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CHAPTER 3 Materials and Methods 3.1.

Physicochemical properties and resistant starch content of green banana and peel starch 3.1.1. Materials One Thai banana cultivar Musa (ABB) sp. (Kluai Namwa) was collected from a local market (Talad Thai). The fruits had green peel and sharp edges. Bananas used for starch isolation were at the first stage of ripening process as described by Von Loesecke (1950). 3.1.2. Extraction of starch from banana and its peel: A modified method of Lii, et al., (1982) will be used to produce starch from green banana and its peel. Peeled green banana and peel were cut into small slice (1cm thick) separately.

Rinsing into 1% citric acid solution for 10 min to avoid browning reaction. Slices will be disintegrated with 0.1% NaOH solution (1:1) using blender at low speed like paste.

Five times of its volume of water was added and screened to pass through 60, 80, and 200 mesh screens to remove fibers and pomace and washed with water until the wash water was clean

Starch slurry was dried at 400C in a hot air oven for 24 hours. Grinding by mortar pestle and passing through 100 mash (250m) sieve to get starch from green banana and peel separately.

Vigorous squeezing of the cheesecloth in order to accelerate the filtration process should be avoided. Carrying out at least two more rounds of grinding and filtration using the residue left on the cheesecloth will improve the recovery of starch. The resulting filtrate containing medium and small fibers is then subjected to further filtration. A series of polypropylene screens (250, 175, 125, and/or 75 m) is normally used. The filtration process yields a milky filtrate that is rich in starch but contains other plant components, such as gums or mucilage, protein, and fine fiber particles that can be separated out by centrifugation.

3.1.3. Chemical characterization: Moisture content, PH, ash, crude protein, fat, amylose content, amylopectin content, total starch, resistant starch, non resistant starch etc. of green banana and its peel starch were determined. Moisture content The moisture content of tested samples was determined using the indirect method, according to the AOAC procedures (2002). Dish was heated to 105C with cover, cooled in desiccators and weighed soon after reaching room temperature. Accurately weight 2.0000 g well-mixed test portion in covered dish. Loosen cover test portion and heat at 105C to constant weight (about 5 h) in hot air oven. Immediately tighten cover on dish, transfer to desiccators and weigh soon after reaching room temperature. Percent moisture content was calculated as follow Ash Ash content was determined by direct ignition method. Five grams of dried sample was weighed to the nearest 0.0001 g and then transferred to the crucible. The crucible containing the sample was placed inside the muffle furnace (Model FSE 621-210D, Sanyo Gallenkamp, UK) set at 550oC and dried for 16 hrs. The crucible was reweighed and the percent ash was calculated as follows:
%ash weight of residues 100 weight of samples

(6) Determination of protein content Regents: 1. 2. 3. 4. 5. 6. Sulfuric acid (H2SO4) Potassium sulfate (K2SO4) Copper sulfate (CuSO4) Sodium hydroxide (NaOH) Bromocresol green Methy red

Method: Total nitrogen content of tested samples was determined using the Kjeldahl method, according to the AOAC procedures (2002). Sample (2.0000 g) was weight in the Kjeldahl digestion tube. Fifteen ml of conc. H2SO4 and 15 g of catalyst (mixed CuSO4 with K2SO4 in a weight ratio of 0.5:1) were added to the sample and digested in a digester at 420C until a clear solution was obtained. Distilled water (55 ml) was added to the tube and the mixture

steam distilled using a distillation unit, while 85 ml of 40% NaOH solution were added automatically via a built in dispensing system. The distillate was collected in a 50 ml saturated boric acid with mixed indicator. The mixed indicator solution was prepared by mixing 0.1% (w/v) of bromocresol green with 0.1% (w/v) of methyl red. Titration was done using 1.0 N HCL until the color changed to pink-red. The total nitrogen content and protein content was calculated as following formula; Total nitrogen = Protein content = (% total nitrogen 5.70) Lipids Total lipids were extracted using petroleum ether in the Soxtec system (Model HT6, Tecator, Sweden). The extract is referred as crude lipid. A 2 g dried sample was weighed to the nearest 0.0001 g into the Whatman filter paper. The sample was wrapped and interested into a thimble holder of soxtec machine. About 60 ml petroleum ether was added into the extraction cups. The heating unit of machine was set at 120 oC with 30 min extraction time. The sample was dried at 100oC and reweighed to calculate percent lipid. Crude fiber Two grams of sample were placed in a beaker and treated with 200 ml boiling sulfuric acid. Then the beaker was placed on a hot plate and boiled for 30 min, with occasional rotating of beaker. The sample was then cooled and filtered. The beaker was rinsed twice with 50 ml of boiling water. The residue was transferred carefully to 200 ml of 1.25% sodium hydroxide and boiled for 30 min. The solution was cooled, filtered and washed with 50 ml boiling water. The residues were washed with 25 ml of 95% ethanol, then dried for 2 h at 130oC, cooled in desiccator and weighed. Crude fiber was calculated as per the following equation:

% fiber

weight loss on ignition weight of sample

(7) Carbohydrates Carbohydrate content was calculated from the difference by subtracting protein, lipids, ash and fiber from banana flour (equation 8).
%carbohydrate 100 % protein % fat %ash % fiber (8) Determination of amylose content of starch: Chemicals and Reagents 1. 95% Ethanol 2. 1N NaOH

3. 1N Acetic acid 4. Iodine - Potassium iodide solution 5. Standard amylose Composition of reaction mixtures A. 1N NaOH solution Dissolve 40g of NaOH in 1000ml distilled water B. 1N Acetic acid solution Dilute 57.5 ml glacial acetic acid to 1000ml using distilled water C. Iodine - Potassium iodide solution Dissolve 0.26 g of Iodine in 10 ml of Potassium iodide solution containing 2.6 g of KI D. Standard Amylose Solution: Take 40mg of pure potato starch (amylose) in a 100 ml volumetric flask and add 1 ml of 95% ethanol and 9.0 ml of 1N NaOH. Shake well and boil over water bath for 10 minutes and make up the solution to 100 ml using distilled water. Procedure 1. Weigh 100 mg well powdered milled rice into 100 ml volumetric flask. 2. Add 1 ml 95% ethanol and 9 ml 1 N NaOH. 3. Heat the sample for 10 minutes in boiling water bath, cool it and make up the volume to 100 ml. 4. Pipette 5 ml from the 100 ml into another 100 ml volumetric flask. 5. Add 1 ml I N acetic acid and then 2 ml iodide solution and make up the volume to 100 ml. 6. Shake, stand for 20 minutes and determine the per cent Transmittance at 620 nm using a colorimeter. 7. Prepare a series of standard starch solution containing 0, 20, 40, 60, 80 and 100% amylose as in the steps 1 to 5 8. Read the transmittance of the standards at 620nm and plot a standard graph. 9. Amylose content of the sample is determined in reference to the standard curve and expressed on percent basis. 10. Making of amylose standards: a. Pipette out 1, 2, 3, 4 and 5 ml of the standard amylose into 100 ml volumetric flasks in three replications. b. Keep one flask as blank without adding anything. c. Add 1.0 ml 1N acetic acid and 2.0 ml I-KI solution to all the flasks including blank. d. Make up all the flasks to 100ml using distilled water and cover all the flasks with a black cloth or aluminum foil to prevent direct light exposure. I-KI disintegrates on exposure to light. e. Keep for 20 minutes and take reading at 620nm in a spectrophotometer. f. The standards including balnk, correspond to 0%, 4%, 8%, 12%, 16% and 20% of amylose. Draw a standard curve using the absorbance reading.

The Absorbance (y) at 620 nm was plotted against concentration (x) of anhydrous amylose (mg/100ml) and the formulae for calculating the amylose concentration in starch sample x=(y-0.0002)/0.178 was deduced from the standard equation y = 0.178x +0.0002.
( ) ( )

Amylose % = Determination of the total starch, digestible starch and resistant starch: Resistant starch (RS), digestible starch (DS) and total starch (TS) content in green banana and peel starch samples were determined by using the methodology of A.O.A.C. 2002.02 (McCleary and Monaghan, 2002) and (McCleary et al., 2002), with modifications.

1. 2. 3. 4. 5. 6. 7.

Sodium maleate buffer (0.1 M, pH 6.0) Sodium acetate buffer (1.2 M, pH 3.8) Sodium acetate buffer (100 mM, pH 5.8) Potassium Hydroxide (2 M) Stock Amyloglucosidase solution Pancreatic - amylase Glucose oxidase-peroxidase-aminoantipyrine reagent

Apparatus a. Bench centrifuge. - Capable of holding 16 x 120 mm glass test tubes, with rating of ca 1500 g (~ 3,000 rpm). b. Shaking water bath (Grant OLS 200)- set in linear motion at 100 revolutions per minute on the dial (equivalent to a shake speed of 200 strokes/min),a stroke length of 35mm and 37oC. c. Water bath.- Capable of maintaining 50 + 0.10C. d. Vortex mixer e. Magnetic stirrer f. Magnetic stirrer bars. 5 x 15 mm g. pH Meter. h. Stop-clock timer (digital) i. Spectrophotometer. - capable of operating at 510 nm, preferably fitted with flow-through cell (10 mm path length) j. Pipettor k. Corning Culture Tubes. - screw cap, 16 x 125 mm l. Plastic lunch box,large enough to hold test-tube rack and serve as an icewater bath Composition of reaction mixtures A. Sodium maleate buffer (0.1 M, pH 6.0). Dissolve 23.2 g maleic acid in 1600 mL of distilled water and adjust the pH to 6.0 with 4M (160 g/litre) sodium hydroxide. Add 0.6 g of calcium chloride (CaCl2.2H2O) and 0.4 g of sodium azide and adjust the volume to 2 L. Stable at 40C for 12 months.

B. Sodium acetate buffer (1.2 M, pH 3.8): Add 69.6 mL of glacial acetic acid to 800 mL of distilled water and adjust to pH 3.8 using 4 M sodium hydroxide. Adjust the volume to 1 litre with distilled water. Stable at room temperature for 12 months. C. Sodium acetate buffer (100 mM, pH 5.8): Add 5.8 mL of glacial acetic acid to 900 mL of distilled water and adjust to pH 4.5 using 4 M sodium hydroxide. Adjust the volume to 1 litre with distilled water. Stable at 4oC for 2 months. D. Potassium Hydroxide (2 M): Add 112.2 g KOH to 900 mL of deionised water and dissolve by stirring. Adjust volume to 1 litre. E. Stock Amyloglucosidase (AMG) solution (12 mL, 3300 U/mL in 50% glycerol): Solution is viscous; for dispensing use positive displacement dispenser. AMG solution is stable for up to 5 years when stored at 4oC. (Note: One unit [U] of enzyme activity is amount of enzyme required to release 1 micromole of glucose from soluble starch per minute at 40oC and pH 4.5). F. Pancreatic -amylase (10 mg/mL) plus AMG (3 U/Ml): Immediately before use, suspend 1 gram of pancreatic -amylase in 100 mL of sodium maleate buffer and stir for 5 min. Add 1.0 mL of AMG (300 U/mL) and mix well. Centrifuge at > 1,500 g for 10 min, and carefully decant the supernatant. This solution should be used on the day of preparation. G. Glucose oxidase-peroxidase-aminoantipyrine reagent (GOPOD): Prepare glucose oxidase-peroxidase-aminoantipyrine reagent (GOPOD) mixture by diluting 50 mL of buffer concentrate to 1.0 L. Use part of this diluted buffer to dissolve the entire contents of the vial containing freeze-dried glucose oxidase peroxidase-amino anti pyrine mixture. Quantitatively transfer the contents of the vial to 1 L volumetric flask containing diluted buffer. This mixture contains; glucose oxidase > 12000 U/L; peroxidase > 650 U/L; and 4-aminoantipyrine 0.4 mM. Reagent is stable 2-3 months when stored at 4oC and > 2 years when stored at -200C. 1) Hydrolysis and solubilisation of non-resistant starch: 1. A sample of 100 mg was weighed into a 50 mL centrifuge tube. 2. Add 4.0 ml of 1.0 M sodium maleate buffer (pH 6.0) containing pancreatic -amylase (10 mg/ml) containing AMG (3 unit/ml to each tube). 3. Tightly cap the tubes, mix them on a vortex mixer and attach them horizontally in a shaking water bath, aligned in the direction of motion. 4. Incubate tubes at 37C with continuous shaking (200 strokes/ min) for exactly 16 hr. 5. Remove the tubes from the water bath and remove excess surface water with paper towel. Remove the tube caps and treat the contents with 4.0 ml of ethanol (99%) with vigorous stirring on a vortex mixer. 6. Centrifuge the tubes at 1,500 g (approx. 3,000 rpm) for 10 min (non-capped).

7. Carefully decant supernatants and re-suspend the pellets in 2 ml of 50% ethanol with vigorous stirring on a vortex mixer. Add a further 6 ml of 50% ethanol, mix the tubes and centrifuge again at 1,500 g for 10 min. 8. Decant the supernatants and repeat this suspension and centrifugation step once more. 9. Carefully decant the supernatant and invert the tubes on absorbent paper to drain excess liquid. Measurement of resistant starch: 1. Add a magnetic stirrer bar (5 x 15 mm) and 2 ml of 2 M KOH to each tube and resuspended the pellets (and dissolve the RS) by stirring for approx. 20 min in an ice/water bath over a magnetic stirrer. 2. Add 8 ml of 1.2 M sodium acetate buffer (pH 3.8) to each tube with stirring on the magnetic stirrer. Immediately add 0.1 ml of AMG (3300 unit/ml), mix well and place the tubes in a water bath at 50C. 3. Incubate the tubes for 30 min with intermittent mixing on a vortex mixer. 4. For samples containing > 10% RS content; quantitatively transfer the contents of the tube to a 100 mL volumetric flask (using a water wash bottle). Use an external magnet to retain the stirred bar in the tube while washing the solution from the tube with the water wash bottle. Adjust to 100 mL with water and mix well. Centrifuge an aliquot of the solution at 1,500 g for 10 min. 5. For samples containing < 10% RS content; directly centrifuge the tubes at 1,500 g for 10 min (no dilution). For such samples, the final volume in the tube is approximately 10.3 ml. 6. Transfer 0.1 mL aliquots (in duplicate) of either the diluted or undiluted supernatants into glass test tubes, treat with 3.0 mL of GOPOD reagent and incubate at 50C for 20 min. 7. Measure the absorbance of each solution at 510 nm against the reagent blank. (Reagent blank solutions were prepared by mixing 0.1 mL of 0.1 M sodium acetate buffer (pH 4.5) and 3.0 mL of GOPOD reagent). 3) Measurement of non resistant (solubilised) starch: 1. Combine the supernatant solutions obtained on centrifugation of the initial incubation with the supernatants obtained from the subsequent two 50% ethanol washings and adjust the volume to 100 mL with water in a volumetric flask. 2. Incubate 0.1 mL aliquots of this solution (in duplicate) with 3.0 mL of GOPOD reagent for 20 min at 50C. 210 3. Measure the absorbance at 510 nm against a reagent blank. 4. Calculate the content of non-resistant (solubilised) starch.

4) Calculations: Resistant starch, non-resistant (solubilised) starch and total starch content (%, on a dry weight basis) in test samples as follows: a) Resistant starch (g/ 100g sample) (Samples containing > 10% RS): = E F 100/0.1 1/1000 100/W 162/180 = E F/W 90. b) Non-Resistant starch (g/ 100g sample) (Samples containing > 10% RS): = E F 100/0.1 1/1000 100/W 162/180 = E F/W 90. c) Total starch content = Resistant starch + Non-resistant starch. Where: E = absorbance (reaction) read against the sample analyzed reagent blank. F = conversion from absorbance to micrograms (the absorbance obtained for 100 g of glucose in the GOPOD reaction is determined and F= 100 (100 g of glucose) divided by the GOPOD absorbance for this 100 g of glucose. 100/0.1 = volume correction (0.1 mL taken from 100 mL). 1/1000 = conversion from micrograms to milligrams. W = dry weight of sample as is weight (100 moisture content)/100. 100/W = factor to present RS as a percentage of sample weight. 162/180 = factor to convert from free glucose, as determined, to anhydro-glucose as occurs in starch. 10.3/0.1 = volume correction (0.1 mL taken from 10.3 mL) for samples containing 0-10% RS where the incubation solution is not diluted and the final volume is 10.3 mL.

3.1.4. Physical characterization: Pasting properties: Pasting properties of the samples will be tested using a Rapid Visco-Analyzer (RVA).

A 4 g sample and 25 g distilled water will be placed in canister in RVA

The RVA pasting curve will be obtained by using a 30 min test profile

Initial equilibrium at 30C for 10 min, heating to 95C over 6 min, holding at 95C for 5 min

Cooling to 50C over 5 min, and holding at 50C for 4 min

The peak viscosity, breakdown viscosity, set back time and final viscosity values will be evaluated with the data analysis.

The Rapid Visco Analyzer (Model 4, Newport Scientific Pvt., Ltd., Australia) was used to gelatinization characteristics of banana flour. About 3.2g of accurately weighed banana flour was added in to about 25.2g of distilled water to prepare a suspension. The banana flour solution was kept at 50oC for 1 min and then heated up to 95oC and held for 3.2 min. It was then cooled to 50oC and a final isothermal step at 50oC. The RVA curve obtained was used to determine in peak viscosity, trough, breakdown viscosity, final viscosity and setback viscosity in Rapid Visco Units (RVU). Swelling power and Solubility pattern: The swelling power and solubility were determined according to the methods of Konik, Kiskelly, and Gras (1993), and Gudmundsson and Aliasson (1991) with some modification. Approximately 1 g starch (d, b) in weighed centrifuge tubes were taken and 50 ml distilled water added. The resultant slurry was heated at the desire temperature (65, 75, 85 and 95 0C) for 30 min in a water bath and thoroughly stirred with a glass rod all through the heating period. The tubes were removed, cooled to room temperature and centrifuged at 2000 rpm for 15 min (Model EBA 8S, Hettich, Germany). The supernatant was carefully sucked into a preweighed crucible and dried in the oven at 100 0C to constant weight. The weight of the pastes was determined and used to calculate the swelling power as weight of sediment paste per gram of dry starch. The difference in weight after drying the supernatant gave the weight of the soluble material. Percentage solubility was calculated as weight of soluble per weight of dry starch.

Solubility (%)= Swelling power (%)= Dry matter weight = Ws (1 MC) Where, W1, W2 = Weight of supernatant and centrifuged swollen granules; Ws = weight of sample; Mc = moisture content of sample, dry basis (decimal); and Wdm = weight of dry matter. %Solubility = (mass of flour in the supernatant / mass of dry flour in the aliquot) x100 Swelling power = mass of wet residue / mass of dry residue Determination of paste clarity: Paste clarity was determined by the method of Singhal and Kulkarni (1990) by measuring the percentage light transmitted by different concentrations of starch (0.54.0%) at 660 nm on a UV/visible spectrophotometer. Distilled water was used in the reference cell. Determination of freezethaw stability: The freezethaw stability was determined according to the method of Singhal and Kulkarni (1990). The determination was carried out by heating 5% (w/v) starch (d,b) in distilled water at 950C for 30 min with constant stirring. This was then removed and stirring continued during cooling to avoid formation of skin. About 10 ml of paste was transferred to weighed centrifuge tubes. The weight of the paste was then determined. This was subjected to alternate freezing and thawing cycles (18 h, 3 h, respectively) for 9 days, centrifuged at 5000 rpm for 10 min after each cycle and the percentage water separated determined as weight of exudates to the weight of paste. Water holding capacity (WHC): Twenty-five millilitres of distilled water were added to 1 g of dry sample, stirred and incubated at 40, 60 or 800C for 1 h. Tubes were centrifuged at 3000 x g for 20 min, the supernatant was decanted and the tubes were allowed to drain for 10 min at a 45 0 angle. The residue was weighed and WHC calculated as g water per g dry sample (Rodrguez-Ambriz, 2008). WHC% = Where, Wwet represents the weight of the wet specimen and W dry represents the weight of the dry specimen. Color Color of banana flour was measured using a Hunter Lab spectro-colorimeter (Model TC-P III A, Tokyo Denshoku Co., Ltd., Japan). The samples (about 10-20 grams) were prepared and put into a clean the petri dish. Petri dishes of sample were kept covered to prevent discoloration due to exposure. A CIELab system was used for determination of L*, a*, b* values of the samples. The parameters determined were L* (L* = 0 [black] and L* = 100

[white]), a* (a* = greenness and +a* = redness) and b* (b* = blueness and +b* = yellowness). 3.1.5. Statistical Analysis Data will be analysed for variance using the MSTAT-C statistical program. When significant differences will be found, the LSD (Least Significant Difference) test will be used to determine the differences among means.

Expt. 3.2. Formation of resistant starch from green banana and its peel starch by enzyme modification and heat treatment 3.2.1. Materials: Green banana starch and peel starch will be obtained from previous experiment. -amylase, pullulanase enzyme pullulanase (Promozyme, 400 PUN/ml) from Bacillus acidipullulyticus, amyloglucosidase, dinitrosalicylic acid, Rochelle salt solution (Potassium sodium tartrate), anhydrous glucose etc. 3.2.2. Enzymatic and heat treatment of starch to produce RS: A modified method of Gonzlez-Soto et al. (2004) will be used. Starch (green banana) suspension (20 %, w/v) will be gelatinized on a boiling water bath at 600 C for 15 min under stirring.

This gel will be autoclaved at 120 C & 1.1 bar pressure for 30 min then re-dissolved it by 50 ml distilled water to obtain a 10% (w/v) gel.

The gel will be cooled to 600C and PH will be adjusted to5 with a solution of 3:1 water/concentrated HCl. Pullulanase will be added (0, 4, 6, 8, and 10% on dry starch weight) and incubated for 24 h with constant stirring. To inactivate the enzyme, samples were heated at1000C for 10 min. Samples will be taken out for different concentrations and reducing sugars will be determined to optimize enzyme concentration for following study. The optimized sample will be autoclaved at 1210C for 30 min, cooled down and stored for 24 h at 40C.

Sample will be divided into 5 and 0%, 20%, 30%, 40% and 50% NaCl (by the weight of solids in solution) will be mixed and heated to 950C and held at that temperature for 24 hours. Cooled to 250C and added ethanol to bring a solution of 50:50 (ethanol: water) to precipitate out the starch product Products will be filtered, washed with 50: 50 (water: ethanol) twice and dried at 45 0C to approximately 13% moisture content and resistant starch will be determined.

3.2.3. Experimental Design: Factor A- amount of pullulanase enzyme 1. 0% of starch 2. 2% of starch 3. 4% of starch 4. 6% of starch 5. 8% of starch Factor B- Amount of NaCl 1. 0% 2. 10% 3. 30% 4. 50% Replication- 3 3.2.4. Chemical properties: Moisture content, total starch and resistant starch will be determined. Procedure is mentioned in the previous experiment. In this experiment, reducing sugar was also been determined. Estimation of reducing sugar by dinitrosalicylic acid method For sugar estmation an alternative to Nelson-Somogyi method is the dinitrosalicylic acid method simple, sensitive and adoptable during handling of a large number of samples at a time. Reagents: 1. Dinitrosalicylic Acid Reagent (DNS Reagent): Dissolve by stirring 1g dinitrosalicylic acid, 200mg crystalline phenol and 50mg sodium sulphite in 100mL 1% NaOH. Store at 4C. Since the reagent deteriorates due to sodium sulphite, if long storage is required, sodium sulphite may be added at the time of use. 2. 40% Rochelle salt solution (Potassium sodium tartrate): 3. Standard glucose solution: Stock: 100 mg in 100 mL distilled water 4. Working standard: 10 mL of stock diluted to 100 mL with distilled water [100 g/mL]. Procedure: 1. Weigh 100mg of the sample and extract the sugars with the hot 80% ethanol twice (5mL each time) 2. Collect the supernatant and evaporate it by keeping it on a water bath at 80C. 3. Add 10mL water and dissolve the sugars. 4. Pipette out 0.5 to 3mL of the extract in test tubes and equalize the volume to 3mL with water in all the tubes. 5. Add 3mL of DNS reagent. 6. Heat the contents in a boiling water bath for 5min. 7. When the contents of the tubes are still warm, add 1mL of 40% Rochelle salt solution. 8. Cool and read the intensity of dark red color at 510nm. 9. Run a series of standards using glucose (0 to 500mg) and plot a graph.

Calculation Calculate the amount of reducing sugars present in the sample using the standard graph 1. Determination of glucose concentration by DNS method: 1 ml glucose sample (2, 4, 6, 8, 10 mg/ml) + 1 ml DNS reagent Vortex Heat the mixture at 95 C for 5 min to develop red-brown color Cool down in a ice water bath Add 8 ml of water Vortex Measure OD at 575 nm Plot a standard graph between OD and sugar concentration 1 ml of stored sample + 1 ml DNS reagent Vortex Heat the mixture at 95 C for 5 min to develop red-brown color Cool down in a ice water bath Add 8 ml of water Vortex Measure OD at 575 nm Determine the glucose concentration by comparing the OD with the standard graph 3.2.5. Physical properties: Pasting properties (Peak viscosity, breakdown viscosity, set back time and final viscosity), swelling and solubility pattern, paste clarity, freeze thaw stability, water absorption capability and colour of different resistant starch samples were determined. Procedure is mentioned in the previous experiment.

3.2.7. Statistical Analysis: Data will be analysed for variance using the MSTAT-C statistical program. When significant differences will be found, the LSD (Least Significant Difference) test will be used to determine the differences among means. Expt. 3.3. Study on the effect of resistant starch from green banana and Hylon VII with SPI for encapsulation of fish oil Materials: Fish oil, whey protein isolate (WPI), soya protein isolate (SPI), high amyloseresistant starch, Hylon VII, will be purchased. Resistant starch product produced from green banana will be obtained from previous experiment. Experimental design: All emulsions will be stabilized by heated protein or protein-resistant starch (1:1) product mixtures containing WPI or SPI and resistant starch product produced from green banana (GBRS) or Hylon VII. Emulsions will be prepared with a constant total solid content of 30% and two different homogenization pressure (40+10) MPa and (20+10) MPa for the production of powders containing constant 50% w/w oil. The formulations of the emulsions are given in Table 1. All emulsions will be prepared in duplicate. Emulsion preparation: Protein powders will be initially dispersed in warm (6065C) distilled water using an overhead stirrer. Resistant starch will be added, and the pH of the resulting solution will be adjusted to pH 7.5 with a 1 M NaOH solution. The aqueous proteinstarch mixtures will be heated in at 100C for 30 min in a retort to promote formation of MRP (Millard Reaction Product). L* a* b* values will be measured using colorimeter. The heated aqueous phase will be cooled and stored overnight at 20C. The oil phase, as formulated in Table 1, will be heated to 65C in a water bath then disperse into the aqueous phase using a high shear mixer-emulsifier at maximum speed for 2 min. The pre-emulsion will be subjected to two stage homogenization (40+10) MPa and (20+10) MPa using a laboratory homogenizer. Spray drying of emulsions: The homogenized emulsions will be spray-dried at a 60C feed temperature, using Spray Dryer with a twin fluid nozzle at 2.0 bar atomizing pressure. Drying will be carried out in a concurrent mode, with inlet and outlet temperatures of 180 and 80C, respectively. Table 1. Formulations and Processing Conditions for the Preparation of Emulsions System Encapsulant Homogenization Emulsions materials Pressure Protein Starch Oil Water Total solid (Mpa) (%) (%) (%) (%) (%) 1 SPI 20+10 15 0 15 70 30 2 SPI- GBRS 20+10 7.5 7.5 15 70 30 3 SPI- Hylon VII 20+10 7.5 7.5 15 70 30 4 SPI 40+10 15 0 15 70 30 5 SPI- GBRS 40+10 7.5 7.5 15 70 30 6 SPI- Hylon VII 40+10 7.5 7.5 15 70 30 GBRS= Green banana resistant starch

Characterization of emulsion: Emulsion stability: The stability of the emulsion was determined by measuring the serum volume fraction [9]. Each emulsion was transferred to a 20 mL cylinder; these were then capped and stored for 12, 24, 36, 48, 60, and 72 h, at 4, 25, and 55C. The emulsions were monitored by visual observation of the height of the serum layer formed at the bottom of the cylinders. The emulsion stability index (ESI) was calculated using the following equation: Emulsion stability index (%) =


Optical microscopy: A drop of emulsion was placed on a microscope slide and then covered with a cover slip. The microstructure of the emulsion was then observed using a conventional optical microscope (FDX-35, Nikon, Japan) equipped with a camera [10]. Turbidity of Encapsulant Components in the Aqueous Phase: The turbidity of aqueous dispersions of protein (SPI, WPI), resistanr starch (GBRS, Hylon VII) and mixtures of these at the concentrations used for preparing the emulsions will be measured. This analysis was carried out using an UV Spectrophotometer in a 1 cm path length optical cell against Milli-Q water at 600 nm. Duplicate assessments will be carried out for each of the prepared duplicate solutions and reported as the mean and standard deviation of the quadruplicate data. Emulsion viscosity: The viscosities of homogenized emulsions will be determined at 25C by using a Brookfield viscometer. Characterization of microcapsules: Texture analysis: Texture of capsules will be analysed by using Lloyd Texture Analyzer. In this test compression load will be applied. A compression test determines behavior of materials under crushing loads. Scanning electron microscopy (SEM): Morphological characterization of the microcapsules will be carried out scanning electron microscopy. The samples will be coated to 200A thickness with gold- palladium using prior to microscopy. Differential scanning calorimetry (DSC): Samples will be analysed by differential scanning calorimeter (Mettler Toledo), which equipped with a liquid nitrogen cooling system. Samples [(5 0.2) mg] will be placed in an aluminum pan with a lid and an empty pan with a lid will be used as a reference. Samples will be heated from 25 to 150C at heating rate 10C/min. Nitrogen will be used as a cooling gas at rate 20 ml/min. DSC thermo grams are analysed to determine the glass transition temperature (Tg), heat capacity change (Cp), melting temperature (Tm), and melting enthalpy (H) using STARe Software.

Estimation of free fat (solvent extractable) in the microcapsules: The free fat in the microcapsules was estimated based on the method by Pisecky (1997) except that petroleum ether was used in place of carbon tetrachloride. Microcapsules (10.0 g) were agitated with petroleum ether (50 mL) for 15 min and the mixture was filtered and the solvent phase was evaporated at 600C. The remaining fat residue was dried at 100 20C for 1 h. The oil obtained was expressed as % (w/w) of microcapsules. Estimation of total fat of the microcapsules: The total fat of the microcapsules was estimated following the SchmidBondzyndkiRatzlaff method (AS 2300.1.3, 1988). The microcapsules (0.5 g) were digested with 36% w/w hydrochloric acid in a Mojonnier tube at 1000C for 10 min. Ethanol (96%, w/v) was added after cooling the sample to room temperature. Solvent extraction of this sample was carried out with petroleum ether and diethyl ether. The extracted solvent was removed using a rotary evaporator and the residue was oven-dried at 10020C for 1 h before weighing. The amount of oil obtained was expressed as the % (w/w) of microcapsules. Microencapsulation Efficiency (MEE): The encapsulation efficiency (EE) and Loading capacity (LC) were calculated from the following equations: EE (%) = LC (%) = Encapsulated oil = Total oil Surface oil

Storage of powder: Fifteen grams of powder will be placed in 125-mL loosely capped brown glass bottle. The samples will be stored at 23C for 4 wk in the dark place. The extent of lipid oxidation in the powder will be monitored by measuring Peroxide Value (PV) and headspace propanal. Peroxide Value (PV): The spectrophotometric method described by Shantha and Decker (1994) will be used with some modifications. The powder will be dissolved in water to obtain an oil-in-water emulsion (5% w/w oil), and the emulsion will be vortexed for 2.5 min at 2,200 rpm and allowed to settle at room temperature (~23C) for 1 h. Then 300 L of this mixture will be mixed with 1.5 mL of isooctane/2-propanol (3:1, vol/vol), and the mixture will be vortexed for 30 s and centrifuged for 2 min at 2,000 g. Next, 200 L of the organic phase was added to 2.8 mL methanol/1-butanol (2:1, vol/vol) mixture, followed by 15 L each of ammonium thiocyanate and Fe2+ solutions. The absorbance of the sample at 500 nm will be determined after 5 min incubation at room temperature (~23C).

3.4. Oxidative stability of encapsulated fish oil in fortified candy bar

Materials: Fully ripen orange, most heat stable beads with probiotics from previous experiments, MRS agar, etc. Preparation of candy with encapsulated fish oil: Sugar will be put in a smooth, clean saucepan with boiling water in 4:1 ratio. Mixture will be heated slowly with stirring until dissolving sugar. It will be boiled without stirring to 240 F or until syrup will form a soft ball when tested in cold water. At the last stage of boiling 30% roasted peanut and 10% microcapsules (encapsulated fish oil) of sugar will be added and stir for homogenous mixing. Mixtures will be shifted into slightly oiled slab or tray. After partial cooling, it will be cut into desired sizes and let them stand few hours for cooling fully and packed in HDP by vacuum sealing. It will be stored at room temperature and oxidative stability will be determined periodically.