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MATERIALS

AND

METHODS

CHAPTER 3

Chapter 3 Materials and Methods

Page 31

The present study was carried out to evaluate the postharvest treatment of

various chemicals as well as edible coatings on postharvest quality maintenance and

shelf life extension of some commercially important perishable horticultural

commodities which includes Climacteric (Guava, Tomato, Mango and Banana) and

Non climacteric (Jamun, Strawberry and capsicum). Selected fruits were harvested at

their commercial maturity stage and transported on the same day to the research

laboratory at B. R. Doshi School of Biosciences, Sardar Patel University, Gujarat,

India. These fruits were graded for their uniformity in size, shape and colour, and the

fruit free from defects and blemishes were selected for further treatment processing.

The very specific coating treatment and/or application pertaining to a

particular fruit is described under respective fruit chapter (Chapter 4), while the

methodology common for all the studied fruits is described herein below:

3.1. Weight Loss Percentage (WLP):

The fruits for each specific condition were randomly selected and the fruits

were weighed during the study with a laboratory weight balance (Schimadzu, BW 320-

H). The weight loss of fruit samples was calculated by considering the differences

between initial weight and final weight of the same samples divided by their initial

weight. The same fruits were evaluated for weight loss each time until the end of the

experiment. Weight loss of fruits was determined at the regular intervals by the

following formula (AOAC, 1994):

WLP = [(A−B)/A] x 100

Where A was the initial weight at harvesting time (i.e. 0 day) and

B was the final weight at different storage intervals.

3.2. Decay Percentage

The decay (%) of fruits was determined by their visual observations. Decayed

fruits (physiological and microbial decay) were discarded and the decay percent was

recorded. The decay percentage of treated and untreated fruits was calculated as the

number of decayed fruits divided by initial number of all fruits multiplied by 100 (El-

Anany et al., 2009). Decay (%) of fruits was calculated by using the following formula:

Decay (%) = [A/B] x 100

Where A was the number of decayed fruits

and B was the number of initial fruits.


Page 32

3.3. Total Soluble Solids (TSS) and pH:

The TSS content of the fruit was determined by using refractrometer (ATAGO

Co., Tokyo, Japan). Homogenous sample was prepared by crushing the fruit pulp in a

mortar and pestle with water. The sample was thoroughly mixed and a few drops were

taken on prism of refractrometer and direct reading was taken by reading the scale in

meter as described in AOAC (1994). The reading of the juice sample as ⁰Brix is

obtained and the amount of TSS is expressed accordingly. The pH of the fruit samples

was assessed using a digital pH meter (Model: Elico, Li 120) as per the method

described by AOAC (1994).

3.4. Titratable Acidity (TA):

Known amount of tissue sample was extracted with distilled water by thorough

crushing. Extract is filtered and the filtrate is made upto a known volume with distilled

water. A known volume of aliquot (1 to 2 ml) was taken in a titration flask and few

drops of phenolphthalein solution were added to this and shaken well. The solution was

titrated against 0.1N sodium hydroxide (NaOH) solution to give a pink colour. The

end-point is determined by the appearance of pink colour and its persistent for atleast

few seconds. Three readings were recorded from each replication of a treatment and

percent acidity as equivalent to citric acid was calculated by using the following

formula (Mazumdar & Majumder, 2003):

TA % = [d x 0.064 (if NaOH solution is 0.1N) x C] x 100

(a x b)

Where, a = Weight of the sample

b = Volume of the aliquot taken

c = Volume made with distilled water

d = Average burette reading for sample

3.5. Starch:

The starch content was determined by anthrone reagent as described by

Thimmaiah (1999). Sample extraction was carried out by adding 10 ml of hot ethanol

(80%) to 0.5g fine grind of fruit tissue. The sample was homogenized and centrifuged

(Remi make) at 5000 rpm for 10 min. The residue was repeatedly washed with hot

ethanol (80%) till the washings do not give colour with anthrone reagent. The residue

was well dried over a water bath (Sedko make) and to the residue 5 ml of distilled

water and 6.6 ml of 52% perchloric acid was added. The solution mixtures were


Page 33

allowed to extract at 0C for 20 min, centrifuged and the supernatant was saved. The

extraction was repeated by addition of fresh perchloric acid and again centrifuged to

pool the supernatant and the final volume was made up to 100 ml by using distilled

water. An aliquot of 0.1 ml was taken and the volume was made upto 1 ml by adding

distilled water. Distilled water was used as a blank. Then, 4 ml of anthrone reagent was

added and boiled for 8 min in a boiling water bath. The samples were rapidly cooled

down under the running tap water and the intensity of green to dark green colour was

measured at 630 nm using spectrophotometer (Model: Elico-SL 207). Glucose (100 μg

ml-1

) was used as the working standard, and the amount of starch present in the sample

solution was calculated using the standard graph and the value obtained was multiplied

by a factor 0.9 to arrive at the starch content.

3.6. Reducing Sugars (RS):

Fruit sample of 0.5 to 1g was extracted with 5 ml of hot 80 % ethanol twice (5

ml each time). The clear supernatant was collected and evaporated on water bath. The

remaining residue was dissolved in the 10 ml of distilled water. Suitable aliquots were

pipetted out in a test tube and volume made up to 3 ml with distilled water. In the

control tube in place of sample distilled water was used. In all the tubes 3 ml of

dinitrosalicylic acid (DNS) reagent was added and mixed thoroughly. The mixture was

heated for 5 min in a boiling water bath. After the colour had developed 1 ml of 40%

sodium potassium tartarate was added when the contents of the tubes were still warm.

After cooling, the optical density was measured using a spectrophotometer at 510 nm

(Miller, 1972). The amount of reducing sugar was calculated using standard curve

prepared from glucose (100 μg ml-1

).

3.7. Non-Reducing Sugars (NRS) and Total Sugars (TS):

100 mg of the fruit tissue was extracted twice with hot 80 % ethanol. The clear

supernatant was allowed to evaporate on a hot water bath and the dried crystals were

dissolved in 10 ml of distilled water. An aliquot of 1 ml of extract was added to the 1

ml of 1N sulphuric acid (H2SO4) solution and hydrolyzed by heating at 50C for 30 min

on a hot plate. The tubes were allowed to cool and 1 or 2 drops of methyl red indicator

was added. The solution was neutralized by adding 1N NaOH drop wise using a

pipette. Later 3 ml of DNS reagent was added to the tubes and placed on boiling water

bath for 5 min. As the colour developed, 1 ml of 40% sodium potassium tartarate


Page 34

solution was added and mixed when still warm. The tubes were cooled under running

tap water and the absorbance was measured using spectrophotometer at 510 nm

(Thimmaiah, 1999). The amount of non-reducing sugars was calculated using a

standard graph prepared from glucose (100 μg ml-1

). The total sugars (TS) were

calculated as reducing sugars (RS) plus non-reducing sugars (NRS).

3.8. Total Phenols:

The total phenols were determined by the Folin-Ciocalteu method as described

by Bray & Thorpe (1954), based on colorimetric oxidation/reduction reaction of

phenols. Fruit sample of 0.5 to 1g was extracted with 10 ml of 80 % ethanol and the

extract was centrifuged at 10,000g for 20 min. The clear supernatant was collected and

the residue was re-extracted with 80% ethanol. The collected supernatant was allowed

to evaporate at room temperature, and the remaining residue was dissolved in a known

volume of distilled water. Suitable aliquots (i.e. 0.2 to 2 ml) of the supernatant were

taken and the volume made up to 3 ml with distilled water. The diluted extract was

mixed with 0.5 ml of Folin Ciocalteu reagent (FCR) (1:1 diluted with distilled water)

and the mixture was allowed to stand for 3 min. Thereafter 2 ml of 20 % sodium

carbonate (Na2CO3) solution was added to each tube and the tubes were placed in the

boiling water for exactly one min. The optical density of the reaction mixture was

measured against the blank at 650 nm with the help of spectrophotometer. The standard

calibration curve was prepared by using the catechol at concentration of 100 μg ml-1

and the results were expressed as mg g-1

.

3.9. Ascorbic Acid:

Ascorbic acid content was determined as per the method of Roe (1964). The

fruit ample (100 mg) was extracted in acetic acid-metaphosphoric acid mixture (15g of

metaphosphoric acid dissolved in a mixture of 50 ml of glacial acetic acid and 450 ml

of distilled water). The extract was centrifuged and clear supernatant was collected.

Suitable aliquots of the supernatant were taken in a test tube containing known amount

of metaphosphoric acid (5%), dinitrophenyl hydrazine (2%) and thiourea (10%)

solutions. The test tubes were kept for incubation for 3h at 37°C in water bath, after

which the reaction was stopped by adding 85% H2SO4. The optical density of the

reaction mixture was measured against blank at 540 nm. Levels of ascorbic acid were

estimated as per the standard curve prepared from pure ascorbic acid (100 μg ml-1

) and

the obtained results were expressed as mg 100 g-1

.


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3.10. Chlorophylls, Total carotenoids and Lycopene:

For chlorophylls, total carotenoids and lycopene estimation, the fruit pulp

tissues were chopped and homogenized with 10 ml of hexane and acetone (60:40)

mixture. The upper organic layer was transferred to the capped tube on ice. Remaining

aqueous layer was re-extracted with 5 ml of same solvent repeatedly and the organic

layer was transferred to the same tube until the aqueous layer becomes colourless. The

supernatant was taken from the total volume of the organic extract and the optical

density of the solution was measured at different wavelengths namely 663 nm and 645

nm (for chlorophylls), 450 nm (for carotenoids) and 502 nm (for lycopene),

respectively on spectrophotometer (Arnon, 1949; Wang et al., 2005). The amount of

chlorophyll ‘a’, chlorophyll ‘b’, total chlorophylls, total carotenoids and lycopene was

calculated by using the following formulas:

Chlorophyll ‘a’ (μg g-1

) =

Chlorophyll ‘b’ (μg g-1

) =

Total chlorophylls (μg g-1

) =

Total carotenoids (μg g-1

) =

Lycopene (μg g-1

) =

Where, A645 - Absorbance values at 645 nm

A663 - Absorbance values at 663 nm



W - Weight of the sample

V – Total volume made by extraction solvent

[(12.7 x A663) – (2.69 x A643)] x V

W

[(22.9 x A643) – (4.68 x A663)] x V

W

[(20.2 x A645) + (8.02 x A663)] x V

W

(4 x A450) x V

W

(3.12 x A502) x V

W


Page 36

3.11. Total Antioxidant Activity:

Total antioxidant activity was measured through estimation of free radical-

scavenging effect on 2, 2- diphenyl-1-picrylhydrazyl (DPPH) radical as per the method

of De Ancos et al. (2000). Briefly, 1g of fruit was extracted with 5 ml of methanol and

centrifuged at 6,000g for 15 min at 4°C, using a cold centrifuge (Eppendorf, 5430R).

The methanolic aliquots of 0.01 ml were mixed with 3.9 ml of methanolic DPPH

(0.025g L-1

) and 0.090 ml of distilled water. The reference was prepared by adding 3.9

ml DPPH to 0.1 ml of distilled water. The content of the tubes were shaken on a vortex

shaker (Genei, SLM- HP- MS- 150) and allowed to stand for 30 min under the dark

condition. Absorbance of the mixture was measured against the blank methanol at 515

nm using visible spectrophotometer. The antioxidant activity is expressed in the form

of the percentage of free radical scavenging by using the following formula:

3.12. Total Anthocyanins and Total Flavanol:

Total anthocyanins and total flavanol estimation was carried out as per the

method described by Lees & Francis (1972). Briefly, 1g of fruit pulp was blended with

20 ml of extracting solvent [95% Ethanol: 1.5N hydrochloric acid (HCl), 85: 15] and

allowed to stand overnight at 4°C. Samples were filtered into a volumetric flask

covered with aluminum foil. The remaining residue was washed with an extractor

solution to completely remove the pigments. The filtrate was made up to a total volume

of 100 ml with the same solvent and after 24 h at room temperature the absorbance was

recorded at 535 nm and 374 nm by using visible spectrophotometer and UV-

spectrophotometer (UV-1800 Shimadzu), respectively, to quantify the total

anthocyanins and total flavanol content. The amount of total anthocyanins and total

flavanol content was calculated by using a standard graph prepared from catechol (100

μg ml-1

) and the obtained results were expressed in μg g-1

.

3.13. Total Proteins:

Total proteins from the fruit tissues were estimated by following the method of

Lowry et al. (1951). The sample (0.5g) was extracted with 10 ml of potassium

phosphate buffer (0.1M, pH 7.2). The homogenate was centrifuged at 5000g for 15

(Absorbance of the reference sample – Absorbance of the test sample) x 100

Absorbance of the reference sample


Page 37

min. The supernatant was used for estimation of total proteins. Fruit tissue (0.5g) was

homogenized with 10 ml of potassium phosphate buffer. An aliquot of 0.2 ml of sample

extract was taken in a test tube and the volume was made up to 1.0 ml with distilled

water. In the sample reaction mixture 5 ml of solution C (mixed 50 ml of solution ‘A’

with 1 ml of solution ‘B’; solution ‘A’ containing 2 % Na2CO3 in 0.1N NaOH; solution

‘B’ containing 0.5 % copper sulphate (CuSO4) in 1 % SPT was added and incubated at

room temperature for 10 min. Finally, 0.5 ml of FCR reagent (commercially available

FCR diluted with equal volumes of distilled water) was added, mixed well and

incubated in dark chamber for further 30 min at room temperature. The absorbance was

measured at 660 nm against blank on spectrophotometer. The amount of protein was

calculated using a standard graph prepared from bovine serum albumin (200 μg ml-1

)

and the obtained results were expressed in mg g-1

.

3.14. Ethylene and Respiration

The levels of ethylene and the rate of respiration of individual fruits were

measured by placing individual fruits in airtight jars (250 ml or 500 ml volume) and

incubated them for 24 h at room temperature. Lids of the jars were modified to fix a

rubber septum for removal of gas to be analyzed using Perkin Elmer Autosystem XL

Gas Chromatography (GC). The levels of ethylene (C2H4) were measured by

withdrawing a gas sample of 10-100 l through the rubber septum fixed on the lid of

airtight jar using a long hypodermic needle and the gas samples were injected into GC

fitted with Flame Ionization Detector (FID), capillary column and N2 (nitrogen) was

used as a the carrier gas. The injector, column and detector temperature used were, 160

°C, 100 °C and 280 °C respectively. The levels of ethylene (C2H4) were expressed as μl

kg-1

h-1

(Ding et al., 1998). The ethylene standard used for the present study was

generated using ethrel (5 ml of ethrel solution containing 2-3 crystals of NaOH).

Similarly the rate of respiration (CO2) was determined by withdrawing a gas

sample of 10-100 l through the rubber septum fixed on the lid of airtight jar using a

long hypodermic needle and injecting them into Perkin Elmer Autosystem XL Gas

chromatography equipped with a Polapak Q column (50-80 mesh, 2 m x 3 mm, 60 °C)

and a Thermal Conductivity Detector (TCD). The rate of respiration (CO2) was

expressed as μl kg-1

h-1

(Teitel et al., 1989). The analytical grade standard CO2 (99 %)

was used as standard.


Page 38

3.15. Enzyme Assays:

3.15.1. Extraction for Polygalacturonase (PG), Cellulase and Invertase:

Fruit tissue (2g) was obtained and homogenized in 15 ml of sodium phosphate

buffer (20mM, pH 7.0) containing cysteine-HCl (20mM), Ethylenediamine Tetra

Acetic Acid (EDTA) (20mM) and Triton X-100 (0.05%). The homogenate was filtered

and centrifuged at 15,000g for 30 min at 4°C in a refrigerated centrifuge (Model:

Eppendorf, 5430R). The clear supernatant was used as enzyme extracts for assaying PG

and cellulase enzyme activities (Srivastava & Dwivedi, 2000).

3.15.2. Assay of PG (EC 3.2.1.15):

PG activity was assayed according to the procedure described by Srivastava and

Dwivedi (2000). The reaction mixture comprised of 0.2 ml sodium acetate (200mM,

pH 4.5), 0.1 ml sodium chloride (NaCl) (200mM), 0.3 ml of 1% polygalacturonic acid

(PGA, pH 4.5) and 0.1 ml of enzyme extract in a total volume of 1.0 ml. The reaction

mixture was held at 37°C for 1h followed by addition of DNS. The reaction was

stopped by heating the reaction mixture in a boiling water bath for 5 min. In control

tubes the substrate was added after the heat treatment. The formation of reducing group

was calculated using D-galacturonic acid (mg ml-1

) as a standard. One unit of PG

activity is defined as the amount of enzyme producing 1 mmol of reducing groups per

min at 37°C. The protein content in the enzyme extract was measured by using the

method of Lowry et al. (1951). The specific activity of enzyme is expressed as unit mg

protein-1

.

3.15.3. Assay of Cellulase (EC 3.2.1.4):

Cellulase activity was measured by following the method of Srivastava and

Dwivedi (2000). The reaction mixture contained sodium acetate buffer (100mM, pH

5.0), Carboxymethyl cellulose (CMC) (1.5%) and enzyme in a final volume of 1.0 ml.

The reaction mixture was incubated at 37°C for 16 h followed by addition of DNS. The

tubes were boiled on a water bath for 10 min and the colour was read at 540 nm using a

visible spectrophotometer. Amount of reducing sugar released was calculated from a

calibration curve drawn using D-glucose as a standard. One unit of cellulase activity

was defined as the amount of enzyme liberating 1 mmol of reducing sugar per h at

37°C. The protein content in the enzyme extract was measured by using the method of

Lowry et al. (1951). The specific activity of enzyme is expressed as unit mg protein-1

.


Page 39

3.15.4. Assay of Invertase (EC 3.2.1.26):

Invertase activity was measured as described by Srivastva & Dwivedi (2000).

The assay mixture contained acetate buffer (100 mM, pH 4.5), sucrose (100 mM) and

enzyme preparation in a total volume of 1.0 ml. The reaction mixture was incubated

for1h at37°C. The substrate was added to control tubes after the incubation and colour

was developed using DNS. Amount of reducing sugar released was calculated from the

calibration graph. One unit of invertase activity was defined as micromoles of reducing

sugars equivalent released per min at 37°C. The protein content in the enzyme extract

was measured by using the method of Lowry et al. (1951). The specific activity of

enzyme is expressed as unit mg protein-1

.

3.15.5. Extraction and assay of Pectin Methyl Esterase (PME) (EC 3.1.1.11):

The procedure for extraction and assay of PME was adapted from Lohani et al.

(2004) with some modifications. For extraction, 2g fruit tissue was homogenized in

Tris–HCl (20mM, pH 7.0) containing cysteine-HCl (20mM), EDTA (20mM) and

Triton X-100 (0.05%). The homogenate was centrifuged at 15,000g for 30 min at 4⁰C

in a refrigerated centrifuge. The clear supernatant was used for enzyme assays. The

reaction mixture contained 1 ml pectin solution (0.01%, pH 7.5), 0.2 ml NaCl (0.15M),

0.1 ml bromothymol blue solution (0.01%), 0.2 ml distilled water and 0.1 ml of enzyme

extract. After adding the enzyme, the cuvette was shaken gently. Absorbance was

measured immediately at 620 nm. The absorbance was again measured after 3 min. The

difference in absorbance between 0 and 3 min was the measure of PME activity. The

protein content in the enzyme extract was measured by using the method of Lowry et

al. (1951), and the specific activity of enzyme is expressed as A620 min-1

mg protein-1

.

3.15.6. Extraction and Assay of β-galactosidase (β-gal) (EC 3.2.1.23):

The procedure for extraction and assay of β-gal was followed in accordance

with Biswas (1985). The fresh fruit sample (1g) was extracted in 10 ml of ice-cold

Tris-HCl buffer (10mM, pH 7.4) containing Triton X-100 (0.1%). The homogenate

was strained through the cheese-cloth and centrifuged at 10, 000g for 20 min at 4⁰C in

a refrigerated centrifuge. The clear supernatant was used as the enzyme assay. The

reaction mixture, containing 0.25 ml of sodium acetate buffer (0.1M, pH 5) and 0.01

ml of PNPG (10mM) at 55°C. The reaction was initiated by adding 0.74 ml of enzyme

extract and incubated for 10 min; similarly the blank was prepared without the enzyme

extract which is replaced by the buffer. The reaction was terminated by adding 4 ml of


Page 40

NaOH (0.1M). The absorbance was measured at 410 nm against the blank. The

standard curve was prepared by using the p-nitrophenol (PNP) (0.1 μmol ml-1

) and the

enzyme activity was expressed as µmol of PNP formed per minute. The protein

content in the enzyme extract was measured by using the method of Lowry et al.

(1951), and the specific activity of enzyme is expressed as µmol min-1

mg protein-1

.

3.15.7. Extraction and Assay of Catalase (CAT) (EC 1.11.1.6):

CAT activity was assayed according to the method of Barber (1980), by

following the titration method. Tissue (2g) was homogenized in 10 ml of 0.1M

potassium phosphate buffer (pH 7.0) and the homogenate was filtered through two

layers of cotton cloth to remove cell debris. The clear supernatant after centrifugation at

15,000g for 30 min at 4⁰C was collected as enzyme extract. The reaction mixture

contained 3 ml of phosphate buffer, 2 ml of freshly prepared 0.005M hydrogen

peroxide (H2O2) and 1 ml of enzyme extract. The reaction mixture was allowed to

incubate at 20⁰C for 1 min. After 1 min the reaction was stopped by adding 10 ml of

H2SO4 (0.7N). The reaction mixture was titrated against 0.01N potassium

permanganate (KMnO4) until the appearance of a faint purple colour and its persistent

for atleast 15 sec. Similarly, the blank was prepared by adding the enzyme extract to an

acidified solution of reaction mixture at zero time. One unit of catalase is defined as the

amount of enzyme which breaks down 1 μmol of H2O2 per min under the assay

conditions. The protein content in the enzyme extract was measured by using the

method of Lowry et al (1951), and the specific activity of enzyme is expressed as unit

min-1

mg protein-1

.

3.15.8. Extraction and Assay of Peroxidase (POD) (EC 1.11.1.7):

POD activity was assessed by following the method described by Guilbault

(1976). Tissue (2g) was homogenized in 10 ml of 0.1M potassium phosphate buffer

(pH 7.2) and the homogenate was filtered through two layers of cotton cloth to remove

cell debris. The clear supernatant after centrifugation at 15,000g for 30 min at 4⁰C was

collected as enzyme extract. The reaction mixture contained 10 µl of enzyme

supernatant, 2.99 ml H2O2 (0.03% in 0.01M potassium phosphate buffer, pH 6.0) and

0.05 ml orthodianisidine dye/6 ml substrate solution (1% in methanol). The reaction

mixture without substrate served as a blank. The changes in absorbance were recorded

at 460 nm for 1min at the interval of 15s. Enzyme activity was expressed as the 1 unit

change in optical density per min per gram of fresh tissue. The protein content in the


Page 41

enzyme extract was measured as per Lowry et al. (1951), and the specific activity of


.

3.15.9. Extraction and Assay of Polyphenol Oxidase (PPO) (EC 1.14.18.1):

The procedure for extraction and assay of PPO was followed in accordance with

Rivas & Whitaker (1973) based on the oxidation of catechol. The fresh fruit sample

(1g) was extracted in 10 ml of ice-cold sodium phosphate buffer (0.1M, pH 6.8). The

homogenate was strained through the cheese-cloth and centrifuged at 10, 000g for 20

min at 4⁰C in a refrigerated centrifuge. The obtained clear supernatant was used for the

enzyme assay. The reaction mixture contained 1 ml of catechol (0.05M prepared in

0.2M potassium phosphate buffer, pH 6.8) and 3.8 ml of 0.2M phosphate buffer at

30⁰C. The reaction was initiated by adding varying amounts of enzyme extract in a

final volume of 5 ml. The rate of increase in absorbance at 410 nm against the blank

(prepared in the absence of enzyme) was measured at every 30 sec upto 3 min by using

a spectrophotometer. PPO activity was calculated as a change in optical density over a

period of 3 minute. One unit of PPO activity was defined as the change in absorbance

of 0.001 per min per gram of fresh tissue. The protein content in the enzyme extract

was measured by using the method of Lowry et al (1951), and the specific activity of


.

3.15.10. Extraction and Assay of Superoxide Dismutase (SOD) (EC 1.15.1.1):

The extraction and activity of SOD was performed according to the method of

Regina & Ranceliene (2008). Fruit flesh (2g) was homogenized in 10 ml of ice-cold

sodium-potassium phosphate buffer (0.05M, pH 7.8) containing 1mM EDTA and

Triton X-100 (0.1%). Homogenates were centrifuged at 15, 000g for 30 min at 4⁰C and

the resulting supernatants were used for the SOD assay. For SOD activity, the reaction

mixture (3 ml) contained sodium phosphate buffer (50 mmol L-1

, pH 7.8), methionine

(13 mmol L-1

), nitroblue tetrazolium (NBT) (75 mmol L-1

), EDTA (10 mmol L-1

),

riboflavin (2 mmol L-1

) and 0.1 ml crude enzyme extract solution. The mixture was

illuminated by fluorescent lamp (60 mmol m-2

s-1

) for 10 min and then the absorbance

was determined at 560 nm. Similar solutions kept in the dark served as blanks. One unit

of SOD activity was defined as the amount of enzyme that caused a 50% decrease of

the SOD-inhibitable NBT reduction. The protein content in the enzyme extract was

measured by using the method of Lowry et al. (1951), and the specific activity of


.


Page 42

3.16. Mineral Analysis:

The analysis of minerals was performed as per the methodology of Jackson

(1973). One gram of dry material was further processed for the wet digestion by the

diacid (1 HClO4: 3 HNO3) mixture and allowed to stand overnight. The samples were

heated on hot plate until solid particles nearly disappeared and heated until a clear

colorless solution is obtained. Once digested, samples were further evaporated to near

dryness. Once the reaction has subsided, samples were cooled and made upto 100 ml

with milliQ water. The solution was allowed to stand overnight, filtered through a dry

paper to remove silica without washing. The solution containing samples was retained

and used for the analysis of minerals against the reagent blank by atomic absorption

spectrophotometer.

3.17. Shelf life or Marketable Period:

The shelf life of the samples was calculated by counting the days required for

them to attain the last stage of ripening, but upto the stage when they remained still

acceptable for marketing (Mondal, 2000).

3.18. Sensory Evaluation:

Sensory evaluation of the fruits for colour, texture, flavour, taste and overall

acceptability for all the samples was done after selected storage period as per the

method described by Bai et al. (2003a) with some modifications. A panel of seven

untrained judges was selected based on their stability and reliability of judgment. All

the panelists were asked to score the differences between the samples where 0-2

represented extreme dislike; 3-5 fair; 6-8 good; and 9 excellent for colour, texture,

flavour and overall acceptability.

3.19. Anatomical Studies:

3.19.1. Histo-chemical Methods:

Selected fruit samples [Mango (from Experiment 1) and Banana (from

Experiment 1) were fixed in fixative mixtures [Formalin (5 ml): Acetic Acid (5 ml):

Alcohol 70 % (90 ml)] until use. The samples were dehydrated in tertiary butyl alcohol

(TBA) series. Thereafter the samples were infiltrated and embedded in paraffin wax

using customary methods described by Berlyn & Miksche (1976). Serial sections of 8-

12 μm thickness were taken from the paraffin embedded materials using rotary

microtome (Zeus, Singapore). For histo-chemical studies, paraffin sections were taken

on slides and stained on staining dishes. The sections were observed and


Page 43

photomicrographed for histo-chemical studies using Olympus Research Microscope

attached with digital camera (Olympus DP 71) and imaging software (Image

Proexpress 6.0).

3.20. Histo-chemical Localization of Metabolites:

3.20.1. Insoluble Polysaccharides

Periodic acid Schiff’s reagent (McManus, 1948) was used for localization of

insoluble polysaccharides for which the sections were deparaffinised and brought down

to water and oxidized in 1 % aqueous periodic acid for 10 min followed by washing in

running tap water for 5 min. The sections were then immersed in Schiff’s reagent for 10

min and again washed in running tap water for 5 min to remove excess stain. The

sections were finally dehydrated, cleared and mounted in DPX for light microscopic

studies.

3.20.2. Starch

The serial sections were deparafinnised and brought down to water and stained

with I2KI (iodine-potassium iodide solution prepared by dissolving 2g of potassium

iodide in 100 ml of water, to this solution 1g of iodine flakes was added) for

localization of starch (Johansen, 1940).

3.21. Scanning Electron Microscopy (SEM):

For SEM studies, coated and control mango fruit samples were fixed in 3%

glutaraldehyde in 0.2M cacodylate buffer (pH 7.2). Samples were cut into blocks of 6-7

mm thickness using a sharp blade, dehydrated in acetone-isoamyl series and coated

with gold using Quaram Sputter Coating Unit, Model SC 7610 and observed with LEO

440i SEM at 10kV.

3.22. Antibacterial Activity:

3.22.1. Zone of Inhibition:

The infusion extraction method (Houghton & Raman, 1998) was used for

extraction, for which 30g of fresh pulp of control and treated fruit samples were soaked

in 125 ml of n-hexane for 24 h at room temperature and then filtered using Whatman

filter paper no. 1. The residue obtained on extraction was dried completely at room

temperature and re-suspended the residue in 125 ml of ethyl acetate, methanol and

water, respectively for 24 h at room temperature. The resulting filtrate solution of each

extract was then centrifuged at 3000 rpm for 10 min to remove any solid debris present


Page 44

and further concentrated using solvent recovery assembly. The concentrated extracts

were dried completely at room temperature and finally stored in refrigerator until

further use.

The following four gram positive and two gram negative bacteria (Table 3.1)

were used for the present study. All the microbial pure cultures used were obtained

from MTCC (Microbial Type Culture Collection, Chandigarh, India). All the bacterial

cultures were grown on nutrient agar medium (pH 7.4) at 37°C. A fresh bacterial

suspension was prepared by sub-culturing the colonies in the nutrient broth medium

(pH 7.4) to maintain uniform growth of organisms. The bacterial culture suspension

was compared with 0.5 Mc Farland turbidity standard (prepared by adding 0.5 ml of

1.175 % barium chloride solution to 99.5 ml of 1 % H2SO4 solution). A 0.5 Mc Farland

turbidity standard is equivalent to approximately 1x108

bacterial cell densities (Perilla

et al., 2003).

Table 3.1: The Bacterial strains selected for the present study.

Sr. No. Gram positive Causes

1. Bacillus cereus (BC) Food poisoning, vomiting, diarrhoea

2. Bacillus subtilis (BS) Food poisoning

3. Micrococcus luteus (ML) Septic shock, septic arthritis

4. Staphylococcus aureus (SA) food poisoning, toxic shock syndrome

Gram negative Causes

5. Klebsiella pneumoniae (KP) Pneumonia, flu, chill and cough

6. Salmonella typhi (ST) Typhoid, enteric fever

The antibacterial activity of coated and uncoated samples of jamun and guava

fruit extracts were screened using agar well diffusion method (Perez et al., 1990) for

which a stock (100 mg ml-1

) of each fruit extract was prepared in Dimethyl sulphoxide

(DMSO). The petri plates and nutrient agar used were sterilized in an autoclave at 15

lbs pressure for 15 min and 20 ml of nutrient agar was poured into each petri plate. The

petri plates were then allowed to solidify at room temperature. The prepared agar plates

were then marked into four equal parts and labeled with the name of organism and

treatments used. A bacterial culture of 100 μl having density of 1x108

CFU ml-1

was

spread on agar plates using a glass spreader. A well of 8 mm diameter was punched off

from the agar plate using sterile cork borer and the prepared well was filled with 100 μl

of fruit extract. The whole process was carried out under sterile condition by using


Page 45

laminar air flow cabinate. The inoculated plates were then placed in refrigerator for 30

min for pre-diffusion of fruit extracts and further incubated at 37 °C for 24 h until the

appearance of inhibition zone. The zone of inhibition formed was measured as a

property of antibacterial activity.

3.23. Statistical Analysis:

The experiment was conducted in a completely randomized design (CRD) with

three replications. All the performed analyses were carried out in triplicate and the

standard deviation (SD) was calculated. Data analyses were performed by analysis of

variance (ANOVA) using IRRISTAT statistical software (v. 3.1, IRRI, Manila,

Philippines). Multiple comparisons among the treatments with significant differences

tested with ANOVA were conducted by using least significant difference (LSD) at P <

0.05 level. Duncan’s multiple range test (DMRT) was used to compare the mean values

in different storage intervals (Bliss, 1967).

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