CHAPTER 3 METHODOLOGY
3.6 Statistical analysis
28
CHAPTER 3
29
3.2 Instruments and equipment
Instruments and equipment used are presented in Table 13 Table 13 Instruments and equipment used in experimental research
No. Instruments/ equipment Details
1 High performance liquid
chromatograph (HPLC) apparatus
Shimadzu LC-20AC
2 Hot air oven Memmert
3 Tray dryer ULM 500, Memmart
4 Spectrophotometer Spectronic Genesys
Shimadzu, Japan
5 pH meter Mettler Toledo
6 Vortex mixer Velp Scientific, Europe
7 Scanning electron microscope (SEM)
JSM-6460L model, JEOL, USA
8 Texture analyzer Model TA-XT2
9 Digital refractometer DBX-55
10 Chroma meter Minolta color meter, model
CR-300
11 Freeze dryer FTS system Dura-DryTm,
USA
12 Electronic balance Precisa 125 A
3.3 Materials
3.3.1 Model food was prepared with agar-agar powder (TM media, Rajasthan, India), sucrose and distilled water (Rózek et al., 2010).
3.3.2 Kaew mango cultivar was purchased from a local market in Bangkok, Thailand from September to November 2017. Mangoes were selected at the full ripe stage.
30
3.4 Chemicals and reagents
Table 14 Chemicals and reagents for experimental research
No. Chemicals/ reagents Types/ Grades Details
1 Gallic acid HPLC Sigma-Aldrich
Chemical Co.
(St. Louis, MO, USA)
2 1, 1-diphenyl-2-
picrylhydrazyl (DPPH)
HPLC Sigma-Aldrich
Chemical Co.
(St. Louis, MO, USA)
3 Acetonitrile HPLC Fisher Scientific
(UK)
4 Methanol HPLC BDH (Poole, UK)
5 Folin-Ciocalteu reagent Analytical Sigma-Aldrich Chemical Co.
(St. Louis, MO, USA)
6 Sodium hydroxide Analytical Carlo Erba,
Thailand
7 Calcium lactate Analytical Sigma-Aldrich
Chemical Co., (St.
Louis, MO, USA)
8 L-ascorbic acid Analytical Chemical-Supply
Pty Ltd, (Australia)
9 Ammonium molybdate Analytical BDH (Poole, UK)
10 Potassium phosphate Analytical BDH (Poole, UK)
11 Gallic acid Analytical Loba Chemie
(India)
12 Other reagents Analytical -
3.5 Methods
Experimental methods were divided into three parts as follows:
3.5.1 Effect of process conditions on mass transfer, volume and porosity of the PVOD model food.
Process conditions included density of vacuum impregnation time (10-30 min), model food (0.65-0.85 g/mL) and sucrose concentrations (30-60 oBrix).
31
Figure 3 Process conditions on mass transfer, volume and porosity of PVOD model food
Preparation of model food
• 3-5% agar-agar + 10%(w/w) sucrose + distilled water (95 oC)
•Mixture was heated for 10 min using a microwave oven
• Cooling
• Stored (6±2oC)
• Cut into cubes (1x1x1 cm)
Vacuum impregnation process (150 mbar)
Analysis Impregnation
(atmospheric pressure, 180 min)
Process optimization
• CCD (18 treatments)
• Regression analysis Draining
(10 min)
Model food characteristics
• Total soluble solids
• Bulk and solid-liquid densities
•Porosity
• Volume
• Water activity
• Moisture content Process variables
• Concentrations (30-60 oBrix) • Vacuum time (10-30 min) • Density (0.65-0.85 g/mL)
• Water loss (WL)
• Solids gain (SG)
•Weight reduction (WR)
• WL/SG
• Porosity
• Volume
• Bulk and solid-liquid densities
• Total soluble solid
• Water activity
• Moisture content
32
3.5.1.1 Preparation of model food
Model food with 0.65-0.85 g/mL density was prepared with 3.5-6.5% (w/w) agar-agar powder (TM media, Rajasthan, India), 10% (w/w) sucrose, and distilled water (95°C). For example, model food with 0.65 g/mL density was prepared with agar-agar powder (3.3 grams) and sucrose (10 grams) dissolved in 86.7 mL of distilled water (95°C) and then heated for 10 min using a microwave oven to achieve completely dissolved agar-agar. The agar gel was cooled to room temperature, stored at 8 ± 2°C and analyzed within 2 days (Rózek et al., 2010b). The agar gel was cut into cubes (1x1x1 cm).
3.5.1.2 Preparation of osmotic dehydration medium (OD medium)
The OD medium was prepared by dissolution with commercial sucrose in distilled water (Mújica-Paz et al., 2003a) Concentrations of OD mediums were 30-60
oBrix (Digital refractometer).
3.5.1.3 Vacuum impregnation process
The model food cubes were weighed (100 g) and impregnated in the sucrose solution (30-60 %) with a 1:2 weight ratio of model food cubes to medium solution. They were then placed into the vacuum chamber (PolyLab, Thailand).
Vacuum pressure was applied at 150 mbar for 10-30 min using a vacuum pump (Gast-model DOA-P504-BN Labmodel, Germany), thereafter the samples were restored in atmospheric pressure for 180 min (Moraga et al., 2009). The impregnated model food cubes were drained for 10 min, weighed and packed in laminated plastic bags. Each treatment was stored at 8 ± 2 oC for further study. All treatments were performed in triplicate. Each treatment was immersed in osmotic solution at a certain concentration for an interval of time according to the central composite design of three factors (Table 15).
3.5.1.4 Analysis
1) Water loss (WL), solids gain (SG) and weight reduction (WR) were examined following (Corrêa et al., 2010).
Analysis of mass transfer parameters included weight reduction (WR), water loss (WL) and solids gain (SG). Changes in weight of the impregnated model food before and after pretreatment were determined individually using a digital balance and calculated according to the following equations:
WL(%) = XoMo -XfMf x 100 (3.1)
Mo
SG (%) = XsfMf - XsoMo x100 (3.2) Mo
WR(%) = Mo-Mf x 100 (3.3) Mo
33
where
Xo is initial moisture content (%)
Mo is initial sample weight (kg)
Xf is final moisture content (%)
Mf is final sample weight (kg)
Xso is initial solids content (%)
Xfo is final solids content (%)
2) Determination of total soluble solids (TSS)
Measurement of total soluble solids was performed using an Atago Hand Refractometer (ATAGO) (Jacob & Paliyath, 2012). Briefly, ten grams of sample were mixed in 20 mL distilled water in a glass tube using a stirrer. The mixture was centrifuged using a centrifuge (Rotina 48R, Universal 320R) at 5,000 g for 15 min.
The aqueous solution was separated from the sediment and analyzed for total soluble solids (TSS) by a refractometer. A drop of clear solution was placed on a refractometer prism and closed with a cover plate before reading the data.
3) Determination of moisture content
Moisture contents of the samples were analyzed according to the standard method (AOAC, 2000) in an oven at 105 oC.
4) Bulk and solid-liquid densities and porosity followed (Nieto et al., 2004).
Measurement of bulk density (b) of the samples was determined by displacement with water. The samples were weighed (~1.5-2.5 g) with a digital balance and put into a glass pycnometer (filling volume 100 mL). The pycnometer was filled with distilled water to investigate the volume of the samples. For solid-liquid density (s) analysis, the pulp was homogenized and de-aerated to remove pores and air using a vacuum pump (Gast DOA-P504-BN Labmodel, Germany) at 150 mbar. Porosity () of samples were calculated following equation 3.4:
(3.4)
5) Determination of volume of model food (Nieto et al., 2004)
The volume of model food was determined from its weight and bulk density (b). Changes in volume were evaluated following equation 3.5:
34
(3.5)
where Vo is initial volume of the sample (m3) and V is the volume of the sample at time t (m3).
3.5.1.5 Statistical experiment design and process optimization
For optimization of the PVOD process for model food, experiments were conducted according to Central Composite Design (CCD) with three variables (density, vacuum impregnation time and sucrose concentrations). The CCD design uniformly predicted all constant distances from their center points. For rotatable designs, variances and co-variances of the estimated coefficients in the fitted model remained unchanged when the design points were rotated about their center. The design was generated by the commercial statistical package, Design-Expert ®v.7 (Stat-Ease, Minneapolis, MN, USA) (Singh et al., 2007). The experimental plan in coded and un-coded form of process variables is presented in Table 15 to Table 17 Results were statistically tested by analysis of variance (ANOVA) at significance level of p = 0.05. The adequacy of the model was evaluated by the coefficient of determination (R2) and model p-value (Šumić et al., 2013).
Table 15 Process factor codes for model food cubes
Process factor Unit -1 Level +1 Level
Density g/mL 0.65 0.85
Concentration % 30 60
Time min 10 30
35
Table 16 Process factor codes for model food cubes
Run Density (g/mL) Concentration (%) Time (min)
1 0.000 0.000 0.000
2 0.000 0.000 -1.682
3 1.000 -1.000 1.000
4 0.000 0.000 0.000
5 0.000 -1.682 0.000
6 0.000 0.000 0.000
7 0.000 1.682 0.000
8 1.000 -1.000 -1.000
9 -1.682 0.000 0.000
10 1.000 1.000 -1.000
11 -1.000 1.000 1.000
12 -1.000 -1.000 -1.000
13 1.000 1.000 1.000
14 1.682 0.000 0.000
15 -1.000 1.000 -1.000
16 0.000 0.000 0.000
17 -1.000 -1.000 1.000
18 0.000 0.000 1.682
36
Table 17 Process factors for model food cubes
Run Density (g/mL) Concentration (%) Time (min)
1 0.75 45 20
2 0.75 45 3.18
3 0.85 30 30
4 0.75 45 20
5 0.75 19.77 20
6 0.75 45 20
7 0.75 70.23 20
8 0.85 30 10
9 0.58 45 20
10 0.85 60 10
11 0.65 60 30
12 0.65 30 10
13 0.85 60 30
14 0.92 45 20
15 0.65 60 10
16 0.75 45 20
17 0.65 30 30
18 0.75 45 36.82
A second order polynomial equation was fitted to the experimental data of each dependent variable (water loss, solid gains, weight reduction, moisture content, total soluble solids, density, porosity, volume change) as given below:
Yk = Ao + A1X1 + A2X2 + A3X3 + A11X12 + A22X22 + A33X32
+ A12X1X2 + A13X1X3 + A23X2X3 + (3.6)
where Yk = response variable, Xi represents the independent variables (X1 = density, X2= osmotic medium concentrations, X3 = VI times), Ao is the value of fitted response at the center point of design, i.e. point (0, 0, 0), A1, A2, A3, A11, A22, A33, A12, A13, A23 are coefficients and is a random error term.
The relative effect of each process parameter was compared from B values by estimated from regression analysis of linear or non-linear model.
3.5.2 Impact of PVOD process conditions on nutraceuticals and health regulation components infusion into mango cubes and model food
Evaluation effect of PVOD process conditions on physicochemical properties of impregnated model food or mango cube compared to osmotic dehydration technique at the same process condition.
37
Figure 4 Impact of PVOD process conditions on nutraceuticals and health regulation components infusion into mango cubes and model food
Composition of infusion medium
• Sucrose
• Gallic acid
• Ascorbic acid
• Calcium lactate Solution from an optimization step
• Density (0.80 g/mL)
• Sucrose concentration (32.58 oBrix)
• Vacuum time (14.34 min) Preparation of mangoes
• Mangoes were peeled and cut into cubes
•Dipped (3 min) in 1% calcium chloride solution containing 1%w/v ascorbic acid and 1% w/v citric acid
• Immersed in water (100 oC, 30 s)
• Immersed in ice water for 5 min
Vacuum impregnation process (150 mbar)
Preparation of model food
• 4.2% agar-agar + 10%(w/w) sucrose + distilled water (95 oC)
•Mixture was heated for 10 min using a microwave oven
• Cooling
Impregnation
(atmospheric pressure, 180 min)
Analysis Draining (10 min)
- Porosity - Volume - Density - Water loss
- Solids gain - Weight reduction
- Moisture content - Water activity - Total soluble solids - Total acidity - Sucrose content - Calcium content
- Ascorbic acid - Gallic acid
- Total phenolic content - DPPH radical
scavenging (IC50)
38
3.5.2.1 Preparation of mango cubes and agar gel 1) Preparation of mango cubes
Kaew cultivar was purchased from a local market in Bangkok, Thailand from September to November 2017. The samples were selected according to their size, color and ripe appearance of skin. Fruits with defects and physiological disorders were discarded. Mango fruits were immediately transported to the laboratory. The fruit was stored in the air-conditioned laboratory for less than 8 hours before analysis of physicochemical properties (total soluble solids, pH, titratable acidity, etc.) and used in the experiment. In each treatment, at least 15 mango samples were used and all experiments were conducted in triplicate.
Mangoes were washed with tap water, then peeled and cut into cubes (1x1x1 cm). Samples were dipped (3 min) in 1 % w/v calcium chloride solution as a firmness stabilizer containing 1 % w/v ascorbic acid and 1 % w/v citric acid as anti- browning agents (Robles-Sánchez et al., 2009). Mango cubes were drained, immersed in water at 100 oC for 30 s and immediately cooled in ice water for 5 min.
2) Preparation of agar gel
The optimum process condition of agar gel density (0.80 mL/g) was used in the preparation of model food to investigate the effect of PVOD process conditions on nutraceuticals and health regulation components infusion into model food. Model food at 0.80 ± 0.08 g/mL density was prepared with 4.2 % (w/w) agar-agar powder (TM media, Rajasthan, India), 10 % (w/w) sucrose and distilled water (95 oC).
The agar gel was cooled down under room temperature, stored at 8 ± 2 oC and analyzed within 2 days (Rózek et al., 2010b). Agar gel was cut into cubes (1x1x1 cm).
All samples were prepared in triplicate.
3.5.2.2 Preparation of osmotic dehydration medium (OD medium) 1) Sucrose solution
The optimum process condition of osmotic solution (32.58 oBrix) was used in the preparation of sucrose solution to study the effect of PVOD and OD process on nutraceuticals and health regulation components infusion into model food. A 32.58
°Brix sucrose solution was prepared by dissolving 32.58 grams of sugar in distilled water (67.42 grams) and then stirring to achieve complete dissolution.
2) Infusion medium
Infusion medium was prepared from the ingredients given in Table 18.
After the ingredient mixing step, the 60 oBrix sucrose solution was used to obtain 32.58 oBrix.
39
Table 18 Composition of infusion medium used for the infusion of mango and model food
Ingredient Quantity
Gallic acid 6.09 g
Ascorbic acid 12.19 g
Calcium lactate Sugar
2.44 g 46.88 g
Water 132.4 mL
3.5.2.3 Vacuum impregnation process
The infusion process condition was selected from the process optimization in Part I. The mango or agar gel cubes were weighed (100 g) and impregnated in the osmotic medium with a 1:2 (w/w) mango or model food cubes/medium ratio before placing in a vacuum chamber (PolyLab, Thailand). Vacuum pressure was applied at 150 mbar using a vacuum pump (Gast-model DOA-P504-BN Labmodel, Germany), thereafter the samples were restored in atmospheric pressure for 180 min (Moraga et al., 2009). The impregnated samples were drained for 10 min, weighed and packed in laminated plastic bags. Each treatment was stored at 8 ± 2 oC for further study.
All treatments were performed in triplicate.
3.5.2.4 Analysis
1) Water loss (WL), solids gain (SG) and weight reduction (WR) followed (Corrêa et al., 2010).
Analysis of mass transfer parameters included weight reduction (WR), water loss (WL) and solids gain (SG). Changes in weight of the impregnated model food agar gels before and after pretreatment were determined individually using a digital balance and calculated according to the following equations:
WL(%) = XoMo -XfMf x 100 (3.7)
Mo
SG (%) = XsfMf - XsoMo x100 (3.8) Mo
WR (%) = Mo-Mf x 100 (3.9) Mo
where
Xo is initial moisture content (%)
Mo is initial sample weight (kg)
Xf is final moisture content (%)
Mf is final sample weight (kg)
40
Xfo is final solids content (%)
2) Determination of total soluble solids (TSS)
Measurement of total soluble solids was performed using an Atago Hand Refractometer (ATAGO). Briefly, ten grams of sample were mixed in 20 mL distilled water in a glass tube using a stirrer. The mixture was centrifuged using a centrifuge (Rotina 48R, Universal 320R) at 5,000 g for 15 min. The aqueous solution was separated from the sediment and analyzed for total soluble solids (TSS) by a refractometer. A drop of clear solution was placed on a refractometer prism and closed with a cover plate before reading the data (Jacob & Paliyath, 2012).
3) Determination of moisture content
Moisture content of samples was determined following the AOAC (2000) method in an oven at 105 oC.
4) Bulk and solid-liquid densities and porosity measurements followed (Nieto et al., 2004)
Measurement of bulk density (b) of the samples was determined by displacement with water. The samples were weighed (~1.5-2.5 g) with a digital balance and put into a glass pycnometer (filling volume 100 mL). The pycnometer was filled with distilled water to investigate the volume of the samples. For solid-liquid density (s) analysis, the pulp was homogenized and de-aerated to remove pores and air using a vacuum pump (Gast DOA-P504-BN Labmodel, Germany) at 150 mbar for solid- liquid density (s) analysis. Porosity () of samples were calculated following equation 3.4:
(3.10)
5) Determination of volume change (Nieto et al., 2004)
The volume of model food was determined from its weight and bulk density (b). Changes in volume were evaluated following equation 3.5:
(3.11)
where Vo is initial volume of the sample (m3) and V is the volume of the sample at time t (m3).
41
6) Determination gallic acid content, total phenolic content (TPC) and antioxidant analysis
6.1) Extraction
The impregnated samples were extracted using the method of (Liu et al., 2013) with some modifications. Homogenized 20 g samples were added to 20 mL of 80 % v/v methanol and the mixture was stirred for 3 h before centrifuging at 4,000 rpm for 10 min at room temperature. The extracts were filtered through Whatman filter paper (No.1). The supernatant was passed through a nylon syringe filter (0.45 µm, GE Healthcare, USA) and kept in a brown glass vial at -20 oC for further study. All treatments were performed in triplicate.
6.2) Gallic acid content
Analysis of gallic acid was conducted using HPLC system (Shimadzu Co., Japan), as described by Butkhup and Samappito (2008) and Loypimai et al. (2016).
The mobile phase for gallic acid assessment was deionized water-acetonitrile (97.8:2
% v/v) containing 0.2 % phosphoric acid (solvent A) and deionized water-acetonitrile (2/97.8, % v/v) containing 0.2 % phosphoric acid (solvent B) with a flow rate of 0.6 mL/min. Operating conditions were as follows: column oven temperature, 40 °C;
injection volume, 20 µl and UV-diode array detector at 278 nm. Gallic acid content in mango samples was calculated using the commercial standard (gallic acid) and expressed as microgram gallic acid equivalent per grams of fresh weight sample (µg GAE/ g sample).
6.3) Total phenolic content (TPC)
Total phenolic content was extracted by the same method as gallic acid content. Total phenolic content was estimated using the Folin-Ciocalteau method followed Liu et al. (2013) as adapted from (Kim et al., 2009) with slight modifications. Briefly, 0.4 mL of sample extracts was mixed with 2 mL of Folin- Ciocalteu reagent (previously diluted 10-fold with distilled water). The mixed solutions were allowed to stand at room temperature for 5 min and 1.8 mL of sodium carbonate solution (7.5 % w/v) was added. The final volume was adjusted to 7 mL and mixed thoroughly. The mixture solutions were measured at 765 nm using methanol (80 % v/v) to zero a spectrophotometer (UV-vis model 1601, Shimadzu Co., Japan). Results were expressed as microgram gallic acid equivalents (GAE) per gram of sample (µg GAE/
g sample).
6.4) DPPH assay
Free radical scavenging activity of the untreated or impregnated samples was determined following Dasgupta and De (2004) with some modifications.
A total of 100 µL of sample extracts were added with 3 mL DPPH solution (0.004 g DPPH radical mixed with 100 mL of 80 % v/v methanol). The mixture was stored in the dark at room temperature for 30 min. Absorbance was measured using a spectrophotometer (UV-vis model 1601, Shimadzu, Japan) at 517 nm. Percentage inhibition activity was calculated as:
42
Inhibition activity (%) = (Ao-As/Ao) x100 (3.12)
where A0 is the absorbance of the control sample and AS is the absorbance in presence of mango or agar gel extract. Results were expressed in IC50, which implies the concentration of sample extract required to scavenge 50 % DPPH free radicals.
7) Determination of ascorbic acid
Ascorbic acid and sucrose content in the samples were extracted following the procedures of Liu et al. (2013) with some modifications. Briefly, twenty grams of untreated or impregnated samples were mixed in 100 mL of 2.5 % w/v metaphosphoric acid solution in a conical flask covered with aluminium foil.
The mixture was extracted using a stirrer (HY-HS11, Korea for 2 h at room temperature. After stirring, all sample solutions were filtered through filter paper (Whatman No.1). The supernatant was filtered through a nylon syringe filter (0.45 µm, USA), contained in a brown HPLC vial and stored at -20 oC before injection into the HPLC column. All samples were analyzed in triplicate.
Ascorbic acid was analyzed as explained by Liu et al. (2013) with some modifications. The extract was injected into the HPLC system (Shimadzu Co., Japan).
The HPLC apparatus consisted of a UV-vis photodiode array detector (PDA, SPD- M20A), a pumping system (LC-20AD), a column oven (CTO-10ASvp), a system controller (CBM-20A) and an autosampler (SIL-10ADvp). A 20 µL extract was injected into an Inertsil® ODS-3 column (4.6 x 250 mm internal diameter, 5 µm particle size, GL Science Inc., Tokyo, Japan). Deionized water was used as the mobile phase with pH value adjusted to 2.2 using 1M metaphosphoric acid solution with a flow rate of 0.8 mL/min. Column temperature was retained at 40 °C. Ascorbic acid was quantified with a UV-vis photodiode array detector at 254 nm. Concentration of ascorbic acid in the samples was calculated using L-ascorbic acid as the external standard and expressed as milligrams (mg) ascorbic acid per 100 grams of fresh weight (FW) sample.
8) Determination of sucrose
Sucrose content was extracted following the procedure of Jacob and Paliyath (2012) with some modifications. A fresh sample (20 g) was extracted with 100 mL of methanol. The homogenate was stirred for 3 h before centrifuging at 10,000 rpm/min for 15 min. The extract was filtered through a nylon syringe filter (0.45 µm). Sucrose content was performed as described by Zhang et al. (2005) and Chen et al. (2012) and modified in our laboratory. Extracts were analyzed using an HPLC system (Shimadzu Co., Japan) composed of a refractive index detector (RID- 10A), a pumping system (LC-20AD), a column oven (CTO-20A) and an autosampler system (SIL-20A). Twenty microliters of clear extracts were injected into an Inertsil®NH2 column (5.0 µm, 4.6 mm x 250 mm, GL Science Inc., Tokyo, Japan).
A refractive index detector was used to identify and quantify sucrose. The mobile phase
43
was composed of acetonitrile/ distilled water (85:15). The flow rate was 1.5 mL/min at 40 °C. Sucrose content in all samples was calculated using sucrose (HPLC grade) as the external standard and expressed as milligrams (mg) sucrose per grams of fresh weight (FW) sample.
9) Determination of calcium content
Calcium content was performed as described by AOAC (2012) with some modifications.
9.1) Preparation of sample solutions
Two grams of untreated or impregnated samples were mixed in 10 mL of the mixed solution with a 2:1 (% w/w) nitric acid/perchloric acid ratio in a glass digestion tube. The reaction mixture solution was maintained at room temperature (1 h) before digestion in the AIM600 Block Digestion System (A.i Scientific, Brisbane, Australia) until a clear solution was obtained. Then, the solution was put into a 50 mL volumetric flask and adjusted to the required volume with deionized water.
9.2) Calcium content analysis
Concentrations of calcium in samples were analyzed by an inductively coupled plasma optical emission spectrometer (ICP-OES Optima 8000 DV, Perkin-Elmer, Wellesley, MA, USA). Operating conditions were as follows: a RF power of 1300 W, a plasma gas flow rate of 15 L/min, a nebulizer gas flow rate of 0.6 L/min, an auxiliary gas flow rate of 0.2 L/min and a pump flow rate of 1.5 L/min.
Heater temperature was retained at 30 °C. Measurements were performed using 206.163 nm spectra line. Concentration of calcium was expressed as milligrams calcium per 100 grams of sample (mg calcium/100 g sample). All treatments were determined according to the following equation:
Calcium content (mg /100 g sample) = AxV
Wx10 (3.13)
where A is the calcium content as read from an inductively coupled plasma optical emission spectrometer (ICP-OES Optima 8000 DV, Perkin-Elmer, Wellesley, MA, USA), V is the final volume of the sample and W is sample weight.
10) Microscopic analysis
Fluorescein diacetate (FDA) staining: Cell viability of impregnated mango was performed employing fluorescein diacetate (Sigma-Aldrich, USA) following Tylewicz et al. (2013) with some modifications. A 100 µM fluorescein diacetate solution was prepared in an isotonic sucrose solution (13 % w/v).
The impregnated mango was cut into thin slices (1 mm) and the slices were submerged in the fluorescein diacetate solution in the darkness for 30 min. Viable cells of the impregnated mango were performed using the Nikon Eclipse Ni-U microscope.