xv y-‘¥"V 5'51var-v1-WI-QMV‘V“T‘VV"’Y‘V -\ n WHHVK qua": -e au 2 Mil E l 7 (o lllillllllill]lilllt'ifilliIlllllllfill 31293 00571 3304 LIBRARY Michigan State L University This is to certify that the thesis entitled Extimation of Iron Availability in Fortified Rice Infant Cereais Prepared Using Drum Dried and Extrusion Processes presented by ZHONG-HAO ZHANG has been accepted towards fulfillment of the requirements for M.S. degmmin Food Science M44494 Major professor €16 ~57? I)ate 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution MSU RETURNING MATERIALS: Place in book drop to LIBRARIES remove this checkout from AlulzjsalL your record. FINES wiII be charged if book is returned after the date stamped beiow. JUL 2 8199? ESTIMATION OF IRON AVAILABILITY IN FORTIFIED RICE INFANT CEREALS PREPARED USING DRUM DRIED AND EXTRUSION PROCESSES Thesis for the degree of M.S. MICHIGAN STATE UNIVERSITY ZHONG-HAO ZHANG 1988 5 (070331- ESTIMATION OF IRON AVAILABILITY IN FORTIFIED RICE INFANT CEREALS PREPARED USING DRUM DRIED AND EXTRUSION PPROCESSES ABSTRACT BY Zhong-hao Zhang Eight cooked rice cereal products obtained from two processes (drum dried and extrusion) supplemented with three sources of iron (ferric ammonium citrate, ferrous sulfate, and electrolytic iron), and a no iron control were evaluated in this thesis. There were two phases at the research; phase I: Rice Infant Cereal Processes; phase II: Analysis of the Products. Analysis of the products comprised four components: 1)nutritional evaluation of the finished products, 2)iron dialyzability study, 3)storage stability study, and 4)amylography analysis. At the nutritional evaluation of the finished products, the nutritional values of all products were similar and within the range of the commercial products. Dialyzability of iron was different among two processes, among three types of iron, and among the iron contents. Iron dialyzability of extrusion products was higher than drum dried products. All correlations of iron content were the ’LOGISTIC’ curve correlations. Storage stability, as measured by hexanal, indicated that drum dried products were less stable than extrusion process products. Iron supplementation increased hexanal evolution, with ferrous sulfate yielding the fastest, and electrolytic iron the slowest rate of rancidity during storage. Color analysis during storage indicated that Hunter b values of extrusion products were higher than those of drum dried products. At amylography study, the viscosity of drum dried products were higher than that of extrusion products. To My Family ACKNOWLEDGMENTS First of all, I would like to express my appreciation to Gerber Products Company for sponsoring my project and offering the facility for my research experiment. Much appreciation is granted to Mr.Bob LaPrad, director of Placement and Alumni, College of Agriculture and Natural Resources,M.S.U. who started the CHINESE VENTURE project. I heartly thank him, his wife,Julie, and their family for their love me since I came U.S.A. A special appreciation is given to my major professor --- Dr.Mark A.Uebersax for his guidance and all of his help. Meanwhile, I would also like to give my appreciation to my guidance committee members; Dr.M.R.Bennink, Dr.M.E.Zabik, Dr.Guy Johhson, and Mr.Albert.D.Bolles. There are so many people who I would like to give my appreciation to for their friendship and helps. Some of them are as follow; Gerber Products Company; Bonita L.Funk, Harold L.Richmann, Robert D.Wallace Timothy J.Bowser, Bruce Vibbert, Julie Wolfe, Carolyn R.Morby, Dexter Fuller, Robert L.Tambin, George Guy, George A.Purvis, Frank Kelly, Nick Hether, Sandra Bartholmey, Grey Joseph, Lange Hunter, Wes Meeuwsen, DeeDee Groenendal,Mike Ganger, Jerry Stroven, Tammy Weaver, Gene Warzak, Scatt Schaefer, II Larry Pekel, Darryl Rasch, Kathi Strand, Luci Rose, Mike Dennis. Mary Carol Farabaugh, Jean Smith. Cathy Knapp, Tom Cotton, Sue LaVigne, Jim Lanciaux, Theresia Cergguint, Brigitte Braathart,Shirley Stanfield, Donite Christoffersen, Michigan State University; Sandra Pfeiffer, Jennifer Carlson, Rod Kroll, Linda Kline, Shari Paulsen, Donna Capp, Sue Jarvi, Mitchell Cohen, Julie Machiorlatti. Lisa Roy, Keshung Liu, Jaffer Dhahir, Andrew Koknhost, Julie Mackey, Songuos Ruengsakulrach, Nruemon Srisuma. Sanir M. Rabie III TABLE OF CONTENTS Page LIST OF TABLES ............................. VI LIST OF FIGURES ............................ VIII INTRODUCTION ............................... 1 REVIEW OF LITERATURE ....................... 4 Iron Requirement for Children ...................... 4 Characteristics of Iron Compounds ...................... 6 In Vitro Estimation of Iron Availability ..................... 8 Drum Dried Cereal Process ...................... 9 Rice Infant Cereal Using Extrusion ...................... 10 Hexanal Analysis during Storage of Rice infant Cereal ...................... 11 MATERIAL AND METHODS ....................... 12 Experiment design .................... 12 Rice Infant Cereal Processes .................... 14 Drum Dried .................... 14 Extrusion .................... 14 Analysis Methods of Products .................... 16 Chemical Analysis ................ 15 Iron Dialyzability ............... 18 Hunterlab Color Values ................ 19 IV Amylography ................ Hexanal and Storage Stability Study ................ Statistics .................... RESULTS AND DISCUSSION .................... Nutritional Profile Analysis Analysis of Iron Dialyzability .................... Color change During Cereals Produts Storage ............... Storage Stability .................... Amylograph .................... CONCLUSIONS .. OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO LIST OF REFERENCE .......................... APPENDIX I - APPENDIX II APPENDIX III APPENDIX IV APPENDIX V APPENDIX VI Methods of Chemical Analysis .................. Method of Iron Dialyzability Study ......... Method of Amylography Analysis ................... Method of Color Change Analysis ............. Method of Rancidity Study ................... Statistics Methods .......... 20 20 21 22 22 24 44 55 63 65 68 72 78 84 86 89 91 Table 1, Requirement of absorbed iron in childhood ............... 2, Production and analysis model of the rice cereals products .......... 3, Nutritional values of the products I ............... Nutritional values of the products II ............... 5, Analysis of Variance of two kinds of iron content at six level of ’gastrointestinal juice’ iron content of drum dried-ferric ammonium citrate product ................ 6, Analysis of Variance of two kinds of iron content at six level of ’gastrointestinal juice’ iron content of drum dried-electrolytic iron product ................ 7, Analysis of Variance of two kinds of iron content at six level of ’gastrointestinal juice’ iron content of extrusion-ferric ammonium citrate product ................ 8, Analysis of Variance of two kinds of iron content at six level of ’gastrointestinal juice’ iron content of extrusion-ferrous sulfate product ................ 9, Analysis of Variance of two kinds of iron content at six level of ’gastrointestinal juice’ iron content of extrusion-electrolytic iron product ................ 10, Correlation coeficients of three correlations of six products ........... 11, Analysis of Variance of slopes of LIST OF TABLES three regressions of three types iron and two processes at six level VI 13 22 23 24 25 25 25 27 29 12, 13, 14, 15, 16, 17, 18, of ’gastrointestinal juice’ iron contents ............... Initial Hunter L, a, b values of eight finished products ............ The order of Hunter L values of finished products Analysis of Variance of Hunter L values at storage time ............... Analysis of Variance of Hunter a values at storage time ............... Analysis of Variance of Hunter b values at storage time ............... Analysis of Variance of hexanal of eight products and different shelf-life ............... Gas chromotograph time program ........ VII OOOOOOOOOOOOOOO 33 44 45 46 47 47 56 90 LIST OF FIGURES Figure 1. 2. 10, ll, 12, 13, 14, Diagram of the experimental procedures ............. Diagram of drum drier process procedures ............. Diagram of extrusion process procedures ............. Diagram of In Vitro estimation of iron dialyzability procedures ...... Correlation coefficients of three correlations of two products .............. Correlation coefficients of three correlations of F-A-C and E-I use drum dried ............ Correlation coefficients of three correlations of three types iron use extrusion ........... % of retentate iron based on ’g-j’ iron of two processes at ’g-j’ iron contents ............ % of dialysis iron based on ’g-j’ iron of two processes at ’g-j’ iron contents ............ % of dialysis iron based on retentate iron of two processes .............. % of retentate iron based on ’g-j’ iron of three types iron .............. % of dialysis iron based on retentate iron of two types iron use drum dried .............. % of dialysis iron based on retentate iron of three types iron use extrusion .............. % of dialysis iron based on VIII Page 12 14 15 19 30 31 32 34 35 36 38 39 40 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, ’g-j’ iron of two types iron use drum dried. % of dialysis iron based on ’g-j’ iron of three types iron use extrusion Correlation CoeffiCient of eight products’ Hunter L values with storage time .............. Correlation Coefficient of eight products’ Hunter a values with storage time .............. Correlation Coefficient of eight products’ Hunter b values with storage time .............. Hunter b values of eight products hold at storage time .............. Hunter L values of drum dried products hold at three storage temperatures .............. Hunter b values of eight products hold at three storage temperatures .............. Hunter b values of three types iron products Iniatial Hexanal of eight products .............. Correlation coefficients of hexanal with shelf-life of eight products Hexanal of two processes products at storage time ............. Hexanal of four drum dried products at storage time ............. Hexanal of four extrusion products at storage time ............. Amylography of drum dried and extrusion processes products .......... IX OOOOOOOOOOOOOO 42 43 48 49 50 52 53 54 55 57 58 59 61 62 64 INTRODUCTION Infant cereals are frequently prescribed as the infant’s first solid food.These cereals are good vehicles for the introduction of iron and other essential minerals and vitamins into the infant’s diet. Rice cereal is usually chosen as the first one among the cereal (Juliane, 1985) A critical characteristic of iron nutrition in infancy, as compared to adults, is the greater dependency of the infant on external sources of iron for daily red cell production. For a 10 kilogram infant, dietary iron must provide 30 % of the needs for hemoglobin iron turnover as compared to only 5 % in adults. This imposes disproportionate requirements of iron for the infant. In addition, infants may consume diets with a low iron content or poor iron availability. Infants are born with decreased iron reserves. Meanwhile, children grow rapidly and have excessive demands for iron (Beard et al 1985). Chemical analysis of fetuses and stillborn term infants have shown a linear relationship between body weight and total body iron, so that prenatural and term infants have an average of 75 mg/kg iron at birth. An important feature of the distribution of this iron is that about 75 % of it is in circulating hemoglobin. Hemoglobin, a major component of iron, is an important transport medium of body gas exchange. The oxygen is carried to the tissues and carbon dioxide to the lungs with hemoglobin. It can not exist without iron. Iron is also an integral component or an essential cofactor of several enzymes that play an important role in metabolic processes and cell proliferation. These include aconitase, catalase, cytochrome C, cytochrome C reductase, cytochrome oxidase, forminotransferase, monoamine oxidase, myeloperoxidase, peroxidase, ribonnucleotidyl reductase, succinic dehydrogenase, tyrosine hydroxylase, tryptophan pyrrolase, and xanthin oxidase. These enzymes are involved in a number of key pathways such as DNA synthesis, mitochondrial electron transport, catecholamine metabolism, neurotransmitter levels, detorification, and other functions(Clydesdale 1985). There are many advantages to thermal extrusion (twin screw) technology in the food industry. These include: 1)versatility --- a wide variety of foods can be produced on the same basic extrusion system using numerous ingredients and processing conditions; 2)high productivity --- an extruder provides a continuous processing system having greater production capability than other cooking/forming systems; 3)low cost --- labor and floor space requirements per unit of production are smaller than for other cooking/forming systems futher enhancing cost effectiveness; 4)product shapes --- extruder can produce shapes not easily formed using other production methods; 5)high product quality --- the high temperature short time(HTST) heating process minimizes degradation of food nutrients and destroys most undesirable factors by heat while improving digestibility by gelatinizing starch and denaturing protein; 6)energy efficient --- extrusion processing systems operate at relatively low moistures and in a closed system; 7) no effluents ~-- the lack of process effluents is an important advantage since stringent controls are being placed upon food processors to prevent their releasing pollutants into the environment. (Harper. 1981). The objective of this study is to evaluate the suitability of using three types of iron supplementation supplied to dehydrated cooked rice cereals produced using traditional drum drying and continuous extrusion processes. The products were evaluated for chemical composition,In Vitro iron dialyzability, storage stability and amylography. REVIEW OF LITERATURE Iron Requirements for Children Numerous previous research has established that the mean requirement of absorbed iron in childhood is about 0.50 mg/day in 0 to 6 months, 0.90 mg/day in 6 to 12 months, 0.7 - 0.8 mg/day in 1 to 8 years (Table 1). It is estimated that infants and children can absorb about 10 z of the iron in a good availability diet and about 5 % in a poor availability diet. On this basis, it would appear that, depending on the quality of the diet, intakes of 10 to 20 mg/day are needed to meet the requirement of 1 mg absorbed iron throughout most of infancy and childhood (Beard et a1 1985). Table 1 Requirement of absorbed iron in childhood mg/day 11;; """"" §;;££;;.;;;£ """ 517653135; """" §;;;E;;;;;;' (years) for Growth Loss for Absorbed 5'76"; ””” 615 """""" 6'53 """"""" 6'36 """"" 0 5 - 1 0 0.40 0 37 0 90 1 0 - 2 0 0 29 0.46 O 7 - 0 8 2 0 - 8 0 0 23 0 56 0 7 - 0 8 * Beard and Finch. 1985 The Committee on Nutrition of the American Academy of Pediatrics (1969) suggested that infants with hemoglobin levels less than 11 grams/100 milliliters of blood, and hematocrit levels less than 33 % be termed anemic. In 1969 and 1976, this Committee recommended an intake of 1.0 mg/kg weight/day up to a maximum of 15 mg/day for infants less than one year old. It was also recommended by the committee (1976) that commercial cereal are fortified with iron at a level of 45 mg/100 grams. Human milk contains an average iron content of 0.2 mg/Liter. Cow’s milk and regular cow’s-milk-based formula have an average iron content of 0.5 mg/IOO Liter. Infant cereals with added iron were first marketed in 1932. Currently, the form of iron added to infant cereals is reduced iron. Reduced iron is a generic term for finely powdered metallic iron, and is manufactured either by reduction with hydrogen or carbon monxide, by electrolytic deposition or by a carbonyl process (Theuer, 1985). Many factors affect iron absorption in man, these include: 1)an individual’s need, 2)composition of the diet, 3)valency of iron, 4)solubility, 5)ease of ionization, and 6)the degree of chelation or complex formation of the iron and food components. There are numerous factors associated with inhibition and promotion of iron availability in food.(Kadan et al, 1987). Smith (1983) found that the chemical state of iron in processed food and its conversion under simulated gastric digestion conditions can provide valuable information about its potential bioavailability. Characteristics of Iron Compounds Electrolytic Iron. Electrolytic iron powder is produced by electrolytic deposition of a hard, brittle metal that is mechanically comminuted. Surface oxide of the electrolytic iron powder is the major "impurity". The surface oxide is present as ferrosoferric, FeO.Fe203. The ferrous oxide portion is very soluble in stomach acid and conveniently provides attack sited for particle dissolution; ferric oxide has little or no bioavailability. The particle shape of this compound is described as irregular. The particle size, surface characteristics, purity, and density of electrolytic iron powder strongly affect the application in the food industry. The most important advantage of electrolytic iron is the stability of the material. This stability guards against off-flavor, odor, caking, and minmizes catalysis of discoloration and rancidity. It contains 96 % or more total iron. The dark gray color, iron insoluble and higher density are major disadvantages of this form of iron. The higher density of iron powder can lead to segregation if incorporation is not carefully performed (Patrick. 1985). Ferrous Sulfate. Ferrous sulfate is the cheapest and most widely used iron source in food fortification. In 1982, all infant formulas in the United State were fortified with ferrous sulfate ,‘as were some 20 % on infant cereals. It has two forms; heptahydrate and dried ferrous sulfate. Ferrous sulfate heptahydrate (FeSOq.7Hzo) contains about 20 % iron that is freely soluble in water. It is odorless and has a slight saline taste. Upon warming to 65°C, the compound forms the monohydrate, which is stable to 300°C. The dried ferrous sulfate (FeSO4.tzO) has a metallic and astringent taste, and is less soluble in water. The iron content is about 33 % (Hurrell.1985). The heptahydrate form of ferrous sulfate has become the reference standard in almost all iron bioavailability studies. In human studies, Brise and Hallberg (1962) found that from less than 5 X to almost 70 % of the initial dose may be absorbed depending simply on the iron status of the subject. Rios et al.(1973) measured the absorption of radioactively labelled ferrous sulfate by infants fed fortified milk-based and soy-based formulas (12 -17 mg of added iron per Liter). The results showed a mean iron absorption of 3.9 % (range = 0.7 - 23.1 X) for milk-based formula and 5.4 % (range = 1.0 - 21.9 %) for soy-based formula. In infant cereals the researchers recorded an absorption of 2.7 % (range = 0.4 - 12.1 %). Ferrous sulfate may catalyze lipid oxidation reactions and cause off-flavors in dehydrated cereals during storage. It also causes unacceptable color changes in infant cereals when hot milk or hot water are added (Disler et al.,1975). Ferric Ammonium Citrate. Ferric ammonium citrate is a reddish brown granular powder that is very soluble in water. It contains 16.5 - 18.5 % iron. Ferric ammonium citrate has similar Relative Bioavailability to ferrous sulfate in rats. A 2 % absorption of ferric ammonium citrate in cereal was reported by Elwood et al.,(1970). It is about five times more expensive than ferrous sulfate heptahydrate for the equivalent amount of iron. In Vitro Estimation of Iron Availability Dr.Miller (1981) introduced the method of In Vitro Estimation of Iron availability which employed enzymatic digestion and membrane dialysis of iron. There are four advantages of this method as compared to the In Vivo methods; 1)cost of the experiment is lower, 2)time of the experiment is shorter, 3)amount of the work is less, and 4)the procedure of the experiment is simpler (Miller. 1981). Correlation analysis indicated significant agreement between the in vitro and human in vivo methods. Correlations between the rat in vivo and human in vivo methods were also significant, but correlations between the in vitro and rat in vivo methods were less significant, and in some cases, not significant (Schricker. 1981) Drum Dried Cereal Process According to the formula, all ingredients are weighed and put in a large container. The dry ingredients are conveyed to a ribbon blender for complete mixing of all ingredients. At the slurry tank inlet, water is metered in through a manifold to produce the wet cereal slurry. The purpose of the slurry tank is to thoroughly mix the water and dry ingredients and to remove any lumps from the slurry. The total solids content and flow rate of the slurry are controled for the rest process. After the slurry tank the cereal slurry is pumped to a surge tank designed to provide a continuous slurry to the rest of the system. Enzyme hydrolysis is used to reduce the cereal slurry viscosity, control reconstitution properties of the finished cereal, and / or produce reducing sugars which add flavor to the cereal. From surge tank, the slurry goes into the enzyme hydrolysis heat system designed to provide optimum enzyme hydrolysis. Cook tank is for mixing the incoming slurry with the hydrolyzed slurry in the tank to insure optimum enzyme distribution and uniform temperature throughout the tank. The slurry will be holded in the cook tank for a certain time so that the enzyme can react with the substract as well as possible. After the cook tank the cereal slurry is pumped through a tangential steam heater. This heater fully gelatinizes all of the starch in the cereal slurry, inactivates the enzyme so that the hydrolysis process is stopped, and sterilizes the slurry. Then the hot slurry enter the flash tank which serves as a reservoir for supplying slurry to the drum drier. Atmospheric double drum drier are used to dry the slurry and produce the thin cereal sheets. There are four basic drum drier controls which the drier operator can use to optimize cereal production; puddle level, drum speed, steam pressure and drum clearance. As the cereal sheets fall from the drier knives, they are carried by side belts to an airlock in the airveying system. The flaker breaks the sheets to consistent size. From the flaker the cereal is conveyed to a sutton seperator which removes the heavy or large particles and fine one. The finished cereal is conveyed from the separator to storage bins until needed. The cereal should pass through another separator before entering the filler and packaging line. Rice Infant Cereals Using Extrusion The extruder can be divided to 5 section. 1,extrusion drive; It includes support, drive motor, speed variation, transmission, thrust bearing. 2,feed assembly; It consistes of hoppers, feeder, slurry tank, liquid feeder, batch feed system, continuous feed system, preconditioner, rotary, and feed transition. 3,extrusion screw; It can divided to feed section, compression section, and metering sections. There are single screw and twin screw two kind of screws. The screw accepts the feed ingredients at the feed port, conveys, cook, 10 and forces them through the die restriction at the discharge. It is the centrol portion of a food extruder. 4,extruder barrel; The extruder barrel is the cylindrical member which fits tightly around the rotating extruder screw. 5,extruder discharge. It normally holds the extruder die, cutters and take-away devices. The extrusion process has been recommended for the production of ready-to-eat weanling foods in developing countries (Mosqueda et al 1986) Many commonly used food processes have been examined for their role in affecting the chemical changes in iron form during processing (Kadan et a1 1987). There are few reports on the effects of the extrusion process. Hexanal Analysis during Storage of Rice Infant Cereal It is well known that the off-flavor of stored rice originates mainly from its lipid deterioration (Myung Gon Shin. et a1 1986). N-hexanal one of the main off-flavor components, may arise from oxidative degradation of unsaturated fatty acids, especially linoleic acid. Studies on the lipid deterioration of stored rice or the development of off-flavor during storage of rice have been carried out by several investigators (Tsugita et al., 1980). However, the correlation between lipid deterioration and development of off-flavor during storage of rice infant cereal has not been studied in detail. 11 MATERIAL AND METHODS Experiment Design Figure 1 illustrates the experimental procedure. There were two primary phases to this research. The first phase was rice infant cereals processing, and the second phase was the product analysis. I Phase I --- Processing I INGREDIENTS / \ / \ DRUM DRIED EXTRUSION \ / \ / PRODUCTS / \ ' Phase II --- Analysis I / / \ \ / / \ \ NUTRITIONAL AMYLOGRAPHY STORAGE IRON DIALYZABILITY ANALYSIS STABILITY / \ COLOR CHANGE RANCIDITY Figure 1 Diagram of the Experimental Procedures Table 2 is the analysis model of the products. There were two processes (drum dried and extrusion), three types 12 of iron (ferric ammonium citrate, ferrous sulfate, and electrolytic iron), and a no iron control that would be produced and analysed. Table 2 Production and analysis model of the rice cereals products agrees; """"""" 5126;135:313 """""" HERE?" 13:33:17: """"""""""" xxx """"""""" xxx """" FERRIC AMMONIUM CITRATE XXX XXX FERROUS SULFATE XXX XXX ELECTROLYTIC IRON XXX XXX 13 Rice Infant Cereal Processes Drum Dried There are four main section in a drum dried rice cereal process; general slurry preparation, enzyme hydrolysis, drum drier rolls (3 feet), and dry cereal handling (Figure 2). Figure 2 Diagram of drum dried process procedures Extrusion The principle components for the extrusion process are illustrated in Figure 3. Extruder is Baker Perkins MPF - 50 14 Figure 3 Diagram of extrusion process procedures 15 Analytical Methods of Products Chemical Analysis Moisture Three grams of the rice infant finished product were weighed into previously dried and tared aluminum dishes and dried to a constant weight at 105°C for 5 hours in an air oven. The sample weight and the moisture was determined from the weight loss on the fresh weight basis (AACC.method 44 - 15): weight before dried - weight after dried Moisture% = ----------------------------------------- * 100 weight before dried Protein 2 grams of each product were analyzed by the Macro- Kjeldahl procedure. Percentage of nitrogen was multiplied by 6.25 to obtain percentage of total protein (AOAC method 2.057) ml of HCI * exact Normality of HCI * 0.014 weight of sample Ash The dried samples obtained from the moisture determinations were incinerated at 525°C for 24 hours in a Barber-Coleman muffle. The uniform white ash was cooled to room temperature in a desiccator prior to weighing (AACC 16 method 08 - 01) (weight of dish + ash) - (weight of dish) (weight of dish + sample) - (weight of dish) Fat The soxhlet extraction procedure was used in the fat determination (AACC method 30 - 25). (weight of dish + fat) - (weight of dish) weight of sample Carbohydrate % = total solid - fat - protein - ash 17 Iron Dialyzability The method of In Vitro Estimation of Iron Availability (Miller, 1987) was used in this research (Figure 4). First, the standardized iron content samples were prepared using the eight products (10, 20, 30, 40, 50, and 60 iron mg/lOOg of sample). Using those samples to make 5 % solid meals (sample + water). Then the meals were put into the vials and ’digested’ with the enzymes. Tubing with buffer was put into the vials to ’absorb’ the iron in the ’gastrointestinal juice’. The meal plus the enzymes (pepsin, pancreatin and bile) before the tubing was added was termed ’gastrointestinal juice’. The juice after ’digestion’ and ’absorbion’ was termed retentate. The buffer the tubing after the absorption was called dialysis solution. Following this preparation procedure, the iron content of ’gastrointestinal juice’, retentate, and dialysis solution were determined using a Varian Atomic Absorption Spectrophotometer model 1250 (APPENDIX II). 18 MEAL PREPARATION ( 5 % solid in meal ) I I l l ACIDIFY THE MEAL TO pH 2.0 USING 6 N HCI / \ / \ pepsin / \ SAMPLE FOR ’GASTROINTESTINAL ’GASTROINTESTINAL JUICE' JUICE’ IRON DETERMINATION INCUBATE IN A 37°C SHAKING WATER BATH FOR 2 h / I / I dialysis tubing / I with buffer / I SAMPLE FOR TITRATABLE INCUBATE FOR 30 MIN ACIDITY DETERMINATION I I pancreatin-bile I INCUBATE FOR 2 h / I / I REMOVE DIALYSIS TUBING AND DETERMINING RETENTATE IRON DETERMINING DIALYSIS IRON USING SUPERNATENTS OF RETENTATE JUICE Figure 4 Diagram of In Vitro estimation of iron dialyzability procedures Hunterlab Color Values The Hunterlab model D 25 A Color and Color Difference 19 Meter standardized with a white tile (L = 95.35, a = - 0.6, b = +0.4) was used to evaluate the color of rice infant cereals. A 100 g of the cereal was placed in an optically inert glass cylinder cup and covered with an inverted, white lined can to keep out extraneous light (APPENDIX IV). Amylography The Visco-Amylo-Graph No.676, type VAl was used in this experiment. 100 g cereal was weighed. The sample was treated at three periods; 1),heating (from 29°C to 95°C for 44 minutes), 2),holding (95°C for 16 minutes), 3),cooling (from 95°C to 29°C for 44 minutes) (APPENDIX III). Hexanal and Storage stability Study The eight finished products were canned in hermetical sealed cans (211 * 208) Each products had 60 cans with 20 grams per can. Those 60 cans were divided to three groups and stored at three temperatures; 21.110C (R.H.: 70 %), 26.6700 (R.H.: 80 %), and 36.6700 (R.H.: 70 %). Hexanal analysis were conducted on the samples stored at 36.6700 to accelerate the storage time (The shelf-life at 36.6700 equate to three time at room temperature). The samples were tested until 21th week for color change analysis. The last samples tested for hexanal was 16th weeks. Tekmar Purge and Trap Concentrator LSC-3 and Gas Chromatograph Hewlett Packard 5890 A was used to determine hexanl (APPENDIX V). 20 Statistics Lotus 1-2-3 was used for the results statistics following the procedure at Appendix VI(Puri. 1971). The data were analysis using ANOVA to see the difference that existed. Then the Tukey’ test analysed the difference among the samples. The correlation and regression analysis were run using Lotus 1-2-3 too following Appendix VI (Drager. 1966). 21 RESULTS AND DISCUSSION Nutritional profile Analysis The fat range of eight products was 2.40 - 3.75 %. Protein range was 6.82 - 7.68 x. Carbohydrate was 79.73 - 82.45 %. Ash range was 2.36 - 2.64 %. Moisture was 5.29 - 6.92 % (Table 3) Table 3 NUTRITIONAL VALUES OF THE PRODUCTS I (%) FAT 3.14 2.51 2.40 2.46 3.75 3.46 2.98 3.05 PROTEIN 6.82 7.29 7.31 7.16 7.50 7.54 7.444 7.68 CARBOHYDRATE 80.58 82.08 81.45 82.45 79.99 79.73 81.20 80.66 ASH 2.54 2.58 2.64 2.64 2.42 2.45 2.36 2.52 MOISTURE 6.92 5.54 6.20 5.29 6.34 6.82 6.02 6.09 Denote; A-I-C, FERRIC AMMONIUM CITRATE. F-S,FERROUS SULFATE E-I, ELECTOLYTIC IRON Most of minerals were natural to the rice flour except iron content because fortification of the rice infant cereal with iron components. The range of iron content in the eight products was 1.90 - 94.75 mg/100 g (Table 4). 22 Table 4 NUTRITIONAL VALUES OF THE PRODUCTS II mg/100 g COMPONENT BLANK- Fe 1.90 Zn 1.20 Ca 1000.00 Phos. 490.00 K 133.00 Denote; A-I-C, FERRIC AMMONIUM CITRATE. E-I, ELECTOLYTIC IRON F-S, FERROUS SULFATE Analysis of Iron Dialyzability Table 5 to 9 shows that retentate iron contents and dialysis iron contents were significant different at different ’gastrointestinal juice’ iron contents (range:2.98 - 28.83 ppm) except extrusion-electrolytic iron product because six level of this product’s ’gastrointestinal juice’ iron content were too close (range:3.13 - 11.12 ppm). Table 5 Analysis of Variance of two kinds of iron content at six level of ’gastrointestinal juice’ iron content of drum dried-ferric ammonium citrate product Retentate iron Dialysis iron Correction Factor 116.3321 0 9243 SS-treatment 7.9042 0.0283 SS-replication 29.2080 0 0819 SS-total 39.4665 0 1277 SS-error 2.3543 0.0176 MS-treatment 1.5808 0.0057 MS-replication 14.6040 0.0409 MS-error 0.2354 0.0018 F-treatment 6.7146 ** 3.2185 ** F-replication 62.0306 ** 23.2834 ** SE 0.8858 0.0765 LSD 4.3496 0 3758 24 Table 6 Analysis of Variance of two kinds of iron content at six level of ’gastrointestinal juice’ iron content of drum dried-electrolytic iron product Retentate iron Dialysis iron Correction Factor 20.1640 0.2538 SS-treatment 2 0112 0.0058 SS-replication 0.2890 0.0148 SS-total 2.5466 0.0297 SS-error 0 2464 0.0091 MS-treatment 0.5028 0.0015 MS-replication 0.2890 0.0148 MS-error 0.0616 0.0023 F-treatment 8 1623 ** 0.6361 F-replication 4 6915 * 6.5018 * SE 0 1754 0.0337 LSD 1 1074 0.2130 Table 7 Analysis of Variance of two kinds of iron content at six level of ’gastrointestinal juice’ iron content of extrusion-ferric ammonium citrate product Retentate iron Dialysis iron Correction Factor 154.6454 6.5510 SS-treatment 6.1878 2.4197 SS-replication 46.5769 0.5734 SS-total 55.4154 3.7791 SS-error 2.6506 0.7860 MS-treatment 1.2376 0.4839 MS-replication 23.2885 0.2867 MS—error 0.2651 0.0786 F-treatment 4.6690 * 6.1573 * F-replication 87.8615 ** 3.6479 * SE 0.9399 0.5118 LSD 4.6152 2.5131 Table 8 Analysis of Variance of two kinds of iron content at six level of ’gastrointestinal juice’ iron content of extrusion-ferrous sulfate product .‘m—~-——---—---—~_—-—-~.—-—u——fi--—~—--n-—--———--~-—nn———~w--——-— Retentate iron Dialysis iron Correction Factor 224.0139 4.8776 SS-treatment 21.5635 2.4300 SS-replication 56.6983 0.2664 SS-total 88.0183 3.0502 SS-error 9.7565 0.3538 MS-treatment 4.3127 0.4860 MS-replication 28.3492 0.1332 MS-error 0.9756 0.0354 F-treatment 4.4203 * 13.7383 ** F-replication 29.0567 ** 3.7648 * SE 1.8033 0.3433 LSD 8.8545 1.6860 26 Table 9 Analysis of Variance of two kinds of iron content at six level of ’gastrointestinal juice’ iron content of extrusion-electrolytic iron product Retentate iron Dialysis iron Correction Factor 69.2665 1 1160 SS-treatment 4 5580 0.0327 SS-replication 9.2137 0 0129 SS-total 17.6377 0 1361 SS-error 3 8660 0.0905 MS-treatment 0.9116 0.0065 MS-replication 4.6068 0.0064 MS-error 0.3866 0 0090 F-treatment 2.3580 0 7230 F-replication 11.9164 ** 0 7113 SE 1 1351 0 1736 LSD 5 5737 0.8527 There are three correlations to be analysed in this paper; 1), retentate iron with ’gastrointestinal juice’ iron; 2),dialysis iron with ’gastrointestinal juice’ iron; 3),dialysis iron with retentate iron. The results were that all of those correlations were found to belong to the "LOGISTIC" curve correlations, and it is a "S" model. The equation of the "LOGISTIC" curve is as follows; a + b * X Ym * 6 Y: --------------------- a + b * X Ym + e DENOTE; Ym ------ Maximum value of Y After we fitted this ”IOGISTIC’ curve to a straight 27 line, the correlation coefficient ( r ) was calculated (Table 10) 28 Table 10 Correlation coefficients of three correlations (1,retentate iron with ’gastrointestinal juice’ iron; 2,dialysis iron with ’gastrointestinal juice’ iron; 3,dialysis iron with retentate iron.) of six products (two processes and three types of iron) retentate iron with dialysis iron with dialysis iron retentate 66 49 42 ’gastrointestinal ’gastrointestinal with juice’ iron juice’ iron iron DRUM DRIER Ferric- Ammonium- Citrate 0.94 0.74 0. Ferrous- Sulfate 0.86 --~- -*~* Electrolytic Iron 0.86 0.37 0. EXTRUSION Ferric- Ammoniu- Citrate 0.92 0.64 0. Ferrous- Sulfate 0.79 0.81 0. Electrolytic iron 0.89 0.48 0. These correlations (Table 10) are illustrated in Figures 5, 6 and 7. The correlation coefficients of the three correlations of most products were medium or strongly positive one except drum drier-ferrous sulfate products and extruder-electrolytic product at the correlation of dialysis iron with retentate iron (Figure 6, 29 do: 23:22 52> :2. 23.220 .5: .To. £3 :2. 22: ram .3: .7... 5.3 :2. 223.2% :32..sz a...“ 5.5 EEO mum-m LL» 23:20:00 02:... < - II.- .- . AIM-IRENE. -, . i o . ., {L I--.- I- 7 I , . .- II. hlrlui l.r-.IOL . 71 I . 1. 1.), .M-Jl. cm. In. J. 4. M . . . t - 4,/ .I f. .. {Idli- ii ATIL-utl. L . ./ . ./ 4 W. 4451-. a fig. v . .. .. I .4 HI... ..- III I IIIIIIIIIJI../+-u..“+- .. .- ..N|...H.r.mw I m x/fitfll . a q . ”77+ .- L x -. if a m . T. I.” a J .r . .. x . +1- . a . o / +- m p m - bib II. V o - w / / 1’ z/ ‘1', off ". Lx.. ' It? i _ ., I- I? i1" / / ,/ 'l ”‘1 - IIll : " / ll... Im .1, ..l. ..'. i. I i 0.0 .I-I Q 77/7 77 . * ' I, ’ I / / / 44+ I t l; ../ /, Z '/ v/ x “I / / / .' , -" ’ / . 757 I . =JIIITI' ’ A; ‘l . LI p,- I—v J i-- r y—o I“ L'—...J-.l-~ 't" ' .2- c .. remiss-emu 53.830 mommoooca oz: no «:28 iotoo 2:5 .0 2205000 5:22.00 m 950...“. 30 5.. 225.2 5.3 5.. 22.220 .5: .79 5.3 :2. so... ..a.u.m .5: .To. 5.3 :2. 225.23 :2. 2.2283 225 52525. a... .- . .-. «5.3.250 02...... . tII . . . x. . u u nil-II l i. a .1. «l« I 1 I u 4 1:22:51. . W. 7:12.... . . . u... ....._... . I . ...._.... . ”.H~.“H,MH_4 . .fiw.ha._k. e . _.. Hm..._..:--. \. .4. If; . . f. ... .MI .... 4...... . . . .mfi—wfl.» ” .4: “om“. . _..... .... .\ fl ..~ H s It ..._...-.i\ _- It... .. ._ . .I...H.v.“.. . ........ . «1%.- .il . . . r.....:...-.-H ------- .1 _.L-I+i.-.- .. . _ .._ _\ x Gui-.n “tr..A\. .5 .q... . .1 _ .. _.. -_ . v.7"..i ...1. x». W...” H...-«I. . .y .2... _k \\ x. 4., or.w A . s _. ..,.._..:..-...__ .x L. .- L- . c ,w . w_ ... t .» x A..._;--.. f . *0 I... _....-..\ .c vx . U..«v no. \ \. . Ether-t. .. + A .. \ .. x \ a . C ..- 0 .\ . roo \ . . \t\ \ v\|l. * v . - . . . \..\ ...o . -§-~.5‘\9\ .L.__ . . . 1. F c I- 0:22:50 5.3.250 5.5 :55 mm: Tm use 0..<..n. 5 25.5 I250 02... .o Eo.o.tooo 5.5.250 0 2:9“. 31 .02. 22:22 5.3 :2. 32.3.0.0 5.. :70. 5.; :2. 2o $.22... .ce.....a..=.3 .3... 23:32... \. . \ \ t . .. \ f . . .. \ .. \ .. . w .I 0 .. . ... .\ \\. .fl . . rm - x L L Nd " .I: .\ . \ x M .I, . r r \\ . \. ‘ MN” J. . \ X. X r / x// \ m . h _// // I \\~ h _ ; . .4. . .0 y \ \ . I . M \x \A .r ,/ /., \\ \\.. V.” V . ».s 1/ . . . . . .\.. n - \ x. - / . _ \\ ~ r /. \. . .. . \ .. \ o . . r L I 0 O . Ln. 7 , . . . ax . .1 . , . .. . , 0 o \H . \l‘uxltvflh M .- - -- - - - z i - - § - - - - iii... I155! : - .. all... r .-.......... 520......000 5.5250 5.0238 00: 5... 02:: 02.: .0 05:0 1.250 02:. .0 E30550 5.5.250 N. 230E 32 Figure 7). Now we calculated the regression equations of each treatment. Table 11 is Analysis of Variance of slopes of three regressions of each treatment(six treatments) at six level of ’gastrointestinal juice’ iron contents. The results of analysis were that there were no significant difference among the treatments at all three correlations. Table 11 Analysis of Variance of slopes of three regressions of three types iron and two processes at six level of ’gastrointestinal juice’ iron contents retentate iron with dialysis iron with dialysis iron ’gastrointestinal ’gastrointestinal with retentate juice’ iron juice’ iron iron SS-treatment 0.0209 0 0202 7.9421 SS-replication 0.0494 0 0103 4.3496 SS-total 0 1104 0 1073 25.4492 SS-error 0.0401 0 0768 13.1575 MS-treatment 0.0042 0.0040 1.5884 MS~replication 0.0247 0.0051 2.1748 MS-error 0.0040 0.0077 1.3158 F-treatment 1.0419 0.5250 1.2072 F-replication 6 1505 * 0 6692 1.6528 SE 0.0365 0.0506 0.6622 LSD 0 1795 0 2484 3.2516 ------—-----———-_—-----’——-—--——_——--—‘-—-—--—-—----------——‘-- The Figures 8, 9, and 10 are percentage of the X iron based on the Y iron in each Y iron content. This research showed us that the difference of amount of iron that intaked had variable percentage of retentate iron and variable percentage of dialysis iron. This variance was also different between drum dried and extrusion products. The percentage of retentate iron based on the ’gastrointestinal juice’ iron, percentage of dialysis iron based on the 33 5... 0.3.2.005 05 32:0 05.5.55 0.20. 5 0555 .. 05.0.. 00000020 500 5.0232 ll 00.20 .55- T. 3.5. 5... .02.... 50.50.52.000. on 0N 0N *N NN ON 9. o— ,v— Nr 0.. o o 0 N 7-.-....-.. ... i........ 5.... . . ...-..O .. on :2. .70. 5 00000 :2. 0550.2 5 8 0.5.50 5.. .Ta. 00 00000020 0...; .0 5... ._...0. 5 00000 5... 0.0.5.2 .0 i w 25.“... 34 duo-P— . on ON ON VN NN ON or or 3. Np 0.. o 02. 0.3.2.020 000 0.2..0 0.200.500 0.20. .0 0022.0 .. 000.0» 00000020 0.00 00.0.2.5. IT 00:0 0.20 -.. .500. 02. .000... 35.00.52.000. . 7 . . . . . . x 0 02. .Ta. 00 00000 02. 2020.0 .0 # 0.00.000 02. .70. .0 00000020 03. .0 02. .10. 00 00000 02. 0.0.00.0 .0 i m 0000...... 35 02. ..>.2.00.0 000 0.2..0 05.02500 0.20. .0 002000 .. 000.0» 00000020 0.00 00.0206 2+-.. 00.00 0.20 It. .500. 02. 0.0.00.0: 0..» h 0.0 0 0.0 0 0.? .v 0.0 0 0.N N 0.9 w .: 4 .2. . . . 2.....- .- .M 2..-- 22.23---.- 52:-.. 2..-}. o m __ .2 . . . . . - . - .0 m b 2P In I. 0 2n 0.1 .. L. . J . J . . / _- . M .. m .2: r {I 2 ,2 .. 2 2 . .11-..- .12- . -..:,..22.:- ll. 0“ .,,._--.o... 23mm? -0... 0.0.0.. 00....--0.0»..0_.0.o 0 00000020 03. .0 02. 0.0.00.2 00 00000 02. 0.0.00.0 .0 3 o. 2:0... 36 ’gastrointestinal juice’ iron, and percentage of dialysis iron based on the retentate iron, the values of extrusion products were all higher than any of drum dried products. Like drum dried and extrusion, the three correlationships, of ferric ammonium citrate, ferrous sulfate and electrolytic iron were LOGISTIC curve correlations. All of the correlations were medium or strongly positive ones excepte the correlation of dialysis iron with retentate iron of extrusion electrolytic iron product ( r = 0.24 ) and two correlations of drum drier ferrous sulfate products (Table 10). The order of the percentages of retentate iron based on ’gastrointestinal juice’ iron at three type iron fortification of products using drum drier and extrusion processes is Ferrous Sulfate > Ferric Ammonium Citrate > Electrolytic Iron (Figure 11). The percentage of dialysis iron based on retentate iron of Ferric Ammonium Citrate fortified product using drum drier was higher than that of Electrolytic Iron’s (Figure 12). At Figure 13, the order of percentage of dialysis iron based on retentate iron of three types of iron fortified products using extrusion process was Electrolytic Iron > Ferric Ammonium Citrate > Ferrous sulfate at the low retentate iron contents. At the higher retentate iron contents, the order was reversed; Ferrous Sulfate > Ferric Ammonium Citrate > Electrolytic Iron. The percentage of dialysis iron based on 37 Tm 2.0:- 0.0.1.1 On<:0u_ .11.. .500. 02. .000... 10.80.52.000. O0 ON ON 0N NN ON 0: 0.. 3 N.. O.. 0 0 v T .. w w 0 w . :2. .2: 2. 4. w .0 . 0.2.. .000. 00 0000.0 02. 0.0.00.0: .0 I 02. 000... 00.... .0 02. .70. 00 00000 02. 0.0.00.2 .0 i 2. 0.50.“. 38 00.. 0_.>_0..00.m 2+:- 0.0...o 05.00.55 0n. 1.! .030. 00.. 0.0.00.0: 0.5 h 0.0 O 6.0 D 0.1 V m5 0 0.N N Dd. r 4: : 2. :0 :0 . 0 . 4: 42:24:22... O :2m 0 -:..J L . . 2. . . b :l+.! : 22+:2- - L N .2::::.::.: : 2:3:.. - -:w.....-:0..0..,00...0.. 00 00.000 0900.030... .0 *0. 0000 0.0.0 00: 00.. 000... 03. .0 00.. 0.0.00.0. 00 00000 00.. 0.0.20.0 .0 i 0. 050.. 39 _.-m i.-- 0... .-.+. 0.0-0. 1.... 0000. 00.. 0.0.00.0: 0.0 s 0.0 0 0.0 0 0... v 0.0 0 0.0 N 0.. . . .. 2 m , _ 0 a 4:. :..:.-:..::.:.:,0 -. 2:22:12 -21. o EI;I*:2 1.: .‘t Ill!!! w.m _ 1WU¥f - I}: .l t: a i L r:-_::...::::,::.-.,-,::: _. , .2:-...:-:.--3.-:-.:3-.. _. -. . : . . _ - . : . . .. : . L UN 0.....0.0.00.0. 00 00000 00.. 0.0.00.0 .0 0. 00.00.08 00: 00.. 000... 00..... .0 00.. 0.0.00.0. 00 00000 00.. 0.0.00.0 .0 s 0.. 050...... 40 ’gastrointestinal juice’ iron of Electrolytic Iron fortified product using drum dried process was higher than that of Ferric Ammonium Citrate’s (Figure 14). Figure 15 looks like Figure 13. The order of percentage of dialysis iron based on ’gastrointestinal juice’ iron of three types iron fortified products using extrusion process was Electrolytic Iron > Ferric ammonium Citrate Iron > Ferrous sulfate’s at low ’gastrointestinal juice’ iron content. But it was again reversed at the higher ’gastrointestinal juice’ iron contents. 41 :2. 0:29:02”.— iT 32:0 5255.5 cm .1! 333 :2. .02.... 353252.30. om mm on em «N on up up 3 «F o. o o e u * . a 1 .. A 1 4| « _ 4 d w BJIIIJIiJO :2. .76. co no.2. :2. 2.22.. .o 1 note Es... on: :2. 32: 25 .o :2. .To. :0 woman :9: 333:. .o .3 3 0.59.“. 42 Tm 1*: 9.... -+ 1 0 Drum dried Ferrous Sulfate Extrusion > Drum dried Ferric Ammonium Citrate Extrusion < Drum dried Electrolytic Iron Extrusion < Drum dried Ferric Ammonium Citrate fortification decreased the Hunter L values of rice infant cereal using drum dried and extrusion processes (Table 12). Ferrous sulfate fortification only decreased the Hunter L values of drum dried products and did not effect extrusion products. Electrolytic iron fortification did not affect the Hunter L values of products from both processes. Three types iron fortification for extrusion products increased the Hunter a values however there were no differences among iron sources. The order of Hunter a values for the four drum dried products is Ferric Ammonium Iron > Ferrous Sulfate > Blank > Electrolytic Iron (Table 12). The Hunter b values of iron fortified extrusion products were lower than the blank product, and it was almost same among them. The order of Hunter b values of drum 45 dried products is Ferric Ammonium Citrate > Blank > electrolytic Iron > Ferros Sulfate (Table 12). There were significant different among the eight treatments (samples) at Hunter L, a, and b at three storage temperatures. Generally, there were no significant different of Hunter L and a when the eight products were stored. But Hunter b had significant different among the storage times (Table 14, 15, and 16) Table 14 Analysis of Variance of Hunter L values at storage time Storage Temperature 21.110C 26.670C 36.6700 CF 563990.70 562382.20 560852.50 SS~sample 1167.68 1245.75 1254.98 SS--time 18.28 30.92 15.82 SS*-total 1279.89 1310.88 1307.27 SS--error 93.92 34.21 36.46 MS--sample 166.81 177.96 179.28 MS~-time 1.66 2.81 1.43 MS-~error 1.21 0.44 0.47 F--sample 136.76 ** 400.53 ** 378.53 ** F-time 1.36 6 32 * 3.03 SE--sample 0.31 0.19 0.19 LSD-~sample 1.41 0.85 0.88 SE--time 0.39 0.23 0.24 LSD-time 1.73 1 04 1.08 46 Table 15 Analysis of Variance of Hunter a values at storage time 21.1100 26.6700 36.6700 CF 1855.92 2059.98 1869.13 SS-sample 67.41 128.64 114.83 SS--time 51.04 26.37 25.36 SS--total 199.74 239.78 218.66 SS--error 81.28 84.77 78.46 MS--sample 9 63 18.37 16 40 MS--time 4 64 2.39 2 30 MS--error 1 05 1.10 1.01 F--sample 9.12 ** 16.69 ** 16.09 ** F--time 4.39 * 2.17 2.26 SE--sample 0.29 0.30 0.29 LSD--sample 1.31 1.34 1.29 SE--time 0.36 0.37 0.35 LSD-time 1.61 1.64 1.58 Table 16 Analysis of Variance of Hunter b values at storage time Storage Temperature 21.1100 26.6700 36.670C CF 36981.35 37430.20 37893.68 SS-sample 270.48 258.89 237.84 SS--time 11.18 12.09 21.15 SS--total 291.60 280.33 269.72 SS--error 9.93 9.34 10.73 MS--sample 38.64 36.98 33.97 MS--time 1.01 1.09 1.92 MS--error 0.12 0.12 0.13 F--sample 299.39 ** 304.68 ** 243.68 ** F--time 7.87 ** 9.06 ** 13.79 ** SE--sample 0.10 0.10 0.10 LSD--sample 0.46 0.44 0.47 SE--time 0.12 0.12 0.13 LSD-time 0.56 0 54 0.58 .----_---..--__---—-.*—-"--t-—--—--—-—--~—‘—-—-———-—---—_— Figure 16, Figure 17, and Figure 18 are correlation coefficients of Hunter L, a, b values with the storage time 47 0.3.0» We 0.5.8 E 0.33 mm COB—:38 3252.. “02.6 Eat m m < m m w < m .231 -,,_ --:-,,-..---.. 3 2 ._;. - _ ; -, . l. l J. 0.0.. ... 0.0.. w ,- 1“ I... .1 «.0- 0 «.0 to > Y l 1 Y 11 I I; \ ‘3 \ \\ ‘ ?\-i\\ \\ ‘ ~\-—-L—\¢~ \ \ \ \ \ "1 IT 11 / T +. \‘\ *— 3 , \ 3- ' 3.. /., I \ _ < H-Jb-o— /,,. \ m6 1. H— 06 1-53 33? t 3 3 -1- 3 3-33-33 _. 0 3.. 20.05000 00:20:00 '\ \ 3‘ 0:5 0923» 5:5 002? ._ 00:5: .30: 1003 £90 “.0 2205000 0020.250 9 2:2“. 48 0.0.0.00 Wm. 0.2.00 mg 0420 Rm 505238 32.08.. 60:“. E95 0 < m m 0 < m at- . -.:-;_ -3-- -3- _ -~..-.----;.--.- .33..- -.m- 3. .- 3-2, - , - 3.3-- 3... 23-311” 0.0... r 3'“ "“ ' I q r 1 F r11, -. 141111 3 3 3-3 . ////’-57; ' 37133333 3- mlnli Q r. .- 4.0 _" ,/ 1 E.- o-f- , V . " :4" 1w 0.0 0 u...- ~=0_0.:0oo 00:20:00 0020., 0 .020: 00000000 220 .0 «0005000 00:20:00 .2 050...“. 49 04.0.00 m/u 0.0.0.00 ES 0.3.3 0. :o_m:=x0 22.8... 0000 E20 w < m m w < \ / .x « 4. o x 0. fixf- fig . \ - ” /7fT. t. s / x. _ \ 1l| «,4.\ . . I / , . ‘ x» - w _/ .LJWIA ‘.. \k ‘ \ / \xL w A 0 .r / 1+ \--+ , \ N. .// \x. \\A ~ H a .r/ / b V x \ I ,- . x... 0 a. .r, . 0-. x N o .\tL- x:/: i, .-.\ a . - w \ Kw ,.-~ U V_\. \~ “ \0 .. x“ a; x; M \ \- a w ./ V.“ \ W. \ \ \ «\l.’ _ /4 fl \ Q . . .. rn. KL \ t “/- \ . ... .0 \ . \- .. . -- ......-.. ’— . \ ‘. ‘ \‘ A_ l .- \ . ‘. v \ L— b X. 0 x \ ‘- ‘ - \ . . .V‘ \ \ “ \ 0 «H- \ \‘k \\\ .\ \ ‘. ‘ \ \->b—\o ~ -\ t—Q— 5—~.—\ A \~ , \ ~\ . x . $ \\ 0 \ . , \0 \. S x ..\ . /. ,\ ., L . it.-- , ; -:- -;.--,;:--- - u -. 2.33;.--1- l-ii-x-I! :f-L P .-.i..--.~.00_0_:00m 00:50:00 00=_0> a 00.00: .300020 390 00 20.05000 00:20:00 0? 950E 50 of eight products. Drum dried-blank, drum dried-ferric ammonium citrate, drum dried-ferrous sulfate and extrusion- blank had medium or strongly positive correlation coefficients at Hunter L values (Figure 16), and extrusion- ferrous sulfate had a negitive correlation coefficient. Most correlations of drum dried products were negitive, but the extrusion products were positove ones at Hunter a values (Figure 17). The eight products of two processes had medium or strongly positive correlations with storage time at Hunter b value (Figure 18). Hunter b values of extrusion products were always higher than that of drum drier products during the storage ( 23 weeks ) (Figure 19). Comparing three storage temperatures ( 21.1100, 26.6700, and 36.6700 ), the slope of Hunter L line of drum dried products were decreased when the storage temperature was increased (Figure 20). The slope of Hunter b line for products both processes were increased when the storage temperature was increased (Figure 21). The three iron fortified products of two processes decreased the Hunter b values during they were storage at all three temperatures(Figure 22). The order of Hunter b values of three iron products during storage is Ferric Ammonium Citrate > Ferrous Sulfate > Electrolytic Iron. The change rate of ferrous sulfate products’ Hunter b values was fast during the storage. 51 =O_¢=.=Km . + - 50—50 Efiun— -ITI $0.00!. 0.0.... 000.05 0000.0000.0.¢0.0.$0.~. .. 0. 0 0 h 0 0 w 0 N . 77.4fiaiifi- 11...), iii-(1.13.4.2; 14. I- A-F-lwrixmjiu {-4,} 4m 1-1. - . - .- 4.. - ii -!-.\ rt. o _ 0 m I. 0. 1. 0. . - - -. 0w tl-L-LT-r-Tk-T+++z~+++++++++++ . .ra-lft. - 1- . -;- $11,. -- .- .l .- :i -. -. - - !.- L mu 00.0.. n 00.5.: 0:... 0003.0 .0 0.0.. 0.00005 .020 .0 00:_0> a 00.00... 0. 200E 52 000.00 + on .. > Oohcdm ima- Oohodw . +- - Oopvfiw 1+1 3.003. 0:... 000.0.» mama—«000. 0. h. 0. m. 30— w. = or 0 0 h 0 m 0 0 N w a fi . 10....- .a: $43.4.- m-z-i..- ..-:.-..I-..---i...1:--J-tl-s.! 31.144 {14.1.14 .6 7 -1. .1 - «.0 - v.0 0.0 .3 ,_ . -. 0.. .-.-:--..-.--..- ..-,w i. t- 2.----- - - - ; : , - z-i . - -. 251.- (Jaw 00.00 .. 3.00.. 00.30.0050. 00000.0 000.. .0 0.0.. 0.000000 0000 0.0.0 .0 000_0> ._ 00.00... 00 200E 53 000.00 + 0. .. > 0.50.00 1%} Ochodu. . 0......3. . .0000). 0.0.. 0020.0 QNNNwNONowo—hwowowzmwuw=90obOUVQN.- fi 0 q 1 A 0 . A . . ~ 0 . J . . . . J . . .- m... N 000.00, 0 00.00: 00030000000. 00000.0 000... .0 0.0.. 0.000000 20.0 .0 000.0> 0 00.00: .0 209... 54 000.00. . 0. .. > 70......- 0-01....- 0.0.0, -r- . 0.5.... . - .2003. 0.0.. 0020.0 auuuwuoua—opto—mvvwopup = o. 0 o h o 0 v 0 u w i 0 4 _ 0 . 4 fl . . J. 4 q q q 0 fl 1 1 . . . o L Dd xm:-m-\mltmlml¢ 0%-: 10: .0 p Eat-$110! .r .+ + + .0.- + 0b; 0.1+ + . -. .. + . IN - - .- . ,. L L. L. ED." -. -. - .0 0 it -- 1 D.” -. ..- --.-i-..-.i iii-.39.. 0 000.9. a 00.00: 0.000000 :00. 000... 000... .0 000_0> 0 00.00: «0 0000...“. 55 Storage Stability Hexanal value of extrusion product before the storage was higher than one of drum dried except drum dried-ferrous sulfate product (Figure 23). The iron fortification increased the hexanal values at the finished products of both processes. The order of hexanal values of three iron fortified product of two processes is Ferrous Sulfate > Ferric Ammonium Citrate > electrolytic Iron (Figure 23) Hexanal had significant difference among eight products and different shelf-life (Table 17). Table 17 Analysis of Variance of hexanal of eight products during storage CF 194.9222 SS--sample 85.93175 SS--time 27.5365 SS--total 145.8477 SS--error 32.3795 MS~-sample 12.27596 MS--time 6.884125 MS--error 1.156410 F--sample 10.61557 ** F--time 5.953010 * SE-sample 0.480918 LSD-sample 3.135585 SE--time 0.380199 LSD--time 1.859173 Both drum dried and extrusion products had medium or strongly positive straight line correlations of hexanal values with the storage time(Figure 24). Figure 25 is hexanal (ppm) of drum dried and extrusion products at the storage period. Hexanal values of both products were increased during the storage, and the 56 Tm M... 0-... m.” w. 0000009... 03... 5.0338 \ \ \ 1 \ \ \ \ \ \\1 .\ X ‘ \ \\ . ‘ \ \ \\ V N ‘4 \ \ \ \ \I \\ \.\ *-\\\‘. _ \XL.\-\ ' » w. «kvmflomox, 43.0% mm... ., ./u.\ \.. \ \ (,A .1 . . r , 2-... - TETFTQ. / HIT-.. .. - ... ' m) . v .\.o.\ .,/ ... ./p .31. , , x *4 , / / / \ \ . \ \ \ \ \ L‘- b...‘. W4 .. . \~. .— x 0 . \. ~ \\ . ._. / [v-41' b % \ ~ \ ..\(.\. ., /¢rul A L. N 4 x. \ \ xx . .2. 0 7K»)... . C. ,, 3...... P- .2. \xx... . I. c . <‘< / . ,Ilvi. . 7) .xx. \ \. .x . \. _ j .1 y ,1 . h. .w 4 1 4 ..\ \\.\\......,.\2. . n .. ~ 1.. It I...- 1.. I A r ( 55555 r4 ."rh‘ . P .F .\\\ L \\:\\..\¥ .1. I a 1: u v \ \ ~\ w / ,.,,/m....._,..h. : X .\ .. . a. l I, . 1.? .W .O \..\ . r . A m I 7 r 70 . L r; \ . _.. f 2 :r..+ ._ f... .. . n... 1. D /r I 11.0.... W1 .- . .Im . *\\.. .\ \. .x \x, o. .. .1“ w .f/ .. ., H . . . x . . . .w . . _ .a p- v- I - ,'/,/ ,1 ‘ \w \ \ \ \ \ \ \ \ \ \ \ ’/, / I/ l \ \ .\ \ 055:.- ~\ J 1 1 . ./ x 4 v .\ / ,1... x _ .- CG 7. ,, x/w \.x \\ .\ R \.. . r/ c- Vin/3L... \\ NIX \ \KL . . . .+:. m i .. i... ‘ 0.5.0.2000 0030.230 22:35 2.90 .o 0:720...» 5.3 .0088: .0 20.2203 00:20:00 0.N 2:9... 57 E .0... 00.00.38 OI ferrous sulfate > ferric ammonium citrate. The order of the value after the crossed point was just reversed; ferric ammonium citrate > ferrous sulfate > electrolitic iron. In color of the products,before they were stored, extrusion products were higher than drum dried Hunter b values.Hunter a values of extrusion’s were lower than drum drier’s. The color change of the extrusion products’ Hunter L values and Hunter b values and drum drier products’ Hunter b values at all three storage temperature had medium or strong positive straight line correlations with storage time. The color change of the blank product’s Hunter L values and Hunter b values and the three iron fortified products’ Hunter b values at all three storage temperatures had also medium or stronge positive straight line correlations with storage time. Hunter b values of extrusion products were always higher than that of drum drier’s during the storage. When increasing the storage temperature, the rate of Hunter b values’ change was increased, but the rate of Hunter L values’ change was decreased. The order of Hunter b values during the storage was Blank > Ferric Ammonium Citrate > Ferrous Sulfate > Electrolytic Iron. Hexanal of extrusion products were higher than drum dried products before they were stored. Hexanal of both Processes products were increased during the storage. The 66 rate of drum dried products ’ hexanal increase were higher than that of extrusion so that the products of drum dried started to undergo rancidity at 12 weeks shelf-life but one of extrusion have not yet ranciditied at 20 weeks. Hexanal of electrolytic iron products were lowest during the storage, and one of ferrous sulfate products were highest during the storage. Ferrous sulfate products started to ranciditity at 8-th week. The viscosity of drum dried products were higher than that of extrusion products at Heating, Holding, and Cooling periods (29°C - 98°C - 98°C - 29°C). The use of extrusion processing with electrolytic iron supplementations provided the product with the greatest storage stability (color and flavor). 67 List of References Beard, J. L., and Finch, C. A., 1985. Iron Deficiency. In ”Iron Fortification of Foods" ed.Fergus M.Clydesdale, and Kathryn L.Wiemer, Academic Press,Inc. Benzo, Z., Schorin, H., and Velosa, H.,. 1986. Simultaneous quantitative determination of manganese, iron, copper and zine by atomic absorption spectroscopy in tropical cereals, fruit and legume materials. J.Food Sci. 51:222 Bothwell,T.H.,Charlton,R.W.Cook,J.D.,and Finch,C.A. 1979. Iron metabolism in man. Blackwell, Oxford. Burghes, D. N., 1981. "Modelling with Diffecential Equations". E.Harwood; New York. Cavill,I., Worwood,M., and Jacobs,A. 1975. Internal regulation of iron absorption. Nature(London) 256, 328- 329. Chauhan, G. S, and Bains, G. S., 1985. effect of granularity on the characteristics of extruded rice snack. J.Food Technology.20:305. Cook,J.D., and Finch, C.A. 1975. Iron nutrition. West. J. Med. 122, 474-481. Dallman,P.R., Siimes,M.,and Stekel,A.1980. Iron deficiency in infancy and childhood. Am. J. Cli. Nutr. 33, 86-118. Dillman, E., Gale, C.,Green, W., Johnson, D.G.,Mackler, B.,and Finch, C. A. 1980. Hypothermia in iron deficiency due to altered triiodothyronine metabolism. Am. J. Physiol. 239, R377-R381. Fritz, G. C., Pla, G. W., Roberts, T.,Boehne, G. W., and Hove,G. C., 1970. Biological avilability in animals of iron from common dietary sources. J. AGR. Food Chem., Vol.18, No.4; 647 Gauthier, S. F., Vachon,C., and Savoie, L., 1986. Enzymatic conditions of an In Vitro method to study protein digestion. J. Food Sci. 51:960 Hallberg, L. 1981. Bioavailability of dietary iron in man. Annu. Rev. Nutr. 1, 123-147. Hallberg, L. 1982. Iron nutrition and food iron fortification. Semin. Hematol. 19(1),31-41. 68 Hallberg, L., 1985. Factors influencing the efficacy of iron fortification and the selection of fortification vehicles. In "Iron Fortification of Foods" ed.Fergus M.Clydesdale, and Kathryn L.Wiemer, Academic Press,Inc. Harper, J. M., 1981. "Extrusion of foods". Volume II. CRC Press,Inc. Boca Raton,Florida. Harper, H. A., 1974. "Review of Physiological Chemistry". Lange Medical Publications. Los Altos, California. Hazell, T., and Johnson, I.T., 1987. In Vitro estimation of iron availability from a range of plant food: influence of phytate, ascorbate and citrate. British J.Nutr. 57:223 Herbert, V., 1987. recommended dietary intakes (RDI) of iron in humans. Am.J.Clin Nutr. 45:679. Hurrell, R. F., 1985. Nonelemental sources. In "Iron Fortification of Foods". ed. Fergus M.Clydesdale, and Kathryn L.Wiemer, Academic Press,Inc. Hurrell, R. F.,Lynch, S.R., Trinidad, T.P., Dassenko, S. A., and Cook, J. D. 1988. Iron absoption in humans: bovine serum slbumin compared with beef muscle and egg white. Am J Clin Nutr 47:102. Juiliano, B. 0. 1985. "Rice --- chemistry and technology". The american Association of cereal Chemists,Inc. St.Paul, Minnesota, USA Kadan, R. S, and Ziegler, G.M., 1987. Changes in iron forms during extrusion processing. Cereal Chem.64(4): 256-259 Kadan, R. S., and Ziegler,G.M., 1985. Iron status in experimental drum-dried rice foods. Cereal Chem. 62(3): 154-158. Kane, A. P., and Miller. D. D., 1984. In Vitro estimation of the effects of selected proteins on iron bioavailability. Am. J. Clin. Nutr 39:393 Ken Lee and F.M.Clydesdale. 1980. Chemical changes of iron in food and drying processes. J. Food Sci. 45:711 Kim, H., and Zemel, M. B., 1986. In Vitro estimation of the potential bioavailability of calcium from sea mustard, milk, and spinach under simulated normal and reduced gastric acid conditions. J. Food Sci. 51:95? Look, S and Bender, A. E., 1980. Measurement of chemically- available iron in foods by incubation with human gastric juice in vitro. Br. J. Nutr. 43, 413 Mercedes B. de Mosqueda, Consuelo M. Perez & Bienvenido 69 O.Juliano, Ricardo R. del Rosario, Donald B. Bechte. 1986. Varietal differences in properties of extrusion- cooked rice flour. Food Chemistry 19: 173-187 Miller, D. D., Schricker, B. R., Rasmussen, R.R., and Campen, D. V., 1981. An in vitro method for estimation of iron availability from meals. Am. J. Clin. Nutr. 34:2248. Myung Gon Shin, Suk Hoo Yoon, Joon Shick Rhee, and Tai-wan Kwon. 1986. Correlation between oxidative deterioration of unsaturated lipid and n-hexanal during storage of brown rice. J.Food Science. Volume 51, No.2; 460. Nathanson, M. H., and Mclaren, G. D., 1987. Computer simulation of iron absorption: regulation of mucosal and systemic iron kinetic in dogs. J.Nutr. 117:1067. Patrick, J., 1985. Elemental sources. In "Iron Fortification of Foods" ed.Fergus M.Clydesdale, and Kathryn L.Wiemer, Academic Press,Inc. Pla, G. W., and Fritz, G. C., 1971. Collaborative study of the hemoglobin repletion test in chicks and rats for measuring availability of iron. J. of The AOAC. Vol.54,No.1 Puri, S. C., 1971. "Applied Statistics for Food and Agricultureal Scientists”. Wiley. New York. Romanik, E. M., and Miller, D. D., 1986. Iron bioavailability to rats from iron fortified infant cereals: acomparison of oatmeal and rice cereals. Nutrition Reports International. Vol.34, No.4: 591 Savoie, L., and Gauthier, S. F., 1986. Dialysis cell for the In Vitro measurement of protein digestibility. J. Food Sci. 51:494. Sayers, M. H., Lynch, 8. R., Charlton, R. W. and Bothwell, T. H.., 1974. Iron absorption from rice meals cooked with fortified salt containing ferrous sullhate and ascorbic acid. Br.J.Nutr. 31:36? Schricker, B. R., and Miller, D. D., 1982. In Vitro estimation of relative iron availability in breads and meals containing different forms of fortification iron. J. Food Sci. 47:723 Schricker, B. R., and Miller, D. D., Rasmussen, R. R.and Campen, D. V., 1981. A comparison of in vivo and in vitro methods for determining availability of iron from meals. Am. J. Clin. Nutr. 34:2257 Shapp, R. N., and Timme, L. H., 1986. Effects of storage time, 70 storage temperature, and packaging method on shelf life of brown rice. Cereal Chem. 63(3):247. World Health Organization (WHO). 1968. Nutrional anaemias. Report of a WHO Scientific Group. W.H.O. Tech. Rep. Ser. 405. 71 APPENDIX I Chemical Analysis PROCEDURE OF PROTEIN DETERMINATION a,On an analytical balance,accurately weigh an appropriate amount of sample ( enough to require 20-30 ml of standard acid ), and transfer to Kjeldahl flask. The amount of sample was 5 gram. b,Add 3 or 4 glass beads. c,Add 1 Kel Pak or Kjeldahl tablet. d,Add 25 ml of sulphuric acid, washing down any particles of sample adhering to the neck of the flask. e,Digest 1 hour longer after the mixture has turned clear and/or blue or bluesgreen. f,Cool flask and contents, then cautiously add 300 ml of distilled water and swirl gently to mix. g,To 500 ml Erlenmeyer flask add 30 ml of the boric acid methyl red solution. Place under condenser so that tip of delivery tube is below the surface of the solution. h,Cautiously pour 70 ml of the 50% sodium hydroxide solution very slowly down the neck of the Kjeldahl flask so as to form 2 distinct layers. Flask may conveniently be hold at a 45* angle. Do not mix! i,Without mixing, add 1-2 gram of zinc granules. Jylmmediately connect flask to distilling apparatus, then SWirl contents well to mix. Be sure water is turned on in 72 distilling unit. k,Collect about 250 ml of distillate in the boric acid solution. l,Lower receiving vessel, allow tube to drain briefly, and rinse with distilled water. m,Titrate with 0.1 N HCI solution. n,Calculations ml HCI * exact Normality of HCI * 0.014 * 100 73 PROCEDURE OF FAT DETERMINATION a,On an analytical balance accurately weigh 2 gram of well ground sample into a 50 ml beaker. b,Add 2 ml of alcohol to moisten sample and mix well with glass rod. c,Add 10 ml of HCI solution and mix well with rod. d,Place in 70-80 C water bath for 30-40 min, mixing well at 5 min.interval(sample should be quite black when ready). e,Cool and add 5 ml of alcohol. Transfer quantitatively to a Mojonnier extraction flask. Rinse beaker with 5 ml more of alcohol, transfer rinsing to flask. f,Rinse beaker with about 15 ml of anhydrous ether, and transfer rinsing to flask. Repeat with remaining 10 ml of ether. Discard beaker. g,Stopper flask and mix well with a rocking motion. h,Add 25 ml of petroleum ether, cork and mix with rocking motion as above i,Allow to stand untill layer separate well, centrifuging if necessary. J.Crefully decant ether layer into a previously dried and weighed fat evaporation dish. k.Evaporate ether slowly on a steam plate under the hood. l,To flask add 25 ml of ethyl ether, cork and mix with rocking motion as above. m.Repeat steps a through k. 74 n.Dry evaporating dish in 100 C vacuum oven for 1 hour, cool in desiccator and weigh. o,Calculation; (weight of dish + fat) - (wieght of dish) weight of sample 75 PROCEDURE OF ASH DETERMINATION a,Ignite ashing dishes at 550 C. Cool in desiccator, and weigh. b,Weigh sample into this dish (5 gram per sample) c,If puree, dry on steam bath first. d,Heat gently over burner in hood to drive off last traces of moisture. Allow sample to char slowly until no more smoke is given off. e,Place sample in muffle furnace at temperature no higher than 425 C. f,Gradually increase temperature to 550 C. g,Incinerate until a white or light grey ash is obtained. h,Cool in desiccator and weigh as soon as room temperature is reached. i,Calculation; (weight of dish + ash) - (weight of dish) Ash % = ------------------------------------------ * 100 (weight of dish + sample) - (weight of dish) 76 CARBOHYDRATE Carbohydrate % = total solid % - fat % - protein % - ash % 77 APPEDIX II “ethod of Iron Dialyzability Study ( In Vitro Method ) Generally, there were two procedure. The first one was the standardizing the sample iron content of the products. Now, we used those eight products to make the standardized samples. The iron content of the standized samples were 10, 20, 30, 40, 50, and 60 mg per 100 gram sample respectively. The way of the standizing the samples was to mix the blank product and one of iron fortificated product according to the ratio of them. The equation is as follows; (1 gram ) * [ Fe )sample = (X gram)blank product * [ Fe Jblank product + (l - X) * [ Fe )one of iron products 1 gram * [ Fe ]sample * [ Fe ]iron product [ Fe )blank - [ Fe Jiron product = gram blank product / 1 gram sample The second procedure was In Vitro Estimation of Iron Availability. The dialysis tubing was Spectrapor Membrane Tubing No.132665 (Spectru Medical Industries,lnc.) ------ vol/cm: 8 ml; m w cutoff: 6,000 - 8,000; dry cylinder dia: 31.8 mm; dry thickness: 0.0012". The instrument that analysed the iron content was Varian Atomic Absorption Spectrophotometer; model 1250; Air-Acetylene Flame. 78 IN VITRO ESTIMATION OF IRON AVAILABILITY 1),REAGENTS AND MATERIALS; a,Pepsin suspension; Prepare just before use. Weigh out 2.8 gm pepsin (Sigma P 7125). Transfer to a 50 ml vol flask, and bring to 50 ml with 0.1N HCI and swirl. b,Pancreatin-bile; Prepare just before use. Weigh out 2.0 gm pancreatin (Sigma P 1500) and 3.0 gm bile extract (Sigma B 8631). Bring to 250 ml with 0.1 M NaHCO in a vol flask and stir. e,Dialysis tubing; Cut Spectrapore I dialysis tubing (No.132650) into 15 cm lengths. Soak in distilled water until needed. d,Other reagents; 0.1 N HCI, 0.1 M NaHCO , 6 N HCI, and 0.5 N KOH. e,Snap cap vials; 120 ml (Fisher No.03-335-10D). f,All glassware must be chromaged. 2),PROCEDURE a,Meal preparation; a),Combine the meal ingredients (rice cereal) and water in a blender ,(beaker, jar or vial) to 100 gm with 5 % solid. b),Meal homogeneous --- Heat the meal in 95 C for 15 mins. While stirring, and cool down for 30 mins. while stirring. prdd 6 N HCI to meal until the pH is 2.0 while stirring. All meal must be cooled down below 37 C. 79 c,Transfer 5 gm meal to the platinum dish to determine the iron content of the meal following the procedure --- BASIC ASHING METHOD. d,Transfer 19 gm meal to each of 4 vials.(If there are too many samples, we can run one replicate of them each time). All samples that can not run on time must be kept in refrigerator. e,Incubate in a 37 C shaking water bath for 2 hours to form pepsin digests. f,Use one of 4 vials to determine the titratable acidity of the sample. Add 5 ml of pancreatin-bile suspension to this vial. titrate it with 0.5 N KOH to pH 7.5. Record the volume of KOH used. N KOH * Vol KOH = N sample * Vol sample g,Prepare dialysis tubings. The dialysis solution is 15 ml with distilled water and an amount of NaHCO, which is equivalent to the amount of KOH used in the titratable acictity titration. Tie the tubing and put it into the vial. h,Incubate the vials with 37 C shaking water bath for 45 mins . Then add 5 ml pancreatin-bile mixture to each vial. Continue incubating with shaking bath for 2 hours. j~aRelnove dialysis bags. Rinse by dipping in distilled water. Cut off one end of each bag and dump the contents into the beaker. (The dialysis solution must be weighed). j’Measure the pH of dialysates and retentate. 1“"Transfer the retentate to the centrifuge tubes and centrifuge at 2,000 rpm for 20 minutes. 80 l,Weigh two 2.5 gm supernatents of retentate to the platinum dish to determine the iron content of supernatent of retentate. Weight two 5.5 gm dialysates to the dish to determine the iron content of the dialysate following the procedure of BASIC ASHING METHOD. 81 BASIC ASHING AND MINERAL METHOD a,Rinse the inside of a platium dish with HCI. In a hood poor off the acid, rinse dish with tap water and then with redistilled water. Wipe dry wish a Kay dry, being careful not to touch the inside of the dish. b,Weigh out the appropriate amount of sample:(generally, if all mineral are run including trace minerals, 25 grams are used; if only the minerals of high content are run, i.e., Ca, Mg, P, Fe, Na, and K, 10 grams are generally sufficient; if the sample is a dry product such as cereal, 2.5 grams are used.) c,Place dishes on the hotplate and char with heat lamp until sample is completely black and it no longer smokes. d,Place dishes in muffle oven overnight at 550 C. a,ROmove dishes from the muffle oven and rinse down the inside with about 5 ml of concentrated HCI using a pipet. f,Steam on the steambath until residue is dry. g,Rinse the inside of the dish with 2 ml of concentrated HCI, cover with a water glass, and steam for five minutes. h,Wash off the inside of the watch glass into the dish with redistilled water. i,Using a funnel, transfer the contents of the dish to a 25 ml volumetric flask quantitatively and make up to volume with redistilled water. Mix well. j,If the sample is not crystal clear, filter through 3 1 82 Whatman filter paper. k,Run the sample in the Atomic Absorption Spectrophotometry. k,Calculation; ml of sample solution Mineral % = --------------------- * dilution ratio weight of sample ppm of standard mineral peak of standard curve * peak of sample 1 = mg / 1000 gm * --- = mg / 100 gm 10 83 APPENDIX III Method of Amylography Analysis After the eight products were made by drum drier and extruder, the amylograph of the products were tested following the procedure as follows. The instrument is Visco- Amylo-Graph; No. 676, Type. VA1; C.W.Brabender Instrument, Inc. South Hackensack, N.J. AMYLOGRAPH PROCEDURE 1),PREPARATION OF SAMPLE a,Measure prescribed amount of distilled water (400 ml) and add all but 100 ml. to a Waring blender bowl. b,Add carefully weighed sample (100.0 grams cereal) to the blender. c,Blend for 60 seconds at slowest speed which will thoroughly disperse sample and break up any lumps. Avoid any foaming or inclusion of air. d,Immediately transfer slurry to amylograph bowl. Use the remaining 100 ml water to thoroughly rinse down sides of blendor bowl, and add rinsing to amylograph bowl. e,Carefully lower the top section of the amylograph, using great care not to jar the thermoregulator or to put sudden strain on the tungsien carbide tip or the sapphire bearing. f,Push the cooling coil all the way down and lock in place. 2),PRE-WARMING PERIOD 84 a,Set the thermoregulator at 29 C, with the clutch in "N" (neutral),position. b,Turn on the cold water solenoid switch. (Be sure water supply is on) c,Turn the instrument on. When the temperature reaches 29 C, red pilot light will flash off, indicating end of the warm-up period. 3),HEATING PERIOD a,As soon as the pilot light flashes off, turn off the water solenoid switch. b,Set the time for exactly 44 minutes. c,Put the clutch in the "U" (up) position. d,Advance chart paper so that pen is recording at “0" time. (The temperature will increase from 29 C to 95 C during this 44 minutes period) 4),HOLDING PERIOD a,As soon as 95 C is reached, set the clutch in the "N“ position. b,Set the timer for 16 minutes. (The temperature of 95 C will be maintained for this period) 5),COOLING PERIOD a,At the end of holding period, set clutch in the "D" (down) position. b,Unless otherwise specified, re-set the timer for 44 minutes. c,Turn on the cold water solenoid switch. (The temperature will automatically decrease from 95 C to 29 C) 85 APPENDIX IV Color Change Analysis Each of eight products was took the samples, and were canned. Each product had 60 cans, and each can had about 20 grams rice cereal. Those 60 cans were divded to three groups, and were separatly stored at three storage temperature; 70 F, 80 F, and 98 F. Each half week of the first month, the samples were tested on the Hunterlab Colorimeter (model D 25 A - 2). After that, the samples were test once a month until five month of the storage. The operation of Hunterlab Colorimeter Model D 25 A - 2 is Appendix IV. Procedure of Hunterlab Colorimeter. Standardizing the Instrument a,Zero Scale Adjustment a),Place black glass, shiny side toward port, at specimen port. Be sure this glass is clean. b),Depress the “Y“ push button. c),Using the small screwdriver supplied with the instrument, adjust the screw accessible through the "Y" zero scale adjustment hole until the digital readout just changes from minus to +00.0. d),Depress the "X" push button, and adjust the screw accessible through the "X" zero scale adjustment hole until the digital readout just changes from minus to +00.0. e),Depress the "Z" push button, and adjust the screw 86 accessible through the "Z" zero scale adjustment hole until the digital readout just Changes from minus to +00.0. b,Standardizing On White Tile a),Select the white calibrated standard from those supplied with the instrument and place it at the specimen port using the lab jack to raise into position. Standards should be centered at the specimen port and the word "HUNTERLAB" on the back of the standard should be facing the operator. b),Depress the "L“ push button. c),Turn the "L-Y“ standardizing control knob until the calibrated “L“ value of the white standard is displayed on the digital readout. If difficulty is encountered in standardizing on any of the calibrated standard values, the standardizing ranges can be increased by adjusting the Y, X, and Z (not Xa or Xb) trimpots located in the optical sensor. This is accomplished by turning the knob for the trimpot for the value where increased range is needed - or if not equipped with the knob control, by using the small screwdriver to turn the screw in the access slot. The two other screws are for the Xa and Xb trimpots and are used when adjusting the amber-blue ratio. The X trimpot can be used to achieve greater standardizing range without affecting the amber/blue ratio. d),Depress the ”a" push button. e),Turn the ”a-X" standardizing control knob until the calibrated "a" value of the white standard is displayed on the digital readout. 87 f),Depress the "b" push button. g),Turn the “b-Z" standardizing control knob until the calibrated "b” value of the white standard is displayed on the digital readout. h),Repeat the zero scale adjustment and standardization on white tile until the zero reading of the black glass and standardization on white tile exist without adjustment. Measuring Specimens a,Measuring a Wide Range of Colors Using White Standard as Reference a),Place the specimen at the specimen port. The position of the light on the specimen, or its flatness can be examined through the veiwing aperture on the front cover of the optical sensor. b),Depress the L push button. Record on data sheet the L value displayed on the digital readout. c),Depress the a push button and record the a value displayed. d),Depress the b push button and record the b value displayed. b,Measuring Specimens of Similar Color. When specimens are all similar in color, long-term accuracy is greatly increased by standardizing the instrument on a calibrated standard of color as close as possible to that of the specimens to be measured. Standardize the instrument using such a standard and the calibrated values on the back and proceed with measurements as above. 88 APPENDIX V Rancidity Study The samples of the eight products were canned , and were stored at 36.670C. The samples were tested each four weeks until sixteen weeks. Hexanal were analysed using Gas Chromatograph technique. The model of the instruments were Tekmar Purge and Trap Concentrator LSC-3 and Gas Chromatograph Hewlett Packard 5890 A; flame ionization detector; column - 50 m * 0.32 mm I.D, 0.25 um OV‘1701 liquid phase. Procedure of Hexanal Determination 1),Working solution preparation; Take 1 ml MIBK (10 mcg/ml) and 5 ml 4- Hephanone (1 meg/ml) to 100 vol flask, and mix well. 2),Weigh 200 mg ground cereal to the Gas Chromotograph test tube; 3),Add 4 ml the working solution to the tube, and mix well. 4),Purge the sample test tube at the TEKMAR PURGE AND TRAP CONCENTRATOR LSC-3 for 7 mins. 5),Make the time program in the Gas Chromotograph; 89 Table 18 Gas Chromotograph Time Program PERIOD RATE TEMPERATURE TIME F minute £003.? "6 """ 16 """""" é """ 1 20 30 2 A 5 50 0 B 15 120 4 6),Run the sample in the Gas Chromotograph with the time program. 7),Record the results from the computer. 90 APPENDIX VI Statistics Method Analysis of Variance and Correlation Analysis and Regression Analysis were used in this experiment. Analysis of Variance --- TWO FACTORS Example; Table 19 21.1100 ---- Hunter b values 1 17.9 18.8 16.5 16.9 20.8 19.5 20.2 19.8 4 19.1 18.7 17.0 17.4 21.9 19.3 21.6 21.4 8 20.0 19.2 17.2 17.7 21.8 19.3 20.9 20.6 12 19 0 19.4 16.9 17.0 21.8 19.8 21.2 21.7 22 19 3 18.6 16.7 17.7 21 2 19.4 21.2 20.2 25 19 5 19.6 17.2 17.3 22 5 19.7 21.9 21.3 29 19 3 19.1 16.9 17.8 21 7 19.7 21.1 20.8 31 19 5 19.4 16.9 17.0 21.1 19.6 21.5 20.6 61 19 1 19.3 17.0 17.1 21 8 19.8 21.9 21.0 92 20.0 19.6 17.4 18.0 22.5 20.3 22.4 21.2 122 19 1 18.9 17.4 17.6 22.9 20.7 22.1 20.3 153 19 8 19.5 17.4 17.3 22.6 19.9 22.1 21.6 CF (Correction Factor) = squaring the total and dividing by the total number of responses = (17.9 + ...... + 21.6)“2/(8 * 12) = 36981.35 SS-treatment 2 adding the squares of the total for each (Sum of Squares) treatment, dividing by the number of time for each treatment, and then subtracting the CF = ((17.9 + ...... + 19.8)“2 + ...... + (19.8 + = 270.4895 91 SS-time = SS-total = SS-error = df-treatment = adding the squares of the total for each time, dividing by the number of treatment for each time, and subtracting the F ((17 9 + ...... + 19.8)“2 + ...... (19.8 + 11.18208 adding the squares of each sample and subtracting the CF (17.9“2 + ...... + 21.6“2) - CF 291.6095 SS-total - SS-treatment - SS-time 9.9379 subtracting one from the number of treatments (Degree of Freedom) df-time = df-total = df—error = MS-treatment = MS-time = MS-error = F-treatment = 8 - 1 = 7 subtracting one from the number of times 12 - 1 = 11 subtracting one from the number of samples 12 * 8 ~ 1 = 95 df-total - df-treatment - df-time 95 - 7 — 11 = 77 dividing the SS of treatment by its df (Mean Square) 270.4895/7 = 38.6414 dividing the SS of time by its df 11.1821/11 = 1.01655 dividing the SS of error by its df 9.9379/77 : 0.12906 dividing the MS of treatment by MS of error (Variance Ratio) 92 38.6414/0.12906 2 299.4065 F-time = dividing the MS of time by MS of error 1.01655/0.12906 = 7.8766 93 Table 20 ANALYSIS OF VARIANCE TABLE SOURCE OF VARIATION df SS MS F TREATMENT 7 270.48950 38.64140 299.4065 ** TIME 11 11.18208 1.01655 7.8766 ** ERROR 77 9.93790 0.12906 TOTAL 95 291.60950 Checking the F Value Table, the possibility of F- treatment and F-time in the distribution of F are less than 10‘(-6). Therefore, there were significant difference among the treatment and among the time. Since there were significant diference among the treatment and among the time, the ones that were different can determined using Tukey’s Test. 94 TUKEY’S TEST Treatment; SE (Standard Error) = taking the square root of the MS of ; error divided by the number of time (0.12906/12)‘(1/2) = 0.1037 LSD (Least Significant difference) = 4.44 * SE 4.44 * 0.1037 0.4604 * 4.44 is obtained in the SIGNIFICANT STUDENTIZED RANGE AT THE 5 % LEVEL TABLE. Table 21 TREATMENT AVERAGE VALUE 1387-3 1387-4 1387-2 1387-1 1388-2 1388-4 1388-3 1387-3 17.042 1387-4 17.400 0.77 1387-2 19 175 4.63 3 85 1387-1 19.300 4.90 4 12 0.27 1388-2 19.750 5.88 5 10 1.24 0 97 1388-4 20.875 8.32 7.54 3.69 3.42 2.44 1388-3 21.508 9.69 8.92 5.06 4.79 3.81 1.37 1388-1 21.967 10.69 9.91 6.06 5.79 4.81 2.37 0.99 )enote; Value = difference between two sample dividinf by the LSD If the Value is > or = 1.00, this means that those two samlpe have significant difference. Time; SE (Standard Error) = taking the square root of the MS of error divided 95 by the number of treatment = (0.12906/8)A(1/2) = 0-1270 LSD (Least Significant difference) = 4.81 * SE = 4.81 * 0.1270 = 0.6109 * 4.81 is obtained in the SIGNIFICANT STUDENTIZED RANGE AT THE 5 % LEVEL TABLE. Table 22 ME AVERAGE VALUE 1 22 29 4 31 8 12 61 25 122 153 1 18.800 2 19.288 0.79 9 19.550 1.22 0.42 4 19.550 1.22 0.42 0 1 19.575 1.26 0.46 0.04 0 04 8 19.588 1.28 0.49 0.06 0.06 0.02 2 19.600 1.30 0.51 0.08 0.08 0.04 0.02 1 19.625 1.35 0.55 0.12 0 12 0.08 0.06 0.04 5 19.875 1.75 0.96 0.53 0 53 0.49 0.46 0.45 0.41 2 19.875 1.75 0.96 0.53 0 53 0.49 0.46 0.45 0.41 0 3 20.025 2.01 1.21 0.78 0.78 0.74 0 72 0.70 0.65 0.25 0.25 2 20.175 2.25 1 45 1.02 1.02 0.98 0 96 0.94 0.90 0.49 0.49 0.25 enote; Value = difference between two sample dividinf by the LSD If the Value is > or = 1.00, this means that those two samlpe have significant difference. 96 Correlation Analysis and Regression Analysis of Straight Line EXAMPLE; Table 23 21.110C ---- Hunter b values Time-day 1387-3 x x22 Y Y"2 x x Y ‘i 1 1 16.5 272.25 16.5 4 16 17.0 289.00 68.0 8 64 17.2 295 84 137.6 12 144 16.9 285.61 202.8 22 484 16.7 278 89 367.4 25 625 17.2 295 84 430.0 29 841 16.9 285.61 490.1 31 961 16.9 285 61 523.9 61 3721 17.0 289 00 1037.0 ; 92 8464 17.4 302 76 1600.8 . 122 14884 17.4 302 76 2122.8 153 23409 17.4 302 76 2662.2 TOTAL 560 53614 204.5 3485 93 9659 1 n * d(X*Y)- d X * d Y { (n * d(X02)‘(d X)“2) * (n * (d(Y“2)‘(d Y)‘2) 12 * 9659.1 — 560 * 204.5 { (12 * 53614 - 560“2) * (12 * 3485.93 - 204.5“2) = 0.7324 Checking the CORRELATION COEFFEIENT TABLE, the 97 possibility of r = 0.7324 is 0.7 %. Therefore, correlation coeffeint was signifacant. REGRESSION ANALYSIS Y = a + b * X 1),Slope --- b n * d(X*Y) - d x x d Y b: ----------------------------------------- n x d (x*2) — (d X)‘2 12 * 9659.1 - 560 * 204 5 _ 12 * 53614 - 560‘2 = 0.0042 2), a d Y — b * d x a: ---------------------------- 204.5 - 0.0042 * 560 12 16.845 : Y = 16.845 + 0.0042 * X { 'LOGISTIC ’ Curve Fits to Straight Line 98 this ’ LOGISTIC ’ curve equation a + b * X Ym * e Y: -------------------- a + b * X Ym + e Denote; Ym ------ Maximum of Y Y in ( ---------- ) = a + b * X 1 - Y/Ym Denote; Y’ : 1n ( __________ 1 - Y/Ym 99 IES "100111All“