Postharvest Changes in Antioxidant Capacity ... - Springer Link

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Oct 18, 2013 - Sunil Kumar & Ramesh Kumar & V E Nambi & R K Gupta. Received: 10 June 2013 /Accepted: 2 October 2013 /Published online: 18 October ...
Food Bioprocess Technol (2014) 7:2060–2070 DOI 10.1007/s11947-013-1212-7

ORIGINAL PAPER

Postharvest Changes in Antioxidant Capacity, Enzymatic Activity, and Microbial Profile of Strawberry Fruits Treated with Enzymatic and Divalent Ions Sunil Kumar & Ramesh Kumar & V E Nambi & R K Gupta

Received: 10 June 2013 / Accepted: 2 October 2013 / Published online: 18 October 2013 # Springer Science+Business Media New York 2013

Abstract Strawberry fruits are highly perishable and cannot be stored for more than 1–2 days at ambient condition. Therefore, it was thought desirable to enhance the shelf-life of harvested strawberry using pectin methyl esterase and divalent ions. The freshly harvested fruits of strawberry were treated with different concentrations of pectin methyl esterase (50– 300 units) and divalent calcium ions (calcium chloride) for 5– 30 min using response surface methodology—Box–Behnken design. The treated and untreated (control) fruits were packed in plastic punnets and stored at two different temperature conditions, viz., 7 °C with 80 % relative humidity (RH) and 25 °C with 60 % RH, for evaluating the shelf-life. Appropriate physico-chemical and microbiological parameters were determined at alternate days during their storage. Overall firmness and color values (L , a , b ) decreased, while physiological weight loss (percent) increased during their storage. No food-borne pathogens, viz., Salmonella , Staphylococcus , and coliforms, were observed during storage. Total antioxidant capacity and ferric reducing antioxidant power decreased, while lipoxygenase and polyphenol oxidase activities increased during storage at 7 and 25 °C. The shelf-life of treated strawberry was found to be 10 days at 7 °C as against 6 days for control fruits under similar conditions. The treated strawberry had shelf-life of 2 days compared to 1 day for control maintained at 25 °C.

Keywords Strawberry . Pectin methyl esterase . Storage . Total antioxidant capacity . Firmness . Microbial load S. Kumar (*) : R. Kumar : V. E. Nambi : R. K. Gupta Division of Horticultural Crop Processing, Central Institute of Post Harvest Engineering and Technology, Malout-Hanumangarh Bye-pass, Abohar, Punjab, India 152116 e-mail: [email protected]

Introduction Traditional preservation techniques greatly affect the sensorial and nutritional quality of fruit. Also, consumer demands for convenient but fresh and healthy foods are motivating the food industries to apply newer and sustainable preservation techniques, which can satisfy the increasing market demands for fewer preservatives, higher nutritive value, and fresh sensory attributes. Strawberry is a good source of vitamins, minerals, and a variety of antioxidants. Its fruits are highly perishable particularly during postharvest storage and are very often susceptible to mechanical injury, decay, and physiological losses (Tulipani et al. 2008; Romanazzi et al. 2013; Luksiene et al. 2013). Ripe strawberries are very soft and have a shelf-life of only 2 days under ambient condition, i.e., at 20 °C (Vicente et al. 2002; Suutarinen and Autio 2004; Zhang et al. 2007). The great susceptibility of strawberries to textural damage is due to their low solids content, large cells, and thin cell walls. A major portion of the softening results from degradation of the middle lamella of the walls of cortical cells accompanied with increased release of pectins from the cell walls (Suutarinen and Autio 2004). Fruit and vegetable cell walls are composed of mainly cellulose and pectins. The main component of pectin backbone is galacturonic acid residues linked by α-1-4 linkages with neutral sugars such as arabinose, galactose, and xylose present in side chains, whereas rhamnose constitutes a minor component of pectin. The carboxyl groups of galacturonic acid are partially esterified by methyl groups and partially or completely neutralized by sodium, potassium, or ammonium ions. Pectins are present in considerable amounts in fruits and vegetables, thus contributing to the strength of these tissues (Mandhania et al. 2010; Kumar et al. 2012). Divalent calcium ions (Ca2+) normally occur between the cells, where they form crosslinks between the carboxyl groups of adjacent

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polyuronide chains (Main et al. 1986). The structure of the pectin “gel” in the middle lamella is generally explained by the “egg box” model in which pectins have few “hairy regions” and a low methoxyl rate. For calcium to be an effective firming agent, a low degree of methoxylation must be present (Suutarinen and Autio 2004). The firmness of strawberry cell walls can be enhanced by giving low methoxyl pectin (attributed to pectin methyl esterase (PME) action) accompanied by use of firming agents (like Ca2+) which, in turn, can prolong shelf-life of strawberry fruits (Baker and Wicker 1996; Micheli 2001; Lamikanra 2002; Suutarinen and Autio 2004; Jyani et al. 2005). Partial hydrolytic demethylation of pectin to pectic acid by pectin methyl esterase in the presence of Ca2+ can result in texture firming due to the formation of crossbridges between Ca2+ and carboxyl groups of the pectic acids (Perera and Baldwin 2001; Martin-Belloso et al. 2006). Thus, the present investigation was carried out to optimize the PME concentration and treatment time for enhancing the shelf-life and maintaining nutritional quality of fresh strawberry fruits during storage at 7 °C with 80 % RH and 25 °C with 60 % RH.

Materials and Methods Raw Material Freshly harvested fruits of strawberry (var. Chandler) were procured from commercial strawberry farm (300 km away) and transported overnight to research laboratory of Horticultural Crop Processing Division of the institute. The next day, calyxes were removed and mature fruits free from any blemishes were used for experimentation. The temperature while transport was 35±2 °C while during dip treatment was 35 °C. Rest of the operations was between 32 and 35 °C. Process Design for the Shelf-life Experiment The experiment was conducted with by using response surface methodology (RSM)—Box–Behnken design (Table 1), and treatments given to the fruit included PME enzyme and divalent ions, with their treatment time as shown in Table 1. The containers, utensils, and probable surfaces to be in contact with the fruits during primary processing were thoroughly washed and sanitized with 0.1 % NaOCl. The experimental fruits were first washed with cold water and then dipped in 0.1 % NaOCl for 1 min. The fruits were dipped in 0.05 M acetate buffer (pH 4.0) containing PME and CaCl2 (different concentrations), and the dip treatments were performed in a water bath having 35±2 °C temperature for defined treatment times as per Table 1. Calcium salts (lactate and chloride) were used as source of divalent cations. Finally, a solution was prepared by mixing all ingredients (PME, calcium chloride, sodium benzoate, kinnow, and pomegranate peel extract) in

2061 Table 1 The experimental domain for pectin methyl esterase and CaCl2 concentration and their time of treatment used during response surface methodology Factor 2 (B): time Standard Run Factor 1 (A): PME units (IU) of treatment (min) (x 2) (x 1)

Factor 3 (C): CaCl2 concentration (%) (x 3)

9

1

175.00 (0)

5.00 (−1)

0.50 (−1)

12

2

175.00 (0)

30.00 (+1)

2.00 (+1)

13

3

175.00 (0)

17.50 (0)

1.25 (0)

3

4

50.00 (−1)

30.00 (+1)

1.25 (0)

4

5

300.00 (+1)

30.00 (+1)

1.25 (0)

14

6

175.00 (0)

17.50 (0)

1.25 (0)

15

7

175.00 (0)

17.50 (0)

1.25 (0)

10

8

175.00 (0)

30.00 (+1)

0.50 (−1)

2

9

300.00 (+1)

5.00 (−1)

1.25 (0)

11

10

175.00 (0)

5.00 (−1)

2.00 (+1)

5

11

50.00 (−1)

17.50 (0)

0.50 (−1)

7

12

50.00 (−1)

17.50 (0)

2.00 (+1)

6

13

300.00 (+1)

17.50 (0)

0.50 (−1)

16

14

175.00 (0)

17.50 (0)

1.25 (0)

1

15

50.00 (−1)

5.00 (−1)

1.25 (0)

17

16

175.00 (0)

17.50 (0)

1.25 (0)

8

17

300.00 (+1)

17.50 (0)

2.00 (+1)

Values in brackets are coded values of three levels

required amount and the fruits were treated with this readymade solution by dip treatment to reduce reaction time and to minimize leaching losses. After preliminary standardizations, a Box–Behnken design based on three levels and three variables (shelf-life of strawberry as a function of PME and CaCl2 concentration and their time of treatment) was used to study the composite influence of the abovementioned independent variables on shelf-life of strawberry. The design consisted of 17 experiments with four equatorial points, four axial points, and nine center points for replication (Table 1). In developing the regression equation, the test factors were coded according to the following equation: xi ¼ X i −X 0 =δX i where x i is the dimensionless coded value of the ith independent variable, x i is the natural value of the ith independent variable, X 0 is the natural value of the ith independent variable at the center point, and δX i is the step change value. Once the experiments were performed, the experimental results were fitted with a second-order polynomial function: Y ¼ b0 þ b1 x1 þ b2 x2 þ b3 x3 þ b12 x1 x2 þ b13 x1 x3 þ b23 x2 x3 þ b11 x21 þ b22 x2 2 þ b33 x23 where Y is the predicted response; b 0 is the intercept; b 1, b 2, and b 3 are the linear coefficient; b 11, b 22, and b 33 are the squared coefficient; and b 12, b 13, and b 23 are the interaction coefficients.

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After treatment, strawberry fruits were drained and dried under shade with forced air having temperature of 32–35 °C. Control fruits were simply washed with chlorinated water. All fruits were then wiped with tissue papers, packed in plastic punnets, and stored under two different temperature conditions, i.e., 7 °C with 80 % RH and 25 °C with 60 % RH for evaluating the shelf-life. Unless stated otherwise, each experiment was thoroughly standardized and replicated thrice. Appropriate physico-chemical and microbiological parameters were determined at each processing step during fruit handling and at alternate days during storage.

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Physico-chemical Parameters Fruit Color Fruit color was measured with hunter color meter. The instrument was calibrated with a white reference tile and expressed as L, a, and b as color coordinates, where L value represents brightness of product, a value represents the green (−ve value) or redness (+ve value) of the fruit while b value represents the change in color from yellowish (+ve value) to purplish/blue (−ve value). Color of fruit surface was taken from distal, blossom, and mid-portion of the fruit and averaged. The manifestations of color were done as per the standard color matrix.

Extractions Methanolic Extraction Methanolic extraction was carried out as per the procedure given by Sreeramulu and Raghunath (2010). For total antioxidant capacity (TAC) and ferric reducing antioxidant power (FRAP), 5.0 g pulp of strawberry fruit was grounded with 25 ml of 60 % methanolic extract containing 0.1 % HCl. For extraction, the resulting broth was covered with aluminum foil and shook at 150 rpm at 30 °C in an incubator for 4 h. The sample suspension was filtered through four layers of cheesecloth and centrifuged (Eltak, India) at 7,000 rpm for 20 min. The supernatant was filtered through grade 4 filter paper and used for analysis of TAC and FRAP.

Buffer Extraction The buffer extraction was prepared at 0–4 °C as adopted by Kumar et al. (2011a). For lipoxygenase (LOX) and polyphenol oxidase (PPO) activity, the extraction was carried out in 0.1 M potassium phosphate buffer (pH 6.8) containing 3 % (w/v) polyvinylpyrrolidone and 1 mM EDTA. The enzymes were extracted by macerating 5.0 g tissue with 15 ml of ice cold extraction medium in a prechilled pestle and mortar using acid washed sand as abrasive. The homogenate was filtered through four layers of cheesecloth and the filtrate centrifuged at 7,000 rpm for 20 min in a refrigerated centrifuge (Eltak, India) at 4 °C. The supernatant was carefully decanted and used as crude enzyme preparation.

Microbiological Extraction For microbiological parameters, 5.0 g of fruit tissue was ground in a chlorinated and sanitized pestle and mortar and transferred to an aseptic plastic vial. Serial dilution up to 10−4 was prepared for determining different microbiological parameters.

Fruit Firmness Fruit firmness was estimated with the help of hand held penetrometer (model: Erma, Italy) using 2 mm probe. The results were expressed in terms of N (newton) force produced per 2 mm of the needle as the maximum force required to penetrate the flesh. Firmness was measured from equatorial region at three different points, and the average was taken as values for fruit firmness. Physiological Weight Loss The physiological weight loss (PWL) of fruit was calculated by taking weight difference at different time interval and is expressed in percentage by using the following formula: PWL ð%; gÞ ¼

Initial weight ðgÞ−Weight at any intervalðgÞ  100 Initial weightðgÞ

Biochemical Estimations Total Antioxidant Activity The total antioxidant capacity (TAC) was determined by evaluating the free radical scavenging effect on 2, 2′-diphenyl-2picrylhydrazyl (DPPH; Himedia, Mumbai) as per the procedure of Yu et al. (2002) and Sreeramulu and Raghunath (2010). To 1.8 ml of phosphate buffer (0.05 M; pH 7.0), 1.0 ml of DPPH reagent (0.1 mM in methanol) and 0.2 ml of methanolic extract were added. The blank (serving as 0) contained 2.0 ml of respective buffer and 1.0 ml of 60 % methanol, while the control contained 2.0 ml buffer with 1.0 ml of 0.1 mM DPPH reagent. The reagents were mixed well and incubated in dark for 30 min at room temperature. The discoloration of DPPH by methanolic extract was measured against blank at 517 nm using UV–visible spectrophotometer (Shimadzu, Japan), while the control (without methanolic extract) retained its color. Percentage inhibition of discoloration of DPPH by sample extract was calculated,

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and the TAC was expressed in terms of nanomoles of ascorbic acid equivalents per gram of fresh weight (f.wt.) of strawberry. %inhibition of DPPH ¼

A517 control−A517 sample  100 A517 control

Ferric reducing antioxidant power FRAP of the fresh as well as stored strawberry was measured with the method described by Benzie and Strain (1996). The reagents included 300 mmol/liter acetate buffer (pH 3.6), 10 mmol/liter 2,4 6-tripyridyl-s-triazine (TPTZ; Himedia, Mumbai), and 20 mmol/liter FeCl3·6H2O. The FRAP reagent was prepared by mixing 50 ml acetate buffer, 5.0 ml TPTZ solution, and 0.1 ml FeCl3·6H2O. The reaction mixture contained 2.5 ml of FRAP reagent, 0.9 ml of acetate buffer, and 0.1 ml of methanolic extract. The blank contained 2.5 ml of FRAP reagent and 1.0 ml of acetate buffer. The contents were mixed thoroughly and allowed to stand for 10 min under dark. The absorbance was recorded at 593 nm. The FRAP was expressed in terms of nanomoles of FeSO4 (Fe2+) equivalents per gram of fresh weight.

at 37 °C for 30 min. After 30 min, the reaction was stopped by adding 0.5 ml of 5 % H2SO4, mixed well, and then the absorbance of the purpurogallin formed was recorded at 420 nm. The activity was calculated using the molar extinction coefficient (2.47 mM−1 cm−1) for purpurogallin (Haddadchi and Gerivani 2009) and expressed as nanomoles of purpurogallin produced per minute per gram of fresh weight. Microbial Load For microbiological examination, the readymade media plates were procured from HiMedia Laboratories Ltd., Mumbai. For total plate count 100 μl of 10-3 dilution, while for coliform and Staphylococcus aureus count, 100 μl of 10-2 dilution of microbiological extraction were used respectively, each on plate count agar, coliform and Chapman stone media. However, for Salmonella count, 100 μl of 10−1 dilution of microbiological extraction was used on Bismuth sulfite medium. The plates were incubated at 30 °C for 24 h, and the colonies developed were counted manually. Data Analysis

Lipoxygenase The LOX (EC 1.13.11.12) was assayed spectrophotometrically at 234 nm by the method of Catherine et al. (1998). The reaction mixture (3.0 ml) contained 2.91 ml of potassium phosphate buffer (0.1 M, pH 6.2), 40 μl of 30 mM linoleic acid in ethanol, and 50 μl of enzyme extract. The reaction was started by the addition of enzyme and increase in absorbance at 234 nm was noted at room temperature for 3 min against buffer blank. The LOX activity was measured by monitoring the formation of conjugated dienes from linoleic acid using molar extinction coefficient of 2.74×104 M−1 cm−1 (Chen and Whitaker 1986). One unit of LOX was defined as amount of enzyme required to produce 1 nmol of conjugated dienes per minute per gram of fresh weight. Polyphenol Oxidase The PPO (EC 1.14.18.1) activity was estimated as described by Kar and Mishra (1976). The reaction mixture contained 1.5 ml of 0.05 M phosphate buffer (pH 6.8), 1.0 ml of 50 mM pyrogallol (made in above buffer), and 0.5 ml of buffer extract. The reaction mixture was mixed well and incubated

Design expert 7.1.4 (Stat-Ease, Inc., Minneapolis, USA) was used for the regression analysis of the experimental data. The quality of fit of the polynomial model equation was expressed by the coefficient of determination, R 2, and its statistical significance was checked by Fisher’s F test. The level of significance was given as p value.

Results and Discussion Standardization of Experiment Using RSM for Strawberry Initially, the experiment was standardized using calcium lactate along with PME (data not shown). Luna-Guzman and Barrett (2000) reported that calcium lactate treatment is a potentialalternativetocalciumchlorideforshelf-life extensionof fresh-cut cantaloupe, since it provided similar or better tissue firming (approx.25–33%firmer than just cut samples) without providing undesirable bitterness. However, calcium lactate imparted bluishness to the product in the present experiment. Hence, it was replaced with calcium chloride for further experimentation. The final experiment was performed as per the

Table 2 Estimated regression coefficients for shelf-life and their p value significance for the experimental domain Term

Constant

x1

x2

x3

x 12

x 22

x 32

x1 x2

x1 x3

x2 x3

Co-efficient p value

5.95 0.0071

−0.0368 0.0035

−0.052 0.0142

3.667 0.3159

0.000112 0.0009

0.0016 0.459

−1.333 0.051

0.00032 0.1705

0.0 1.0

0.0 1.0

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Table 3 Observed and predicted shelf-lives of treated strawberries during RSM Run number

Actual value (coded level)

Response (shelf-life in days)

Residual error

Factor 1 (A): PME units (IU) (x 1)

Factor 2 (B): time of treatment (min) (x 2)

1

175.00 (0)

5.00 (−1)

0.50 (−1)

5

4.5

0.5

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

175.00 (0) 175.00 (0) 50.00 (−1) 300.00 (+1) 175.00 (0) 175.00 (0) 175.00 (0) 300.00 (+1) 175.00 (0) 50.00 (−1) 50.00 (−1) 300.00 (+1) 175.00 (0) 50.00 (−1) 175.00 (0) 300.00 (+1)

30.00 (+1) 17.50 (0) 30.00 (+1) 30.00 (+1) 17.50 (0) 17.50 (0) 30.00 (+1) 5.00 (−1) 5.00 (−1) 17.50 (0) 17.50 (0) 17.50 (0) 17.50 (0) 5.00 (−1) 17.50 (0) 17.50 (0)

2.00 (+1) 1.25 (0) 1.25 (0) 1.25 (0) 1.25 (0) 1.25 (0) 0.50 (−1) 1.25 (0) 2.00 (+1) 0.50 (−1) 2.00 (+1) 0.50 (−1) 1.25 (0) 1.25 (0) 1.25 (0) 2.00 (+1)

6 6 8 10 6 6 6 7 5 5 6 8 6 7 6 9

6.5 6 7.25 10.25 6 6 6 7.75 5 5.75 6.25 7.75 6 6.75 6 8.25

−0.5 0 0.75 −0.25 0 0 0 −0.75 0 −0.75 −0.25 0.25 0 0.25 0 0.75

Factor 3 (C): CaCl2 concentration (%) (x 3)

matrix of RSM (Table 1). The range and levels of experimental variables investigated are presented in Table 1. The application of RSM yielded the following regression equation, which is an

Observed

Predicted

empirical relationship between shelf-life and the test variables in actual units:

Shelf life ¼ þ5:95−0:0368 EC−0:052 Time þ 3:667 CaCl2 þ 0:00032 EC:Time þ 0:0 EC:CaCl2 þ 0:0 Time:CaCl2 þ 0:000112 EC2 þ 0:0016 Time2 −1:333 CaCl2 2

The significance of each coefficient was determined by p values, which are given in Table 2. The predicted shelflife was compared with the experimental (observed) values and data given in Table 3. Statistical testing of the model was carried out by Fisher’s statistical (F ) test for analysis of variance (Table 4). If the model is a good predicator of the experimental results, the calculated F values should be fairly greater than tabulated F value. In the present case, the calculated F value of 7.55 was greater than tabulated F 9,7 (1 %= Table 4 ANOVA table for shelf-life of strawberries Source

SS

DF

MS

F value

p value

Model Residual Total

29.12 3.0 32.12

9 7 16

3.24 0.43

7.55

0.0071

SS sum of square, DF degree of freedom, MS mean square, R 2 =90.7 %; table F 9,7(1%) =3.68

3.68) one, which implies that model is significant and there is a quadratic relationship between the independent variables and response variables (Table 4). The coefficient of determination, R 2, calculated as 0.907 implies that the fitted model explains nearly 90.7 % of total variation in the shelf-life (Table 4). The value of R 2 suggested fair agreement between the experimental and predicted values obtained from the model. It was deduced from the results that 300 units of enzyme applied for 30 min with 1.25 % CaCl2 was found optimum for maximum shelf-life of strawberry (10 days) when stored at 7 °C with 80 % RH (Fig. 1a–c; Table 3). Figure 1a–c represents interaction effects between the three parameters for whole strawberry as deduced by RSM. Maximum shelf-life was predictable with time of treatments of more than 15 min and up to 30 min of PME having near about 290 to 300 IU activity (Fig. 1a). It is clear from the data presented in Fig. 1b that the maximum shelf-life (10 days) was predictable using RSM interaction between PME (295–300 IU) and CaCl2 (0.9– 1.5 %). Between 70 to 170 IU PME activity, a horse saddle

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ƒFig. 1

Interaction effects of pectin methyl esterase (IU) with time of treatment (minute) (a) and with calcium chloride concentration (percent) (b ), and of time of treatment (minute) with calcium chloride concentration (percent) (c), on shelf-life of strawberry maintained at 7 °C and 80 % RH. The squares represent the shelf-life in days (6, 7, 8, 9, and 10), while the red dot indicates the mid-value for shelf-life (i.e., 5) during quadratic relationships of variables used during the experiment

was observed that means nothing could be predictable in that range. Time of treatment near 28–30 min with a CaCl2 range of 0.9–1.5 % resulted in a shelf-life of 10 days or so (Fig. 1c). Response surface methodology has been instrumental in a variety of research activities say microbiological media optimization, enzyme characterization, recovery of oil/active ingredient, etc. using software mediated speculations and minimizing the workload and effort paid in any research (Ghafoor et al. 2010; Kumar et al. 2011b; Radojkovic et al., 2012).

Physico-chemical Parameters Changes in physico-chemical parameters were assessed during storage of strawberry fruits at 7 and 25 °C. The changes in color values of strawberry are given in Table 5. The brightness (L value) of fruit decreased with the increase in storage period under both the conditions (Table 5). However, it was marginally improved in strawberry on 10th day. Similarly a value which represents the red color of the fruit decreased during storage (Table 5). However, this decrease was more in control samples at 7 °C (decreased from 51.92 to 44.81) as compared to treated sample (decreased from 51.48 to 45.60). Decrease in b value is an indicative of decrease in yellowness with a shift towards bluishness. The b value decreased drastically in control samples (decreased from 30.57 to 16.91), while the loss in

yellowness was very less in enzyme-treated (decreased from 29.13 to 24.73) fruits maintained at 7 °C (Table 5). The data of fruit firmness are given in Fig. 2a. The firmness of the strawberry decreased with the advancement of storage period. The firmness of strawberry stored at 25 °C reduced drastically, and the control samples were lost within 1 day of storage due to fungal attacks and tissue liquefication. The fruits treated with enzyme retained some acceptable firmness by 2nd day of storage although tissue liquefication in these samples, too, made them unfit for consumption on the 3rd day. Strawberry fruits maintained at 7 °C reduced this loss in fruit firmness. Fruit firmness of cold stored (7 °C) fruits was found to be 0.872 N on 6th day for control sample, while the corresponding values for enzymetreated samples was observed to be 0.987 N on the 6th day and it reached at the stage of control sample on the 10th day (0.881 N) of storage (Fig. 2a). So, the enzyme-assisted calcium chloride treatment was found to extend shelf-life by more than 4 days compared to control. The calcium chloride pretreatment with PME in a vacuum (Suutarinen et al. 2002), or of calcium lactate or other calcium salt pretreatments in a vacuum before freezing and thawing (Garcia-Berbari et al. 1998), enhanced firmness independent of the species. The treatments did not affect the sensory quality of the fruits (Suutarinen and Autio 2004). Baker and Wicker (1996) infused free stone peaches with PME and calcium which resulted in four times firmer fruits. Activation of PME increases the number of calcium-binding sites in the pectins and thus allow for increased calcium cross-linking and better texture. It has been shown that adding PME, purified either from tomatoes (Castaldo et al. 1995) or a microbial source (Grassin 2002), to diced tomatoes enhanced the ability of calcium to improve firmness (Anthon et al. 2005). The increase in tissue firmness with an elevation of tissue calcium is caused by the interaction of the calcium ions with pectin polysaccharides in both the middle lamellae and parenchyma cell walls (Balla and

Table 5 Changes in color scale (L, a, b values) of strawberry fruits stored at 7 and 25 °C Storage condition

Day(s)

Color scale L

7 °C and 80 % RH

25 °C and 60 % RH

0 2nd 4th 6th 8th 10th 0 2nd

b

C

T

C

T

C

T

42.23±0.457 40.83±0.722 37.62±0.266 36.08±0.486 – – 42.23±0.457 35.99±0.488

41.91±0.457 41.88±0.265 41.42±0.217 39.30±0.182 36.45±0.284 37.08±0.213 41.91±0.457 40.27±0.241

51.92±0.375 50.95±0.993 45.86±0.502 44.81±0.306 – – 51.92±0.375 49.06±0.608

51.48±0.551 50.83±0.333 48.52±0.634 49.23±0.481 46.09±0.635 45.60±0.320 51.48±0.551 49.93±0.324

30.57±0.729 29.01±0.531 19.19±0.798 16.91±0.137 – – 30.57±0.729 26.83±0.350

29.13±0.358 27.76±0.181 26.88±0.967 25.68±0.826 25.33±0.546 24.73±0.775 29.13±0.358 26.96±0.141

L, a, b represent the color scales. Scale value±SEM; n =3 C control fruits, T treated fruits

a

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Fig. 2 Changes in firmness (a), physiological weight loss (b), and total plate count (c) of strawberry fruits stored at 7 and 25 °C

Farkas 2006). Our results are in conformity to that of Baker and Wicker (1996) in case of stone peaches and of Anthon et al. (2005) in case of diced tomatoes. PWL is an important indicator of shelf-life of perishables. The changes in PWL were more or less same for the control (4.9 %) as well as treatment (3.9 %) for the strawberries stored at 25 °C; however, the significant differences were observed when the fruits were stored at 7 °C (Fig. 2b). Control fruits lost

12.53 % of their weight at the 6th day of cold storage (7 °C) while the corresponding value of 12.04 % was observed for treated fruits on the 10th day of storage (Fig. 2b). This suggested that the simultaneous application of PME and calcium chloride was able to make the cell wall/ strawberry tissue firmer enough to prevent weight losses for longer times and to extend the shelf-life by 10 days compared to control under similar conditions (Micheli 2001; Balla and Farkas 2006).

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Fig. 3 Total antioxidant capacity (a) and ferric reducing antioxidant power (b) of strawberry fruits stored at 7 and 25 °C

Microbial Load No Salmonella, Staphylococcus, and coliforms were present in fresh as well stored strawberry samples during storage of fruits under both conditions. However, total plate count increased sharply for control samples (33×104 cfu/g), while it was low for treated samples (15×104 cfu/g) at the 2nd day of storage at 25 °C with 60 % RH (Fig. 2c). Similarly, less total plate count was observed for treated strawberry (11×104 cfu/g) compared to control (34×104 cfu/g) under low temperature storage (7 °C with 80 % RH). This may be due to firmer cell wall, low pH of strawberry fruits, and less spoilage because of treatment effect (Baker and Wicker 1996; Anthon et al. 2005). This low pH, combined with the presence of organic acids, generally prevents the growth of pathogenic bacteria (Beuchat 1996). Biochemical Parameters The TAC is an indicator of the capacity of total antioxidants to counter oxidative stress mediated by biotic and abiotic factors. Overall, TAC decreased during storage at both conditions of storage (Fig. 3a). At 25 °C, the decrease in TAC was more for

control samples (from initial value of 3,578.82 to 2, 204.65 nmols of ascorbic acid equivalents/g f.wt.), while the corresponding decrease for treated samples was found to be in the range of 3,578.82 to 2,565.41 nmols of ascorbic acid equivalents/g f.wt. At 7 °C, the TAC protected the control samples up to the 6th day, but the treated samples survived up to the 10th day of storage though the respective decrease in TAC from initial value (3,578.82 nmols of ascorbic acid equivalents/g f.wt.) was 1,297.42 (control) and 1,367.83 (treated) (Fig. 3a). FRAP, an important part of TAC, however, showed a differential response during storage (Fig. 3b). The FRAP activity decreased during both conditions of storage. However at 7 °C, the FRAP activity first decreased up to the 2nd day and then increased slightly at the 4th day before being finally decreased in both control and treated strawberries. Further, this decrease was noticed much earlier in controls, thus suggesting less shelflife of control fruits in comparison to treated ones (Fig. 3b). Kumar et al. (2011a) studied oxidative stress and antioxidant system during storage in two ber varieties and found that oxidative stress increased and overall antioxidant capacity decreased during storage of ber. A similar observation while on

Food Bioprocess Technol (2014) 7:2060–2070

2069

Fig. 4 Changes in polyphenol oxidase (a) and lipoxygenase (b) activities of strawberry fruits stored at 7 and 25 °C

storage has been made by Mondal et al. (2006) in case of tomato fruits. However, Alothman et al. (2010) observed concomitant increase in TAC and FRAP by ozone treatment in pineapple and banana while a corresponding decrease in guava. The increase in TAC and FRAP may be attributed to increase in phenolic content (Alothman et al. 2010). Polyphenol oxidase activity is considered as negative indicator of shelf-life of fruit as any rise in its activity will result in oxidation of polyphenols which can produce undesirable colors contradictory to the natural variants. The surface browning is usually caused by the enzyme PPO that converts phenolic compounds in to dark colored pigments in the presence of atmospheric oxygen (Oms-Oliu et al. 2010). The activity of PPO increased continuously during storage at both conditions. The PPO activity increased from an initial value of 56.78 nmols of purpurogallin produced/min/g f.wt. to 216.28 in control fruits and 190.97 in treated fruits during storage at 25 °C (Fig. 4a). Whereas, the corresponding values for control and treatment were 252.43 and 251.05 nmols of purpurogallin produced/min/g f.wt., respectively, during storage at 7 °C (Fig. 4a). Lipoxygenase enzyme causes oxidative degradation of unsaturated fatty acids to conjugated dienes (Catherine et al. 1998). The LOX activity also showed the similar trend with the storage period (Fig. 4b). During storage at 25 °C, the

LOX activity changed from a minimum value of 145.97 to 672.81 and 531.40 nmols, while the LOX activity increased from a minimum value of 145.97 to 556.49 and 517.72 nmols of conjugated dienes produced/min/g f.wt., respectively, for control and treated strawberry samples during storage at 7 °C. Calcium in plant tissues is involved in the delaying of senescence, reducing respiration, decreasing ethylene production, increasing tissue firmness, and preventing enzymatic browning (Balla and Farkas 2006). Thus, the simultaneous effect of PME and calcium ions might be responsible for less LOX and polygalacturonase (due to improved tissue firmness) and PPO (due to prevention of enzymatic browning) activities in treated strawberries compared to untreated ones as advocated by Balla and Farkas (2006).

Conclusion From the ongoing results, it may be inferred that by applying 300 units of PME along with 1.25 % CaCl2 for 30 min, the shelf-life of strawberry could be extended by 10 and 2 days, respectively, during storage at 7 °C with 80 % RH and 25 °C with 60 % RH. Untreated fruits lasted for 6 days at 7 °C with 80 % RH as against only 1 day at 25 °C with 60 % RH.

2070 Acknowledgments The work being submitted for publication is the output of project no. 8192 approved by the institute and the authors are thankful to Indian Council of Agricultural Research for research funding through institute project. Authors are also thankful to M/s Advanced Enzymes Technologies Limited, Thane, India, for providing complimentary sample of enzyme pectin methyl esterase for this research.

References Alothman, M., Kaur, B., Fazilah, A., Bhat, R., & Karim, A. A. (2010). Ozone-induced changes of antioxidant capacity of fresh-cut tropical fruits. Innovative Food Science and Emerging Technologies, 11, 666– 671. Anthon, G. E., Blot, L., & Barrett, D. M. (2005). Improved firmness in calcified diced tomatoes by temperature activation of pectin methyl esterase. Journal of Food Science, 70, 342–347. Baker, R. A., & Wicker, L. (1996). Current and potential application of enzyme infusion in the food industry. Trends in Food Science and Technology, 7, 279–284. Balla, C., & Farkas, J. (2006). Minimally processed fruits and fruit products and their microbiological safety. In Y. H. Hui, J. Barta, M. P. Cano, T. W. Gusek, J. S. Sidhu, & N. K. Sinha (Eds.), Handbook of fruits and fruit processing (pp. 115–128). Germany: Blackwell. Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Analytical Biochemistry, 239, 70–76. Beuchat, L. R. (1996). Pathogenic microorganisms associated with fresh produce. Journal of Food Science, 59, 204–216. Castaldo, D., Servillo, L., Laratta, B., Fasanaro, G., Villari, G., De Giorgi, A., et al. (1995). Preparation of high-consistency vegetable products: tomato pulps (part II). Industrial Conserve, 70, 253–258. Catherine, N. S. P. S., Perez-Gilabert, M., Vander-Hijden, H. T. W. M., Veldink, G. A., & Vliegenthart, J. F. G. (1998). Purification, product characterization and kinetic properties of soluble tomato lipoxygenase. Plant Physiology and Biochemistry, 36(9), 657–663. Chen, A. O., & Whitaker, J. R. (1986). Purification and characterization of a lipoxygenase from immature English peas. Journal of Agriculture and Food Chemistry, 34, 203–211. Garcia-Berbari, S. A., Nunes-Nogueira, J. N., & Silva-Campos, S. D. (1998). Effect of different pre-freezing treatments on the quality of frozen strawberry variety chandler. Ciénc and Technology Aliment, 18, 82–86. Ghafoor, K., Park, J., & Choi, Y. (2010). Optimization of supercritical fluid extraction of bioactive compounds from grape (Vitis labrusca B.) peel by using response surface methodology. Innovative Food Science and Emerging Technologies, 11, 485–490. Grassin, C. (2002). Firm up your fruit! Fruit Processing, 12, 208–211. Haddadchi, G. R., & Gerivani, Z. (2009). Effects of phenolic extracts of canola (Brassica napuse L.) on germination and physiological responses of soybean (Gycine max L.) seedlings. International Journal of Plant Products, 3(1), 63–73. Jyani, R. S., Saxena, S., & Gupta, R. (2005). Microbial pectinolytic enzymes: a review. Process Biochemistry, 40, 2931–2944. Kar, M., & Mishra, D. (1976). Catalase, peroxidase and polyphenol oxidase activities during rice leaf senescence. Plant Physiology, 57, 315–319. Kumar, S., Jain, V., & Malhotra, S. P. (2011a). Superoxide dismutase from Zizyphus mauritiana Lamk.: characterization and stability as a function of temperature and pH. Journal of Food Biochemistry, 35(5), 1407–1412. Kumar, S., Yadav, P., Jain, V., & Malhotra, S. P. (2011b). Evaluation of oxidative stress and antioxidative system in ber (Zizyphus mauritiana L.) fruits during storage. Journal of Food Biochemistry, 35(5), 1434–1442. Kumar, S., Jain, N. K., Sharma, K. C., Mishra, B. K., Srinivasan, R., & Paswan, R. (2012). Pectinolytic profile of various fungal strains. Journal of Mycology and Plant Pathology, 42(2), 213–217.

Food Bioprocess Technol (2014) 7:2060–2070 Lamikanra, O. (2002). Fresh-cut fruits and vegetables: science, technology and market. Washington: CRC. Luksiene, Z., Buchovec, I., & Viskelis, P. (2013). Impact of high power pulsed light on microbial contamination, health promoting components and shelf life of strawberries. Food Technology and Biotechnology, 51(2), 284–292. Luna-Guzman, I., & Barrett, D. M. (2000). Comparison of calcium chloride and calcium lactate effectiveness in maintaining shelf stability and quality of fresh-cut cantaloupes. Postharvest Biology and Technology, 19, 61–72. Main, G. L., Morris, J. R., & Wehunt, E. J. (1986). Effect of preprocessing treatments on the firmness and quality characteristics of whole and sliced strawberries after freezing and thermal processing. Journal of Food Science, 51, 391–394. Mandhania, S., Jain, V., & Malhotra, S. P. (2010). Culture optimization for enhanced production of microbial pectin methyl esterase under submerged conditions. Asian Journal of Bichemistry, 5, 12–22. Martin-Belloso, O., Soliva-Fortuny, R., & Oms-Oliu, G. (2006). Freshcut fruits. In Y. H. Hui, J. Barta, M. P. Cano, T. W. Gusek, J. S. Sidhu, & N. K. Sinha (Eds.), Handbook of fruits and fruit processing (pp. 129–144). Germany: Blackwell. Micheli, F. (2001). Pectin methylesterase: cell wall enzymes with important roles in plant physiology. Trends in Plant Science, 6, 414–419. Mondal, K., Sharma, N. S., Malhotra, S. P., Dhawan, K., & Singh, R. (2006). Oxidative stress and antioxidative systems in tomato fruits stored under normal and hypoxic conditions. Physiology and Molecular Biology of Plants, 12, 145–150. Oms-Oliu, G., Rojas-Grau, M. A., Alandes, G. L., Varela, P., SolivaFortuny, R., Hernando, H. M., et al. (2010). Recent approaches using chemical treatments to preserve quality of fresh-cut fruit: a review. Postharvest Biology and Technology, 57, 139–178. Perera, C. O., & Baldwin, E. A. (2001). Biochemistry of fruits and its implications on processing. In D. Arthey & R. P. Ashurst (Eds.), Fruit processing. nutrition, products, and quality management (2nd edition). New York: Aspen. Radojkovik, M., Zekovic, Z., Zokic, S., Vidovic, S., Lepojevic, Z., & Milosevic, S. (2012). Optimization of solid–liquid extraction of antioxidants from black mulberry leaves by response surface methodology. Food Technology and Biotechnology, 50(2), 167–176. Romanazzi, G., Feliziani, E., Santini, M., & Landi, L. (2013). Effectiveness of postharvest treatment with chitosan and other resistance inducers in the control of storage decay of strawberry. Postharvest Biology and Technology, 75, 24–27. Sreeramulu, D., & Raghunath, M. (2010). Antioxidant activity and phenolic content of roots, tubers and vegetables commonly consumed in India. Food Research International, 43, 1017–1020. Suutarinen, M., & Autio, K. (2004). Improving the texture of frozen fruit: the case of berries. In D. Kilcast (Ed.), Texture in foods: solid foods (volume II). Cambridge: Woodhead. Suutarinen, M., Honkapää, K., Heiniö, R. L., Autio, K., Mustranta, A., Karppinen, S., et al. (2002). Effects of calcium chloride-based prefreezing treatments on the quality factors of strawberry jams. Journal of Food Science, 67, 884–894. Tulipani, S., Mezzetti, B., Capocasa, F., Bompadre, S., Beekwilder, J., Ric de Vos, C. H., et al. (2008). Antioxidants, phenolic compounds and nutritional quality of different strawberry genotypes. Journal of Agricultural and Food Chemistry, 56, 696–704. Vicente, A. R., Martinez, G. A., Civello, P. M., & Chaves, A. R. (2002). Quality of heat-treated strawberry fruit during refrigerated storage. Postharvest Biology and Technology, 25, 59–71. Yu, L., Haley, S., Perret, J., Harris, M., Wison, J., & Qian, M. (2002). Free radical scavenging properties of wheat extracts. Journal of Agriculture and Food Chemistry, 50, 1619–1624. Zhang, H., Wang, L., Dong, Y., Jiang, S., Cao, J., & Meng, R. (2007). Postharvest biological control of gray mold decay of strawberry with Rhodotorula glutinis. Biological Control, 40, 287–292.