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					APPENDIX A PHYSICAL EXAMINATION PROCEDURES

A.1 A.2

Determination of Diameter and Thickness Determination of Weight

APPENDIX A.1 Determination of Diameter and Thickness

A. Measurement 1. Using a Vernier Caliper, measure the diameter and thickness of the product. 2. For thickness, get four measurements at different point or sections of the product and get the average.

B. Reading Measurementsa 1. A measurement is obtained by adding the reading of the vernier scale to that of the main scale. 2. Take the vernier scale reading at the graduation which coincides with one on the main scale.

A: Main Scale Reading B: Vernier Scale Reading C: Reading (= A+B)

a

Mitutoyo Vernier Caliper Instructional Manual. Japan: Mitutoyo Corporation.

APPENDIX A.2 Determination of Weighta

1. Turn on the balance/scale. Self-test performed. 2. Place container on balance scale. 3. Tare the balance/scale. 4. Place sample in container on balance/scale. 5. Record the reading.

a

Acculab. 2003. Operating Instructions ALC Models Electronic Analytical Balances and Precision Scales. Germany: Acculab.

APPENDIX B PROXIMATE ANALYSES PROCEDURE

B.1. B.2. B.3. B.4. B.5. B.6.

Determination of Moisture Content Determination of Crude Protein Content Determination of Ash Content Determination of Fat Content Determination of Crude Fiber Determination of Carbohydrate Content

APPENDIX B.1 Determination of Moisture Contenta

1. Place the disposable sample dish on the dish retainer and press the ENTER key. TAR will appear on the screen followed by the weight readout 0.000 g. 2. Place the sample in the disposable dish, making sure that it is evenly spread. 3. Lower the hood. 4. END will displayed after moisture content determination is finished. 5. Read the percentage moisture content value flashed on the screen.

a

Scaltec Moisture Analyzer SMO 01 Instruction Manual. (2000). Scaltec Instruments GmbH.

APPENDIX B.2 Determination of Crude Protein by Kjeldahl Methoda

A. Apparatus Kjeldahl Flask Kjeldahl Digestion Unit Graduated Cylinder Burette Analytical Balance B. Reagents Concentrated Sulfuric Acid Catalysts (selenium mixture of copper sulfate and potassium sulfate {1:9}) 45% Sodium Hydroxide Solution or 5% Sodium Thiosulfate Solution 4% Boric Acid Solution Standard 0.1 N HCl Indicator (methyl red-bromcresol green solution) C. Procedure I. Digestion 1. Weigh accurately the sample based on the expected protein level. Place the weighed sample into the Kjeldahl flask. 2. Add boiling chips, 5.5 g of selenium mixture and 20 ml concentrated sulfuric acid to the sample in the Kjeldahl flask. 3. Digest until clear solution will be observed from the sample using the digestion apparatus in the Laboratory. II. Distillationb 1. After digestion, cool the flask. 2. Add cautiously 100 ml of distilled water. 3. Transfer the diluted digest into the distilling flask. Kjeldahl Distillation Unit Pipette Erlenmeyer Flask Beaker

a

Madamba, L. S. P. (1993). Laboratory Manual for Technical Analysis: Foods and Feeds. Laguna: UP Los Baños. b Note: In actual analysis, automatic distillation unit was used.

4. Rinse the Kjeldahl flask twice with 50 ml quantities of distilled water and combine washing with the diluted digest. 5. Set up distillation assembly. 6. To the receiving flask, add 50 ml of boric acid with methyl red-bromcresol green indicator. 7. Place the receiving flask at the end of the condenser with the tip of the adaptor dipping below the boric acid solution. 8. Make the diluted digest of alkaline by adding 80-85 ml of 45% NaOH taking care to cool down the mixture by placing the flask in cooling water. Place a long stem funnel to prevent the NaOH from running out of the side arm of the distilling flask. 9. After the alkali addition, immediately stopper the distilling flask and connect to the distilling set-up. 10. Distill the ammonia into the boric acid solution. Do not over boil to prevent the violent bumping in the flask. 11. Collect approximately 150 ml of the distillate. 12. Stop the flame and connect the condenser and adaptor. Wash them down with distilled water into the receiving flask. III. Titration 1. Titrate the distillate with 0.1 N HCl. Perform the procedure on a blank. 2. Calculate the % Nitrogen in the sample by the following formula:

% Nitrogen =

(VHCl for the sample - VHCl for the sample) x N acid x meq Nitrogen Weight of the sample

X 100

3. Calculate the % Protein in the sample by the following formula:

% Protein = % Nitrogen x Protein factor

APPENDIX B.3 Determination of Ash by Dry Ashing Methoda

A. Apparatus Muffle Furnace Crucible and lids Thong Tirrill Burner Iron Ring Air Oven B. Procedure 1. Clean crucible and lid with soap and water. Dry in an oven. 2. Place the crucible and the lid in the furnace at 550°C overnight to ensure that impurities on the surface of the crucible are burned off. Cool the crucible in the dessicator for about 30 minutes. 3. Weigh the crucible and the lid to four decimal places. 4. Weigh about 5 grams of sample into the crucible. Heat over low Bunsen flame with the lid half covered. When fumes are no longer produced, place the crucible and the lid in the furnace. 5. Heat at 550°C overnight. During heating do not cover with the lid. Place the lid on after complete heating to prevent loss of fluffy ash. Cool down in the dessicator. 6. Weigh the ash with crucible and lid to four decimal places. 7. Ash must be white or light grey. If not, return the crucible and lid to the furnace for further ashing. Gloves Dessicator Analytical Balance Iron Stand Clay Triangle

a

Madamba, L. S. P. (1993). Laboratory Manual for Technical Analysis: Foods and Feeds. Laguna: UP Los Baños.

APPENDIX B.4 Determination of Fat Content by Soxhlet Methoda

A. Apparatus Soxhlet Flask Analytical Balance Thong Extraction Apparatus B. Reagent Petroleum Ether C. Procedure 1. Wash, dry and weigh the Soxhlet flask. Fill with 150 ml solvent. 2. Weigh about 1-2 g of the sample (previously oven-dried) in the thimble or filter paper. 3. Place the dried sample in the extractor and extract for 16 hours. 4. After the extraction has been completed, recover the solvent and remove the sample from the extractor. 5. Place the flask containing the fat in a hot plate (50°C) for 5 minutes or until the solvent has been removed. 6. Cool and weigh to constant weight. Air Oven Dessicator Thimble Hot Plate

a

Madamba, L. S. P. (1993). Laboratory Manual for Technical Analysis: Foods and Feeds. Laguna: UP Los Baños.

APPENDIX B.5 Determination of Crude Fiber by Crude Fiber Methoda

A. Apparatus Hot Plate Crucible (as weighing bottle) Graduated Cylinder Analytical Balance Rubber Policeman Iron Stand Red and Blue Litmus Paper Muffle Furnace B. Reagents 1.25% Sulfuric Acid Solution 2.5% Sodium Hydroxide Solution C. Procedure 1. Boil 1 liter of distilled water. Keep hot. 2. Flute no. 54 filter paper. Place in a weighing bottle and dry at 105°C for 1 hour. Determine the weight of the filter paper. 3. Weigh 2g (defatted or residue from fat extraction) add 200 ml of 1.25% H2SO4 in a 500ml beaker. Stir to break any lumps. The glass rod should be tipped with a rubber policeman. 4. Cover the beaker with watch glass and boil for 30 minutes. Make up any lost in volume during boiling with distilled water. 5. Filter the hot solution through the Whatman No. 41 filter paper, washing the residue well with distilled water until the washings are no longer acidic to litmus paper. 6. Wash the residue back in the beaker with the total of 100 ml hot distilled water. Add 100 ml of 2.5% NaOH solution. This procedure can be carried out using beakers which have been previously marked to indicate the volume. Boil for 30 minutes making up any loss in volume with distilled water.
a

Beaker Whatman No. 41 Filter Paper Oven Drier Stirring Rod Watch Glass Iron Ring Clay Triangle Tirrill Burner

Madamba, L. S. P. (1993). Laboratory Manual for Technical Analysis: Foods and Feeds. Laguna: UP Los Baños.

7. Filter through the weighed filter paper. Wash any residue from the side of the beaker (aiding the removal with the policemen tipped string rod), using hot distilled water, into the filter paper. Wash the hot distilled water until the washings are no longer alkaline to litmus paper. 8. Allow to drain and transfer the weighing bottle. Dry at 105°C for 3 hours and weigh. Redry for 15 minutes and weigh to a constant weight. 9. Incinerate (ignite) the paper and contents to an ash at a dull red heat. 10. Subtract the weight of the ash from the increase in weight on the filter paper due to the insoluble material and report the differences as the weight of the crude fiber.

APPENDIX B.6 Determination of Carbohydrate Content by Difference Methoda

Obtain the available carbohydrate content by calculation having estimated all the other fractions by proximate analysis:

% Available Carbohydrates = 100 – (%Moisture + %Ash + %Fat + %Protein + %Fiber)

a

Nielsen, S. S. (1994). Introduction to the Chemical Analysis of Foods. England: Jones and Barlett, Inc.

APPENDIX C PHYSICO-CHEMICAL ANALYSES PROCEDURE

C.1. C.2. C.3. C.4.

Determination of Water Activity Determination of Moisture Content Determination of pH Determination of Peroxide Value

APPENDIX C.1 Determination of Water Activitya

1. Place the sample inside the disposable sample cup with the cover on. 2. Place the cup(s) in the same general area as the probe. 3. Allow for sufficient time for the samples to come to the temperature of the probe. 4. Turn on the power and set to AwQuick mode. 5. Place the disposable sample cup without the cover in the sample holder. 6. Seal the container by placing the probe on top of the sample holder. 7. Press on the ENTER key on the keypad of the HygroPalm AW1. 8. Wait for five (5) minutes until the projected value of Aw stabilizes. 9. Read and record data. 10. Press ENTER key again to exit.

a

HygroPalm AW1 Portable Water Activity Indicator Instruction Manual, vol. 3.

APPENDIX C.2 Determination of Moisture Contenta

1. Place the disposable sample dish on the dish retainer and press the ENTER key. TAR will appear on the screen followed by the weight readout 0.000 g. 2. Place the sample in the disposable dish, making sure that it is evenly spread. 3. Lower the hood. 4. END will displayed after moisture content determination is finished. 5. Read the percentage moisture content value flashed on the screen.

a

Scaltec Moisture Analyzer SMO 01 Instruction Manual. (2000). Scaltec Instruments GmbH.

APPENDIX C.3 Determination of pHa

A. Determination 1. Weigh five (5) grams of the sample. 2. Place the sample in a blender. 3. Add 50 ml distilled water and homogenize. 4. Transfer to 250-ml beaker and immerse the pH meter electrode. 5. Read the pH value directly from the pH meter.

A. Using the Oakton pH 5/6 pH Meterb

1. Rinse the electrode and temperature probe with distilled water. 2. Power on the meter using the ON key. 3. Press MODE key to select your desire mod of operation (pH, mV, ion, or temperature). 4. Dip and stir both probes gently into the aqueous test samples. 5. Swirl gently and wait for the reading to stabilize. 6. Note the reading. 7. Rinse probes with distilled water thoroughly before taking next sample measurement.

a b

Gould, W. (1979). Food Quality Assurance. Connecticut, USA: AVI Publishing Co., Inc. pH 5/6 Instruction Manual. (1999). Eutech Instruments Pte Ltd/Oakton Instruments.

APPENDIX C.4 Determination of Peroxide Value

A. Reagents Acetic Acid – Chloroform Solution: Mix 3 volumes acetic acid with 1 volume chloroform Saturated Potassium Iodide Solution: Dissolve excess potassium iodide in freshly boiled water. Excess solid must remain. Store in the dark. Test daily by adding 0.5 ml to 30 ml HOAc-CHCl3, then add two drops 1% starch solution. If solution turns blue, requiring > 1 drop 0.1 N Na2S2O3 to discharge color, prepare fresh solution. 0.1 N Sodium Thiosulfate (Na2S2O3) Solution 1% Starch Soultion B. Sample Preparation 1. Take out 10 g sample and homogenize for about two (2) minutes with 45 ml chloroform. 2. Filter through a Buchner funnel with Whatman No. 41 filter paper with suction. 3. Mix thoroughly fat solution. 4. Evaporate solvent in a water bath and determine the weight of the residual fat. C. Procedure 1. Weigh 5.00 ± 0.05 g of fat sample into 250 ml Erlenmeyer flask. 2. Add 30 ml HOAc-CHCl3 and swirl to dissolve. 3. Add 0.5 ml saturated KI solution from Mohr pipette, let stand with occasional shaking for 1 minute, and add 30 ml water. 4. Slowly titrate with 0.1 N Na2S2O3 with vigorous shaking until yellow is gone. Add about 0.5 ml 1% starch solution and continue titration, shaking vigorously to release all iodine from CHI3 layer, until blue just disappears. If < 0.5 ml 0.1 N Na2S2O3 is used, repeat determination with 0.01 N Na2S2O3. 5. Conduct blank determination (must not be > 0.1 ml 0.1 N Na2S2O3). Subtract from sample titration.

6. Calculate the peroxide value using the following formula: Peroxide Value (meq/kg sample) = S x N x 1000 Weight of the Sample (g)

APPENDIX D MICROBIOLOGICAL ANALYSES PROCEDURE

D.1 D.2

Yeast and Mold Count Cultivation and Morphological Examination of Fungi

APPENDIX D.1 Yeast and Mold Counta

A. Preparation of Acidified Potato Dextrose Agar (PDA) 1. Dissolve 39 g of PDA in 1 liter distilled water using the hot plate with stirrer. 2. Sterilize at 121°C under 15 psi for 15 minutes. 3. Before using, melt the agar and acidify with 10% sterilized tartaric acid to pH 3.5 ± 1 (approximately 3 ml per 100 ml PDA). Do not reheat to preserve the solidifying capacity of the medium. B. Quantitative Plating Procedure 1. Using separate sterile pipettes, prepare decimal dilutions of 10-2, 10-3, 10-4, and appropriate other dilutions of food homogenate by transferring 1 mL of the previous dilution to 9 mL diluent. 2. Shake all dilutions 25 times in one-foot arc (30 cm) within seven seconds. 3. Pipette 1 mL of each dilution into separate appropriately marked Petri plates. If the dilution stands more than three minutes, re-shake the dilution bottle 25 times in one-foot arc (30 cm) within seven seconds. 4. Add 20 to 25 ml acidified PDA (cooled to 44 to 46°C) to each plate within 15 minutes of the time of the original dilution. 5. Immediately mix the sample dilutions and agar medium thoroughly and uniformly by alternate rotation and back-and-forth motion of the plates on a flat level surface. 6. Let the agar solidify and incubate promptly for 3 to 5 days at 30°C. Do not stack plates higher than 3 and do not invert. 7. After incubation, count triplicate plates in the suitable range (25 to 250 colonies) and reports the results as yeast and mold count per gram sample.

Speck, M. L. (Ed.). (1984). Compendium of Methods for the Microbiological Examination of Foods, 2nd ed. Washington: American Public Health Association.

a

APPENDIX D.2 Cultivation and Morphological Examination of Fungia

A. Cultivation of Fungi 1. Aseptically weight 25 g sample. 2. Add into 225 ml sterile, distilled water and homogenize. 3. Perform serial dilution up to 10-3. 4. Inoculate 1 ml of each dilution in a sterile Petri plate. 5. Aseptically pour 15 ml of the acidified PDA. 6. Incubate at room temperature until growth is visible. B. Isolation of Fungal Growth 1. In a sterile Petri plate, pour 15 ml of melted acidified PDA and allow it to solidify. 2. Cut out the hardened agar into 1 cm blocks. 3. Transfer a block of agar to a sterile slide kept inside a sterile Petri plate and positioned on top of a sterile U-shaped bent glass. 4. Inoculate the centers of both slides of the agar block with the fungus from the culture. 5. Cover the block with sterile cover glass and place about 8 ml of sterile water in the bottom of the Petri plate. 6. Cover and incubate at room temperature until sporulation occurs. 7. When sporulation occurs, remove the cover glass from the agar block and apply a drop of 95% ethanol to its center. 8. Add a drop of lactophenol blue mounting fluid when nearly dry and lower the cover glass onto a separate clean slide.

a

Raymundo, A. K., Zamora, A., and Dalmacio, I. F. (1991). Manual on Microbiological Techniques. UPLB: Technology and Livelihood Resource Center.

9. Lift the agar block from the slide and discard. 10. Place a drop of lactophenol blue on the slide and cover with a clean cover glass. 11. Blot away excess mounting fluid from both preparations and air-dry. 12. Seal the edges of the preparations with fingernail polish. C. Microscopic Examination 1. Examine the prepared slides under the microscope. 2. Describe the mycelium, asexual spores, and fruiting heads. 3. Note the presence of special structures (stolons, rhizoids, foot cell, columnella, sclerotica, chlamydospores, etc.) and their locations.

APPENDIX E SENSORY EVALUATION SCORE SHEETS AND RATING KEY

E.1. E.2. E.3.

Scoresheet for Triangle Test Scoresheet for the General Acceptability Test for Stored Samples Scoresheet for Attribute Rating Test

APPENDIX E.1 Scoresheet for Triangle Test

Name: _________________________________

Date: ______________ Panelist No: ________

Instructions: You are presented with three coded samples of flat piaya. Two of these samples are identical while the third is odd or different. Smell and taste each sample. Indicate the code of the odd sample by placing an X mark across the code. If no difference is apparent, you might guess. Please write down any comments. Sample Code Odd Sample

Comments: ______________________________________________________________ ________________________________________________________________________ ________________________________________________________________________

Thank You Very Much!

APPENDIX E.2 Scoresheet for General Acceptability Test for Stored Samples

Name: _________________________________________ Batch No.: ___ Panelist No.: _________ Day: ___ Date: _______________ Instructions: You are given three (3) coded samples of flat piaya. Please evaluate the acceptability of the samples by checking the corresponding space of your choice.

_____ 1. Can you see any manifestation of microbial growth? Yes _____ No _____

Sample Codes _____

_____

_____ _____

_____ _____

If yes, you can stop here and please give your comments. _______________________ ______________________________________________________________________ If no, please proceed to No. 2. 2. Please smell the product. Can you detect evidence of spoiled odor? Yes _____ _____ No _____ _____

_____ _____

If yes, you can stop here and please give your comments. _______________________ ______________________________________________________________________ If no, please proceed to No. 3. 3. Please assess the general acceptability of the samples Like extremely _____ Like very much _____ Like moderately _____ Like slightly _____ Neither like nor dislike _____ Dislike slightly _____ Dislike moderately _____ Dislike very much _____ Dislike extremely _____

_____ _____ _____ _____ _____ _____ _____ _____ _____

_____ _____ _____ _____ _____ _____ _____ _____ _____

Thank You Very Much!

APPENDIX E.3 Scoresheet for Attribute Rating Test

Name: _________________________________________ Batch No.: ___ Panelist No.: _________ Day: ___ Date: _______________ Instructions: You are given three (3) coded samples of flat piaya. Please evaluate the samples based on the given sensory attributes by checking the corresponding space of your choice.

_____ 1. PIAYA ODOR Strong piaya odor Moderate piaya odor Slight piaya odor Perceptible piaya odor Absence of piaya odor 2. OFF-ODOR Strong off-odor Moderate off-odor Slight off-odor Perceptible off-odor Absence of off-odor 3. PIAYA FLAVOR Strong piaya flavor Moderate piaya flavor Slight piaya flavor Perceptible piaya flavor Absence of piaya flavor 4. OFF-FLAVOR Strong off-flavor Moderate off-flavor Slight off-flavor Perceptible off-flavor Absence of off-flavor _____ _____ _____ _____ _____

Sample Codes _____ _____ _____ _____ _____ _____

_____ _____ _____ _____ _____ _____

_____ _____ _____ _____ _____

_____ _____ _____ _____ _____

_____ _____ _____ _____ _____

_____ _____ _____ _____ _____

_____ _____ _____ _____ _____

_____ _____ _____ _____ _____

_____ _____ _____ _____ _____

_____ _____ _____ _____ _____

_____ _____ _____ _____ _____

Thank You Very Much!


				
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