et al., 2016). The range and centre point values of the independent variables were given in Table 1. The dependent variables as a function of independent variables were expressed using the following second order polynomial equation.

Y = β0 +

k

∑j=1

βi Xj +

k

∑j=1

βjj X2j +

∑ ∑i < j

βij Xi Xj

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) were also performed to establish optimum conditions for ultra-sonication treatment for Docynia indica. The surface plotting’s for the optimized results were shown in Fig. 1–3. 2.4. Analogy experiment Optimal conditions (ultrasonic amplitude: 80%, temperature: 25 °C and treatment time: 5.82 min) obtained from RSM was used to compare with untreated fruits as a control sample (Tables 5 and 6). After ultrasonication fruits were then immediately analysed for total plate count, firmness, respiration rate and vitamin C. Triplicates of 120 ± 5 g of fruits each per treatment, and the same experimental combinations was conducted in duplicate. Finally treated fruits were subjected to storage study after vacuum and normal packaging for 30 days (unpublished data).

2. Materials and methods 2.1. Materials The freshly ripened Docynia indica fruits i.e. partly matured fruits were harvested and collected from forest area of Manipur, North-East India in the month of September 2015. The good quality fruits were selected for the experimentation i.e. uniform size, absence of severe defects and visual wounds. Then the fruits were taken to the laboratory within 24 h from the time of harvest. The selected Docynia indica had an initial total soluble solid (TSS) of 9 ± 1 oBrix and moisture content (M.C) of 78.00 ± 1.00% w.b. (wet basis).

2.5. Total plate count Total plate count (TPC) was examined according to Pao and Brown, 1998 with minor modification. 120 g of ultra-sonicated sample was placed into sterilized bags consist of 1 l of 0.1% (w/v) peptone solution. Then the sterilized sample bags were mixed thoroughly with the help of

2.2. Optimization of ultrasonic treatment Probe sonicator (BBI-8535027, Sartorius Labsonic M, Germany) with a working constant frequency of 30 kHz having a maximal output power density of 600 W/cm2 was used for treatment of Docynia indica. A 7-mm titanium probe with a maximum amplitude of 220 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 distilled water used as a solvent by 25 mm. While the Cut-off cycle time of 2 s was fixed for all the experiment to control the temperature of the solvent. 600 ml of solvent was used to

Table 1 Independent variables and their level used for central composite design. Independent variables

Ultrasonic amplitude (X1) Treatment time (X2) Temperature (X3)

164

level −1

0

+1

40 5 25

60 10 37.5

80 15 50

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Fig. 1. Effect of ultrasonic amplitude and treatment time on total plate count.

constant speed of 3 mm s−1 to a depth of 5 mm. The peak puncture force was treated as firmness of Docynia indica and is measured in netwton (N) (Meng et al., 2014). While the vitamin C concentration was measured by 2,6 – dichlorophenolindophenol titration method. The results of vitamin C were expressed as mg/100 g of FW (Pal et al., 2015). Severity of decay was visually evaluated using four grade scale (‘0’ represents no decay, ‘1’ represents slight decay (25% of fruit surface), ‘2’ represents moderate decay (25–50%) and ‘3’ represents severe decay (more than 50%)) for 50 fruits. The fruit Decay index was calculated using the following formula (Cao et al., 2010).

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. 2.6. Rate of respiration Respiration rate was measured by sealing 100 ± 1 g fruits into 1 l plastic container. The container used was fitted with an airtight rubber septum and held at 25 ± 1 °C for 1 h. Rate of respiration was measured as explained by Vivek et al., 2016b 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).

Fruit decay index =

∑

((decay scale ) × (number of fruits at the decay scale )) total number of fruits in the treatment

Total soluble solids (TSS) was measured using the hand held refractometer and titratable acidity (TA) was measured using automated titrimeter and the results were expressed in terms of citric acid equivalent. Sample was titrated to an endpoint of pH 8.1 using 0.1N NaOH.

2.7. Firmness, vitamin C, decay index, Tss and TA

2.8. Statistical analysis

Firmness of the ultra-sonicated Docynia indica were measured accordance with Vivek et al., 2016b. 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

All the experimental results obtained were statistically analysed by applying independent sample t-test using SPSS v16 for inspecting the Fig. 2. Effect of ultrasonic amplitude and treatment time on Firmness.

165

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Fig. 3. Effect of ultrasonic amplitude and treatment time on respiration rate.

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).

Table 2 Box behnkan design matrix and response values. Experimental no

X1

X2

X3

Total plate count (log CFU/ cm2)

Respiration Rate (mg CO2 kg−1 h−1 FW)

Firmness (N)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

−1 +1 −1 +1 −1 +1 −1 +1 0 0 0 0 0 0 0 0 0

−1 −1 +1 +1 0 0 0 0 −1 +1 −1 +1 0 0 0 0 0

0 0 0 0 −1 −1 +1 +1 −1 −1 +1 +1 0 0 0 0 0

2.96 2.89 2.89 2.59 2.92 2.70 2.75 2.60 2.94 2.85 2.82 2.60 2.87 2.84 2.89 2.89 2.83

35.79 48.44 43.05 72.65 45.74 56.51 35.79 72.65 35.79 43.05 35.79 45.74 53.82 45.74 53.82 56.51 48.44

66 65 64 60 67 64 66 64 66 63 66 63 66 65 65 66 66

MAE =

1 N

N

∑i =1 N

RMSE =

⎧∑

i=1

Rreal − Rp (1) 1/2

(Rp − Rreal )2 ⎫

⎨ ⎩

N

⎬ ⎭

(2)

Where Rp is the predicted value; Rreal is the experimental/observed value; N is the number of points. 3. Result discussion 3.1. Model fitting 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). The Lack of fit (LoF) for all the responses (total plate count, respiration rate and firmness) were found to be insignificant, this indicates the error analysis obtained from RSM among centre points in the experimental combinations were minimum. The linear terms of all the factors were found to be significant (p < 0.05) for all the response variable, 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 2. Identical results were presented by Cao et al., 2010 and Vivek et al., 2016b for kiwifruit and strawberry fruits, respectively. The total number of experimental trails required to assess and construct the model for different variables and their interactions was easily done by using RSM. Finally, the models constructed were statistically measured to describe the deviation in the data.

Table 3 Regression coefficient for the responses. Coefficients

Total plate count

Respiration

Firmness

β0 X1(β1) X2(β2) X3(β3) X1X2(β4) X1X3(β5) X2X3(β6) X12(β7) X22(β8) X32(β9) R2 Adj R2 Pred. R2 Adq. prec Std dev C.V RMSE MAE Lack of fit

2.86 −0.092*** −0.085*** −0.080*** −0.058** 0.017 −0.032** −0.046 0.014 −0.076 0.98 0.95 0.86 18.14 0.026 0.93 0.001 0.014 N.S

51.66 11.23*** 6.09*** 1.11 4.24* 6.53 0.67** 5.45** −7.13*** −4.44* 0.95 0.88 0.71 12.56 3.94 8.08 3.20 2.10 N.S

65.60 −1.21*** −1.65*** −0.22 −0.75*** 0.17 −0.050 −0.46** −1.39 0.24** 0.97 0.96 0.94 22.91 0.39 0.60 0.03 0.2 N.S

3.2. RSM analysis

* Significant at p < 0.1. ** Significant at p < 0.05. *** Significant at p < 0.001.

3.2.1. Total plate count The overall model for the total plate count displayed significant 166

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Table 4 Responses and limits of optimizer for optimization using numerical optimization in design expert. Constraints Name

Goal

Lower limit

Upper limit

Lower weight

Upper weight

Importance

Total plate count Respiration rate Firmness

Minimize Minimize Maximize

02.60 35.79 60.00

3.00 72.65 67.11

1 1 1

1 1 1

5 4 4

3.2.2. Respiration rate The overall model for the Respiration rate displayed significant difference at p < 0.05 with F value 14.10. And the lack of fit for respiration rate had showed non-significant difference. All three independent variables i.e. ultrasonic amplitude, treatment time and treatment time displayed positive effect on respiration rate. As these independent variables increases with increase in respiration rate. Ultrasonic amplitude and treatment time also displayed the significant difference at p < 0.001. The chosen two synergy (interaction) terms i.e. ultrasonic amplitude and treatment time displayed the significant positive effect on respiration rate at p < 0.100. The other interaction terms between treatment time and temperature also displayed the significant positive effect on respiration rate at p < 0.05. The quadratic terms for ultrasonic amplitude showed significant positive effect at p < 0.05 while quadratic terms for treatment time and temperature had showed significant negative effect at p < 0.001 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. From the above obtained results one can easily claim that the model equation fits extremely well. The RMSE and MAE values were also calculated (RMSE = 3.20 and MAE = 2.10), which tells the deviation in the experimental data. The actual and predicted values of RR is shown in Table 8. The maximum respiration rate was observed at 80% ultrasonic amplitude, 15 min and 37.5 °C and the minimum respiration rate was observed at 40% ultrasonic amplitude, 5 min and 25 °C. Ultra-sonication at higher power and longer treatment time for kiwifruits have showed higher respiration rates (Vivek et al., 2016b). This may be due to the rupturing of fruit tissues and cell wall degradation (Gonzalez and Barrett 2010; Pieczywek et al., 2017). While lower respiration rates were observed for the ultra-sonicated samples after 2 days of storage period at refrigeration temperature compared to the control sample (Fig. 5). Similar kind of results were shown by Chen and Zhu (2011). Where the ultra-sonicated samples showed less respiration rate compared to control plum fruits during storage period at refrigeration temperature. In case of Prunus nepalensis, the respiration rate data does not follow any trend with increase in ultra-sonication time and amplitude (unpublished work). Therefore, the obtained results may vary from fruit to fruit. The fruit respiration is a major factor contributing to the postharvest quality losses, involves a series of oxidation-reduction reactions where various substances within the cells are oxidized to CO2 (Bhande et al., 2008). The initial higher respiration rates of fruits immediately after ultra-sonication treatment is may be due to the minor damage in fruit cell wall or tissue rupturing (Pieczywek et al., 2017). The higher respiration rates of fruits indicate a more active metabolism and usually a faster deterioration rate (Cantwell and Suslow, 1999). Heat treated mango fruits showed higher respiration rate (Ketsa et al., 1999).

Table 5 Optimized solution – response optimizer in design expert. Variables

Optimized conditions

Ultrasonic amplitude (%) Treatment time (min) Temperature (°C) Total plate count (log CFU/cm2) Respiration rate (mg CO2 kg−1 h−1 FW) Firmness (N)

80.00 05.82 25.00 02.82 43.30 65.22

Table 6 Effect of ultrasonic treatment under the optimized conditions on responses. Responses

Ultrasound treated sample (optimized conditions)

Un treated sample

p-value

Total plate count (log CFU/ cm2) Respiration rate (mg CO2 kg−1 h−1 FW) Firmness (N) Vitamin C (mg/100 g of FW)

2.84

3.43

0.01*

43.67

32.33

0.04*

65.00 67.33

67.00 71.00

0.015* 0.235

p value corresponds to Student’s t-test to related samples (paired). * Significant at p < 0.05.

difference at p < 0.001 with F value 37.27. And the lack of fit for total plate count had showed non-significant difference. All three factors i.e. ultrasonic amplitude, treatment time and treatment time displayed the negative effect on total plate count. Increase in individual values of both the ultrasonic amplitude and treatment time significantly (p < 0.001) decreases the total plate count. Similarly, the synergy terms between these independent variables (ultrasonic amplitude and treatment time) also displayed the significant negative effect on total plate count at p < 0.001. The other interaction terms (treatment time and temperature) also displayed the significant negative effect on total plate count at p < 0.100. While the individual quadratic terms had showed a non-significant difference at p < 0.05. The coefficient of determination and adjusted coefficient of determination values (R2 = 0.98 and Adj R2 = 0.95) resulted high for TPC. From the above obtained results one can easily claim that the model equation fits extremely well. The RMSE and MAE values were also calculated (RMSE = 0.001 and MAE = 0.014), which tells the deviation in the experimental data. The actual and predicted values of TPC is shown in Table 8. The maximum total plate count was observed at 40% ultrasonic amplitude, 5 min and 37.5 °C and the minimum total plate count was observed at 80% ultrasonic amplitude, 15 min and 37.5 °C. Similar kind of results were obtained for kiwifruit and strawberry (Cao et al., 2010; Vivek et al., 2016b). This microbial reduction may be due to cavitation bubbles, localized temperature, formation of free radicals and pressure occured in solvent during ultra-sonication (Cao et al., 2010). The mechanical effects induced by pressure near the microbes during the collapse of cavitation bubbles leads to microbial cell disintegration (Joyce et al., 2011). There is no much significant decrease in total plate count was observed for Prunus nepalensis (unpublished work).

3.2.3. Texture-firmness The overall model for the firmness displayed significant difference at p < 0.05 with F value 33.47. And the lack of fit for firmness had showed non-significant difference. All the factors i.e. ultrasonic amplitude, treatment time and temperature displayed the pessimistic effect on texture. i.e. As these independent variables increases firmness of the Docynia indica decreases significantly at p < 0.001. The synergy factors (ultrasonic amplitude and treatment time) had also displayed the 167

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fruit firmness, which indicates the model fits well. The RMSE and MAE values were also calculated (RMSE = 0.03 and MAE = 0.2), which tells the less deviation in the experimental data. The actual and predicted values of fruit firmness (FF) is shown in Table 8. The maximum firmness was observed at 40% ultrasonic amplitude, 10 min and 25 °C and the minimum firmness was observed at 80% ultrasonic amplitude, 15 min and 37.5 °C. Various authors have reported that the cell wall degradation and tissue rupturing of fruit is mainly due to the disturbances in cell wall constituents i.e polygalacturonase and pectin methylesterase (Nogata et al., 1993; Ariel et al., 2007) and also due to the mechanical effects induced by pressure (Joyce et al., 2011). Hence this may be the reason for decrease in firmness of ultra-sonicated fruits.

Table 7 Shelf life study and Quality analysis of treated and un-treated sample for 3 weeks. Storage period (Days)

Treatment

Total plate count (log CFU/ cm2)

Decay index (%)

TSS (oBrix)

TA (%)

Firmness (N)

0

Treated control Treated control Treated control Treated control Treated control Treated control Treated control Treated control

2.85a 3.40a 2.90a 3.42a 3.07a 3.52b 3.20a 3.71b 3.46a 4.77b 4.07 – 4.11 – 4.69 –

0 0 0 0 0.5a 14.5b 02.00a 26.50b 06.00a 31.00b 11.00 – 17.00 – 19.50 –

9a 9a 9a 9a 10a 11b 11a 12b 11a 14b 12 – 13 – 15 –

0.730a 0.729b 0.726a 0.725a 0.701a 0.681b 0.685a 0.660b 0.650a 0.593b 0.613 – 0.602 – 0.587 –

65.00a 67.10a 65.00a 66.40a 64.60a 62.41b 64.00a 58.00b 61.20a 54.00b 59.30 – 58.00 – 55.00 –

3 6 9 12 15 18 21

3.3. Optimization of ultrasonic treatment conditions The desirability function of 0.70 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 (Harrington, 1965). Importance of ‘5’ was provided to total plate count, while importance level of ‘4’ were given to both firmness and respiration rate. Importance of ‘3’ was given to all the independent (ultrasound amplitude, treatment time and temperature) variables based on the analogous contribution and 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 obtained for all the independent variables viz. Ultrasound amplitude, treatment time and temperature of the solvent (water) were shown in Table 5.

a, b represents the significant (p < 0.05) difference along the column. 6 5 4 3

3.4. Ultrasonic treatment on microbial load and quality of docynia indica at optimum conditions

2 1

The optimum values predicted by the RSM i.e. 80% – ultrasonic amplitude, 25 °C – temperature and 5.82 min – treatment time were considered for determining the microbial population on the surface of the fruit. Other quality aspects like firmness and respiration rate of 100 g’ samples were also evaluated for ultra-sonicated samples then finally compared with the reference samples (Table 5). Ultra-sonication treatment does not induce any change in TSS and TA (Ordóñez-Santos et al., 2017). But significant difference was observed from 6th day of storage due to the senescence of fruit between ultra-sonicated and control sample. The average values of total plate count, firmness, respiration rate and vitamin C for both the ultra-sonicated samples and reference samples were presented in Table 6. Ultra-sonication significantly impedes the microbial growth on the surface by 0.6 log cycle at p < 0.05. Similarly, both the respiration rate and firmness of the fruit also showed significant (p < 0.05) difference with the reference sample (Table 6). Insignificant difference was noticed for vitamin C at p > 0.05 for both ultra-sonicated and reference samples. Similar kind of results was given for kiwi fruits (Vivek et al., 2016b). The degradation of vitamin C may be due to the oxidation processes generated during ultrasonic treatments (Lešková et al., 2006; Ordóñez-Santos et al., 2017). The respiration rate for the ultra-sonicated Docynia indica samples resulted higher (26%) compared to reference samples. Initially the respiration rate of ultra-sonicated sample was obtained higher compared to control sample due to the disturbances in cell wall constituents. But after 2nd day of storage period the respiration rate of ultra-sonicated samples got declined significantly compared to control sample (Fig. 5). This shows that the ultra-sonication treatment delays the senescence of fruit and extends its shelf life to 3 weeks. While the total plate count, firmness and vitamin C for the ultra-sonicated Docynia indica samples were decreased (17.2%, 3%, 5%) compared to control samples. The validity and adequacy of the predicted models were reviewed with the optimised results obtained from the experiments. Cao et al., 2010 showed similar kind of results for the total bacterial count.

0 0

5

10

15

20

Fig. 4. Total plate count of ultra-sonicated sample (optimized conditions) Vs control sample.

Fig. 5. Respiration rate of ultra-sonicated sample (optimized conditions) Vs control sample.

significant pessimistic effect on firmness at p < 0.001 i.e. this combined effect decreased the fruit firmness due to the pressure produced by ultrasound. Similar kind of results were reported for strawberry and kiwifruit (Cao et al., 2010; Vivek et al., 2016a,b). The other interaction terms showed non-significant difference on fruit firmness. The quadratic terms for ultrasonic amplitude and temperature had also showed significant negative and positive difference on fruit firmness at p < 0.05. The coefficient of determination and adjusted coefficient of determination values (R2 = 0.97 and Adj R2 = 0.96) resulted high for 168

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Table 8 Actual and predicted values of all dependent variables. Exp. no

TPC (Actual) (log CFU/ cm2)

TPC (Predicted) (log CFU/ cm2)

RR (Actual) (mg CO2 kg−1 h−1 FW)

RR (Predicted) (mg CO2 kg−1 h−1 FW)

FF (Actual) (N)

FF (Predicted) (N)

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

2.96 2.89 2.89 2.59 2.92 2.70 2.75 2.60 2.94 2.85 2.82 2.60 2.87 2.84 2.89 2.89 2.83

2.95 2.88 2.89 2.59 2.93 2.71 2.73 2.58 2.93 2.83 2.84 2.60 2.86 2.86 2.86 2.86 2.86

35.79 48.44 43.05 72.65 45.74 56.51 35.79 72.65 35.79 43.05 35.79 45.74 53.82 45.74 53.82 56.51 48.44

36.90 50.89 40.60 71.54 46.85 56.27 36.02 71.54 33.57 44.40 34.44 47.96 51.66 51.66 51.66 51.66 51.66

66.00 65.00 64.00 60.00 67.11 64.44 66.00 64.00 66.00 63.00 66.00 62.80 65.78 64.98 65.33 66.00 65.90

65.86 64.94 64.06 60.14 66.98 64.23 66.21 64.13 66.27 63.07 65.93 62.53 65.60 65.60 65.60 65.60 65.60

The firmness of ultra-sonicated kiwi fruits was occurred 5.42% less compared to the NaOCl treated samples, while the TSS of remains unaffected (Vivek et al., 2016b). Similarly, the TSS of the ultra-sonicated Docynia indica samples also remains unaffected. Shelf life study and quality analysis of ultra-sonicated fruits for three weeks at refrigeration temperature (6–8 °C) was determined and compared with the untreated sample (Table 7). Control samples were spoiled after 12th day of storage and were not considered for further study while the ultra-sonicated samples last till 21 days of storage further storage spoils the fruit and cannot be considered for consumption. Ultrasound treatment significantly increased the firmness of fruit by 26% during storage till 12 days of storage (Fig. 4). Decay index on 15th day of storage is greater than 50% hence the sample was rejected from the study. Therefore, from the above results it can be clearly seen that the ultrasonicated samples clearly reduce the senescence of the fruit and increases the shelf life by 10 days compared to the untreated samples (Table 7).

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