1. 1. Current Protocols in Food Analytical Chemistry Table of contents Foreword Preface Contributors A Water B Proteins C Enzymes D Lipids E Carbohydrates F Pigments and Colorants G Flavors H Textural/Rheology Appendices Index Detailed table of contents A Water A1 Gravimetric Measurements of Water A1.1 Gravimetric Determination of Water by Drying and Weighing A1.2 Karl Fischer Titration A1.3 Application of Low-Resolution NMR for Simultaneous Moisture and Oil Determination in Food (Oilseeds) A2 Vapor Pressure Measurements of Water A2.1 Factors to Consider When Estimating Water Vapor Pressure A2.2 Dew-Point Method for the Determination of Water Activity A2.3 Measurement of Water Activity using Isopiestic Methods A2.4 Direct Manometric Determination of Vapor Pressure A2.5 Measurement of Water Activity by Electronic Sensors (New, February 2002) B Proteins B1 Measurement of Protein Content B1.1 The Colorimetric Detection and Quantitation of Total Protein B1.2 Determination of Total Nitrogen B1.3 Spectrophotometric Determination of Protein Concentration (New, May 2002) B2 Biochemical Compositional Analyses of Proteins B2.1 Analyses of Protein Quality B2.2 Evaluation of the Progress of Protein Hydrolysis (New, May 2002) B3 Characterization of Proteins B3.1 Electrophoresis Analysis B3.2 Electroblotting from Polyacrylamide Gels B3.3 Detection of Proteins on Blot Membranes
2. 2. B3.4 Immunoblot Detection B4 Purifcation of Proteins B4.1 Overview of Protein Purification and Characterization (New, February 2002) B4.2 Overview of Conventional Chromatography (New, February 2002) C Enzymes C1 Strategies For Enzyme Activity Measurements C1.1 Strategies for Enzyme Activity Measurements C2 Proteolytic Enzymes C2.1 Activity Measurements of Proteases using Synthetic Substrates C2.2 Peptidase Activity Assays Using Protein Substrates (New, February 2002) C3 Lipolytic Enzymes C3.1 Lipase Assays C4 Oxidoreductases C4.1 Polarographic and Spectrophotometric Assay of Diphenol Oxidases (Polyphenol Oxidase) C4.2 Analysis of Lipoxygenase Activity and Products D Lipids D1 Lipid Composition D1.1 Extraction and Measurement of Total Lipids D1.2 Analysis of Fatty Acids in Food Lipids D1.3 Cholesterol D1.4 Oil Quality Indices D1.5 Analysis of Tocopherols and Tocotrienols (New, May 2002) D2 Lipid Oxidation/Stability D2.1 Measurement of Primary Lipid Oxidation Products D2.2 Chromatographic Analysis of Secondary Lipid Oxidation Products D2.3 Measurement of Oil Stability for Lipids D2.4 Spectrophotometric Measurement of Secondary Lipid Oxidation Products D3 Physical Properties of Lipids D3.1 Determination of Solid Fat Content by Nuclear Magnetic Resonance D3.2 Lipid Crystal Characterization D3.3 Emulsion Droplet Size Determination D3.4 Emulsion Stability Determination (New, February 2002) E Carbohydrates E1 Mono- and Oligosaccharides E1.1 Colorimetric Analyses E2 Starch and Starch Derivatives E2.1 Overview of Laboratory Isolation of Starch from Plant Materials
3. 3. E2.2 Enzymatic Quantitation of Total Starch in Plant Products E2.3 Determination of Total Amylose Content of Starch E3 Cell Wall Polysaccharides E3.1 Isolation of Plant Cell Walls and Fractionation of Cell Wall Polysaccharides E3.2 Determination of Neutral Sugars by Gas Chromatography of their Alditol Acetates E3.3 Determination of the Uronic Acid Content of Plant Cell Walls Using a Colorimetric Assay E3.4 Determining the Degree of Methylation and Acetylation of Pectin F Pigments and Colorants F1 Anthocyanins F1.1 Extraction, Isolation and Purification of Anthocyanins F1.2 Characterization and Measurement of Anthocyanins by UV-Visible Spectroscopy F1.3 Separation and Characterization of Anthocyanins by HPLC F2 Carotenoids F2.1 Extraction, Isolation and Purification of Carotenoids F2.2 Detection and Measurement of Carotenoids by UV/VIS Spectrophotometry F2.3 Chromatographic Techniques for Carotenoid Separation F2.4 Mass Spectroscopy of Carotenoids F3 Miscellaneous Colorants F3.1 Betalains F3.2 Spectrophotometric and Reflectance Measurements of Pigments of Cooked and Cured Meats F3.3 Measurement of Discoloration in Fresh Meat F4 Chlorophylls F4.1 Overview of Chlorophylls in Foods F4.2 Extraction of Photosynthetic Tissues: Chlorophylls and Carotenoids F4.3 Chlorophylls and Carotenoids: Measurement and Characterization by UV-VIS Spectroscopy F4.4 Chromatographic Separation of Chlorophylls F4.5 Mass Spectrometry of Chlorophylls F5 Strategies for Measuring Colors and Pigments F5.1 Overview of Color Analysis (New, February 2002) G Flavors G1 Smell Chemicals G1.1 Direct Sampling G1.2 Isolation and Concentration of Aroma Compounds G1.3 Identification and Quantitation of Aroma Compounds
4. 4. G1.4 Stereodifferentiation of Chiral Odorants Using High-Resolution Gas Chromatography G2 Acid Tastants G2.1 Titratable Activity of Acid Tastants G2.2 Liquid Chromatography of Nonvolatile Acids H Textural/Rheology H1 Viscosity of Liquids, Solutions, and Fine Suspensions H1.1 Overview of Viscosity and Its Characterization H1.2 Measurement of Viscosity of Non-Newtonian Fluids H1.3 Viscosity Determination of Pure Liquids, Solutions, and Serums Using Capillary Viscometry H1.4 Measuring Consistency of Juices and Pastes H2 Compressive Measurement of Solids and Semi-Solids H2.1 General Compressive Measurements H2.2 Textural Measurements with Special Fixtures H2.3 Texture Profile Analysis Appendices A.1 Abbreviations and Useful Data A Abbreviations Used in This Manual A.2 Laboratory Stock Solutions, Equipment, and Guidelines A Common Buffers and Stock Solutions B Laboratory Safety C Standard Laboratory Equipment A.3 Commonly Used Techniques A Introduction to Mass Spectrometry for Food Chemistry Supplier Appendix
5. 5. FOREWORD Accurate, precise, sensitive, and rapid analytical determinationsare as essentialinfood science and technology as in chemistry, biochemistry, and other physical and biological sciences. In many cases, the same methodologies are used. How does one, especially a young scientist, select the best methods to use? A review of original publications in a given field indicates that some methods are cited repeatedly by many noted researchers and analysts, but with some modifications adapting them to the specific material analyzed. Official analytical methods have been adopted by some professional societies, such as the Official Methods of Analysis (Association of Official Analytical Chemists), Official Methods and Recommendation Practices (American Oil Chemists Society), and Official Methods of Analysis (American Association of Cereal Chemists). The objective of Current Protocols in Food Analytical Chemistry is to provide the type of detailed instructions and comments that an expert would pass on to a competent technician or graduate student who needs to learn and use an unfamiliar analytical procedure, but one that is routine in the lab of an expert or in the field. What factors can be used to predetermine the quality and utility of a method? An analyst must consider the following questions: Do I need a proximate analytical method that will determine all the protein, or carbohydrate, or lipid, or nucleic acid in abiologicalmaterial? Or do I need to determine one specific chemical compound among the thousands of compounds found in a food? Do I need to determine one or more physical properties of a food? How do I obtain a representative sample? What size sample should I collect? How do I store my samples until analysis? What is the precision (reproducibility) and accuracy of the method or what other compounds and conditions could interfere with the analysis? How do I determine whether the results are correct, as well as the precision and accuracy of a method? How do I know that my standard curves are correct? What blanks, controls and internal standards must be used? How do I convert instrumental values (such as absorbance) to molar concentrations? How many times should I repeat the analysis? And how do I report my results with appropriate standard deviation and to the correct number of significant digits? Is a rate of change method (i.e., velocity as in enzymatic assays) or a static method (independent of time) needed? Current Protocols in Food Analytical Chemistry will provide answers to these questions. Analytical instrumentation has evolved very rapidly during the last 20 years as physicists, chemists, and engineershaveinvented highly sensitivespectrophotometers,polarometers, balances, etc. Chemical analyses can now be made using milligram, microgram, nanogram, or picogram amounts of materials within a few minutes, rather than previously when grams or kilograms of materials wererequiredbymultistepmethodsrequiringhours or days of preparation and analysis. Current Protocols in Food Analytical Chemistry provides state-of-the-art methods to take advantage of the major advances in sensitivity, precision, and accuracy of current instrumentation. How do chemical analyses of foods differ from analyses used in chemistry, biochemistry and biology? The same methods and techniques are often used; only the purpose of the analysis may differ. But foods are to be used by people. Therefore, methodology to determine safety (presence of dangerous microbes, pesticides, and toxicants), accept- ability (flavor, odor, color, texture), and nutritional quality (essential vitamins, minerals, amino acids, and lipids) are essential analyses. Current Protocols in Food Analytical Chemistry is designed to meet all these requirements. John Whitaker Davis, California Contributed by John Whitaker Current Protocols in Food Analytical Chemistry (2001) Copyright 2001 by John Wiley & Sons, Inc. i Current Protocols in Food Analytical Chemistry Current Protocols in Food Analytical Chemistry
6. 6. PREFACE Accurate and state-of-the-art analysis of food composition is of interest and concern to a divergent clientele including research workers in academic, government, and industrial settings, regulatory scientists, analysts in private commercial laboratories, and quality control professionals in small and large companies. Some methods are empirical, some commodity specific, and many have been widely accepted as standard methods for years. Others are at the cutting edge of new analytical methodology and are rapidly changing. A common denominator within this diverse group of methods is the desire for detailed descriptions of how to carry out analytical procedures. A frustration of many authors and readers of peer-reviewed journals is the brevity of most Materials and Methods sections. There is editorial pressure to minimize description of experimental details and eliminate advisory comments. When one needs to undertake an analytical procedure with which one is unfamiliar, it is prudent to communicate first-hand with one experienced with the methodology. This may require a personal visit to another laboratory and/or electronic or phone communication with someone who has expertise in the procedure. An objective of Current Protocols in Food Analytical Chemistry is to provide exactly this kind of detailed information which personal contact would provide. Authors are instructed to present the kind of details and advisory comments they would give to a graduate student or technician who has competent laboratory skills and who has come to them to learn how to carry out an analytical procedure for which the author has expertise. Some basic food analytical methods such as determination of Brix, pH, titratable acidity, total proteins, and total lipids are basic to food analysis and grounded in procedures which have had wide-spread acceptance for a long time. Such methods cannot be ignored and are included in this manual. Others, such as analysis of cell-wall polysaccharides (Chapter E3), analysis of aroma volatiles (Chapter G1), and compressive measurement of solids and semisolids (Chapter H2), require use of advanced chemical and physical methods and sophisticated instrumentation. These methods are particularly prone to rapid change and evolution. Current Protocols capitalizes on todays electronic communication technolo- gies and provides a mechanism for updates, additions, and revisions. The publication is available in loose-leaf binder, CD-ROM, or Online formats. Supplements are published quarterly. Users have the opportunity to provide feedback so that individual units can be clarified, modified, and expanded. Thus Current Protocols in Food Analytical Chemistry should be viewed as a dynamic resource which will be constantly up-dated. In organizing Currrent Protocols in Food Analytical Chemistry we chose to categorize on a disciplinary rather than a commodity basis. Included are chapters on water, proteins, enzymes, lipids, carbohydrates, colors, flavors, and textural components. We have made an effort to select methods which are applicable to all commodities. However, it is impossible to address the unique and special criteria required for analysis of all commodi- ties and all processed forms. There are several professional and trade organizations which focus on their specific commodities, e.g., cereals, wines, lipids, fisheries, and meats. Their methods manuals and professional journals should be consulted, particularly for special- ized, commodity-specific analyses. With respect to our Editorial Board, we have selected scientists who are widely regarded as being authorities in their field. Their research productivity is impressive, and they are all professors who are experienced in training undergraduate and graduate students, technicians, post-doctoral students, and visiting scientists in analytical procedures. This common experience provides insight regarding the types of experimental details needed to clarify how procedures are to be conducted. Supplement 10 Contributed by Ronald E. Wrolstad, Terry E. Acree, Haejung An, Eric A. Decker, Michael H. Penner, David S. Reid, Steven J. Schwartz, Charles F. Shoemaker, Denise M. Smith, and Peter Sporns Current Protocols in Food Analytical Chemistry (2003) iii-vi Copyright 2003 by John Wiley & Sons, Inc. iii Current Protocols in Food Analytical Chemistry
7. 7. HOW TO USE THIS MANUAL Format and Organization This publication is available in looseleaf, CD-ROM, and Online formats. For loose-leaf purchasers, a binder is provided to accommodate the growth of the manual via the quarterly update service. The looseleaf format of the binder allows easy insertion of new pages, units, and chapters that are added. The index and table of contents are updated with each supplement. Purchasers of the CD-ROM and Intranet versions receive a completely new disc every quarter and should dispose of their outdated discs. The material covered in all versions is identical. Subjects in this manual are organized by sections and chapters, and protocols are contained in units. Units generally describe a method and include one or more protocols with listings of materials, steps and annotations, recipes for unique reagents and solutions, and commentaries on the hows and whys of the method; there are also overview units containing theoretical discussions that lay the foundation for subsequent protocols. Page numbering in the looseleaf version reflects the modular arrangement by unit; for example, page D2.1.1 refers to Section D (Lipids), Chapter D2 (Lipid Oxidation/Stabil- ity), UNIT D2.1 (Measurement of Primary Lipid Oxidation Products), page 1 of that particular unit. Many reagents and procedures are employed repeatedly throughout the manual. Instead of duplicating this information, cross-references among units are used extensively. Cross-referencing helps to ensure that lengthy and complex protocols are not overbur- dened with steps describing auxiliary procedures needed to prepare raw materials and analyze results. Certain units that describe commonly used techniques and recipes are cross-referenced in other units that describe their application. Introductory and Explanatory Information Because this publication is first and foremost a compilation of laboratory techniques in food analytical chemistry, we have not offered extensive instructive material. We have, however, included explanatory information where required to help readers gain an intuitive grasp of the procedures. Some chapters begin with overview units that describe the state of the art of the topic matter and provide a context for the procedures that follow. Section and unit introductions describe how the protocols that follow connect to one another, and annotations to the actual protocol steps describe what is happening as a procedure is carried out. Finally, the Commentary that closes each protocol unit describes background information regarding the historical and theoretical development of the method, as well as alternative approaches, critical parameters, troubleshooting guidelines, anticipated results, and time considerations. All units contain cited references and many indicate key references to inform users of particularly useful background reading, original descriptions, or applications of a technique. Protocols Many units in the manual contain groups of protocols, each presented with a series of steps. The Basic Protocol is presented first in each unit and is generally the recommended or most universally applicable approach. Alternate Protocols are given where different equipment or reagents can be employed to achieve similar ends, where the starting material requires a variation in approach, or where requirements for the end product differ from those in the Basic Protocol. Support Protocols describe additional steps that are Preface Supplement 10 Current Protocols in Food Analytical Chemistry iv
8. 8. required to perform the Basic or Alternate Protocols; these steps are separated from the core protocol because they might be applicable to other uses in the manual, or because they are performed in a time frame separate from the Basic Protocol steps. Reagents and Solutions Reagents required for a protocol are itemized in the materials list before the procedure begins. Many are common stock solutions, others are commonly used buffers or media, whereas others are solutions unique to a particular protocol. Recipes for the latter solutions are supplied in each unit, following the protocols (and before the commentary) under the heading Reagents and Solutions. It is important to note that the names of some of these special solutions might be similar from unit to unit (e.g., SDS sample buffer) while the recipes differ; thus, make certain that reagents are prepared from the proper recipes. On the other hand, recipes for commonly used stock solutions and buffers are listed once in APPENDIX 2A. These universal recipes are cross-referenced parenthetically in the materials lists rather than repeated with every usage. Commercial Suppliers In some instances throughout the manual, we have recommended commercial suppliers of chemicals, biological materials, or equipment. This has been avoided wherever possible, because preference for a specific brand is subjective and is generally not based on extensive comparison testing. Our guidelines for recommending a supplier are that (1) the particular brand has actually been found to be of superior quality, or (2) the item is difficult to find in the marketplace. The purity of chemical reagents frequently varies with supplier. Generally reagent grade chemicals are preferred. Special care must be paid to procedures that require dry solvents. Different suppliers provide special anhydrous grade solvents which may vary in water content depending on the supplier. Addresses, phone numbers, facsimile numbers, and web addresses of all suppliers mentioned in this manual are provided in the SUPPLIERS APPENDIX. Safety Considerations Anyone carrying out these protocols will encounter hazardous or potentially hazardous materials including toxic chemicals and carcinogenic or teratogenic reagents. Most governments regulate the use of these materials; it is essential that they be used in strict accordance with local and national regulations. Cautionary notes are included in many instances throughout the manual, but we emphasize that users must proceed with the prudence and precaution associated with good laboratory practice, and that all materials be used in strict accordance with local and national regulations. Reader Response Most of the protocols included in this manual are used routinely in our own laboratories. These protocols work for us; to make them work for you we have annotated critical steps and included critical parameters and troubleshooting guides in the commentaries to most units. However, the successful evolution of this manual depends upon readers observa- tions and suggestions. Consequently, a self-mailing reader-response survey can be found at the back of the manual (and is included with each supplement); we encourage readers to send in their comments. Current Protocols in Food Analytical Chemistry Supplement 10 v Current Protocols in Food Analytical Chemistry
9. 9. ACKNOWLEDGMENTS This manual is the product of dedicated efforts by many of our scientific colleagues who are acknowledged in each unit and by the hard work of the Current Protocols editorial staff at John Wiley and Sons. The publishers commitment and continuing support for a food analytical chemistry manual were essential for realizing this ambitious project. We are extremely grateful for the critical contributions by Ann Boyle and Elizabeth Harkins (Series Editors) who kept the editors and the contributors on track and played a key role in bringing the entire project to completion. Other skilled members of the Current Protocols staff who contributed to the project include Tom Cannon Jr., Davide Dickson, Michael Gates, Tuan Hoang, Alice Ro, Liana Scalettar, Mary Keith Trawick, and Joseph White. The extensive copyediting required to produce an accurate protocols manual was ably handled by Allen Ranz, Amy Fluet, Tom Downey, and Susan Lieberman. KEY REFERENCES Association of Official Analytical Chemists (AOAC): Official Methods of Analysis, 2000. AOAC,Arlington, Va. A compilation of analytical methods which have been collaboratively tested and approved as official methods by the AOAC. Available as a monograph, in loose-leaf binder or CD-ROM. Food Chemicals Codex. Fourth Edition, 1996. Committee on Food Chemicals Codex, National Academy of Sciences, National Research Council, Washington D.C. National Academy Press. Available on CD-ROM, CRC Press, Boca Raton, Fla. Provides quality standards for an extensive list of food chemicals along with chemical and physical methods for their determination. Journal of the Association of Official Analytical Chemists. AOAC. Arlington, Va. Peer-reviewed journal providing original articles on methods of analysis, compositional data and collabo- rative studies. AACC Approved Methods, 10th Edition, 2000. American Association of Cereal Chemists, St. Paul, Minn. Standardized, approved methods for analysis of cereal grains and cereal-based ingredients. Available in printed form or CD-ROM. American Journal of Enology and Viticulture. American Society of Enology and Viticulture, Davis, Calif. Journal contains peer-reviewed articles giving analytical methods for analysis of wines and grape products. American Oil Chemists Association (AOCS): Official and Tentative Methods of Analysis. American Oil Chemists Society, Champaign, Ill. A compilation of standard methods for the analysis of fats and oils approved by the American Oil Chemists Association. American Society of Brewing Chemists. Methods of Analysis of the American Society of Brewing Chemists 1992. 8th edition, ASBC, St. Paul, Minn. Standard methods of analysis recommended by the American Society of Brewing Chemists. Ronald E. Wrolstad, Terry E. Acree, Haejung An, Eric A. Decker, Michael H. Penner, David S. Reid, Steven J. Schwartz, Charles F. Shoemaker, Denise M. Smith, and Peter Sporns Preface Supplement 10 Current Protocols in Food Analytical Chemistry vi
10. 10. UNIT A1.1Gravimetric Determination of Water by Drying and Weighing Water (moisture) in a sample is measured gravimetrically by determining the weight loss in a sample after it has been placed in an appropriate oven (convection, vacuum, or microwave) for a given time. In addition, there are automatic moisture analyzers available that utilize infrared lamps as a heat source. These types of moisture analyzers are fast but many times are matrix dependent, which requires some trial-and-error testing to deter- mine the correct settings (power and time). Water and moisture are used interchangeably in the description of these protocols. In addition, it is assumed in the gravimetric method that only water is removed in the drying process, when in fact there may be volatile loss in some samples. Although the measurement of weight loss due to evaporation of water is frequently used to calculate moisture content, it should be pointed out that the value obtained may not be a true measure of water content. In some samples, only a proportion of the water present is lost at the drying temperature. The balance (bound water) is difficult to remove completely. In addition, the water lost may actually increase as the temperature is raised. Some samples with high fat content may exhibit volatile oil loss at drying temperatures of 100C. Weight loss may also be dependent on such factors as particle size, weight of samples used, type of dish used, and temperature variations in the oven from shelf to shelf. Thus, it is important to compare results obtained using the same drying conditions. This unit provides three protocols for which there are established procedures for various matrices. The Basic Protocol describes water removal and quantitation after a sample is placed in a convection oven. It is probably the method of choice when one does not know which method to choose when dealing with an unknown matrix, or when one looks at samples that foam excessively in the vacuum oven method or react, such as popcorn under vacuum. Alternate Protocol 1 describes water removal and quantitation after a sample is placed in a vacuum oven. Because it is at reduced pressure, drying times are slightly reduced compared to the convection method. In addition, drying temperatures