Food Chemistry Pratical Manual

FOOD CHEMISTRY
A PRACTICAL MANUAL
Bruce R. DArcy Geoff Hawes Ian Bentley
A University of Queensland Publication

2006
Copyright 2006 by The University of Queensland All rights reserved
The University of Queensland
Food Chemistry A Practical Manual
Food Chemistry
CONTENTS
PRACTICAL EXERCISE NO.
Course References
Page No.
5
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
pH and Titratable Acidity of Orange Juices Salt Determination Carbohydrate Analysis Moisture Determination Protein Determination Properties of Enzymes Properties of Lipids Nonenzymic Browning Maillard Reaction Ascorbic Acid Determination Ash Determination Enzymic Browning: Activity of Fruit Homogenates
7 11 15 19 23 29 33 37 41 45 49
Appendix 1 Tables for Converting Specific Gravity to % TSS (Brix) Appendix 2 Lane Eynon Titration Tables
53 55
All practical exercises take one week except No. 4. A number of practicals will be done concurrently, ie. two 1 week practical exercises are done concurrently over 2 weeks. The practicals exercises will not be done in the order above but as assigned each year by the lecturer in charge.
Course References
The following references are a list of chosen references available in UQ libraries that may be useful for preparing for practicals and for practical report preparation. Some of the references listed below are specifically highlighted as part of particular practical exercises. AOAC International. (2001). Official Methods of Analysis. 17th Ed. (AOAC International: Gaithersburg, MD) Aurand, L. W., Woods, A. E. and Well, M. R. (1987). Food Composition and Analysis. (AVI: New York). Australian and New Zealand Food Standards Code (available in UQ Biological Sciences Library). Australian Food Composition Tables (available in UQ Biological Sciences Library). Belitz, H. D. and Grosch, W. (1999) Food Chemistry 2nd Ed. (Springer-Verlag : New York). Charalambous, G. (1990) Flavors and Off-Flavours. (Elsevier: New York). Christian, G.D. (1994) Analytical Chemistry 5th Edition (John Wiley and Sons Inc. : New York, USA) Christen, G.L. and Smith, J.S. (Ed.) (2000) Food Chemistry: Principles and Applications (Science Technology Systems, West Sacramento, CA, USA) Clark, N. (1992). Food Chemistry. (Food Trade press : Westerham, England) Coultate, T. P. (2002). Food - The Chemistry of its Components 4th Ed. (Royal Society of Chemistry: London). de Mann, J. M. (1999) Principles of Food Chemistry 3rd Ed. (Aspen Publishers, Inc.: Gaithersburg, Maryland, USA). Dewdney, P. A., Burke, C. S. and Crawford, C. (1975). Commercial Use of Enzymes in the Food Industry. (BFMIRA : Leatherhead, Surrey) Dickinson, E. and Stainsby, G. (1982). Colloids in Foods. (Applied Science : London). Fennema, O. (1996). Food Chemistry 3rd ed. (Marcel Dekker: N.Y.)
Food Chemistry
Gruenwedel, D. W. and Whitaker, J. R. (1984) Food Analysis: Principles and Techniques, Vol. 1 and 2. (Marcel Dekker: New York). Heimann, W. (1980). Fundamentals of Food Chemistry. (Ellis Horwood: Chichester, England). Holland, B., Welch, A. A., Unwin, I. D., Buss. D. H., Paul, A. A. and Southgate, D. A. T. (2002) McCance and Widdowsons The Composition of Foods. (Royal Society of Chemistry: Cambridge). James, C.S. (1999) Analytical Chemistry of Foods (Blackie Academic and Professional: London) Kirk, R. S and Sawyer, R. (1991) Pearson's Chemical Analysis of Foods 9th Ed. (Longman Scientific and Technical; Harlow, Essex, UK). Lawrence, J. R. (1984). Food Constituents and Food Residues: Their Chromatographic Determination. (Marcel Dekker: New York). Lee, F. A. (1983). Basic Food Chemistry 2nd Ed. (AVI: Westport, Conn.). Maarse, H. and Van Der Heij, D. G. (1994) Trends in Flavour Research. (Elsevier: New York). Maynard, (1970). Methods in Food Analysis, 2nd Ed. (Academic Press, London). Miller, D. D. (1998) Food Chemistry: A laboratory manual. (John Wiley & Sons. Inc. : New York, USA). Morton, I. D. and McLeod, A. J. (1982). Food Flavours - Part A Introduction. (Elsevier: Amsterdam). Nielsen, S. S. (ed.) (1998) Food Analysis 2nd Ed. (Aspen Publishers, Inc.: Gaithersburg, Maryland, USA). Nollet, L. M. (ed.) (1992) Food Analysis by HPLC (Food Science and Technology Vol 52) Paul, A. A. and Southgate, D. A. T. (1988). The Composition of Foods 4th Rev. Ed. (Elsevier: Amsterdam). Pomeranz, Y. and Meloan, C. E. (1994) Food Analysis: Theory and Practice 3rd Ed. (Chapman and Hall: New York, USA). Robinson, D. S. (1987). Food-biochemistry and nutritional value. (Longman: London).
Schwimmer, S. (1981). Source Book of Food Enzymology. (A. V. I. : Westport, Conn.). Sikorski, Z. E. (1996) Chemical and Functional Properties of Food (Technomic Publishing Co, (Tech)) Skoog, D.A. and Leary, J.J. (1998). Principles of Instrumental Analysis. 4th Edition. (Saunders College Publishing: New York) Skoog, D. A. and West, D. M. (1992). Principles of Instrumental Analysis. (Holt, Rinehart and Winston, Inc.: New York). Souci, S. W. Fauchman, W. and Kraut, H. (2000) Food Composition and Nutrition Tables. 6th Edition. (Medpharm Scientific Publishers: Stuttgart). United States Department of Agriculture. (2002) Nutrient Data Laboratory. (online). (www.usda.gov.). Wong, D. W. S. (1989). Mechanism and Theory in Food Chemistry. (Van Nostrand Reinhold : New York).
Food Chemistry
PRACTICAL EXERCISE NO 1
pH AND TITRATABLE ACIDITY OF ORANGE JUICES

INTRODUCTION The determination of pH and titratable acidity are routine Q.C. analyses used to give an indication of the quality of the fruit juice during processing. Because of the natural buffering capacity of many food materials, pH is only a very mediocre measure of changes in the acidity of a substance. A more accurate measure is a quantity called the titratable acidity. The major acid in orange juice is critic acid and this can be measured titrimetrically. However, before titratable acidity of a fruit juice can be determined, the sodium hydroxide used to titrate the natural acidity must be standardized.
PART A
PREPARATION AND STANDARDIZATION OF 0.1 M SODIUM HYDROXIDE
The techniques used in quantitative chemistry to measure out volumes of liquids are called volumetric techniques. Titration is an important volumetric technique. The purpose of a titration is to measure the volume of one solution required to react completely with a known volume of some other solution. This knowledge can then be used to prepare solutions of standard concentration which are used then in other volumetric analyses. In this experiment, the volume of sodium hydroxide (NaOH) required to react with 20 mL of hydrochloric acid (HCl) will be measured. The end-point of the titration ie. the point at which exactly enough acid is added to neutralise the sodium hydroxide, is made visible by means of an indicator. An indicator is a substance which changes colour at the end-point of a titration. In this experiment, the indicator is phenolphthalein. Two drops of this indicator are added to the hydrochloric acid solution before titration, the solution will remain colourless. At the end-point the solution will be a pale pink colour. If the colour is bright pink or pink-purple, the titration has gone too far and it will be necessary to repeat the titration. NaOH (Sodium Hydroxide) METHOD 1. Preparation of Approx. 0.1M NaOH Weigh approximately 1 g of the sodium hydroxide pellets in a 100 mL beaker. approximately 50 mL of distilled water and dissolve. Add + HCl (Hydrochloric Acid) NaCl (Salt) + H2O (Water)
Quantitatively transfer the solution to a 250 mL volumetric flask via a funnel. Rinse the beaker several times with distilled water and transfer the washings to the volumetric flask. Make up to the mark with distilled water and mix thoroughly. 2. Standardization of the 0.1 M NaOH 1. Check that the burette is clean and the tip clear. Rinse it out with the approximately 0.1 M sodium hydroxide solution. Clamp the burette in a vertical position and fill it with the sodium hydroxide solution using a small funnel. Remove the funnel when the burette is full but do not let the burette overflow. Rinse out a 20 mL pipette with the 0.1 M hydrochloric acid solution and a 250 mL conical flask with distilled water from the wash bottle. Pipette 20 mL of 0.1 M hydrochloric acid solution into the conical flask. Add 2 drops of phenolphthalein indicator to the flask. Take the initial burette reading (it does not have to be zero). Rapidly add about 18 mL of sodium hydroxide (about 2 mL less than the amount needed) to the conical flask. Adjust the burette tap to deliver the acid dropwise. Note: This is only true if M1V1 _____ 1 = M2V2 _____ 1
2.
3.
4. 5. 6. 7.
8.
When the end point is reached (ie. when the pink colour just appears and remains for 15-20 seconds), take the final burette reading. Repeat steps (4) - (8) at least two more times or until 2 consecutive values are within 0.1 mL.
9.
Calculation: Calculate the molarity of the sodium hydroxide solution given that the molarity of the hydrochloric acid is 0.1M using the formula: MAVA a where MB VA VB a b MA = = = = = MBVB b
= Molarity Acid Molarity Base Volume Acid Volume Base Balancing factor Acid Balancing factor Base
Food Chemistry
QUESTIONS 1. 2. Why are the burette and pipette rinsed out with the solution to be used in them? Why does it not matter if the conical flask is wet with water?
PART B METHOD
ANALYSIS OF ORANGE JUICES
A number of commercial orange juices are supplied. In addition, prepare freshly squeezed orange juice. (a) pH Determination
Determine the pH (hydrogen ion concentration) of the various orange juice samples using the pH meter. Be sure to calibrate the pH meter before use using the supplied buffers.
(b)
Titratable Acidity Determination
Determine the titratable acidity of these juices using your standardized 0.1 M NaOH from Part A and the procedure below: (i) Dilute the juice sample(s) (1 : 4) using a 100 mL volumetric flask; and then pipette 25 mL of the diluted juice into a 250 mL conical flask and add a few drops of phenolphthalein indicator. (ii) Titrate with the standardized solution of 0.1 M NaOH until a slight pink end-point is reached which persists for about 15-20 seconds. Repeat until titration volumes are within 0.1 mL. (iii) Calculate the per cent citric acid in the various juice samples using your NaOH molarity determined in Part A and given that the balanced equation of the reaction between citric acid and sodium hydroxide is: CH2 - COOH HO - C - COOH CH2 - COOH (Citric Acid) CH2 - COONa OH -C - COONa CH2 - COONa (Sodium Citrate)
3NaOH
3H2O (Water)
(Sodium Hydroxide)
Ensure you do not forget about the dilution factor.
10
QUESTIONS 1. 2. 3. 4. Define titratable acidity. Outline the procedure for correct maintenance of pH electrodes. What is an indicator? Why is it important to select the correct indicator for an acid-base titration?
REFERENCES Shugar, G.J.; Shugar, R.A.; Bauman, L. & Bauman, R.S. (1981) Chemical Technicians Ready Reference Handbook 2nd ed. (McGraw Hill: New York) Kirk, R. and Sawyer, R. (1991) Pearson's Chemical Analysis of Foods 9th Edition. (Longman Scientific and Technical: Harlow, Essex, UK)
11
Food Chemistry
PRACTICAL EXERCISE NO. 2
SALT DETERMINATION
INTRODUCTION Salt is added to foods to enhance the flavour, act as a preservative or in some cases both. Samples that can be analysed include butter, cheese, margarine and bottle spring water. The method varies slightly for the analysis of different products. Silver nitrate is the most common reagent used in precipitimetry, being suitable for the estimation of chloride (Cl-), bromide (Br-), iodide (I-) and thiocyanate (SCN-). ClBrISCN+ + + + Ag+ Ag+ Ag+ Ag+ AgC1 AgBr AgI AgSCN
As well as direct titrimetric methods, silver nitrate solutions may be used in conjunction with thiocyanate solutions using a back titration technique eg. the Volhard's Method. In the latter, halides are precipitated by the addition of excess silver nitrate solution and the excess Ag+ back titrated with standard thiocyanate. Silver nitrate may be used as a primary standard provided it is freshly recrystallised and has not been allowed to photo-decompose. However, because of the extreme cost of A.R. reagent, the usual practice is to prepare approximate solutions from commercial reagent and then standardize using sodium or potassium chloride. End point detection is achieved using either potassium chromate (Mohr's method) or a fluorescein-type indicator (Fajan's Method). N.B. Avoid contact with silver nitrate as it produces brown stains on skin.
PART A STANDARDIZATION OF AgNO3 WITH SODIUM CHLORIDE Sodium chloride (NaCl = 58.46) is a good primary standard but has the disadvantage of possessing a low molecular weight, (thus leading to possible weighing errors) and a slightly hygroscopic nature. The latter may be overcome by drying for 1-2 hours at 250-350C before using. To overcome the former problem when using sodium chloride for the standardization of 0.1 M AgNO3 solutions, a standard NaCl solution is used instead of the direct titration technique.
12
METHOD Obtain the solution of approx. 0.1 M AgNO3 in a clean conical flask that has been washed chloride-free with distilled water.
1.
Prepare the standard NaCl solution by accurately weighing about 0.40 g of AR NaCl into a clean dry beaker. Dissolve and quantitatively transfer the solution to a 250 mL volumetric flask then make up to the mark with distilled water. Calculate the molarity of this solution. Transfer 20 mL aliquots using a pipette to 250 mL conical flasks and add 3 drops of fluorescein indicator. Titrate with 0.1 M AgNO3 solution, with constant agitation, until AgCl formed coagulates and settles to the bottom of the flask. The end point lies within 1 mL of this point and is detected by the first observable pink colouration in the precipitate (after settling). Repeat the titration to 0.1 mL agreement. Determine the molarity of the silver nitrate solution.
2. 3.
4.
5. 6.
Notes: (i) Constant agitation is necessary throughout the titration to promote coagulation of the colloidal AgCl. The reason for this is that the organic fluorescein molecule is absorbed preferentially on to the AgCl particle at the end point where it produces a colour change which is best observed in the coagulated precipitate. Avoid areas of intense natural light for completion of this exercise since photodecomposition in the AgCl will results and cause problems. The obvious sign of this effect is a blue-black appearance in the precipitate. All silver residues from titrations must be returned to the Silver Residues Bottles located in the fume cupboard. Silver chloride deposits on the sides of working flasks may be removed by rinsing with a few mL of bench ammonium hydroxide, then thoroughly washing with deionised water.
(ii)
(iii)
(iv)
13
Food Chemistry
PART B SALT CONTENT OF BUTTER AND MARGARINE This determination will use potassium chromate as indicator. METHOD Accurately weigh approximately 2 g of butter or margarine or low salt butter or margarine into a 250 mL conical flask and add 100 mL boiling distilled water. Shake to melt sample, cool to 50 - 55 C and add 2 mL K2CrO4 indicator. Titrate with the standardized 0.05 M AgNO3 until a faint permanent red-brown colour is produced. AgNO3 + NaCl AgCl + NaNO3 (white) 2Ag+ + CrO4 2 Ag2CrO4 (red brown)
Titre:
1 mL 0.1 M AgNO3 = 0.005846 g NaCl.
Determine the % salt content for butter, normal margarine, and a low salt margarine or butter.
QUESTIONS 1. 2. What is a standard solution? What are the major characteristics of a primary standard and what is the importance of using a primary standard? What is a meniscus and what is the correct procedure for reading a meniscus? What does it mean to accurately weigh approximately 4 g?
3. 4.
14
5.
What are the limits for salt stated by the ANZFA Food Standards Code for butter and margarine? Do the samples comply with these limits? Compare the calculated salt content of butter and margarine to the value in the Australian Food Composition Tables. Compare the chloride level (mg/mL) in bottled spring water found in this exercise with that on the package label.
6.
7.
REFERENCES Shugar, G.J.; Shugar, R.A.; Bauman, L. & Bauman, R.S., (1981) Chemical Technicians Ready Reference Handbook 2nd ed. (McGraw Hill: New York) Kirk, R. and Sawyer, R. (1991) Pearson's Chemical Analysis of Foods 9th Edition. (Longman Scientific and Technical: Harlow, Essex, UK)
15
Food Chemistry
CARBOHYDRATE ANALYSIS
PART A. DETERMINATION OF TOTAL SOLUBLE SOLIDS (TSS)

INTRODUCTION A term that will often be encountered in a QC laboratory is total soluble solids or per cent soluble solids. This refers to the amount of solute (eg. sugar, organic acids) dissolved in a solvent (most often water). Generally, soluble solids refer to sugar(s) dissolved in water. Brix corresponds directly to the total soluble solids expressed as a per cent, eg. 10 Brix = 10% total soluble solids. However, this is true only for pure sugar solutions. There are a number of ways of measuring the total soluble solids, and two of the more important ones include: (i) Hydrometer. The specific gravity may be measured using a specific gravity hydrometer, and then by using conversion tables, the specific gravity can be converted to Brix. Additionally, Brix hydrometers can be used to measure the Brix directly. Refractometer. In some refractometers, TSS (%) can be read off directly. In others, the refractive index is measured and by reference to conversion tables, the TSS (%) can be obtained.
(ii)
METHOD (i) Prepare 5%, 10% and 20% w/w and w/v sucrose solutions using the following method, and label each of these carefully: For w/v examples, for the 5% w/v sucrose solution, sucrose (5 g) in a 100 mL beaker is dissolved in distilled water (30-40 mL) and then transferred quantitatively to a 100 mL volumetric flask using 10 mL washings of distilled water. The flask is then made up to the mark with distilled water, a plastic stopper added, and the flask inverted several times to mix. Do not use your finger as a stopper. For w/w examples, for the 5% w/w sucrose solution, tare a 250 mL conical flask and weigh in sucrose (5 g), followed by distilled water to make up to 100 g total mass. Mix the sucrose solution well by swirling before analysis. Similarly prepare the 10% and 20% w/v and w/w solutions. Once the 20% w/w solution has been prepared, pour it into a 100 mL volumetric flask and note your observation of its volume relative to the final volume of the 20% w/v solution.
16
Using the electronic refractometer only, measure the TSS (%) of each solution using the following procedure: Place a few drops of the liquid on the main prism surface. DO NOT TOUCH PRISM WITH SHARP OBJECTS. Record the Brix. After determination, clean the prism with tissue wool soaked in distilled water. (ii) Using BOTH the electronic refractometer and the S.G. and Brix hydrometers, measure the soluble solids contents of the three (3) unknown solutions provided. Lower the hydrometer into the sample contained in a sufficiently large measuring cylinder. Depress slightly and then note the reading at the top of meniscus. The hydrometer must not touch the side of the cylinder. To convert the specific gravity readings provided by the SG hydrometer to per cent soluble solids, use the table provided in the Appendix 1. The Brix hydrometers give the per cent soluble solids directly. (iii) Using the refractometer, measure the TSS (%) of the three samples of fruit juice provided.
(iv) Measure the temperature of the solutions.
QUESTIONS 1. 2. Why are the soluble solids contents of the w/w and w/v solutions different? Why is temperature important when measuring the Brix of a solution using a refractometer? Comment on the advantages and disadvantages of both instruments in measuring TSS.
3.
REFERENCES AOAC International. (1995). Official Methods of Analysis. 16th Ed. (AOAC International: Gaithersburg, MD) Kirk, R. and Sawyer, R. (1991) Pearson's Chemical Analysis of Foods 9th Ed. (Longman Scientific and Technical: Harlow, Essex, UK) Nielsen, S. S. (ed.) (1998) Food Analysis 2nd Ed. (Aspen Publishers, Inc.: Gaithersburg, Maryland, USA). Shugar, G.T., Shugar, R.A., Bauman, L. & Bauman, R.S. (1981) Chemical Technicians Ready Reference Handbook 2nd Ed. (McGraw Hill, New York).
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Food Chemistry
PART B
METHOD
DETERMINATION OF % INVERT SUGAR
Weigh accurately (to 2 decimal places) about 0.8 1 g (any weight in this range is suitable) of jam into a clean 100 mL beaker. Add a small volume (30 mL) of distilled water to dissolve the jam, then quantitatively transfer (using several 20 mL washings with distilled water after initial transfer of the 30 mL jam solution from the beaker) the solution to a 250 mL volumetric flask, and then make the solution up to the mark with distilled water. Use this diluted jam solution for the following titrations (Lane and Eynon method). Filter the solution before placing it in the burette.
Preliminary Titration Pipette 5 mL each of Fehlings A and B solution into a clean 250 mL conical flask, and add from a burette the diluted jam solution (15 mL). Boil the liquid using a hot plate and add further quantities of the diluted jam solution (1 mL at a time) to the boiling liquid until the blue colour is nearly discharged. Then add 3 - 5 drops of 1% aqueous methylene blue solution and continue titrating as above until the blue colour is completely discharged giving a brick-red colour. This will give an approximate measure of the titration value. N.B. The titration must be completed quickly (maximum time is 3 min) while keeping the liquid boiling. If the colour is discharged before the addition of 15 mL of dilute jam solution, then a new jam solution diluted by a factor of 2 must be prepared, and used in a retitration. If the colour is not completely discharged after the addition of 50 mL of dilute jam solution, then a new jam solution must be prepared with its strength increased by a factor of 2, which is then used in a retitration. Accurate Final Titration Repeat the titration, this time adding before heating almost all of the diluted jam solution required to effect near-complete reaction (the preliminary titration minus 3 mL). This is done to ensure the titration is completed quickly. Boil gently for 2 min, add 3 - 5 drops of the methylene blue indicator and complete the titration within a total boiling time of 3 min. It is important to keep the solution boiling during the titration, since it can revert to the blue colour on cooling. At the end point, the blue colour should be completely discharged and the liquid should be brick-red. Record the volume of diluted jam solution required to titrate, and calculate the concentration of invert sugar from tables (see Appendix 2). If this concentration = y mg/100 mL and the original mass of jam taken was x g, then % invert sugar in jam = y / 4x %.
18
REFERENCES Kirk, R. and Sawyer, R. (1991) Pearson's Chemical Analysis of Foods 9th Ed. (Longman Scientific and Technical: Harlow, Essex, UK).
Miller, D. D. (1998) Food Chemistry: A laboratory manual. (John Wiley & Sons. Inc. : New York, USA).
Nielsen, S. S. (ed.) (1998) Food Analysis 2nd Ed. (Aspen Publishers, Inc.: Gaithersburg, Maryland, USA).
Rauch, G.H., (1965), Jam Manufacture. (Leonard Hill: London).
19
Food Chemistry
MOISTURE DETERMINATION
INTRODUCTION The determination of moisture is one of the most important and widely used analytical measurements in the processing and testing of food products. The moisture content is frequently an index of stability and quality, and is also a measure of yield and quantity of food solids. It is closely concerned with the economics and legal aspects of food processing. The moisture content of foods varies considerably: for fresh fruits, from 65% in ripe avocados to 95% in rhubarb; for fresh vegetables, from 66% in green lima beans to 96% in cucumbers; for fresh meat and fish, from 50 - 75%. The moisture content of processed foods varies even more from about 7 - 12% for dried vegetables to 27 -35% for jams and jellies. The particular methods used for determining moisture content will depend on the nature of the food product and on whether speed of execution or accuracy of result is desired. During processing, such as dehydration or concentration, a rapid but not very accurate determination is usually preferable, while the marketing of perishable products requires more accurate moisture determination to meet legal and storage requirements. The accurate determination of moisture in foods is extremely difficult, since during the removal of all water present, many other volatile substances may also removed. Thus, for most foods there is a standard method from which other methods are calibrated.
METHOD All methods below are to be done in duplicate. NOTE: Only use tongs when handling dishes during moisture determination. Samples should be finely grated (where possible) and kept in a covered container before use.
1.
Direct Drying in an Oven
Weigh accurately 2 - 3 g (to 2 decimal places) of grated carrot (in duplicate) and skim milk powder (in duplicate) into numbered weighed moisture dishes (do not using taring), which have been previously dried in a hot air oven and cooled in a desiccator. Place in a hot air oven and dry over 1 h intervals at a temperature 100 - 102. Remove and cool to room temperature in a desiccator and reweigh. DO NOT COMPLETELY SEAL THE DESICCATOR. Repeat until the loss in mass does not exceed 0.1% per 1 h drying period.
20
Calculation: Moisture and volatile matter % w/w = Loss in mass x 100 Mass of sample
2.
Direct Drying in an Oven with Sand
This method is suitable for butter, margarine, condensed milk, sausages, cheese, etc. Two foods are to be analysed in duplicate by each student. METHOD Place about 20 g of purified sand and a short glass stirring rod in a numbered dish made of nickel or aluminium. Record the total weight. Dry to constant mass, eg. 30 min in an oven at 100 - 102C. Cool in a desiccator to room temperature and weigh. Tilt the sand to one side of the dish and accurately weigh, into the clear space, between 1.5 - 3g (to 2 decimal places) of butter. Stir. Place on a warm hotplate for 20 min, stirring at intervals in order to obtain a well ventilated mass. Transfer dish and rod to the oven and dry at 100 for 1 h. Cool in a desiccator (do not seal completely) and reweigh. Transfer dish to the oven for a further 1 h, remove, cool and reweigh. Repeat until loss in mass does not exceed 0.1%. Calculation: . Moisture and volatile matter % w/w = 3.
Loss in mass x 100 Mass of sample
Determination of Moisture by the Vacuum Oven Method
This is an accurate method suitable for nearly all foods, and is often used as the official method. However, the method is time consuming and should only be used for accurate determination. If the food sample has a high moisture content, the samples should be first dried in a hot air oven at approximately 60-70 C for a short time to remove the majority of the moisture. This is to ensure that a large amount of water is not drawn into the vacuum pump, particularly if the pump does not have a trap between it and the vacuum oven. METHOD a. Weigh accurately about 5 g of well mixed dry skim milk powder (in duplicate) and 5 g of grated carrot in duplicate) into separate weighed numbered moisture dishes (do not use taring) which has been previously dried in an oven and cooled in a desiccator. Dry in a vacuum oven at approximately 75 C. Remove from the oven, cool in a desiccator to room temperature, and reweigh.
b. c.
21
Food Chemistry
d.
Constant mass is attained when the loss is not more than 0.1% in successive 1.5 h drying periods.
Calculation: Moisture and volatile matter % w/w = Loss in mass x 100 Mass of sample
4.
Determination of Moisture by the Infrared Balance Method
This is a rapid method suitable for drying powders, such as skim milk powder. Each student will determine the moisture content of 5 foods of choice in duplicate, one of which is skim milk. Instruction is the use of the two automated infrared balances will be given by the tutors. These calibrated instruments give the % moisture directly with no calculations required.
5.
Determination of Moisture by the Hot-Plate Method
The two foods, butter and margarine must be analysed in duplicate. Weigh accurately 5-10 g (to 2 decimal places) of butter, margarine or similar fat in duplicate, which has been thoroughly mixed, into a dry 100 mL beaker containing a small thermometer which reads to 240 C. The beaker and thermometer must be weighed together before adding the food sample. Gently heat the sample in the beaker on a hot-plate. Stir the contents gently with the thermometer to avoid spluttering, which may result from too rapid ebullition of moisture. The approach of the end-point may be judged by the cessation of rising bubbles of steam, as well as by the advance of foam. Another good method of judging the end-point is to place a clean dry watchglass on top of the beaker. The evolution of steam is indicated by condensation on the watch glass. The temperature of the sample should at no time be permitted to exceed 130C, except at the end of the test. Calculation Moisture and volatile matter % w/w = Loss in mass x 100 Mass of sample
6. a.
Determination of Moisture by the Toluene Distillation Method Weigh into a 250 mL round-bottom flask 30 g of dry skim milk powder. Connect the flask to the appropriate Dean-Stark receiver and connect to the condenser. Add sufficient toluene to cover the sample completely (about half full) by pouring toluene through the condenser. Insert a loose cotton plug in the top of the condenser to prevent condensation of atmospheric moisture within the tube.
22
b.
In a fume cupboard, heat the flask to boiling in a heating mantle, and distil slowly (about 2 drops per s) until most of the water has come over. Next, increase the distillation rate to 4 drops per s until no more water comes over. Ask for assistance at this point of the experiment. Wash down the condenser (brush down with a burette brush saturated with toluene while at the same time pouring toluene through the top of the condenser). Continue the distillation for a short time to determine if any more water comes over, then repeat the washing. Remove from heat, and allow the collecting tube to come to room temperature. If drops of water adhere to the sides of the collecting tube, force them down by means of a rubber tubing slipped over the end of a glass rod.
Calculation Moisture % w/w = Volume of water x 99.7 Mass of sample
Note For the Whole Practical Exercise: The entire apparatus to be used should be cleaned with an acid cleaning solution to minimize the adherence of water droplets to the sides of the condenser and receiver. Rinse thoroughly and dry completely before using. All this has been done prior to the practical class.
QUESTIONS 1. Correlate your results for each food and suggest the most suitable method in each case. Check your suggestions against the normal official methods used. Why keep samples for moisture determination in a sealed container before use? Include a diagram of the Dean-Stark apparatus used in this experiment.
2. 3.
REFERENCES AOAC International. (1995). Official Methods of Analysis. 16th Ed. (AOAC International: Gaithersburg, MD) Kirk, R. and Sawyer, R. (1991) Pearson's Chemical Analysis of Foods 9th Ed. (Longman Scientific and Technical: Harlow, Essex, UK) Nielsen, S. S. (ed.) (1998) Food Analysis 2nd Ed. (Aspen Publishers, Inc.: Gaithersburg, Maryland, USA).
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Food Chemistry
PROTEIN DETERMINATION
INTRODUCTION
Nitrogen is the most distinguishing chemical element present in proteins, which are the most common organic nitrogen compounds among foodstuffs. The determination of nitrogen is thus often used to give protein level, but this is only a crude estimate, as many other nonprotein nitrogenous substances are also present in foods. The protein content of foods varies widely: most fresh fruits and vegetables have less than 6%; white flour and fresh eggs about 12%; and most meats, fish and cheese about 25%.
PART A SEMI-MICRO KJELDAHL DETERMINATION OF TOTAL ORGANIC NITROGEN

The Kjeldahl (1883) and Dumas (1831) methods determine the total organic nitrogen, which is used to calculate the protein present through the use of previously determined factors. There have been many modifications to the Kjeldahl method, which is the most universally used and probably the most accurate. The Kjeldahl method is carried out in two distinct phases. Firstly, the nitrogen compounds are digested with conc. H2SO4 to produce (NH4)2SO4 with the aid of catalysts such as mercury or selenium and K2SO4 to raise the boiling point. Secondly, ammonia is formed after neutralization of (NH4)2SO4 with NaOH, which is then analysed by steam distilling it into a weak acid (boric acid) and titrating against standard HCl, or colorimetrically with ninhydrin reagent. METHOD A. Digestion Perform duplicate analyses, and a blank (reagents only but no flour). Weigh out accurately (to 2 decimal places) on a low ash paper (cigarette paper), an amount of each flour sample (0.1 g) that contains approximately 10 mg of protein. Transfer to a numbered digestion tube and add approx. 2 g K2SO4 and a catalyst tablet. Add 3 mL of concentrated H2SO4 and place the rack and tubes in the digestion apparatus. Connect the exhaust manifold onto the tubes and turn on the water vacuum pump. Set the thermostat to 400 C and turn on for 45 min. After the stated time, with the assistance of the tutor and while wearing the safety shield provided, lift the rack out of the digestion block and place on the stand to cool. Leave the water pump and manifold connected.
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If the samples still contain black particles, wash down the sides of the tubes with distilled water and digest for a further 15 min. When the tubes are cool, remove the exhaust manifold, add approximately 5 mL distilled water carefully to each tube, and finally place a mark on the digestion tube about 40 mm above surface of the liquid. B. Distillation There are many distillation assembles suitable for recovering ammonia from the digest. In essence, most of these employ steam distillation.
OPERATING INSTRUCTIONS FOR GERHARDT DISTILLATION UNIT 1. Turn on cooling water.
2. Ensure that the appropriate tubes are in the distilled water and sodium hydroxide containers. 3. 4. 5. 6. 7. Turn on white power switch. Steam setting switch set to low. Timer set to 2.0. The apparatus cannot be used until the green start light is on. Check there is a mark on the digestion tubes approximately 40 mm above the surface of the liquid. Place digestion tube in the distillation apparatus. Add boric acid (10 mL) plus indicator to a 250 mL conical flask and place it in the right hand side of the apparatus, ensuring the exit tubing is below the level of the liquid in this collecting flask.
8. 9.
10. Close the safety screen. 11. Depress the NaOH control in small amounts to add NaOH to the digest up to the required mark.
12. Depress start button. 13. After 3 min distillation, a buzzer indicates that distillation has been completed. To cancel the buzzer, depress the stop button. This automatically resets the timer.
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Food Chemistry
14.
The green-yellow distillate is then titrated to a pale pink/red colour with 0.02 M HCl from a semi-micro burette (10 mL). When all distillations have been completed, add distilled water (20 mL) to a clean digestion tube and place in the apparatus. Add about 10 mL of NaOH by pressing the NaOH control. Press the start button to clean out the system.
15.
Calculation % (w/w) nitrogen = (mL std acid for sample - mL of std acid for blank) x molarity x 14 x 100 mg of sample Multiply by the appropriate factor to obtain % protein Eggs, peas, meat, beans, corn Wheat flour Milk and products 6.25 5.70 6.38
PART B BIURET DETERMINATION OF SOLUBLE PROTEIN

Compounds containing the peptide bond (-CO-NH-) give a violet colour with NaOH and dilute copper sulphate (Biuret reagent). This is quantitative since the colour depends on the number of peptide bonds. In this experiment, the amount of albumin in liquid egg white will be determined. Liquid egg white contains 10-11 % protein of which albumins (ovalbumin and conalbumin) constitute 67%.
PREPARATION OF BULK EGG WHITE PROTEIN SOLUTION Separate the liquid white from the yolk of an egg into a pre-weighed beaker. Liquid egg white contains 10-11 % protein, ie. 100-110 mg protein / g egg white. Weigh the beaker and egg white, and determine the mass of liquid egg white separated. Add in approximately an equal amount of 0.1 M NaOH and dissolve the egg white using a magnetic stirrer. Using distilled water, wash the dissolved egg white into a 100 mL volumetric flask (filter if cloudy), rinse the beaker several times, and dilute to the volume with distilled water. Shake the solution to mix. Calculate the approximate concentration of this protein solution in mg protein / mL for use below. PREPARATION OF PROTEIN STANDARDS Standard Gelatin Protein Solution: 10 mg protein/mL distilled water. Note, gelatin is 88% pure so this has to taken into account when calculating the amount to prepare 100 mL of the protein solution. Use an analytical balance.
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Weigh the calculated amount into a beaker and dissolve in distilled water (60 mL). Gentle heat will be required to dissolve the gelatin. Do not over heat. When dissolved, cool the solution, and transfer quantitatively to a 100 mL volumetric flask. This can be achieved by rinsing the beaker several times with small amounts of distilled water (10 mL) and adding the washings to the volumetric flask. Then, make up to the 100 mL mark with distilled water. Set up twelve test tubes in six pairs.
The biuret reagent MUST NOT be added until all of the solutions are in the twelve test tubes.
BLANK (Std 1)
Std 2
Std 3
Std 4
Std 5
Final Egg White Sample (Soln. 6) ? mL ? mL 8 mL ??
H2O Protein Solution (Gelatin) Biuret Reagent Conc. Diluted Gelatin Protein Solution (mg/mL)
2 mL 0 mL 8 mL 0
1.6 mL 0.4 mL 8 mL 0.4
1.2 mL 0.8 mL 8 mL 0.8
0.8 mL 1.2 mL 8 mL 1.2
0.4 mL 1.6 mL 8 mL 1.6
All the test tubes must have a total of 10 mL so the maximum amount of the bulk egg white solution is 2 mL.
PREPARATION OF FINAL EGG WHITE PROTEIN SAMPLE (SOLUTION 6) Using the concentration of the bulk egg white protein solution calculated above, calculate the volume (mL) of this bulk egg white solution required to fit on the calibration curve (ie. between the minimum and maximum gelatine protein concentrations). Add this amount and the appropriate volume of distilled water (to bring to a total of 2 mL) for Solution 6 in the right hand column of the table above. Please check this calculation with the tutor before proceeding. Then add this amount to the final pair of test tubes (Solution 6).
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Food Chemistry
PROTEIN DETERMINATION Now add biuret reagent (8 mL) to all of the tubes and mix each well on a vortex mixer. Leave the tubes to stand for 30 min and read absorbance on a spectrophotometer at 550 nm. Plot a best fit straight line (going through zero-zero) of protein concentration (x axis) versus absorbance (y axis) at 550 nm for the gelatin protein standards. Using this graph, determine the protein concentration (mg/mL) of the diluted egg white solution (Solution 6), followed by the concentration of the bulk egg white solution (100 mL) taking account of the dilution factor. From this concentration, determine the protein concentration (% protein) of the original liquid egg white used, taking account of the dilution factor for this experimental step. Finally, calculate the albumin concentration (% albumin) of the liquid egg white used in this practical exercise, and compare to that in the literature.
QUESTION Why is it necessary to cool the standard protein solution before making it up to volume in a volumetric flask?
REFERENCES AOAC International. (1995). Official Methods of Analysis. 16th Ed. (AOAC International: Gaithersburg, MD) de Mann, J. M. (1999) Principles of Food Chemistry 3rd Ed. (Aspen Publishers, Inc.: Gaithersburg, Maryland, USA). Kirk, R. and Sawyer, R. (1991) Pearson's Chemical Analysis of Foods (Longman Scientific and Technical: Harlow, Essex, UK) 9th Edition.
Nielsen, S. S. (ed.) (1998) Food Analysis 2nd Ed. (Aspen Publishers, Inc.: Gaithersburg, Maryland, USA).
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Food Chemistry
PROPERTIES OF ENZYMES
Part A HYDROLYTIC ENZYMES

INTRODUCTION Hydrolytic enzymes are those which break down large polymer molecules into smaller molecules by insertion of water, ie. hydrolysis. These are by far the most important group of enzymes, as far as foods and food processing are concerned, and include prototypic enzymes or proteases, lipases, carbohydrases, pectinases, and sugar splitting enzymes. 1. RENNIN
The stomach of a calf and other young mammals contains a proteolytic enzyme (hydrolysed proteins) called rennin, which, if allowed to act upon milk, will cause it to clot (in the presence of calcium ions). The enzyme rennin is present in commercial rennet for making cheese. The chief proteins in milk are casein and lactalbumin. Casein is a phosphoprotein which is soluble in the presence of calcium ions, also present in milk. If the calcium ions are removed from the milk, eg. by precipitation with oxalic acid as calcium oxalate, and the oxalated milk is acted on by rennin, the casein is still converted into para-casein, but it now remains in solution, in the absence of the calcium ions and no clotting occurs. Calcium ions are required for clotting. METHOD Commercial rennet containing the enzyme rennin is required. A solution of rennet is available. 1. Set up the following test tubes - (use same size tubes). Tube A with 10 mL of milk + 1 mL of rennet solution. Tube B with 10 mL of milk + 1 mL of boiled rennet solution.
Shake the tubes and place in a water bath at 37 C (place each set of tubes in the water bath at the same time). Observe changes in both tubes with gentle shaking every min. Explain the results.
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2. Next set up three tubes as below, mix on a vortex mixer, and incubate at 37 C. Observe changes in each tube every min for 5 min. Add 1% potassium oxalate (6 mL) to milk (20 mL) before preparing these three solutions.
Oxalated Milk (mL)
2.5% Rennet Soln. (mL) 1 1 1 boiled
1% CaC12 (mL) 1 Nil Nil
A B C
4 4 4
After 5 min, boil solutions B and C then cool and add 1 mL of 1% CaC12 solution to both. Observe and explain. QUESTION Give a general overview of the mechanism of action of the enzyme, rennin on milk showing chemical structures where appropriate.
2.
LIPASE
Neutral fats are esters of glycerol and are hydrolysed by lipases (eg. in pancreatic juice and milk) to glycerol and fatty acids. This reaction is reversible and is helped by the presence of bile salts, which enable the fat to form smaller droplets, and so be more accessible to the enzyme. Lipase catalyses fat hydrolysis (lipolysis) in milk leading to the development of off odours; this process is called hydrolytic rancidity.
METHOD Take about 20 mL of milk and boil in order to destroy bacteria that might produce lactic acid from lactose present. Cool and add 15 drops of 1% aqueous phenol red indicator (changes colour in the presence of acid). Add just enough 2% Na2CO3 to give a definite red colour eg. colour of strawberry milk. Make up the following mixtures. The lipase should be added last.
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Food Chemistry
Phenol red/milk mixture (mL)
0.2% Lipase extract (mL) 2 2 boiled 2
Water (mL) 1 1 Nil
2% Bile Salts (mL) Nil Nil 1
A B C
5 5 5
Shake the tubes and place in a water bath at 37 C and examine the colour changes at 30 sec intervals.
QUESTIONS 1. 2. Outline the mechanism of lipase activity using chemical structures where appropriate. What would happen if too much 2% Na2CO3 is added during the lipase test?
PART B
PEROXIDASE ACTIVITY DETERMINATION
INTRODUCTION Enzyme activity is widely used as a test of adequate heat treatment (blanching) during processing. Peroxidase is one of the most heat resistant plant tissue enzymes and its extent of thermal inactivation has been shown to parallel that of enzymes responsible for off-flavour formation and undesirable browning that occur in unblanched or under blanched, frozen or dehydrated vegetables and fruits. The likelihood that enzymic browning will occur in these products can be determined using the peroxidase test. The peroxidase test is used to determine the adequacy of blanching of frozen vegetables by determining the activity of the enzyme peroxidase. The method is based on the oxidation of guaiacol by peroxidase to give a reddish-brown pigment.
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METHOD Perform test on unblanched (ie. fresh) and blanched vegetables (ie frozen).
To ensure adequate sampling, take 50 g of the largest pieces or parts of the vegetables since these are the most likely to be under blanched. Blend with 150 mL distilled water for 1 min at moderate speed, and then filter through cotton wool. Sample: Add 2 mL of filtrate to 20 mL distilled water for sample. Blank: Add 2 mL of filtrate to 22 mL distilled water for blank. To the sample add 1 mL of 0.05% guaiacol solution (in 50% alcohol) and 1 mL of 0.08% hydrogen peroxide solution. Mix well and watch for any colour difference from blank. If no difference after 3.5 min, then blanching is adequate. Disregard colour developing after 3.5 min.
QUESTIONS 1. Outline the mechanism of the reaction of the enzyme, peroxidase with the substrate, guaiacol that leads to the formation of the red pigment. Why are some enzymes more stable to thermal treatment than others? Name three enzymes that cause problems in frozen vegetables if they are not inactivated. Describe the reactions involved.
2. 3.
REFERENCES DArcy, B.R. Web/CD Notes for Food Chemistry, de Mann, J. M. (1999) Principles of Food Chemistry 3rd Ed. (Aspen Publishers, Inc.: Gaithersburg, Maryland, USA). Fennema, O. (1996). Food Chemistry 3rd Ed. (Marcel Dekker: N.Y.) Miller, D. D. (1998) Food Chemistry: A laboratory manual. (John Wiley & Sons. Inc. : New York, USA). Nielsen, S. S. (ed.) (1998) Food Analysis 2nd Ed. (Aspen Publishers, Inc.: Gaithersburg, Maryland, USA).
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Food Chemistry
PROPERTIES OF LIPIDS
INTRODUCTION The lipids are a naturally occurring group of substances insoluble in water, but soluble in organic solvents. They consist of fats, oils, waxes, steroids, fatty acids, phospholipids, etc. There is no general chemical test for the presence of lipids, but a greasy diethyl ether extract residue and the absorption of the red dye Sudan III by an diethyl ether extract/water emulsion indicates their presence. Fats, fatty acids and lecithin give a translucent stain when smeared on paper (Grease Spot Test), while steroids, phospholipids and carotene have individual identification tests. Lipids of all types are widely distributed both in plant and animal material. From a food science point of view, the most important group is that of the fats and oils (differing only in their melting point) which not only occur widely in food but are also extracted and incorporated in the production of other foods such as baked goods, spreads, etc. The physical and chemical properties of these lipids differ widely and are of great importance in food processing as they influence the texture, taste and storage stability of the food. Fats and oils and higher lipids are characterized by their extreme insolubility in water and their solubility in organic solvents such as diethyl ether or petroleum spirit (alkanes). Most other compounds in foods are insoluble in these solvents and thus the term "crude fat" or "ether extract" is employed to cover all the compounds that are solvent extracted. These include small amounts of waxes, resins, plant pigments, steroids etc., together with fats and oils. The fat content of fresh food varies greatly. Most fruits and vegetables, except avocados (26%) and olives (17%), have less than 1% crude fat while fish and meat vary from less than 1% in cod and haddock to over 20% in some fish and most meat cuts. Milk contains about 3.6% fat while some dairy products, such as cheese and butter, contain up to 82% fat. Nuts are traditionally high in fat (35 - 70%).
METHOD A. SOLVENT EXTRACTION METHOD All methods use extraction with organic solvents; thus, grinding and thorough mixing gives a better extraction and a more representative sample. N.B. Take great care when using solvents especially diethyl ether and petroleum spirit as a flame is not necessary for their ignition; a hot plate is sufficient.
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Soxhlet Extraction Weigh out approx. 10 g of the biscuit sample accurately (to 2 decimal places) into a soxhlet thimble. Close the thimble top with cotton wool. Weigh again. Extract the thimble in the soxhlet apparatus for 2 h with petroleum spirit (50 - 70) contained in a round-bottom flask. N.B. Weigh the round-bottom (RB) flask containing a small amount of anti-bumping granules (2-3 boiling chips) before the extraction. After extraction, distil off the solvent from the RB flask using the rotary evaporator, then dry the oil in the flask in an oven at 70 C for a maximum of 10 min, remove from the oven, cool to room temperature, and weigh the flask and the extracted oil.
B. CHEMICAL QUALITY TESTS FOR FATS AND OILS 1. IODINE VALUE (IV)
The iodine value of an oil or fat is defined as the mass of iodine absorbed by 100 g of the sample. The unsaturated fatty acid residues of the glycerides react with iodine, and thus the iodine value indicates the degree of unsaturation of the fatty acid residues of the glycerides. It is constant for a particular oil or fat, but depends on the method used. Animal fats (butter, dripping, lard) Non-drying oils (olive, almond) Semi-drying oils (cottonseed, sesame, soya) Drying oils (linseed, sunflower) 30 - 70 80 - 110 80 - 140 120 - 200 Iodine Value Iodine Value Iodine Value Iodine Value
The iodine value is often most useful in identifying the source of an oil. Generally, the higher iodine values indicate oils and the lower values fats. Iodine values are normally determined using Wigs or Hanus methods. HANUS METHOD Weigh out accurately a suitable quantity of oil or fat (0.2 g fat or 0.1 g oil) into a 250 mL conical flask, and then add 10 mL of chloroform to dissolve the oil. In a second 250 mL conical flask prepare a blank of 10 mL of chloroform (no oil). Using a safety pipette, add 10 mL of Hanus iodine solution to both flasks, and allow to stand for exactly 30 min with occasional shaking. Then add 15 mL of 15% potassium iodide solution and 40 mL of distilled water. Titrate the unreacted iodine with 0.1 M Na2S2O3, adding it gradually with constant shaking until the yellow colour has nearly disappeared. Then, add a few drops of starch indicator (Vitex) and continue titration until the blue colour has disappeared, shaking well all the time to take up residual iodine. Repeat with blank (no oil or fat present). The difference between the blank (largest titration volume) and the test sample gives the amount of iodine absorbed by the oil. Calculate Hanus Iodine Value as g iodine taken up per 100 g oil. (Titre: 1 mL 0.1 M Na2S2O3 = 0.0127 g iodine)
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Food Chemistry
2.
SAPONIFICATION VALUE (SV)
The saponification value of an oil or fat is defined as the number of mg of potassium hydroxide (KOH) required to neutralize the fatty acids resulting from the complete hydrolysis of 1 g of the sample. Its value is inversely proportional to the mean molecular mass of the fatty acids present. Most oils have similar values (eg. olive oil series 188 - 196) but the presence of coconut oil (SV 255), palm-kernel oil (SV 247) and butter fat (SV 225) which contain a high proportion of the lower molecular weight fatty acids may be detected. Weigh 2 g of oil into a 250 mL quick fit round-bottom flask. Add 25 mL (using a volumetric pipette) of 0.5 M alcoholic potassium hydroxide solution and 2-3 boiling chips. Boil gently under reflux for 40 min. Make sure there is one phase in the contents; if not, continue refluxing. Add 1 mL of phenolphthalein (1%) solution and titrate while hot (in the round-bottom flask) the excess alkali with 0.5 M HCl. Next, carry out a blank determination (use only 25 mL 0.5 M alcoholic KOH with no fat or oil present); heat using a hot plate (refluxing as for the oil sample is not required). Calculate the saponification value expressed as mg KOH required per g of oil, by first determining the difference between the blank (largest titration volume) and the test sample. Titre: 1 mL of 0.5 M HCl = 28.05 mg KOH
The saponification value/number gives an indication of the average molecular mass of the glycerides, while the iodine value/number gives an indication of the degree of unsaturation (the number of double bonds) of the fatty acid residues of the glycerides in the oil.
3.
FREE FATTY ACIDS (FFA) OR ACID VALUE
The acid value of an oil and fat is defined as the number of mg of KOH required to neutralize the free fatty acid in 1 g sample, or the % by mass (g/100 g) of free fatty acid present. With freshly refined oils and fats, the FFA should be very low but oils having a FFA (as oleic acid) of about 0.3 - 1.5% are noticeably rancid to the palate. Weigh out 6 g oil or 20 g of fat into a 250 mL conical flask. In a second 250 mL conical flask heat and neutralize 100 mL of 95% ethanol with 0.1 M KOH (1-2 drops of KOH should be enough), using 0.5 mL of 1% phenolphthalein indicator. Add the hot neutralized alcohol (50 mL) to the fat, boil and titrate hot with 0.1 M KOH (from a 10 mL burette) using vigorous stirring until a definite pink colour persists for 15 s. Express result as: Acid Value: mg KOH required per g of oil. (Titre: 1 mL 0.1 M KOH = 5.6 mg KOH)
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or as % Free Fatty Acids calculated as oleic acid (g oleic acid per 100 g of oil) (Titre: 1 mL 0.1 M KOH = 0.028 g oleic acid) ie. % FFA = Acid Value / 2 (for oleic acid)
Determine if the oil or fat is rancid enough to be discarded by using the level of % FFA (as oleic acid).
QUESTION Comment on the quality of the oil tested by referring to the literature for the values for foods studied.
REFERENCES AOAC International. (1995). Official Methods of Analysis. 16th Ed. (AOAC International: Gaithersburg, MD) Fennema, O. (1996). Food Chemistry 3rd ed. (Marcel Dekker: N.Y.) Nielsen, S. S. (ed.) (1998) Food Analysis 2nd Ed. (Aspen Publishers, Inc.: Gaithersburg, Maryland, USA).
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Food Chemistry
NONENZYMIC BROWNING MAILLARD REACTION
INTRODUCTION Nonenzymic browning is purely chemical and occurs by several mechanisms involving reactive sugars and amino compounds (eg. lysine). Nonenzymic browning is found in food processing operations involving dehydration/concentration (powdered / condensed milk). The extent of this browning will increase with concurrent increase in process pH and/or temperature. In both cases, complex "melanoidin" browning polymers are formed. These may have a significant effect upon the colour, flavour and nutritive value of the processed food product. When reducing sugars (eg. glucose, fructose, lactose, maltose) and amino compounds (eg. amines, amino acids, proteins) are present together in a system (eg. a food), browning reactions may occur. These reactions are not enzyme-catalysed but are affected by temperature, concentration of reactants, pH, inhibitors or preservatives, etc. Browning reactions may be desirable (eg. crust on baked goods, colour of caramel, flavour, colour of maple syrup) or detrimental (darkening of dehydrated fruits, eggs, vegetables, canned or dried milk) to the food. The following experiments attempt to gauge the effect of several variables and the rate of browning in a model sugar-amine browning system.
METHOD
1. Trial of the Model System A model browning system is as follows: To a test tube, add 4 mL 50 Brix invert sugar + 1 mL 10% glycine + 1 mL pH 8 buffer. Mix well, cover the tops of the tubes with foil and incubate in a water bath at 100 C for 30 min. The colour produced may be read at 420 nm on the spectrophotometer. The tubes may have to be diluted so that a reading can be obtained on the spectrophotometer. If this is necessary, add the same amount of dilutent to all tubes, about 10 mL distilled water is usually needed. The above is only a model for the 10 mini experiments to be performed below.
2. Final Experiments Using the Model System Using the above model system, set up the following 10 tubes (all of the same size) and record your results in a table.
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Tubes 1-16: Effect of pH: 3, 4, 4.5, 5, 6, 7, 8, 8.5 at 100 C for 30 min in duplicate. After heating for 30 min, quickly place in a beaker of cold water for 5 mins than in an ice bath for 15 mins to bring the temperature to room temperature as quickly as possible. All the buffers used were prepared using phosphate solutions. The preparation of the reagents for Tubes 1-16 should be done in a randomized manner, and the final absorption measured in the same randomized manner using the spectrophotometer at 420 nm. Tubes 1-16, 18 and 19 should go in the one large 2 L beaker of boiling water on a hotplate. Following the 30 min heating at 100 C, the tubes should be removed from the hot water, placed in tap water in a large beaker for 5 mins and then in an ice bath for 15 min to cool quickly to room temperature. Do not cool to below room temperature as this will lead to problems with the spectrophotometric measurements (fogging of the cuvette).
Tubes 17:
Effect of temperature: 70C C at pH 8 for 30 min.
Place tube 17 in a constant temperature water bath set at 70 C.
Tube 18:
4 mL 50 Brix invert sugar + 1 mL of 10 % glycine + 1 mL pH 8 buffer + 0.1 g sodium metabisulphite, at 100 C for 30 min.
Tube 19:
4 mL 50 Brix invert sugar + 1 mL of distilled water (instead of glycine) + 1 mL pH 8 buffer, at 100 C for 30 min.
Tube 20 (blank): 4 mL 50 Brix invert sugar + 1 mL of distilled water (instead of glycine) + 1 mL pH 8 buffer, at 25 C for 30 min. The blank is used to calibrate the absorbance to zero on the UV/Vis spectrophotometer before each measurement.
RESULTS From your results and from the literature, discuss the effect of the variables tested on nonenzymic browning. Determine at what pH the Maillard reaction rate reaches a constant level.
QUESTIONS 1. 2. 3. Use chemical structures to outline the major mechanisms of non-enzymic browning. What are the possible nutritional implications of non-enzymic browning? (Use chemical structures.) Plot absorbance versus pH, and explain the effect of pH.
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Food Chemistry
4.
Give three examples each of desirable and undesirable nonenzymic browning reactions in food systems.
REFERENCES Coultate, T. P. (1995). Food: The Chemistry of its Components. 3rd ed. (Royal Society of Chemistry: London). DArcy, B.R. Web/CD Notes for Food Chemistry, de Mann, J. M. (1999) Principles of Food Chemistry 3rd Ed. (Aspen Publishers, Inc.: Gaithersburg, Maryland, USA). Fennema, O. (1996). Food Chemistry 3rd ed. (Marcel Dekker: N.Y.) Miller, D. D. (1998) Food Chemistry: A laboratory manual. (John Wiley & Sons. Inc. : New York, USA).
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Food Chemistry
ASCORBIC ACID DETERMINATION

DETERMINATION OF ASCORBIC ACID BY REDOX TITRATION Besides being an important vitamin, ascorbic acid is a sugar acid and a powerful reducing agent. The most common assay of ascorbic acid in foods is based on its oxidation to dehydroascorbic acid by the redox dye 2,6-dichlorophenol (indophenol) which is blue in neutral solution and pink in acid. The titration is carried out in acid conditions and at the end point the dye appears rose pink.
1.
VITAMIN C DETERMINATION OF FRUIT JUICES
Fruit juice samples (2 mL) are to be analysed.
METHOD
Extracting Solution - 10% acetic acid. This will keep in the refrigerator for 7 - 10 days. Ascorbic Acid Standard - 1 mg / 1 mL freshly made up. Each student prepares their own 100 mL of standard. Indophenol Solution - 50 mg in 200 mL distilled water (this solution is prepared). a. Standardize in triplicate the indophenol solution by rapidly titrating against 2 mL aliquots of standard ascorbic acid solution plus 5 mL of extracting solution until a distinct rose-pink persists for at least 5 s. Express standardized indophenol solution as mg ascorbic acid equivalent per 1 mL of dye.
b.
Titrate in triplicate 2 mL aliquots of the fruit juices (which should contain between 10 100 mg ascorbic acid / 100 mL) plus 5 mL extracting solution using the standardized indophenol solution. Take the average of the three titrations, subtract a blank titration (no food sample), and calculate the ascorbic acid as mg / 100 mL by the following method: (mean sample titration volume blank titration volume) x (standardized mg AA/mL dye) x 100 2 2. VITAMIN C DETERMINATION OF POTATOES
The above method for Vitamin C determination is very useful for fruit juices etc, but a modified method will be used for determining the Vitamin C content in a potato.
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METHOD Standardize the indophenol dye as per above method (no need to repeat if already done in (1) above at the same practical session). Note, perform a blank. Peel a potato and cut into two pieces. Weigh and cook one piece in distilled water until soft (15 - 20 min). Weigh the other piece and blend for 1 min with approximately twice its mass of extracting solution, eg. if the potato weighed 32.65 g, blend with 65 mL or 70 mL of extracting solution. It is important to know the exact amount of extracting solution used. Filter using a buchner funnel and cotton wool. Titrate 20 mL aliquots of the extract using the standardized indophenol solution. Blend the cooked potato in extracting solution equal to 4 times its mass, and titrate a 20 mL aliquot as above for the raw potato. Continue as above for the raw potato.
CALCULATIONS FOR ASCORBIC ACID DETERMINATION 1. * STANDARDIZATION OF DYE (TITRE DETERMINATION) STD ascorbic acid solution is prepared = 1 mg / mL. * 2 mL aliquot of STD ascorbic acid solution is titrated with indophenol. in 2 mL there are 2 mg of ascorbic acid (the exact amount will depend on how much you actually weighed out). * If the volume of indophenol used = 17 mL
then 1 mL of indophenol reacts with 2 mg ascorbic acid 17 = 0.1176 mg ascorbic acid ie. Titre: 1 mL indophenol = 0.1176 mg ascorbic acid
2.
CALCULATION OF ASCORBIC ACID (AA) IN POTATOES Volume of dye = 5 mL (from titration) mass of ascorbic acid in final sample solution (20 mL) = 5 x 0.1176 = 0.588 mg
If 20 mL sample taken from a solution of 30 g potatoes + 60 mL acetic acid extracting solution (total 90 mL):
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Food Chemistry
Then mass of AA in original solution
= 0.588 x 90 mg 20 = 2.65 mg
But this 2.65 mg is actually in 30 g of potato: 2.65 x 100 mg in 100 g potato 30 = 8.83 mg/100g Calculate the ascorbic acid content of raw and cooked potatoes in units of mg / 100 g
3.
% Vitamin C Loss
mg/100 g RAW - mg/100 g COOKED x 100 mg/100 g RAW
Calculate the % loss in vitamin C on cooking potatoes. QUESTIONS 1. How does your values for ascorbic acid content of raw and cooked potatoes compare to food composition tables? Comment on the effect of boiling potatoes in water on the Vitamin C content, and its possible nutritional importance.
2.
REFERENCES Australian Food Composition Tables. DArcy, B.R. Web/CD Notes for Food Chemistry, de Mann, J. M. (1999) Principles of Food Chemistry 3rd Ed. (Aspen Publishers, Inc.: Gaithersburg, Maryland, USA). Holland, B., Welch, A. A., Unwin, I. D., Buss. D. H., Paul, A.A. and Southgate, D. A. T. (1991) McCance and Widdowsons The Composition of Foods. (Royal Society of Chemistry: Cambridge). Miller, D. D. (1998) Food Chemistry: A laboratory manual. (John Wiley & Sons. Inc. : New York, USA). Paul, A. A. and Southgate, D. A. T. (1988). The Composition of Foods 4th Rev. Ed. (Elsevier: Amsterdam).
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Food Chemistry
ASH DETERMINATION
INTRODUCTION Ash may be regarded as the inorganic residue remaining after oxidation of the organic matter in a sample. The composition of ash will vary according to the nature of the food material but usually consists of the more common metals ions in the form of their oxides, sulphates, phosphates, silicates and chlorides. Oxidation may be conducted by incineration over an open flame, in a muffle furnace or by wet combustion with mixtures of sulphuric, nitric and perchloric acids. The average ash contents of fresh food ranges from 0.0% for eggs, white sugar and oils, to as high as 6.0% for green olives and bacon. Ash content can be an index of the quality of a product such as in flour, or indicate adulteration of the sample, as in spent tea. For these purposes, incineration at fairly low temperatures (550C - 600C) is used to avoid loss of volatile chlorides. Wet ashing is used when determining mineral constituents since less loss of volatiles occurs. The official method states that ashing is required on meat products before a salt analysis can be performed.
METHOD You are provided with samples of two different types of common flour. Use the methods below to find the adulterated samples and the type of adulteration. Use two flour samples in duplicate (four samples) 1. DETERMINATION OF TOTAL ASH CONTENT
AOAC recommends using a quantity of material representing at least: 2 g of dry matter for fish products, grain and stock feed. 3 5 g of cereal food, milk or cheese. 5 10 g of sugar, sugar products, meat or vegetable products. 25 g of fruit juice, fresh or canned fruit. 10 g for jellies, syrups, preserves. NOTE: Use the four decimal place analytical balances throughout this practical exercise. The weight differences are too small and will not be measurable if a two decimal place balance is used.
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Using a marker pen, number four silica crucibles. Weigh into the numbered crucibles the flour sample (3 g) in duplicate. Char the sample (until no visible smoke is present) on a hot plate in the fume cupboard. Once charred, place the crucibles on a heat resistant mat and transfer to a muffle furnace heated to 550 C. DO NOT place the hot crucibles on the painted surface of the fume cupboard. Place the dishes in the muffle furnace in a known order in case the numbers are erased. Leave in the muffle furnace for 4 h until the ash is white or greyish-white. If the material fails to ash properly, cool the dish and moisten the ash with distilled water prior to reheating. After incineration, cool in a desiccator and reweigh (use tongs). Determine the percentage of ash in the sample. 2. WATER SOLUBLE ASH CONTENT
This is of value in determining added mineral matter, particularly in the analysis of fruit and sugar products. Add 20 mL of distilled water to the flour ash and heat until nearly boiling. Filter through an ashless filter paper into a numbered conical flask, and wash with an equal volume of hot distilled water. Keep the filtrate for determining alkalinity of ash. Place the filter paper in the silica crucible and ignite in the fume cupboard. If the paper catches fire, cover the crucible with a watch glass (or other suitable container). Care must be taken to ensure that the ash does not float out of the crucible. Once the papers have ashed, cover the crucible with a watch glass and transfer them to the muffle furnace for 10 min heating at 550 C. Remove the watch glasses before placing the crucibles in the furnace. Cool and reweigh. From the mass, calculate the amount of water insoluble ash, and by difference, the amount of water soluble ash. Next determine the % water soluble ash in the flour sample(s) used.
3.
ACID INSOLUBLE ASH CONTENT
This is of value in detecting adulteration of spices and confectionery with dirt and talc etc. Add 25 mL of 10% HCl (not 0.1 M HCl) to the water insoluble ash. Cover with a watch glass and boil gently over low heat for 5 min. Filter through ashless filter paper and wash with hot distilled water. Follow the procedure for water soluble ash for igniting the paper and determine the amount of acid insoluble ash. Finally, calculate the % acid insoluble ash in the flour(s). Note, 10% HCl is not the same as 0.1 M HCl.
4.
ALKALINITY OF ASH
This is of value in detecting adulteration of foods with minerals and in determining the acidbase balance of the food.
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Food Chemistry
Add a measured excess of standard 0.1 M HCl (usually 25 mL and use a volumetric pipette) to the filtrate from the water soluble ash. Add a few drops of methyl orange and back titrate the excess acid with standard 0.1 M NaOH. By difference, determine the volume (ml) of 0.1 M HCl required to neutralize the alkali in the flour ash (1 mL of NaOH neutralizes 1 mL of 0.1 M HCl). Express the alkalinity as the number of mL of 0.1 M acid required to neutralize the ash from 100 g of sample.
QUESTIONS 1. 2. Why use standard solutions and volumetric glassware for alkalinity of ash? Determine the AOAC Method Number for the ash determination of flour using the most up-to-date AOAC reference in the UQ libraries.
REFERENCES AOAC International. (1995). Official Methods of Analysis. 16th Ed. (AOAC International: Gaithersburg, MD) Maynard, (1970). Methods in Food Analysis, 2nd Ed. (Academic Press, London). Nielsen, S. S. (ed.) (1998) Food Analysis 2nd Ed. (Aspen Publishers, Inc.: Gaithersburg, Maryland, USA).
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Food Chemistry
ENZYMIC BROWNING: ACTIVITY OF FRUIT HOMOGENATES

INTRODUCTION Enzymatic browning of cut or damaged fruits and vegetables is due to the action of polyphenol oxidases (collectively called phenolase) on phenolic substrates; the reaction occurs at room temperature. These enzymes catalyse the oxidation of phenolic substrates to quinones which subsequently polymerize to give brown-black pigments. For oxidative browning to occur, three components must be present, namely: enzyme, substrate and oxygen. A number of methods are used both commercially and domestically to inhibit this reaction. These include destruction of the enzyme by heat, elimination of oxygen, control of pH, and the use of chemical inhibitors. The standard method for examining enzymic browning in-vitro is to use a model system consisting of fruit homogenate as the enzyme preparation and the phenolic compound, catechol as the substrate.
METHOD Each student is to do tests on one fruit extract at three different pHs. 1. Tissue Extract Preparation Prepare tissue extracts of fresh fruits, eg. Apple (e.g. a pink lady), pear, or banana etc., by blending 10 g of fruit with 100 mL of distilled water, then filtering using a buchner funnel. It is not necessary to filter the whole extract since only a few mL are required. Extracts should be clear and MUST be stored in an ice bath for a short time (same practical session only). 2. Measurement Oxidation Rate The method is based on the oxidation of catechol at pH 6 and 25C, with the colour development being assessed by absorption at 420 nm in a spectrophotometer. All buffer solutions need to be removed from the refrigerator at least 3 h prior to use, and then allowed to warm to room temperature. Switch on a spectrophotometer and allow to warm up for 10 minutes. Set the wavelength to 420 nm, and zero the instrument using a blank consisting of 10 mL 0.01 M citrate buffer pH 6, 1 mL 0.5 M catechol, and 0.5 mL distilled water. Once the instrument is zeroed, prepare the sample test tubes near the spectrophotometer so that there is no delay in placing the sample in the instrument. Place in a clean test tube, 10 mL 0.01 M citrate buffer, 1 mL catechol and 0.5 mL of the enzyme solution of clear tissue extract. It is important to add the fruit extract last.
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Mix well, quickly transfer the solution to a cuvette, place the cuvette in the spectrophotometer, and read the absorption every 0.5 min for the first 3 min, including at 0 min. Then record every minute until 10 min when activity should have ceased. Start timing from when the enzyme solution is first added to the test tube (time zero). You must be quick with the transfer to the cuvette to record the 0 min and 0.5 min initial readings.
3. Effect of pH Change on Enzyme Activity Repeat the exercise using pHs 3, 4, 5 and 7, 8 buffer solutions instead of the pH 6 one. New separate blanks (with each pH) are required. Perform these parts of the practical exercise for one fruits only. Plot these results on the same graphs as those for the pH 6 part, with one graph (ie. pHs 3-7) for each fruit extract. Next share the results with other members of your practical group so you have all the data for the three different fruit homogenates (i.e. apple, pear and banana).
4. Effect of Added Sodium Bisulphite and Sodium Chloride on Enzyme Activity Next, repeat the exercise twice at pH 6, once with added sodium bisulphite (50 ppm) and once with sodium chloride (1000 ppm). All solutions must be freshly prepared. (a) Effect of added sodium chloride on enzyme activity: In one tube, add 0.2 mL of the concentrated NaCl solution (115,000 ppm) to the model system sample to achieve 2000 ppm Cl ions. Perform this exercise for one fruit. Additionally, a new blank (1000 ppm chloride) needs to be prepared by adding 0.2 mL of the concentrated NaCl solution (115,000 ppm) as part of the preparation of the pH 6 blank detailed earlier. (b) Effect of added sodium bisulphite on enzyme activity: In another tube, add 0.2 mL of the concentrated sodium bisulphite solution (5750 ppm) to the model system sample to achieve 100 ppm sulphite. Perform this exercise for one fruit. Additionally, a new blank (100 ppm sulphite) needs to be prepared by adding 0.2 mL of the concentrated sodium bisulphite solution (5750 ppm) as part of the preparation of the pH 6 blank detailed above.
RESULTS 1. Plot absorbance (y axis) versus time (x axis) for each pH and each fruit homogenate (each fruit on a different graph). Also include the plots for the NaCl and bisulphite trials with the plots of the 5 different pHs. Next, determine and tabulate the polyphenol oxidase activity (where the slope is greatest) of the three fruit extracts at pHs 3-7 (express as increase in absorption / minute for the first 2 minute period, i.e. A2 min A1 min) from the graphs (results for each fruit homogenate are to be shared between group members).
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Food Chemistry
2.
Determine the effect of increased acidity on polyphenol oxidase activity, eg. effect of pH by plotting activity (A2 min A1 min) versus pH (x axis) on another graph for each fruit. What is the optimum pH for each fruit homogenate? Determine the effect of added sulphite and chloride ions on polyphenol oxidase activity. Why is there a difference in the inhibition of sulphite ions relative to chloride ions? Discuss why the graph obtained in (1) above flattens off with time. Comment on your results.
3.
4. 5.
QUESTIONS 1. Quote the Australian and New Zealand Food Standards Code for the permitted levels of sodium bisulphite in various food stuffs. Show the chemical structure of sodium bisulphite and the mechanism of action as an anti-browning agent. Show the mechanism of action of polyphenol oxidase in the formation of enzymic browning pigments. Is bisulphite an effective inhibitor of enzymic browning, and why?
2.
3.
REFERENCES Coultate, T. P. (1995). Food: The Chemistry of its Components. 3rd ed.(Royal Society of Chemistry: London). DArcy, B.R. Web/CD Notes for Food Chemistry, de Man, J. (1990). 'Principles of Food Chemistry'. 2nd ed. (Van Nostrand Reinhold: New York). Fennema, O. (1996). Food Chemistry 3rd ed. (Marcel Dekker: N.Y.) Furia, T. E. (1972). 'Handbook of Food Additives'. 2nd ed. (C.R.C. Press: Ohio). Miller, D. D. (1998) Food Chemistry: A laboratory manual. (John Wiley & Sons. Inc. : New York, USA).
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Food Chemistry
APPENDIX 1
TABLES FOR CONVERTING SPECIFIC GRAVITY TO % TOTAL SOLUBLE SOLIDS (BRIX)
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Food Chemistry
APPENDIX 2
LANE EYNON TABLES FOR CONVERTING TITRATION VOLUMES OF SUGAR SOLUTION TO INVERT SUGAR CONCENTRATION
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Food Chemistry

Food Chemistry Pratical Manual

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FOOD CHEMISTRY

Bruce R. DArcy Geoff Hawes Ian Bentley

A University of Queensland Publication

Copyright 2006 by The University of Queensland All rights reserved

The University of Queensland

Food Chemistry A Practical Manual

Food Chemistry A Practical Manual

The University of Queensland

Food Chemistry A Practical Manual

Food Chemistry A Practical Manual

The University of Queensland

Food Chemistry A Practical Manual

pH AND TITRATABLE ACIDITY OF ORANGE JUICES

PREPARATION AND STANDARDIZATION OF 0.1 M SODIUM HYDROXIDE

Food Chemistry A Practical Manual

The University of Queensland

Food Chemistry A Practical Manual

ANALYSIS OF ORANGE JUICES

Titratable Acidity Determination

Ensure you do not forget about the dilution factor.

Food Chemistry A Practical Manual

The University of Queensland

Food Chemistry A Practical Manual

PRACTICAL EXERCISE NO. 2

Food Chemistry A Practical Manual

The University of Queensland

Food Chemistry A Practical Manual

1 mL 0.1 M AgNO3 = 0.005846 g NaCl.

Food Chemistry A Practical Manual

The University of Queensland

Food Chemistry A Practical Manual

PART A. DETERMINATION OF TOTAL SOLUBLE SOLIDS (TSS)

Food Chemistry A Practical Manual

The University of Queensland

(iv) Measure the temperature of the solutions.

Food Chemistry A Practical Manual

DETERMINATION OF % INVERT SUGAR

Food Chemistry A Practical Manual

The University of Queensland

Rauch, G.H., (1965), Jam Manufacture. (Leonard Hill: London).

Food Chemistry A Practical Manual

Direct Drying in an Oven

Food Chemistry A Practical Manual

The University of Queensland

Direct Drying in an Oven with Sand

Loss in mass x 100 Mass of sample

Determination of Moisture by the Vacuum Oven Method

Food Chemistry A Practical Manual

Determination of Moisture by the Infrared Balance Method

Determination of Moisture by the Hot-Plate Method

Food Chemistry A Practical Manual

The University of Queensland

Calculation Moisture % w/w = Volume of water x 99.7 Mass of sample

Food Chemistry A Practical Manual

PRACTICAL EXERCISE NO. 5

PART A SEMI-MICRO KJELDAHL DETERMINATION OF TOTAL ORGANIC NITROGEN

Food Chemistry A Practical Manual

The University of Queensland

OPERATING INSTRUCTIONS FOR GERHARDT DISTILLATION UNIT 1. Turn on cooling water.

Food Chemistry A Practical Manual

PART B BIURET DETERMINATION OF SOLUBLE PROTEIN

Food Chemistry A Practical Manual

The University of Queensland

Final Egg White Sample (Soln. 6) ? mL ? mL 8 mL ??