Food Analysis - Laboratory Manual

VIETNAM NATIONAL UNIVERSITY, HCMC INTERNATIONAL UNIVERSITY FOOD ANALYSIS LAB MANUAL Instructor: Assoc Prof Pham Van Hung Teaching Assistant: BEng Nguyen Dang Tam CONTENTS Session Determination of Moisture contents Session Determination of Ash contents Session Determination of Lipid Content Session Determination of Protein Content in Food Session Determination of Total Carbohydrate in foods Session 11 Determination of Crude Dietary Fibre in Foods 11 Session 13 Determination of Total Flavonoid Content (TFC) 13 Session 14 Determination of Vitamin C in foods 14 Session1 Determination of Moisture contents I Objective Determine the moisture content (using moisture balance analyzer versus forced draft oven versus) of rice flour and liquid milk II Materials • Rice flour, 50 g • Fat-free liquid milk, 50 ml • Volumetric pipette, 10 ml • Plastic gloves • Tong • Spoon • Aluminium pan • Tray • Desiccators • Analytical balance • Moisture balance analyzer • Forced draft oven III Method A Moisture content determination a1 Using moisture balance analyzer *Note: test rice flour and liquid milk separately Follow operating instructions from manufacturer • Milk: pipette 5mL into the aluminium disk • Rice flour: weigh g • Turning on the power switch and waiting for instrument warming up • Specifying settings o Automatic operation mode for both rice flour and liquid milk • Placing an aluminium pan in the tester • Tarring the tester • Placing test material on the pan (record the weight) • Start the measurement • Obtain results a2 Using forced draft oven • • Milk: o Label and weigh the crucible o Pipette 10mL into a crucible o Evaporate majority of water on a hot stirring plate o Place the crucible containing samples in a forced draft oven at 130oC o Weigh the crucible after each 30 until no change in weight observed Rice flour: o Label and weigh the crucible o Weigh g of sample o Place samples in the crucibles o Place crucibles containing samples in a forced draft oven at 130oC o Weigh the crucible after each 30 until no change in weight observed • Store samples in a desiccator until samples are weighed • Weigh IV Questions What is the difference between moisture content and water activity measurements? (20%) Why the milk sample was partially evaporated on a hot plate before being dried in the hot oven? (20%) Compare the rice flour and liquid milk moisture content results obtained by moisture balance analyzer and forced draft oven methods Explain any differences (40%) Method Moisture balance analyser Forced draft oven % Moisture Rice flour Liquid milk 1 2 3 𝑿= 𝑿= SD = SD = 1 2 3 𝑿= 𝑿= SD = SD = What are advantages and disadvantages of each method? (10%) What we need to pay attention during sample preparation in order to limit errors? (10%) References AOAC International (2007) Official methods of analysis, 18thedn, 2005; Current through revision 2, 2007 (On-line).AOAC International, Gaithersburg, MD Bradley RL Jr (2010) Moisture and total solids analysis, Ch.6.In: Nielsen SS (ed) Food Analysis, 4thedn Springer, New York Nielsen SS (2010) Determination of moisture content, Ch.3.In: Nielsen SS Food analysis laboratory manual, 2ndedn Springer, New York Session Determination of Ash contents I Objective Determine the ash content of rice flour and liquid milk using muffle furnace II Materials • Rice flour, 50 g • Fat-free liquid milk, 50 ml • Volumetric pipette, 10 ml • Plastic gloves • Tong • Spoon • Aluminium pan • Tray • Crucible (preheated at 550oC for 24 h) • Desiccators (with dried desiccant) • Analytical balance • Muffle furnace III Method A Moisture content determination • Milk: • o Label and weigh the crucible o Pipette 10mL into a crucible o Evaporate majority of water on a hot stirring plate o Place the crucible containing samples in a forced draft oven at 130oC o Weigh the crucible after each 30 until no change in weight observed Rice flour: o Label and weigh the crucible o Weigh g of sample o Place samples in the crucibles o Place crucibles containing samples in a forced draft oven at 130oC o Weigh the crucible after each 30 until no change in weight observed • Store samples in a desiccators until samples are weighed • Weigh B Ash content determination • Re-weigh crucible containing dried sample • Primary burning the content of materials by flame in a fume hood • Place all the crucibles in muffle furnace at 550oC for 3h until only white matters can be seen • Cool crucibles in a desiccator • Weigh IV Questions What is ash? What does ash consist of? Give examples to illustrate your answer (30%) How many types of ashing procedures? What are they? (30%) Calculate percentage ash (wt/wt) as shown on the table below Refer your findings to the ones in literature (20%) Sample Rep Crucible Crucible + Wet Crucible + Ashed (g) sample (g) sample (g) Rice flour Liquid milk % Ash 𝑿 = SD = 𝑿 = SD = Why the samples need to be placed in desiccators, but not outside, before weighed? (10%) What we need to pay attention during performing this experiment in term of lab safety? (10%) References AACC International (2010) Approved methods of analysis, 11thedn (On-line) AACC International, St Paul, MN AOAC International (2007) Official methods of analysis, 18thedn, 2005; Current through revision 2, 2007 (On-line).AOAC International, Gaithersburg, MD Bradley RL Jr (2010) Moisture and total solids analysis, Ch.6.In: Nielsen SS (ed) Food Analysis, 4thedn Springer, New York Nielsen SS (2010) Determination of moisture content, Ch.3.In: Nielsen SS Food analysis laboratory manual, 2ndedn Springer, New York Wehr HM, Frank JF (eds) (2004) Standard methods for the examination of dairy products, 16 thedn American Public Health Association, Washington Session Determination of Lipid Content I Objective Determine the lipid content of snack foods and rice bran by the Soxhlet method II Materials • Rice bran, 30g • Snack food, 30 g • Hexane • Aluminium weighing pan, pre-dried in 70oC oven over night • Filter paper, pre-dried in 70oC oven over night • Plastic gloves • Spatula • Tape • Tongs • Beaker, 250 ml • Graduated cylinder, 500 ml • Desiccators • Mortar and pestle • Pumice • Analytical balance • Soxhlet extractor, with glassware • Forced draft oven III Method • Record the fat content and serving size of snack food product as shown on the package label The fat content on the label Name of Snack Food Label g fat / serving Label serving size (g) Label g fat / 100 g product • Grind approx 10 g sample with mortar and pestle • Correct moisture content: o Determine moisture content of g sample using moisture balance analyser • Weigh filter paper • Prepare samples: place g of snack or rice bran in the pre-weighed filter paper Tightly wrap it up and reweigh • Prepare control • Place the wrapped filter paper in a Soxhlet extractor Put 300 ml hexane in the flask Add some – pumices Extract for h • Remove wrapped filter paper from the Soxhlet extractor using tongs, air dry overnight in a hood, and thenopen filter paper to expose the content; dry in a drying oven at 70 oC for 24 h • Cool dried samples in a desiccator then reweigh IV Questions Calculate % moisture and % fat and report on the tables below (25%) Data from Soxhlet extraction Dried sample Sample Snack Rice bran Filter Wet Wet sample + + Filter paper paper (g) sample (g) Filter paper (g) (g) 1 1 1 2 2 2 % moisture % fat 𝑿 = 𝑿 = 𝑿 = 𝑿 = 𝑿 = 𝑿 = SD = SD = SD = SD = SD = SD = 1 1 1 2 2 2 𝑿 = 𝑿 = 𝑿 = 𝑿 = 𝑿 = 𝑿 = SD = SD = SD = SD = SD = SD = Compare your findings to fat content reported on the nutrition label Explain any similarities and/or differences between the two (30%) What were the advantages of using the Soxhlet extraction method? Compare Soxhlet extraction method with another lipid extraction method you know to illustrate your answer (30%) What we need to pay attention in this experiment in term of lab safety? (10%) How to treat the organic waste? (5%) References AOAD International (2007) Official Methods of Analysis, 18thedn, 2005 ; Current through Revision 2, 2007 (On-line) AOAC International, Gaithersburg, MD Min DB, Ellefson WC (2010) Fat analysis.Ch.8.In : Nielsen SS (ed) Food analysis, 4thedn Springer, New York Nielsen SS (2010) Determination of fat content, Ch.4.In: Nielsen SS, Food analysis laboratory manual, 2ndedn Springer, New York Wehr HM, Frank JF (eds) (2004) Standard methods for the examination of dairy products 17thedn American Public Health Administration, Washington, DC Session Determination of Protein Content in Food I Objective Determine the protein content of rice flour using the Kjeldahl method II Materials • Rice flour, 30 g • NaOH 32% (wt/v) • H3BO3 4% (wt/v) • Tashiro indicator • H2SO4, 0.1 N • H2SO4, concentrated • CuSO4 • K2SO4 • Digestion tubes • Erlenmeyer flasks, 500 ml • Spatula • Weighing paper • Analytical balance • Burette with ring stand • Kjeldahl digestion and distillation system III Method Digestion *Note: Digestion takes place in a hood with fan on • Turn on digestion block and heat up to 80% energy (about 370oC) • To prepare sample o Weigh approx g rice flour Place rice flour in a digestion tube o Add 0.2 g CuSO4, g K2SO4, and 20 ml concentrated H2SO4to each tube with rice flour • To prepare blank: o Add 0.2 g CuSO4, g K2SO4, and 20 ml concentrated H2SO4to a tube with weighing paper • Place rack of digestion tubes on digestion block • Let samples digest until white fumes can be seen Continue heating for about 60 – 90 minutes The samples should be clear with no charred material remaining • Take samples off the digestion block and hang them on hanging rack and allow to cool • Dilute digest with 50 ml of distilled water Swirl each tube Distillation • Start-up distillation system follows instruction manual o Rinse the distillation block throughout using distilled water automatically pumped by the system (Program 0) • Add 50 ml H3BO3 4% into the 500 ml receiving flask • Insert the digestion tube and receiving flask in to distillation block • Start measurement (Program 1) o Automatically pump 50 ml NaOHto digestion tube o Heat the digestion sample to boiling point o Distilling sample for minutes Titration • Using a colorimetric endpoint: o Fill 25 ml burette with H2SO4 0.1 N solution; record the initial volume o Assemble burette into ring stand o Place the receiving flask after distillation under the burette o Titrate each sample and blank with the H2SO4 0.1 N solution to the colour before distillation process Record volume of H2SO4 0.1 N solution used IV Questions Calculate the percent nitrogen and the percent protein (wet weight basis (wwb) and dry weight basis (dwb)) (20%) Assume moisture content of 10% Use 6.25 for the nitrogen to protein conversion factor % 𝑁 = 𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 𝐻2𝑆𝑂4 × 𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑎𝑐𝑖𝑑 𝑣𝑜𝑙 𝐿 𝑔 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 × 14 𝑔 𝑁 × 100 𝑚𝑜𝑙 % Protein = % N X Protein Factor Could phenolphthalein be used as an indicator in the Kjeldahl titration? Why or why not? (15%) Describe the function of the following chemicals used in this determination (20%) (a) Catalyst pellet (b) Borate (c) H2SO4 (d) NaOH Why was it not necessary to standardize the boric acid solution? (20%) For each of the disadvantages of the Kjeldahl method, give another protein analysis method that overcomes that disadvantage (15%) What we need to pay attention in this experiment in term of lab safety? (10%) References Chang SKC (2010) Protein analysis Ch In : Nielsen SS (ed) Food analysis, thedn Springer, New York AOAC International (2007) Official methods of analysis, 18thedn, 2005 ; Current through revision 2, 2007 (On-line) Method 960 :52 (Micro-Kjeldahl method) and Method 992.23 (Genetic combustion method) AOAC International, Gaithersburg, MD Nielsen SS (2010) Protein nitrogen determination, Ch.5.In: Nielsen SS, Food analysis laboratory manual, 2ndedn Springer, New York Session Determination of Total Carbohydrate in foods I Objective Determine the total carbohydrate content of soft drinks and beers II Materials • Beer, regular; opened and stored in 10oC fridge over night • Soft drink, regular; opened and stored 10oC fridge over night • Erlenmeyer flask, 100 ml, for distilled water • Erlenmeyer flasks, 500 ml, for beverages • Bottle to collect waste • Cuvettes for spectrophotometer • Gloves • Mechanical pipettes, 5000 µm, 1000 µm and 100 µm (or 200 µm), with plastic tips • 20 Test tubes, 16-20 mm internal diameter • Test tube rack • Volumetric flasks, 1000 ml • Volumetric pipette, ml • Volumetric pipettes, 10 ml • Spectrophotometer • Vortex mixer • Water bath, maintained at 25oC III Method Prepare standard curve tubes: • Using the glucose standard solution (100 µg glucose/ml) and distilled water as indicated in the table below µg Glucose/10 ml ml glucose stock solution (100 àg/ml) ml distilled water ã 20 40 60 80 100 10 10 Record caloric content from labels of soft drinks and beers Prepare sample tubes: • • Dilute sample tubes as 1:2000 dilution by taking: o 1ml sample + 9ml distilled water => 10ml A (dilute 10 times) o 1ml A + 9ml distilled water => 10ml B (dilute 100 times) o 0.5ml B + 9.5ml distilled water => 10ml C (dilute 2000 times) Pipette 1.0 ml sample and 1.0 ml of distilled water into a test tube *Note: Phenol and H2SO4 additions are carried out in a hood with fan on • Phenol addition: add 0.05 ml 80% phenol to each tube of Standard curve tubes and Sample tubes containing a total volume of ml Vortex • H2SO4 addition: after adding phenol, add 5.0 ml H2SO4 to each tube Mix on a Vortex test tube mixer Let tubes stand for 10 and then place in to cool them to room temperature Vortex • Reading absorbance: transfer samples from test tubes into cuvettes Do not rinse cuvettes with water between samples • Zero the spectrophotometer with the blank • Read absorbance of all other samples at 490 nm IV Questions Construct a standard curve for your total carbohydrate determinations, expressed in terms of glucose (A490 versus µg glucose/ 2ml) Determine the equation of the line for the standard curve (20%) Calculate the concentration of glucose in your soft drink samples and beer samples in terms of g/ L (20%) Calculate the caloric content (based only on carbohydrate content) of your soft drink samples and beer samples in term of Cal/ L and compare to the label Explain any differences (30%) Compare and explain any differences between the calories that carbohydrates contributed to soft drinks and to beers (10%) What are the advantages, disadvantages, and sources of error for this method to determine total carbohydrates? (20%) References BeMiller JN (2010) Carbohydrate analysis Ch 10 In: Nielsen SS (ed) Food Analysis, 4thedn Springer, New York Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances Anal Chem 28: 350 – 356 Nielsen SS (2010) Phenol-Sulphuric acid method for total carbohydrates, Ch.6.In: Nielsen SS, Food analysis laboratory manual, 2ndedn Springer, New York 10 Session Determination of Crude Dietary Fibre in Foods I Objective Determine total dietary fibre in rice bran using Weeden method II Materials • Defatted rice bran, 10g • H2SO4 1.25% • KOH 1.25% • Acetone • n-octanol • Deionized water • Oven • Dessicator • Muffle furnace • Hot plates • Beakers 600mL • Cylinders 100mL, 50mL III Methods Sample preparation • Dry-mill test sample to mm particles Total dietary fibre determination Determine moisture content of sample separately in oven set at 1050C Weigh 1g sample in each glass crucible Record the weight of sample plus crucible (F0) • Add 150mL pre-heated H2SO4 1.25% (up to the 2nd notch) • Add 3-5 drops of n-octanol • Boil exactly 30 • Connect to vacuum to drain sulfuric acid • Wash with 30mL (fill up to the top of crucible) of hot deionized water Connect to compressed air to stir the content of crucible and vacuum to drain water Repeat times Add 150mL pre-heated KOH 1.25% (up to the 2nd notch) • Add 3-5 drops of n-octanol • Boil exactly 30 • Connect to vacuum to drain potassium hydroxide • Wash with 30mL (fill up to the top of crucible) of hot deionized water Connect to compressed air to stir the content of crucible and vacuum to drain water Repeat times Wash with cold deionized water to cool the crucibles Add 25mL acetone Connect to compressed air to stir the content of crucible and vacuum to drain water Repeat times 11 Remove the crucibles Dry at 1050C for an hour Let crucibles cool in desiccators Weigh the crucible and contents inside (F1) Place and heat crucibles in a muffle furnace up to 5500C for hours Let crucibles cool in desiccators Weigh the crucible and contents inside (F2) IV Questions What are dietary fibers? (15%) What are included in F1 and F2? (15%) Determine the crude fiber content of rice bran (20%) Explain the purpose of each step in the procedure (30%) Recommend another method for determination of dietary fiber (20%) References AOAC International (2007) Official methods of analysis, 18th ed (2005) Current through revision 2, 2007 (On-line) AOAC International, Gaithersburg, MD 12 Session Determination of Total Flavonoid Content (TFC) I Objective Determine the total flavonoid content of tea using the aluminium chloride colorimetric method II Materials • Tea, 10g • AlCl3 10% • CH3COOK 1M • Ethanol 95% • Distilled water • Rutin stock 100μg/mL III Method Prepare tea samples • Brew 1g tea with 100mL boiling water in a covered beaker • Let the sample stand for 30 at room temperature • Take supernatant and dilute times Do in duplicate Prepare rutin standard at different concentration Concentration (μg/mL) 20 40 60 80 100 mL rutin stock 0.2 0.4 0.6 0.8 1.0 mL distilled water 1.0 0.8 0.6 0.4 0.2 Prepare standard curve and determine total flavonoid contents of tea For each test tube: • Add 0.5mL of rutin standard or diluted tea • Add 1.5mL of ethanol 95% • Add 0.1mL of AlCl3 10% • Add 0.1mL of CH3COOK 1M • Add 2.8mL of distilled water Let standard/sample stand for 30 and measure absorbance at 415nm IV Questions What are flavonoids? What are their health effects on human? List some food sources for flavonoids (20%) What is the principle of aluminium chloride colorimetric method? (20%) Construct the standard curve for your TFC determination (15%) Based on your standard curve, calculate TFC of different tea in term of μg rutin equivalent per g of tea Discuss your results (30%) How to treat the chemical waste in this test? (15%) Reference Van Hung, P., & Morita, N (2008) Distribution of phenolic compounds in the graded flours milled from whole buckwheat grains and their antioxidant capacities Food chemistry, 109(2), 325-331 Yao, L H., Jiang, Y M., Shi, J., Tomas-Barberan, F A., Datta, N., Singanusong, R., & Chen, S S (2004) Flavonoids in food and their health benefits Plant foods for human nutrition, 59(3), 113122 13 Session Determination of Vitamin C in foods I Objective Determine the Vitamin C content of orange juice using the indicator dye iodine in a titration method II Materials • Orange • Soluble starch • KI • KIO3 • H2SO4, 3M • Beaker, 150 ml • Beaker, 250 ml • Burette, 25 ml • Graduated cylinder, 25 ml • Graduated cylinder, 100 ml • Erlenmeyer flasks, 125 ml • Fluted filter paper, pieces • Filtering cloth • Funnel, approximately – cm diameter (to hold filter paper) • Funnel, approximately – 3cm diameter (to fill burette) • Glass stirring rods • Pipette bulb or pump • Ring stand • Spatulas • Volumetric flask, 50 ml • Volumetric flask, 200 ml • Volumetric flask, 250 ml • Volumetric pipettes, ml • Volumetric pipettes, ml • Volumetric pipette, 10 or 20 ml • Analytical balance III Method Prepare 1% starch indicator solution • Add g soluble starch to 100 ml distilled water • Mix the solution well on a hot stirring plate until the solution is clear • Allow the solution to cool Prepare iodine solution • Dissolve g KI and 0.27 g KIO3 in 200 ml of distilled water • Add 30 ml of M sulphuric acid in a hood with fan on • Dilute the solution with distilled water to 500 ml in a 500 ml graduated cylinder • Vortex Prepare vitamin C standard solution 14 • Dissolve 500 mg vitamin C tablet in 200 ml distilled water • Dilute to 500 ml with distilled water Standardize vitamin C solution • Add 25 ml of vitamin C solution to a 250 ml flask • Add 10 drops of 1% starch solution • Titrate with iodine solution until the colour of solution changes and persists for at least 20 seconds • Record the initial and final readings and calculate the difference to determine the amount of dye used for the titration • Do triplicate Prepare orange juice solution • Cut an orange and swirl the pieces to extrude the juice out Note: swirl the fruits slowly and gently to get the juice without taking too much pulp (the pulp will block the filter paper and take longer time to filter the juice) • Filter the juice through filter paper Titrate juice samples • Add 25 ml of juice to a 250 ml flask • Perform the same as standardizing vitamin C solution above • Do triplicate Titrate blank solution • Add 25 ml of water to a 250 ml flask • Perform the same as standardizing vitamin C solution above • Do triplicate IV Questions Using the data obtained in standardization of the dye, calculate the titre (20%) Calculate the ascorbic acid content of the juice sample in mg/ ml (20%) By comparing results obtained for various orange juice products, did heat and/or oxygen exposure during processing and storage of the samples analysed seem to affect the vitamin C content? (20%) Why was it necessary to standardize the iodine solution? (20%) Why was it necessary to titrate blank samples? (20%) References AOAC International (2007) Official methods of analysis, 18th ed (2005) Current through revision 2, 2007 (On-line) AOAC International, Gaithersburg, MD Nielsen SS (2010) Vitamin C determination by indophenol method, Ch.7.In: Nielsen SS, Food analysis laboratory manual, 2ndedn Springer, New York Pegg RG, Landen WO, Eitenmiller RR (2010) Vitamin analysis Ch 11 In: Nielsen SS (ed) Food analysis, 4thedn Springer, New York 15 ... total solids analysis, Ch.6.In: Nielsen SS (ed) Food Analysis, 4thedn Springer, New York Nielsen SS (2010) Determination of moisture content, Ch.3.In: Nielsen SS Food analysis laboratory manual, 2ndedn... Phenol-Sulphuric acid method for total carbohydrates, Ch.6.In: Nielsen SS, Food analysis laboratory manual, 2ndedn Springer, New York 10 Session Determination of Crude Dietary Fibre in Foods... Ch.7.In: Nielsen SS, Food analysis laboratory manual, 2ndedn Springer, New York Pegg RG, Landen WO, Eitenmiller RR (2010) Vitamin analysis Ch 11 In: Nielsen SS (ed) Food analysis, 4thedn Springer,