CHAPTER 3 MATERIALS AND METHODS - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/42103/12/12_chapter3.pdfCHAPTER 3 MATERIALS AND METHODS The study on various aspects and objectives

Materials and Methods

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CHAPTER 3

MATERIALS AND METHODS

The study on various aspects and objectives related to the present research

work was carried out during the year 2008 - 2012 at Department of Food Processing

Technology, A.D. Patel Institute of Technology, New Vallabh Vidyanagar, Anand and

Sophisticated Instrumentation Center for Applied Research and Testing, Vidyanagar,

Anand.

In the present investigation, advanced maturity stages of banana i.e. stage 5

(yellow with a trace of green), stage 6 (all yellow) and stage 7 (all yellow with a

brown speckles); according to Standard Banana Colour Chart as shown in Plate 3.1

have been selected to optimize the enzymatic clarification treatment and thereby to

develop the process technology for banana pulp clarification. Prepared clarified juices

of selected ripening stages have been further studied for physic-chemical parameters

and sensory evaluation. Another important aspect of current research project was to

inhibit the browning in clarified banana juice by applying some anti-browning

treatments. This chapter deals with the description of materials, experimental set-ups,

analytical techniques and processing techniques used in various experiments.

3.1 Materials

Banana Fruits

Bananas (Musa sp.) of variety ‘Robusta’ were used in the present study and for

every trial bananas were procured at the maturity stage 1(beginning to ripen) from

selected banana orchards near Anand city.

Commercial Enzymes

Commercial pectinolytic enzyme i.e. Pectinex Ultra SP-L and cellulase

enzyme i.e. Celluclast 1.5L were obtained from Novozymes South Asia Pvt. Ltd.,

Bangalore.

Ethrel (2-chloro ethyl phosphonic acid) used for ripening of banana and other

branded chemicals and reagents namely sodium phosphate, polyvinyl pyrrolidone,

Triton X-100, sulfuric acid, hydrochloric acid, silver nitrate, acetic acid, calcium

chloride, petroleum ether used during analysis were purchased from local chemical

supplier.


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3.2 Methodology for Ripening of Banana

Banana (Musa sp var. ‘Robusta’) bunches harvested at optimum stage of

maturity (stage 1) were brought from orchard and banana hands were separated.

Selected banana hands were washed thoroughly with potable water and dipped in 500

ppm ethrel (2-chloro ethyl phosphonic acid) solution for 5 min. Treated fruits were

dried by gentle air to remove surface moisture. Thereafter, fruits were covered with

polyethylene sheet and packed in ventilated plastic baskets to create high RH 80%

atmosphere and kept for ripening at 20±1°C (Kulkarni et. al., 2010; Saeed et al.,

2001). As the ripening progressed, fruit passes through various ripening stages. But

fruits of advanced ripening stages viz. stage 5, 6 and 7 (as shown in Plate 3.1) were

selected for the present study. Hence, after ripening initiation at some specific

intervals when the bananas attained the maturity stage 5, 6 and 7; bananas of each

particular stage has been drawn and pulp of each selected stage has been used for juice

clarification study. Also fruits of each selected ripening stage were tested on the

following aspects.

Physico-chemical properties:

1. Pulp/peel ratio

2. PPO activity

3. Moisture

4. Ash

5. Fat

6. Protein

7. Titratable Acidity

8. Pectin

9. Total sugar

10. Starch

11. TSS

Mechanical properties:

1. Firmness

2. Cohesiveness

3. Chewiness

4. Fracture force

5. Stiffness


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Plate 3.1 Standard Colour Chart by SH Pratt & Co (Bananas) Ltd.

(Luton)


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3.2.1 Pulp/Peel Ratio

Pulp to peel ratio of fruits for each treatment was ratio of mass of pulp to peel.

3.2.2 Polyphenol Oxidase (PPO) Activity

Extracted and assayed according to the method described by Galeazzi et al,

(1981). About 25 g of pulp was homogenized for 60 sec with 50 ml of 0.2 M sodium

phosphate buffer solution of pH 7, containing 1% insoluble polyvinyl pyrrolidone

(PUP) and 0.5% Triton X-100. The homogenate was centrifuged at 40C for 15 minute

at 15000 x G. The enzyme activity was assayed in the supernatant by measuring the

rate of increase in absorbance at 420 nm by spectrophotometer using catechol solution

(0.1M) as substrate. The enzyme activity was calculated from slope of the initial

straight section of reaction curve and expressed as U min-1

m-1

3.2.3 Moisture

Moisture content in the pulp of banana fruits was determined by drying in oven

at 70±100C for 12 h as described by Ranganna (2000).

3.2.4 Ash

Ash content was determined according to AOAC (1984) method. About 10 g

of sample was weighed in a silica dish and ignited on flame till fuming ceased. Then

the dish was transferred to a muffle furnace at 5500C for 6 h. The dish was then cooled

in a desiccator. The weight of the residue was then noted and the % ash was calculated

as follows:

------------- [3.1]

3.2.5 Fat

Fat content was determined according to AOAC (1984) method. About 50g of

sample was dried and transferred to a thimble after grinding. Petroleum ether (boiling

point 40-600C) was used as a solvent which was subsequently evaporated and the

extracted fat was weighed after complete drying in an oven at 600C. The % crude fat

was calculated as follows:

----------- [3.2]


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3.2.6 Protein

Micro-kjeldhal method (AOAC, 1984) was used for protein estimation. About

10g of the sample was transferred to a digestion flask followed by the addition of 3g

of catalyst mixture (K2SO4: CuSO4:SeO2 in 100:20:2.5 ratio) and 20 ml of

concentrated sulfuric acid.

The content was then digested till a blue or green transparent liquid was

obtained. The volume of digested material was made up to 100 ml with distilled water.

Carry out a blank digestion without the sample and make the digest to 100 ml.

A 20 ml aliquot of digested material was distilled with excess of 40% NaOH

solution and the liberated ammonia was collected in 20 ml of 2 % boric acid solution

containing 2-3 drops of mixed indicator (10 ml of 0.1 per cent bromo cresol green + 2

ml of 0.1 per cent methyl red indicators in 95 per cent alcohol). The entrapped

ammonia was titrated against 0.01N hydrochloric acid. A reagent blank was similarly

digested and distilled. Nitrogen content in the sample was calculated as follows and a

factor of 6.25 was used to convert nitrogen to protein.

------------ [3.3]

Protein % = Nitrogen % X 6.25

3.2.7 Titratable Acidity

Titratable acidity (TA) was determined by direct titration of diluted pulp with

0.1N NaOH as per Ranganna (2000) method and TA was expressed as a percentage

of malic acid.

3.2.8 Pectin

Pectin was determined as calcium pectate (AOAC, 1975) method.

Reagents:

1 N Acetic acid (approx.): Dilute 30 ml of glacial acetic acid to 500 ml with water

1 N Calcium chloride : Dissolve 27.5 g of anhydrous CaCl2 in water and dilute to

500 ml.

1% Silver Nitrate : Dissolve 1 g of AgNO3 in 100 ml of water

0.05N Hydrochloric acid solution


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Procedure:

Blended pulp (50 g) was transferred to a 1L beaker. Extraction was carried out with

400 ml of 0.05N HCl for 2 h at 80-900C. The water lost by evaporation was replaced.

It was then cooled and the content were transferred to a 500 ml volumetric flask and

made up to mark with water. The content were properly mixed and filtered through a

Whatman No. 4 filter paper into a 500 ml conical flask.

Two aliquots of 200 ml each were drawn into two 1000 ml beakers. Water was

added at the ratio of 250 ml per aliquot. The acid was neutralized with 1N NaOH

solution using phenolphthalein indicator. About exact 10 ml excess 1N NaOH was

added with constant stirring. This was allowed to stand overnight. Then 50 ml of 1N

acetic acid was added followed by the addition of 25 ml of 1N calcium chloride after 5

minute and allowed to stand for 1h. After that heated to boiling for 1 minute and

filtered through a previously prepared filter paper (wet the filter paper in hot water,

oven dried for 2h at 1000C, cooled in desiccators and weighed in a covered weighing

dish). The precipitate was washed with almost boiling water until chloride-free.

Chlorides were detected by 1% silver nitrate solution. The filter paper containing the

calcium pectate was transferred to the original weighing dish and dried overnight at

1000C. It was cooled in a desiccator and weighed. The pectin content was determined

as calcium pectate by the following formula:

----------- [3.4]

3.2.9 Total Sugar

Total sugar content was determined by following the Shaffer-Somogyi method

as described by Ranganna (2000).

3.2.10 Starch

After the sugars present in the sample have been leached out, starch in the

alcohol insoluble solids was hydrolyzed using HCl and the resultant reducing sugar

was determined by Shaffer-Somogyi method given in Ranganna (2000).

3.2.11 Total Soluble Solid (TSS)

TSS was measured as 0Brix at 20

0C by using Hand Refractometer (measuring

range 0-320Bx). The necessary corrections were made when the measurements were

carried out at temperatures other than 200C.


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3.2.12 Mechanical Properties

Mechanical properties viz. firmness, cohesiveness, chewiness, fracture force, stiffness

of whole fruit were determined by using Texture Analyzer (Lloyds, England)

controlled by a PC- based data acquisition card in a personal computer with the

software Nexgen V 4.5. A 8mm cylindrical probe (Saeed et al., 2006), the cross head

speed was 50 mm/min and deformation % was kept at 30. The samples were placed on

a fixed plate on its lateral surface and the puncture test was carried out at 2 cm away

from the middle of the fruit. Firmness values were expressed as the maximum force

(N) required until tissue failure.

3.2.13 Statistical Analysis

For each stage, three samples were randomly selected and the average values

of three experiments were reported. Experimental data were analyzed using analysis

of variance (ANOVA). Tukey’s Multiple Comparison Test was employed for mean

separation. The level of significance was at 5%. The analysis was performed using the

Daniel's XL Toolbox version 4.00.

3.3 Optimization of Process Technology for Clarification of

Banana Pulp

3.3.1 Experimental Design and Statistical Analysis

RSM was used to generate the experimental designs, statistical analysis and

regression model with the help of Design Expert Software Version 8 (Statease Inc.).

The Central Composite Design (CCD) with a quadratic model (Box and

Draper, 1987) was employed. Three independent variables namely enzyme

concentration (x1), temperature (x2) and time (x3) were chosen. Each independent

variable had 3 levels which were coded as (-)1, 0 and (+)1. A total of 20 different

combinations (including six replicates of the of the centre point each signed the coded

value 0) were chosen in random order according to a CCD for three factors (Cochran

and Cox, 1957). The experimental design in the coded (x) and actual (X) levels of

variables is shown in Table 3.1.

The responses function (y) measured were the yield, viscosity and clarity of

the banana juice. These values were related to the coded variables (xi, i = 1, 2 and 3)

by a second degree polynomial using the equation below.

y = b0 + b1x1 + b2x2 + b3x3 + b12x1x2 + b13x1x3 + b23x2x3 + b11x12 + b22x2

2 +

b33x3 2 -------------------------------- [3.5]


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Plate 3.2 ‘Texture Analyzer’ used for Textural Studies

Plate 3.3 Texture Probe Used for Textural Studies


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Table: 3.1 The Central Composite Experimental Design Employed For

Enzymatic Clarification of Banana Juice

Sr. No.

Enzyme

Concentration (%)

Temperature

(0C)

Time

(min)

X1 (x1) X2 (x2) X3 (x3)

1 0.15(+1) 50(+1) 60(-1)

2 0.1(0) 40(0) 120(0)

3 0.15(+1) 30(-1) 180(+1)

4 0.1(0) 40(0) 120(0)

5 0.05(-1) 50(+1) 180(+1)

6 0.05(-1) 30(-1) 60(-1)

7 0.05(-1) 30(-1) 180(+1)

8 0.1 (0) 40(0) 120(0)

9 0.1(0) 40(0) 120(0)

10 0.05(-1) 50(+1) 60(-1)

11 0.15(+1) 50(+1) 180(+1)

12 0.15(+1) 30(-1) 60(-1)

13 0.1(0) 40(0) 120(0)

14 0.15(+1) 40(0) 120(0)

15 0.1(0) 40(0) 120(0)

16 0.1(0) 50(+1) 120(0)

17 0.1(0) 40(0) 60(-1)

18 0.05(-1) 40(0) 120(0)

19 0.1(0) 40(0) 180(+1)

20 0.1(0) 30(-1) 120(0)

x represents the coded level of variables.

X represents the actual level of variables.

The coefficients of the polynomial were represented by b0 (constant term), b1,

b2 and b3 (linear effects), b11, b22 and b33 (quadratic effects), and b12, b13 and b23

(interaction effects). The analysis of variance (ANOVA) tables were generated and the

effect and regression coefficients of individual linear, quadratic and interaction terms

were determined. The significances of all terms in the polynomial were judged


45

statistically by computing the F-value and compared with standard significance level

of 0.1%, 1% and 5%. The regression coefficients were then used to make statistical

calculation to generate contour maps from the regression models.

3.3.2 Banana Pulp Clarification Process

Banana pulp of each selected maturity stage i.e. stage 5, 6 and 7 were tested

separately for clarification process. Pulp clarification has been done by using the

commercial pectinolytic enzyme i.e. Pectinex Ultra SP-L and cellulase enzyme i.e.

Celluclast 1.5L as shown in Fig 3.1

The banana fruits were washed, peeled manually and cut into small pieces.

Pieces were then pulped using a kitchen blender for 2 minute into pulp. After the

pulping, the pulp was divided into equal portions for enzyme treatment. For each

experiment 500g pulp was subjected to different enzyme treatment conditions as

shown in Table 3.1. The required quantity (0.05-0.15%) of pectinase enzyme i.e.

Pectinex Ultra SP-L pulp was added to 500 g batches of banana pulp and incubated at

different temperature (30-500C) and time (60-180 min) and then treated with 0.05%

cellulase enzyme i.e. Celluclast 1.5L. At the end of the treatment, the enzymes in the

pulp was inactivated by heating at 900C for 5 minute and immediately cooled to room

temperature. At this stage viscosity of the pulp samples were measured. The enzyme

treated pulp was centrifuged at 2900 G/15 minute and the supernatant was filtered

through a fine mesh nylon cloth spread on glass funnel and juice was collected.

The following parameters of the clarified juice were used as basis for optimization

study:

1. Viscosity of treated pulp

2. Yield of clarified juice

3. Clarity

3.3.3 Methodology for Measurement of Effectiveness of Enzymatic

Clarification Treatments

3.3.3.1 Yield

The juice yield was estimated as percentage of the juice obtained based on the

initial pulp.


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Sorted Banana Fingers (maturity stage 5, 6 or 7)

-Enzyme: Pectinex Ultra SP-L

0.05-0.15%

-Time 60-180 min, temp 30-500C

-Enzyme: Celluclast 1.5L

(0.05%, 60 min)

-Enzyme Inactivation (900C/5min)

Clarified juice

Fig. 3.1 Process for Banana Pulp Clarification

Washing & Peeling

Pulping

Mixing

Mixing

Incubation

Heating

Cooling (450C)

Centrifugation (2900 G/ 15 min)

Supernatant Filtration


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3.3.3.2 Viscosity

The viscosity of clarified banana juice was determined by using a Brookfield

viscometer (Model LVDV-II+, Brookfield Engineering Laboratory, Inc.) at 100 rpm

with spindle No. LV3 and LV4.

3.3.3.3 Clarity

The clarity of the juice obtained was determined by measuring % transmittance

at a wavelength of 660 nm using Shimadzu UV–VIS spectrophotometer. Distilled

water was used as the reference.

3.4 Study of Clarified Banana Juice

Banana pulp treated with different enzymatic conditions were optimized by

using RSM tool and for the pulp each ripening stage viz. stage 5, 6, 7 one best

enzymatic combination was identified as shown in Table 3.2 to obtain clarified juice

with maximum yield and clarity and minimum viscosity. Most desirable enzymatic

combination for ripening stage 5, 6 and 7 were denoted as A, B and C respectively.

Table 3.2 Optimized Enzymatic Combination for Clarification of Banana

Pulp of Different Ripening Stages Using RSM

The optimized combination of a particular ripening stage was applied to the

pulp of other ripening stages and the clarified juices thus obtained were used during

subsequent investigation to study the following physico-chemical parameters:

1. TSS (o Brix)

2. Sugar/Acid ratio

3. Total Sugar %

4. Reducing Sugar %

5. % Titratable Acidity (as malic acid)

6. Pectin (as calcium pectate) %

Enzymatic

Combination

Ripening

stage

Enzyme

Conc. (%)

Temperature

(0C)

Incubation

Time (min)

A 5 0.15 35.0 180

B 6 0.12 40.0 135

C 7 0.11 40.0 145


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3.4.1 Methodology for Physico-Chemical Parameters of Clarified

Banana Juices

3.4.1.1 TSS (o Brix)

As per the method described in section 3.2.11

3.4.1.2 Sugar/Acid ratio

The sugar acid ratio also termed as Brix : Acid ratio was calculated by taking

the ratio of total soluble solid and acidity of respective samples.

3.4.1.3 Total Sugar %


3.4.1.4 Reducing Sugar %

The reducing sugar content were determined by following the Shaffer-

Somogyi method as described by Ranganna 2000.

3.4.1.5 % Titratable Acidity (as malic acid)


3.4.1.6 Pectin (as calcium pectate) %



Analysis of variance (ANOVA) was used to detect treatment effect. Mean

separation was performed by using least significance difference (LSD) at the p< 0.05

level.

3.4.3 Sensory Evaluation of Clarified Banana Juices Prepared by

Optimized Enzymatic Treatment of Each Ripening Stage

Clarified banana juices prepared by optimized enzymatic combination i.e.

enzymatic combination A, B and C for maturity stage 5, 6 and 7 respectively were

evaluated for various sensory parameters. Hedonic scale 1-9 (1: dislike extremely, 9:

like extremely) (Larmond, 1977) was used to indicate the preference for quality

factors viz. colour, aroma, taste and acceptability of clarified banana juices. Trained

panelist was presented all the coded samples and they were requested to show their

preferences for the quality attributes using Hedonic scale 1-9 (Appendix A). The

mean scores were analyzed using Analysis of Variance and their comparison with

Duncan’s Multiple Range Test was performed. For quality description of sensory


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evaluated samples, juice samples scored below 5 were considered as poor quality, 5 to

7 as fair and above 7 as good quality.

3.5 Prevention of Enzymatic Browning in Clarified Banana

Juice

Discolouration was the major problem in clarified banana juices. It was

observed that within 3 to 4 minutes of pulping the peeled banana, the surface of the

macerated pulp lost its original colour turning into brown colour which ultimately

affected the colour of prepared clarified juice. This may be due to enzymatic reactions

occurred in the pulp (oxidation of native phenolics by PPO). Therefore it was

necessary to find a suitable method to inhibit the activity of PPO in banana and

prevent discolouration during the course of juice clarification. Hence in the present

investigation browning inhibition study was undertaken with one highly acceptable

clarified juice sample which was selected on the basis of sensory evaluation study of

clarified juices.

3.5.1 Anti-browning Pretreatments to Pulp

The following experiments were carried out to inhibit the PPO activity and

enzymatic browning during preparation of highly acceptable clarified banana juice.

a) Addition of Ascorbic acid at concentrations of 200-1200 ppm (Galeazzi and

Sgarbieri, 1981) to the peeled fruits during blending and clarified juices were

prepared as per the process described in section 3.3.2.

b) Blanching the peeled fruits in hot water temperature 900C (Sims and Bates, 1994)

for different periods up to 10 minutes. These heated samples were quickly cooled to

room temperature by dipping in cold water then blended and clarified juices were

prepared as per the process described in section 3.3.2.

Browning of the juices obtained was measured immediately after extraction

and at intervals of 2, 4, 6 and 24 h.

3.5.2 Evaluation of Effectiveness of Anti- browning Pretreatments

Effectiveness of anti-browning pretreatments to the pulp was evaluated by

measuring the browning in terms of Browning Index in prepared clarified juices. For

measuring the enzymatic and non enzymatic browning, 20 ml of the juice was mixed

with 30 ml of ethyl alcohol in a glass stoppered flask (Ranganna, 2000). The mixture

was shaken thoroughly and filtered through Whatman No.42 filter paper. The


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absorbance of the filtrate was measured at 420 nm against 60% aqueous alcohol in a

spectrophotometer. Browning index (A at 420 nm) was expressed as absorbance

value.


Analysis of variance (ANOVA) was used to detect the effect of pretreatment

during different time intervals. Tukey’s Multiple Comparison Test was employed for

mean separation. The level of significance was at 5%. The analysis was performed

using the Daniel's XL Toolbox version 4.00.

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CHAPTER 3 MATERIALS AND METHODS - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/42103/12/12_chapter3.pdfCHAPTER 3 MATERIALS AND METHODS The study on various aspects and objectives