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Thermal degradation of thiamine in bread Nadeau, Louise 1982

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THERMAL DEGRADATION OF THIAMINE IN BREAD by LOUISE NADEAU B.Sc., Universite de Montreal, 1977 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Food Science) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February 1982 © Louise Nadeau, 1982 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Food Science  The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date March 16, 1982 DE-6 (2/79) ABSTRACT Thiamine is an important nutrient found in significant amounts in wheat flours. This vitamin is heat labile thus destruction occurs during bread baking. Using a kinetic ap proach, the effect of heat and pH on thiamine degradation in a model system were studied. In order to compare the sta bility of thiamine from natural (whole wheat) and synthetic (enriched white) sources, thermal destruction of thiamine in the two breads was investigated. Destruction rates of thiamine hydrochloride in phosphate buffer at pH 6.0 and temperatures between 80 and 120°C were measured. The breakdown reaction could be described by first order kinetics. An energy of activation of 3^-2. kcal/mole was obtained. Destruction rates of thiamine hy drochloride in phosphate buffer at 120°C were measured for pH values between 4.0 and 7.0. The reaction rate increased as the system was made more alkaline, with greater destruc tion at pH 6.0 and above. Thiamine losses in an enriched white flour system baked at a nominal temperature of 246°C (475°F) for 60, 75 and 90 min were found to be 2.4, 27.9 and 29.2%, respectively. Two experiments were carried out with 450 g (1 lb) enriched white loaves baked at 221°C (430°F). Baking times were 30 - i -min for the first experiment, and 15, 37 and 60 min for the second experiment. No appreciable thiamine destruction were found in either experiment. The main investigation was with a semi-model system of 12g bread loaves made from enriched white and whole wheat flours. Four different nominal oven temperatures of 177, 221, 246 and 288°C (350, 425, 475 and 550°F) were used with four different baking times for each run. The pH of the dough and baked bread were determined. Oven, crust and loaf center temperatures were monitored. The mass average temp erature data of the bread during baking showed a changing rate of temperature rise, and because of this, it was not possible to obtain kinetic data. However, a linear rela tionship was obtained when the logarithm of the percent thi amine retention was plotted against time. This experiment showed a lower thiamine stability with higher oven tempera ture. Thiamine was less stable in whole wheat bread than in enriched white bread. This might be explained by higher pH and ash content in whole wheat bread. Thiamine losses dur ing normal baking of whole wheat and enriched white bread were found to be in the range of 28.3 to 47.8%. - ii CONTENTS ABSTRACT i ACKNOWLEDGMENTS ix page I. INTRODUCTION 1 II. LITERATURE REVIEW 2 Nomenclature and function 2 Thermal destruction of thiamine 5 Thiamine losses in bread 14 III. MATERIALS AND METHODS 18 Model System 1Vitamin analysisPreparation of buffered thiamine solutions . 19 Treatment of samples 1Come-up times . . 20 Temperature effectpH effect 21 Bread System .22 Thiamine analysis ... 22 pH determination . 23 Moisture determination 2Apparatus for heatingFlour samples 2Enriched white flour slurry assay 23 One pound loaves (enriched white bread) . . 24 Experiment 1Experiment 2 .... 212 g loaves (enriched white and whole wheat bread) .24 Treatment of samples 25 Experiment 1 26 Experiment 2IV. RESULTS 31 Model System 3Temperature effect 31 pH effect 6 Bread System 42 - iii -Enriched white flour slurry assay 42 One pound loaves (enriched white bread) ... 42 Experiment 1 4Experiment 212g loaves (enriched white and whole wheat bread) 5 Experiment 1 4Experiment 2 6 V. DISCUSSION 64 VI. CONCLUSION 72 LITERATURE CITED 3 Appendix page A. THIAMINE LOSSES IN BREAD 77 B. STATISTICAL ANALYSIS - 12G LOAVES (EXPERIMENT 2) . 81 t-test 8F-statistic 2 SNK test 3 Enriched white bread 8Whole wheat breadDeviation from linearity 3 C. EXAMPLE OF CALCULATION FOR % THIAMINE RETENTION . . 86 Model system . 8Bread systemiv -LIST OF TABLES Table page 1. Review of kinetic studies for the thermal degradation of thiamine 9 2. Retention of thiamine hydrochloride in phosphate buffer (ph 6.0) at different heating times between 80 and 120°C 32 3. k, r and T* 1/2 values for thiamine HCl in phosphate buffer (pH 6.0) in the temperature range 80 to 120°C 35 4. Retention of thiamine hydrochloride in phosphate buffer (120°C) at different heating times between pH 4.0 and 7.0 38 5. k, r and f' 1/2 values for thiamine HCl in phosphate buffer (120°C) in the pH range 4.0 to 7.0. ... 40 6. Thiamine retention for enriched flour slurry for different baking times at 246°C (475°F) (nominal) 43 7. Thiamine retention in 450 g (1 lb) loaves of enriched white bread baked at 221°C (430°F) for different times 44 8. Thiamine retention of white and whole wheat bread baked at 4 different temperatures for several baking times (Exp.1) 47 9. pH values for white and whole wheat dough and bread baked at 252°C (485°F) (nominal) for 17 min (Experiment 2) 48 10. Oven temperatures for enriched white and whole wheat bread baked in different loads at 4 nominal oven temperatures 49 11. Thiamine retention of white and whole wheat bread baked at 4 different temperatures for several baking times (Exp.2) 54 - v 12. Slope and r values of thiamine destruction curves for white and whole wheat bread baked at 177, 218, 246 and 288°C 60 13. Half-life values for thiamine in enriched white and whole wheat bread baked at 177, 218, 246 and 288°C (nominal) 63 - vi LIST OF FIGURES Figure . page 1. Enriched white and whole wheat loaves baked at 4 different baking time periods 27 2. Retention curves for thiamine hydrochloride in phosphate buffer (pH 6.0) 33 3. Retention curve for thiamine hydrochloride in phosphate buffer (pH 6.0) 34 4. Arrhenius plot for the thermal degradation of thiamine hydrochloride in phosphate buffer (pH 6.0) 37 5. Retention curves for thiamine hydrochloride in phosphate buffer at 120°C at various pHs 39 6. The effect of pH on the destruction rate constant for thiamine hydrochloride in phosphate buffer at 120°C 41 7. Heat penetration curves for enriched white bread baked at 4 nominal oven temperatures 50 8. Heat penetration curves for whole wheat bread baked at 4 nominal oven temperatures 51 9. Heat penetration curves for the middle and crust of enriched white bread baked at 218°C (425°F) (nominal) 53 10. Thiamine retention curves for enriched white and whole wheat bread baked at 177°C (350°F) (nominal) . . . 56 11. Thiamine retention curves for enriched white and whole wheat bread baked at 218°C (425°F) (nominal) 7 12. Thiamine retention curves for enriched white and whole wheat bread baked at 246°C (475°F) (nominal) 58 - vii -13. Thiamine retention curves for enriched white and whole wheat bread baked at 288°C (550°F) (nominal) 59 14. Thiamine retention curves for enriched white bread baked at 177, 218, 246 and 288°C (nominal). ... 61 15. Thiamine retention curves for whole wheat bread baked at 177, 218, 246 and 288°C (nominal). ... 62 16. A review of pH/k curves for thiamine in buffered solutions 66 - viii -ACKNOWLEGMENTS First, I would like to express my gratitude to Dr. John Vanderstoep for his constant support throughout this study. I wish to thank H.S. Ramaswamy, Estella Lee, Lynne Robin son and especially Sherman Yee for their help in technical matters . I must thank my fiance Robert Rempel for his help in the statistical analyses and in the preparation of the computer ized manuscript and for his constant encouragement. Finally, I would also like to thank my committee members for their suggestions. This study was made possible through a scholarship to the author and a grant to Dr. John Vanderstoep from the Natural Sciences and Engineering Research Council of Canada. - ix -I INTRODUCTION Thiamine is an important nutrient. The wheat kernel con tains significant amounts of thiamine and therefore baked goods made from wheat flour are potentially a good source of this nutrient. However, this vitamin is heat labile and some destruction occurs during the baking process. It is desirable therefore to determine the stability of this nut rient in these foods during the production procedures, par ticularly the baking period. Knowledge about the mechanisms of thiamine degradation can be obtained by studying the behaviour of thiamine under different conditions of temperature, time and pH in bread baking as well as in model systems. Such studies can lead to the definition of optimum conditions for thiamine reten tion. It would also be interesting to know whether differ ent bread systems, that is, breads made from whole grain flours or enriched white flours, have any effect on the re tention of thiamine. The most reliable and satisfactory ap proach to obtaining insight into this is through simple re action kinetics. - 1 -II LITERATURE REVIEW 2.1 NOMENCLATURE AND FUNCTION Vitamin activity was recognized in 1890 by the Dutch physician Eijkman (Neal and Sauberlich, 1973)- Prof B.C.P. Jansen of Amsterdam with W.F. Donath first isolated the substance from a natural source in 1926 (Williams, 1938; Dwivedi and Arnold, 1973; Neal and Sauberlich, 1973). Jan sen suggested the trivial name Aneurine, which has come into extensive use in Europe. However, because of therapeutic implications, this name, aneurine, was eventually replaced by thiamine (thiamin) (Williams, 1938; Neal and Sauberlich. 1973). It was only in 1936 that R.R. Williams and his col leagues isolated sufficiant quantities of the vitamin to fully identify its structure. They established it as being composed of a pyrimidine and a thiazole moiety and as 3-(2 -methyl -4'-amino -5 '-pyrimidylmethyl) -5-(2 -hydroxye-thyl)-4 - methylthiazole (Williams, 1938; Harper, 1973; Neal and Sauberlich, 1973). The formula below shows the thiamine hydrochloride form : - 2 -3 NH2.HC1 CH3 Pyr imidine moiety CH3 CH2 Cl-Methylene bridge -CH2-CH20H Thiazole moiety The rest of this section is documented from Holvey (1972), Harper (1973) and Neal and Sauberlich (1973). Thia mine is readily absorbed from the small intestine with no appreciable storage in the tissues. All excess intake is eliminated in the urine. The active form of the thiamine is TPP (thiamine pyrophosphate), diphospho thiamine or cocar-boxylase: 0 0 1 i CH2 - CH2 - 0- P- O- P-OH i i OH OH Phosphorylation, which is reversible, occurs in the intesti nal mucosa, kidney and liver (Harper, 1973; Neal and Sauber lich, 1973). TPP participates as a coenzyme in the oxidative decarbox ylation of alpha keto acids to aldehydes. These reactions play a major role in energy production (ATP) in plants and animals. In yeasts, TPP promotes alcoholic fermentation. 4 TPP is also the coenzyme of transketolase that participates in the direct oxidation of pentoses. Some of the first signs of deficiency are loss of appe tite (anorexia), weight loss, loss of muscular tone and de pression. Beri-beri, the classical syndrome of thiamine de-ficiency, is characterized by peripheral neurological changes and cardiovascular troubles with oedema (wet beri beri) or without (dry beri-beri). Accumulation of pyruvic acid and lactic acid, as well as pentoses, in the tissues is probably responsible for these troubles. Beri-beri is main ly found in the countries where polished rice is the princi pal food. In the occidental world, a deficiency is observed with alcoholism, particularly where the food intake is very low. This leads to the Wernicke-Korsakoff syndrome (mental confusion, psychosis) when the deficiency is severe and chronic. Thiamine deficiency can be detected by depletion of thiamine in urine and blood, reduction of the activity of erythrocyte transketolase, a thiamine pyrophosphate-requir-ing enzyme, and abnormal elevation of pyruvic acid and alpha ketoglutarate in blood and urine. The requirements for thiamine in human nutrition are usu ally based on caloric intake. They are 0.23 to 0.50 mg per 1000 kilocalories. For added safety, the Food and Nutrition Board (1980) recommends 0.5 mg of thiamine for each 1000 ki-localories in the diet. Therefore the recommended average daily intake for men is 1.2 to 1.4 mg and 1.0 mg for women. 5 Lipids seem to have a saving effect on thiamine requirements while carbohydrates have an opposite effect. In the different regions of the world, thiamine is pro vided by different foods. In North America, cereals provide 35% of the thiamine intake; meat (especially pork), 30%; fruits and vegetables, 15% and dairy products,10%. 2.2 THERMAL DESTRUCTION OF THIAMINE Many researchers have worked on the thermal degradation of thiamine for several years. At first, the results were not very accurate because of the poor methods of thiamine determination, for example, rat growth and diazotation meth ods. The thiochrome procedure is now the most widely used method. Only recently has the kinetic approach been used to determine the degradation of thiamine in several food sys tems. (Farrer, 1955). Some of the degradation reaction products in food or in model systems have just been identified (Dwivedi and Arnold, 1973). The main mechanism involves hydrolytic cleavage of the C-N bond of the methyl bridge between thiazole and pyri-midine moieties of thiamine, leading to a pyrimidine deriva tive, probably 2- methyl- 4- amino- 5- hydroxymethyl- pyri midine, and 4- methyl- 5- (B hydroxyethyl) thiazole (Williams, 1938; Dwivedi and Arnold, 1972, 1973). A second reaction involves the breakdown of the thiazole ring with hydrogen sulfide as the major degradation product and other volatile products (Dwivedi and Arnold, 1972, 1973). Many different factors affect the thermal degradation of thiamine, and among them, temperature, time of heating and pH are the most important ones. According to a number of different authors, as reviewed by Farrer (1955),1 Feliciotti and Esselen (1957) and Mulley et al. ( 1975), the degradation reaction for thiamine can be described as following first order kinetics and adhering to the Arrhenius equation. The rate of a first order reaction is directly proportional to the concentration of the reac tant. The rate expression which describes a first-order re action is: - dc = k c dt where c = concentration of reactant t = time k = reaction rate constant or velocity coefficient (expressed in units of time ~\) Upon rearranging: - dc — = k dt c and integrating, this becomes: - In c = k t + constant This author as well as Coppock et al. (1956) have written excellent reviews and much of the information obtained prior to 1955 is in these reviews. 7 Integrating this equation between limits of concentration c1 at t1 and c2 at a later time t2, gives: C2 - Cdc c1 - In c2 - (- In c.j) = k (t2 - t ^) 2.303 log c1 This can be modified to: t2-tl c. 2.303 log cQ (1) where Cq = concentration at the beginning (when t is 0) c = concentration after time t This equation written in exponential form is: - kt c = cQ e The period of half-life, f 1/2, which is the time necessary for half a given quantity of material to decompose, can be calculated by the substitution of the appropriate numerical values into equation (1): 2 .303 log 1 k = Y 1/2 1/2 0.693 << 1/2 k 4 1/2 is usually independent of the concentration of the initial substance. 8 The rate constant generally depends on the absolute temperature following the law first proposed by Arrhenius in 1889: - Ea/RT k = A e where k = reaction rate constant A = frequency factor Ea = energy of activation (kcal/mole) R = gas constant (1.987 kcal/mole K°) T = absolute temperature (K°) (Daniel and Alberty, 1958; Boudart, 1968; Capellos and Biel-ski, 1972; Adamson, 1979) A review of the kinetics of thiamine in buffer systems and in foodstuffs is given in Table 1. Many researchers reported the well known fact that thia mine is rapidly destroyed in neutral and alkaline medium. Farrer (1941) plotted log k against the corresponding pH and showed an abrupt increase in the slope of the curve, forming two distinct straight lines. He concluded that the reaction rate constant was inversely proportional to the hydrogen ion concentration. Lincoln et. _al. (1944) studied the loss of thiamine during cooking (at 100°C) of flour enriched with thiamine hydrochloride and cocarboxyla se at five different pH values ranging from 5.8 to 7-5. Cooking losses increased with pH. They also looked at the same two forms of thiamine in phosphate buffer solutions at pH values of 3-5 to 7.0 at Table 1 Review of kinetic studies for the thermal degradatation of thiamine Reference Median pH Temperature range Ea range Kcal/tole (min-1) Watanabe (1939)1 aqueous solution 248°F Greenwood et al. (1944) pork (lunch meat) 210-250°F Rice and Beuk (1945) pork (lean) 49-121°C Farrer and Morrison (1949) phosphate buffer 120-230°F Bendix et aJL_ (1951) whole peas natural PH 220-270°F 21.2 0.0058-0.0351 Farrer (1953) potatoes carrots peas cabbage 6.0 5.7 6.5 5.5 212°F 212 F 212°F 212 F 0.0026 0.0022 0.0021 0.0027 Garrett (1956)2 Bi-HCl liquid multivitamin prep. 3.2 39-158°F 26 0.00118a Feliciotti and Esselen (1957) aqueous solution phosphate buffer carrot puree green bean purge pea puree spinach puree beef heart puree beef liver puree lamb puree pork puree 3.5 4.5-7.0 6.1 5.8 6.8 6.7 6.1 6.1 6.2 6.2 228-300°F 228-300°F 228-300°F 228-300°F 228-300°F 228-300°F 228-300 F 228-300°F 228-300 F 228-300°F 0.0026-0.0944 28.8 0.0024-2.092 27.0 27.0 27.0 27.0 0.0049-0.2326 27.0 27.0 27.0 27.0 VO Table 1 (continued) Reference Medium pH Temperature Ea range range Kcal/frtole (min x) Gillepsy (1962)2 20.0 Mulley et al. (1975a) phosphate buffer 6.0 250-280°F 29.4 0.015-0.067a pea puree natural 250-280 F 27.5 0.0093-0.038a beef puree pH 250-280°F 27.4 0.0091-0.037a peas-in-brine " 250-280°F 27.0 0.010-0.039a puree aCalculated from the D values (k=2.303/TJ). "Steferenced in Farrer (1955). 2Referenced in Lund (1975). 11 120°C. Losses increased with pH, with greater losses be tween pH 6.25 and 7.0. Pace and Whitacre (1952) studied the pH effect in corn bread. At some pH value between 6.2 and 6.6 for the batter (corresponding to pH 7.2 to 8.9 for the resultant breads) there appeared to be a critical point at which thiamine was rapidly destroyed during baking. With increasing pH from 4.5 to 7-0 in a phosphate buffer at 228, 246, 264 and 282°F (109, 119, 129 and 139°C), Feliciotti and Esselen (1957) observed an increase in the rate of thiamine destruction. The most pronounced change was between pH 6.0 and 6.5, as indicated by a change in the slope of the log k versus pH curves (similar to Farrer, 1941). The most recent study on the pH effect in a phosphate buffer solution is by Mulley e_t al. ( 1 975b). The rates of destruction curves at 265°F (129°C) were determined for thiamine hydrochloride, cocarboxylase and mixtures of the two at pH 4.5, 5.0, 6.0 and 6.5- Log D values (time for 90% destruction) were plot ted against pH. the D values for pH 4.0 and 5-0 were simi lar for each system individually. However, when the pH ex ceeded 6.0, the D values decreased sharply showing a decreased stability of the thiamine molecule. In the case of cocarboxylase, the change in the slope of the curve oc curred at a lower pH than it did for the thiamine hydrochlo ride. Dwivedi and Arnold (1972) suggested that thiamine would be more stable at pH 3.5 than pH 5.0 or 6.0 because the protonated form of thiamine, which predominates at pH 3.5, is less prone to thermal destruction. 12 It is well recognized that thiamine can occur in three forms: free thiamine, pyrophosphate ester (cocarboxylase) and protein-bound thiamine (Booth, 1943; Greenwood e_t al. , 1943; Lincoln et al., 1944; Farrer, 1955). Greenwood et al. (1943) showed that cocarboxylase was slightly more resistant than thiamine, however other workers (Booth, 1943; Lincoln et al., 1944; Farrer, 1945; Mulley et al., 1975b) found the opposite to be true, i.e. cocarboxylase was much more sus ceptible to degradation whether in buffer solutions or in foodstuffs. Mulley e_t a_l. ( 1975b) also mentioned that con centrations of less than 35% of the cocarboxylase form (which is the amount normally present in foods) did not af fect the kinetics of the thermal destruction of this vita min. The protein-bound thiamine has a greater thermal sta bility, according to Farrer (1955). However, Feliciotti and Esselen (1957) mentioned that the combined form (probably the protein-bound form) was found to be less stable, at a given pH, than the free form. These authors suggested that thiamine degradation in foods may be dependent on the inter relationship of pH and the proportion of the free and the combined form of the vitamin. The work of these different researchers has demonstrated that thiamine in food products is notably more resistant to heat than pure thiamine in aqueous or buffer solutions. This would indicate that many factors other than the ones mentioned previously could affect the thermal degradation of 13 thiamine. These are still not very well established, how ever they cannot be ignored, and they will be discussed briefly. Farrer (1955) and several others since then reported that acids, gelatin, albumin, proteins, a and g amino acids, cal cium hydrogen phosphate, soluble starch, gums, dextrins, fructose, invertase and inositol have a protective or stabi lizing effect upon thiamine. Borate, thiosulfate, acetate, carbonate, monohydrogen phosphate, potassium dihydrogen phosphate, oxidizing agents (like potassium bromate, a dough improver), y , 6 , c -aliphatic amino acids, p-aminobenzoic acid, copper, gamma radiation, ultraviolet light and ultra sonic waves would accelerate thiamine destruction. Fe, Zn, Al, Sn and Ni could modify the destruction reaction or be without effect, depending on the conditions obtained in the solutions. No effect on thiamine stability has been ob served with lithium, sodium, potassium, chloride, potassium nitrite, sulfate, iodide, magnesium ions, glucose, ethyl al cohol, glycine, xanthine and riboflavin. Thiaminases, en zymes found mainly in fresh water fish, shellfish and mol luscs, are capable of destroying thiamine. The percent of moisture in the food, together with other factors, also in fluences thiamine destruction. The rate of destruction is more likely to increase as the product becomes more concen trated. Finally, Farrer(1955) commented that under normal conditions found in foods, most of these factors do not play a very significant role in thiamine degradation. 14 2.3 THIAMINE LOSSES IN BREAD Even though no work has been done on thiamine kinetics in bread, many studies on thiamine losses in bread and baked products were reported. The data of these studies are pre sented in Appendix A. A review of the data in the literature (as presented in Appendix A) allows one to conclude that the thiamine losses of baked bread are of the order of 20%, whatever the condi tions. The only exceptions are the 43 and 47% losses found by Tabekhia and D'Appolonia (1979). Among all these au thors, two different conclusions were reached. Some au thors, Schultz et al. (1942) and Coppock et al. (1956). agreed that bread made from flours of different extractions show similar retention rates, whereas Farrer (1949) and Daw son and Martin (1942) found higher losses from high-extrac tion flours. Goldberg and Thorpe ( 1946) cited in Coppock e_t al. (1956) also agreed with the latter, but attributed this result to a longer baking time for the wholemeal (100% ex traction) bread. There are several theories explaining the differences in thiamine retention between low and high extractions flours. Dawson and Martin (1942) suggested two hypotheses. The first was that these differences were due to changes in pH, although Farrer (1949) cited in Coppock et al. (1956) com mented that in Dawson and Martin's study little difference in the pH values were detected in the bread. The second hy-15 pothesis was that the free thiamine was absorbed by the yeast during fermentation and then was protected during sub sequent baking. They suggested that this would explain higher losses at high extraction rates, especially if the vitamin in the yeast was less susceptible to heat and if thiamine was bound to bran, and therefore less available to the yeast. Here again Farrer (1949) criticized them by say ing that thiamine in yeast was in the cocarboxylase form, which was more thermolabile than the free form. An interesting explanation for higher losses at higher extraction rates is related to the ash or mineral content of the flours. Dawson and Martin (1942) and Farrer (1955) showed a linear relationship when ash content, which is higher as extraction rates increased, was plotted against thiamine destruction. Farrer (1955) also observed that the pH increased with the percent of extraction (from pH 5.68 with 75% extraction to pH 5-98 with 100%). He concluded that because he had shown in previous work (Farrer, 1945) only a slight increase in the k values between pH 5.0 and 6.0 in phosphate buffer solutions (which is the predominant anion in cereals) then the increase in the destruction rates may be due not only to the pH but also to the increase in inorganic constituents. Coppock et al. (1956) commented that they disagreed with the ash theory since they showed similar thiamine losses for all the different extractions. 16 Only one study by Van der Mijl Dekker (1951) cited in Coppock et al. (1956) showed smaller losses at higher ex traction rates. This was attributed by other authors (Far rer, 1955; Coppock et. al. , 1956) to the use of the diazota-tion procedure, a very poor method for thiamine analysis. The source of thiamine would be a factor that affects its stability. As previously mentioned, the cocarboxylase form (which is the form of thiamine in yeast) is more vulnerable to heat. Farrer (1955) suggested that more cocarboxylase could be present in the higher extraction flours, which would explain the higher thiamine losses. However, some au thors referenced by Farrer (1955), agreed that wheat con tains little cocarboxylase and little or no protein bound thiamine. The cocarboxylase content of 74.9, 85, 98 and 100% extraction flours were reported to be 8.2, 12.9, 11.3 and 10% respectively. A study described by Coppock e_t al. ( 1956) using inter nal temperatures of various 450 g (1 lb) bread loaves, cakes and biscuits found a linear relationship between the product of minutes heated above 80°C and final temperature minus 80°C versus average percent loss. This experiment took into consideration the bulk of the product. And finally, Coppock e_t _al. ( 1956) concluded from this latter study, as well as other studies and their own work, that 'thiamine destruction in baked products is mainly ther mal and only to a relatively small extent affected by other factors'. 17 Although a number of studies have been carried out on thiamine losses in bread, these have been conducted over a wide range of conditions, making it difficult to compare them. In addition, there have been only a few studies at tempting to investigate the mechanism of thiamine degrada tion in food using the kinetic approach, and none have been done on bread. As stated earlier, the kinetic approach al lows one to obtain standardized data to make comparative studies. The objectives of this thesis were to study the destruc tion of thiamine (using the kinetic approach) in: 1. a phosphate buffer solution at pH 6.0 over the temp erature range 80 to 120°C. 2. a phosphate buffer solution at 120°C over the pH range 1.0 to 7.0. 3- in a semi-model enriched white and whole wheat bread system at natural pH over the nominal oven tempera ture range 177 to 288°C (350 to 550°F). Furthermore, thermal destruction in bread was to be stud ied to determine whether any difference in the destruction of the natural and synthetic sources of thiamine occurred by comparing their rate of destruction. Ill MATERIALS AND METHODS All thiamine analyses were done by the thiochrome method described by the Association of Vitamin Chemists ( 19 6 6). The fluorescence was measured with an Aminco-Bowman Spectro-fluorometer (American Instruments Co. Inc, Silver Spring, MD) and was recorded on a photomultiplier- microphotometer. The wave lengths used were 365 nm for excitation, and 435 nm for the output. Blanks were used in each run. An Accumet Model 230 pH/ion meter (Fisher Co., Pittsburg, PA) was used to check pH values. All chemicals used were reagent grade and/or prepared as specified in the procedure. 3. 1 MODEL SYSTEM 3.1.1 Vitamin analysis Because the analyses were made on pure solutions, the ex traction and purification steps were omitted from the thio chrome method. For each run, two standards and two refer ence controls (unheated samples representing 100% thiamine) were measured. - 18 -19 3.1.2 Preparation of buffered thiamine solutions Intermediate buffered thiamine solutions were made by adding 5.0 ml of stock thiamine solution (see Thiochrome method, p.130 in Assoc. of Vit. Chem., 1966) to a buffer so lution and diluting to 100 ml. Two different working buffer solutions were used. One, for the test tubes, was made by adding 5.0 ml of intermediate thiamine solution to a buffer solution and diluting to 100 ml. The concentration of this working buffer was 0.25 yg thiamine/ml. The other working buffered thiamine solution, for the TDT (thermal death time) tubes, was made with 30.0 ml of intermediate buffered thia mine diluted to 100 ml with a buffer solution, giving a con centration of 1.5 yg thiamine/ml. 3-1.3 Treatment of samples The samples heated at temperatures lower than 100°C were heated in a water bath using test tubes closed with a metal cap. Those heated at temperatures higher than 100°C were heated in a oil bath using TDT tubes sealed in an oxy/ace-tylene flame. Both baths were equipped with temperature controls. After heating, the tubes were cooled immediately in an ice bath. HCl (0.01N) solution was added to the sam ples to adjust the pH to between 3-5 and 4.5. The final volume of all samples was 6.8 ml. The final concentration of all unheated samples was 0.2193 JJ g thiamine/ml. The sam ples were then stored at 4°C in an ice bath until needed for analysis. 20 Preliminary work showed that the ice holding time and storage at 4°C up to 30 hr did not have any significant ef fect on thiamine concentration. For each heating time, a total of 2 to 6 replicates were done over several days. 3.1.4 Come-up times Come-up times for 80, 90 and 100°C were estimated from heat penetration data obtained from two test tubes monitored with thermocouples. The come-up times were found to be 3-6 min for 80°C, 4.0 min for 90°C, and 2.3 min for 100°C. In the assays using the TDT tubes, the come-up times were esti mated to be 0.6 min according to the procedure described by Sognefest and Benjamin (1944). In the present study the come-up time corrections were not applied to the data, since the estimated come-up times were very small and the cooling lag factor will compensate for the heating lag factor. 3.1.5 Temperature effect The buffer system used was a 0.2 M phosphate buffer (Sor-ensen) (Gomori, 1955) at pH 6.0. Destruction rates were de termined at temperatures of 80, 90, 100, 110, 115 and 120°C. The kinetic rate data were obtained graphically by plot ting the logarithm of percent thiamine retention against time for different temperatures. The lines were all forced through 100% thiamine retention. Slopes and coefficient of determination (r2s were calculated by linear regression 21 equation using appropriate formula for slopes forced through the origin (Zar, 1974, p.214). The destruction rates, k, expressed in reciprocal minutes, were calculated by the for mula: k = - 2.303 slope Half-life values (I* 1/2) were calculated by the formula: 0.693 Y 1/2 = k Log k values were plotted against the reciprocal of the absolute temperature (°K) x 1000. Slopes were determined by the method of least squares. The energy of activation, Ea, expressed in kilocalories per mole, was calculated by the formula: - Ea/RT k = A e Ea = - 2.303 R (slope) where R (gas constant) = 1.987 kcal/mole K° 3.1.6 pH effect The destruction rates were determined at 120°C and pH of 4.0, 4.5, 5.0, 6.0 and 7.0. This temperature was taken as being representative of average bread temperature while bak ing. The pH 6.0 data were previously determined in section 2.1.5. Phosphate buffer (0.2M) (Sorensen) (Gomori, 1955) was used for pH 6.0 and 7.0 and 0.2M phosphate-0. 1M citric acid buffer (Mcllvaine, 1921) for the pH below 6.0. The ki netic rate data and ^ 1/2 values for the pH effect were ob-22 tained in the same way as for the determination of the temp erature effect. To evaluate the effect of the pH on the stability of thi amine, log k values were ploted against pH. 3.2 BREAD SYSTEM 3.2.1 Thiamine analysis The enzyme used for the extraction step was acid phospha tase (0.4 units/mg) at a concentration of 0.25 g/100 ml. This step was not omitted since preliminary work showed higher thiamine recovery for commercial enriched white and whole wheat breads when using this enzyme. The purification procedure was carried out as well, because preliminary de termination showed that without this step, the solutions were not clear and blank values were very high (see thio-chrome procedure 2 (b)(6), in Assoc. of Vitamin Chemists, 1966). Ionac C-102 (modified alumino silicate resin 30-80 mesh-85%, by MCB, Los Angeles, CA) and activated Decalso (Permutit-T by the Fisher Co., Pittsburg, PA) were used as column material. Two standards were used for each run. Two samples of dough after proofing (before baking) were analysed at each run as the reference representing 100% thiamine retention. Each sample was finely ground in a Cuisinart Food Processor in preparation for the extraction procedure. 23 3.2.2 pH determination In the 12 g bread assays, the pH of the bread was meas ured in a constantly stirred slurry made of bread crumbs, or dough, and distilled water. 3.2.3 Moisture determination The moisture in the samples (dough and baked product) were determined by the standard method (AOAC, 1980) using the ground up material left over from the extraction step. Results could therefore be calculated on dry weight basis. 3.2.4 Apparatus for heating A Despatch laboratory electric oven (Minneapolis, MN) with Robertshaw thermostat control was used to heat all the samples. 3.2.5 Flour samples The commercial enriched all purpose white flour used in all assays was labelled as containing 0.45 mg of thiamine per 100 g of flour . 3.2.6 Enriched white flour slurry assay Mixtures of 100 g of enriched flour and 150 g of tap wa ter were heated at a nominal oven temperature of 246°C (475°F) for 60, 75 and 90 min. Duplicate determinations for each baking time were used. Moisture analysis was done on 24 each baking time mixture for each pair of duplicates. Anal ysis was performed right after cooling the samples in an ice bath. Percent thiamine retentions were calculated. 3.2.7 One pound loaves (enriched white bread) The bread formula and procedure were those used by Tabek-hia and D'Appolonia (1979). Bread loaves were also baked at an oven temperature of 221°C (430°F). Two slices from each loaf were stored at -10°C prior to analysis. Experiment 1 Two loaves of bread were baked for 30 minutes. An aver age moisture content, using duplicates, was determined on slices from both loaves. Percent thiamine retentions were recorded for each bread. 3.2.7-2 Experiment 2 Three loaves of bread were baked for 15, 37 and 60 min. No moisture analyses were done. Percent thiamine retentions were recorded for each duplicate for the different baking times . 3.2.8 12 g loaves (enriched white and whole wheat bread) The following formula, similar to straight-dough formula from AACC (1969), was used: Flour 120.0 g Water 70.0 g 25 Sugar 6.5 g Shortening 4.0 g Yeast 2.7 g Salt 1.5 g A straight-dough method with 3 hr fermentation and 55 min proofing done in a fermentation cabinet at 36-38°C was used. After the fermentation period, the dough was allowed to rest for 10 minutes, then it was divided into approximately 20 g loaves (12 g flour each), and finally molded by hand and placed into bread pans (dimensions: top: 5.8 cm x 3.1 cm; bottom: 5.0 cm x 2.3 cm; height: 2.3 cm). Treatment of samples Bread loaves were baked for 4 different time periods at 4 different nominal oven temperatures. Enriched white and whole wheat bread loaves were baked in different batches. The bread was considered to be underbaked for the 2 first time intervals. For the 3rd time, it was normal baked, and the last one was overbaked. Normal bake refers to the stage during baking where the colour of the crust and degree of doneness are optimum for consumption in terms of its func tional and organoleptic properties. Underbaked means when the bread has not been baked long enough to reach the normal bake stage, and overbaked when it has been baked beyond the normal bake stage. An illustration, on the following page (Figure 1), of the enriched white and whole wheat bread 26 shows the loaves baked at the 4 different baking time peri ods . After each baking time, the loaves were removed from the oven, allowed to cool at room temperature, and then were frozen (-10°C). Just before the extraction procedure, the bread was thawed at room temperature, or at 37°C The en tire bread loaf was used for the extraction step in the thiochrome procedure. Each run included two replicates of each baking time at a given temperature for each type of bread . 3-2.8.2 Experiment 1 The nominal oven temperatures used were 163, 191, 218 and 246°C (325, 375, 425 and 475°F) . Two replicates, baked in the same oven load, for each time and temperature treatment were used. No moisture analyses were done. Only the oven temperature was monitored, using thermocouples. pH of bread after baking was recorded. The percent thiamine retentions were calculated from these experimental results. 3.2.8-3 Experiment 2 The nominal oven temperatures used were 177, 218, 246 and 288°C (350, 425, 475 and 550°F). Four replicates, baked in the same oven load, were used for each time and temperature treatment. An average moisture content, using duplicates, was determined on each pair of replicates analysed in the 27 Figure 1: Enriched white and whole wheat loaves baked at 4 different baking time periods. 28 the same run. pH of dough before proofing, dough after proofing and bread after baking were recorded. Heat penetration data. The oven, crust and bread crumb temperatures were moni tored with a 0.12 mm diam. (crust) and 0.60 mm diam. (middle and oven) copper-constantan thermocouple on a Digitec Datal ogger (United Systems Corporation, Dayton, OH), which re corded the temperature at one minute intervals. In each oven load, two bread pans were equipped with thermocouples. One thermocouple was placed in the middle of the loaf, and another was inserted just beneath the top surface of the dough. One thermocouple monitored the oven temperature in the middle of the oven, just above the loaves. The mass av erage temperatures (MAT) at minute intervals for each bread treatment were calculated by using the Stumbo (1965) formula that applies to a cylinder shaped mass: MAT (°C) = °Tcrust - 0.27(°Tcrust - °Tmiddle) This Stumbo formula is only an approximation of the real av erage bread temperature for a given time. Heat penetration curves (mass average temperature versus time) were plotted for each oven load, taking the average of the two bread MAT data. In addition, the heat penetration data for the crust and the middle were plotted for enriched white bread baked at 218°C (425°F) (nominal temperature). The average oven temperature for each load was also calculated. 29 Treatment of data. Degradation reaction rates were obtained graphically by plotting the logarithm of percent thiamine retention against time for the various baking temperatures. All the lines were forced through 100% thiamine retention. All slopes and r were calculated by the method of least squares (Zar, 1974, p. 214). Half-life values (Y1/2) were calculated by the formula: 0.301 < 1/2 = slope Interpretation of data. The slopes were used to compare the stability of thiamine in 1) the enriched white and whole wheat breads for each different temperature; 2) the same type of bread at differ ent oven temperatures. Statistical analysis Slopes for the results of data pertaining to bread types (1 above) were compared using the Students' t-test. Slopes for the results of data for different oven temperatures (2 above) were compared using F-test and SNK (Student-Newman-Keul) . Deviation from linearity, viz. whether Y is a linear function of X, was tested for each slope. The test is anal ogous to a one-way ANOVA. Essentially, the total variance is divided into between groups SS and within groups SS, and the between groups SS was further divided so that the devia-30 tion from linearity SS is equal to the between groups SS minus the regression SS. If the F value is high, viz. the deviation from linearity MS is greater than the within groups MS, then the null hypothesis that the regression is linear is rejected. Results were considered to be signifi cant at the maximum limit of the 5% probability level. (Zar, 1974, chap.16 and 17, and Appendix B) IV RESULTS 4.1 MODEL SYSTEM A sample calculation of percent thiamine retention is given in Appendix C. 4.1.1 Temperature effect Table 2 shows the average percent retention - the stan dard deviation for thiamine hydrochloride in phosphate buff er (pH 6.0) for different times at temperatures ranging from 80 to 120°C. Coefficients of variation for the percent thi amine retention data varied from 0.0 to 29.8%. Thermal de struction curves for these data are given in Figure 2 and 3-The extremely good fit of the experimental points to a straight line is strong evidence that the thermal destruc tion of thiamine in phosphate buffer is first order in na ture. Table 3 gives the k values and the coefficients of deter-mination (r ) for each of these destruction curves. The very high r values also demonstrate the excellent fit of the curves. These results reveal that thiamine stability decreases as temperature increases. - 31 -Table 2 Retention of thiamine hydrochloride in phosphate buffer (pH 6.0) at different heating times between 80 and 120 C. Thiamine retention (%) Time 80°C 90°C 100°C 110°C 115°C 120°C 10 min 20 min 30 min 40 min 50 min 60 min 87.0+1.9(6) 77.6±3.8(4) 80 min 90 min 100 min 120 min 80.6±4.2(4) 66.9±2.6(4) 3 hrs 76.Oil.5(4) 55.5±3.4(4) 4 hrs 64.9±1.8(3) 47.1±4.6(4) 5 hrs 38.3±5.1(4) 6 hrs 58.9±4.0(4) 32.4±2.1(4) 7 hrs 49.2±3.5(4) 8 hrs 80.0±4.4(4) 12 hrs 32.8±3.8(2) 24 hrs 45.5±1.8(4) 46 hrs 22.4±3.5(4) 72 hrs 9.8±1.8(4) 72.6±3.8(4) 49.9±2.2(3) 37.3±5.7(4) 25.9±1.8(4) 13.6±1.2(4) 69.4±2.5(3) 49.4±8.6(4) 30.6±6.0(4) 29.1±0.3(2) 17.1±1.7(3) 9.610.7(2) 72.0+0.0(2) 51.814.5(4) 37.011.8(4) 27.218.1(4) 24.113.9(3) 15.612.2(3) Values are presented as means 1 standard deviations. Numbers in parentheses refer to number of replicates. 33 Figure 2. Retention curves for rMamine hydrochloride in phosphate buffer (pH 6.0). 34 TIME (HR) LEGEND i TEMP°C • • • 60 Figure 3. Retention curve for thiamine hydrochloride in phosphate buffer (pH 6.0). 35 TABLE 3 k, r2 and < 1/2 values for thiamine HCl in phosphate buffer (pH 6.0) in the temperature range 80 to 120°C. (°C) (min-1) (min) 80 0.0005477 0.9952 1265 90 0.001602 0.9862 433 100 0.003199 0.9894 217 110 0.01116 0.9952 62 115 0.01827 0.9871 38 120 0.03132 0.9845 22 Table 3 gives ttllae half—life values for the differ emit heating temperatures. In Figure 4, ttoe linearity of the ^srhenius plot indi cates that the data conform to the Arrheinius equation. Ttoe energy of activation, Ea, ©Ibtained from this curve for t'ftie phosphate buffer-system is .3-4.2 kcal/mole, with a r valuae of 0.8375, which shows a good fit of t'he data to the curve. 4.1.2 pH effect The average percent thiamine retention i the standard de viation for thiamine hydrochloride in phosphate buffer at piH 4.0 to 7-0 heated at 120°C for different, lengths of time are given-in Table 4. Coefficients of variation- for the percent thiamine retention values among the different replicates vary from 0.0 to 25.5%. Figure 5 stows the destruction curves plotted from these data. Like ita the temperature as say, the experimental points fit very well to the line. Ttae o k values of these destruction curves wifcih the r values are '2 given in Table 5- High r values confirm the good fit ©f the lines. Half-lives for the different pH are given in Table 5. When the k valuaes are plotted against pH (Figure 6), tthe graph shows that tihiamine stability decreases with pH, with greater losses when the ;piH reaches 6-©. One can observe that the experimental points fit on a irregular curve shaped line. 37 i i i ' 2.54 2.58 2.62 2.66 2.70 2.74 2.78 2.82 1/T X 1000 (°K~1) Figure 4. Arrhenius plot for the thermal degradation of thiamine hydrochloride in phosphate buffer (pH 6.0). Table 4 at different heating times between pH 4.0 and 7.0. Thiamine retention (%) 4.0 4.5 5.0 5.5 6.0 7.0 (Min)  3 48.4±3.9(3) 7 11.2±1.2(310 72.0±0.0(2) 5.6±1.1(2) 12 3.7±0.2(215 83.2±4.9(4) 20 51.8±4.5(4) 30 63.0±4.6(3) 63.1±2.8(4) 37.0±1.8(4) 32 61.5 (1) 38 58.3±0.5(2) 40 62.8±2.5(4) 27.2±8.1(445 55.7±0.7(2) 50 24.1±3.9(360 39.2+7.4(6) 42.3±4.1(6) 15.6±2.2(3) 78 36.8+1.5(2) 80 43.9±0.8(3) 90 25.5±3.1(6) 36.1±3.9(3) 110 27.1±6.9(2) 120 32.312.3(2) 21.5il.8(2) 19.6+3.4(5) 160 21.812.5(3) Values are presented as means 1 standard deviations. Numbers in parenthesis refer to number of replicates. Figure 5. Retention curves for thiamine hydrochloride in phosphate buffer at 120°C at various pHs. 40 TABLE 5 p k, r and Y 1/2 (120 values for °C) in the thiamine pH range HCl in phosphate 4.0 to 7.0. buffer PH k 1 (min ]) r2 < 1/2 (min) 4.0 0.009733 0.9942 71 4.5 0.01261 0.9915 55 5.0 0.01459 0.9838 47 5.5 0.01299 0.9769 53 6.0 0.03132 0.9845 22 7-0 0.2870 0.9951 3 41 Figure 6. The effect of pH on the destruction rate constant for thiamine hydrochloride in phosphate buffer at 120°C. 42 4.2 BREAD SYSTEM A sample calculation of percent thiamine retention is given in Appendix C. i 4.2.1 Enriched white flour slurry assay Results for the average thiamine retention are shown in Table 6. After 60 minutes of baking at 246°C (475°F), no appreciable thiamine destruction was found, whereas for both 75 and 90 minutes of baking, about 30% of thiamine was de stroyed . 4.2.2 One pound loaves (enriched white bread) Experiment 1 After baking 450 g (1 lb) loaves of bread for 30 minutes at 221°C (430°F) (oven temperature), no destruction of thia mine was found. The percent thiamine results were actually slightly higher than 100%: 103-3%, for loaf 1, and 105.2%, for loaf 2. Experiment 2 Table 7 shows the mean percent thiamine retention of 450 g (1 lb) loaves made from enriched white bread and baked for different times. The results are inconclusive because they are on a wet weight basis. Because bread becomes dryer as baking time increases, and because percent thiamine results 43 TABLE 6 Thiamine retention for enriched flour slurry for different baking times at 246°C (475 F) (nominal). Baking time Thiamine (min) retention* 60 97-6 ± 6.0 75 72.1 + 16.7 90 70.8 ± 6.9 *Mean of duplicates ± standard deviations. 44 TABLE 7 Thiamine retention in 450 g (1 lb) loaves of enriched white bread baked at 221°C (430°F) for different times. Baking time Thiamine (min) retention* (%) 15 126.3 ± 4.8 37 112.3 ± 21.3 60 97.9 ± 1.3 *Means of duplicates ± standard deviations. 45 are based on the bread dough, which has a higher moisture content than the baked bread, each percent thiamine result is therefore calculated on a different weight basis and thus cannot be compared to each other. However, if the dry weight data of the dough and bread at 30 min baking from Ex periment 1 are considered in the calculation of the % thia mine retention for the 37 min baking, the result is 97.6% retention. This result agrees very well with the one from Experiment 1. 4.2.3 12g loaves (enriched white and whole wheat bread) Experiment 1 The mean pH value of two replicates (one baked at 191°C and the other one at 246°C - nominal temperatures) was 5.08 for enriched white bread and 5.63 for whole wheat bread. pH of whole wheat bread was slighty higher than enriched bread. Table 8 shows the mean percent thiamine retention for en riched white and whole wheat bread at four different oven temperatures, 163, 191, 218 and 246°C (325, 375, 425 and 475°F) , for different baking times. Because the results were calculated on a wet weight basis instead of on a dry weight basis, they are inconclusive (see explanation in pre vious section). This is shown by the very irregular pat terns of thiamine destruction with time and different oven temperatures. The greatest thiamine destruction obtained was 26.1% for enriched white bread at 163°C (325°F) for 45 minutes. 46 Experiment 2 The pH values of dough and baked bread for enriched and whole wheat bread are given in Table 9. Whole wheat bread as well as the dough have a slightly higher pH than enriched white bread and dough. The dough after proofing and the baked bread, for each type of bread, have essentially the same pH values. The higher degree of acidity demonstrated by the dough after proofing, compared to the dough before proofing, is a result of yeast activity during the fermenta tion process. Heat penetration data. The recorded average oven temperatures during the baking of the enriched white and whole wheat bread are shown in Ta ble 10. The oven temperatures for both types of bread at each nominal temperature are in good agreement. Consider ing that the oven doors were opened four times during each baking period and that the oven has its own temperature cy cle, the standard deviations are quite small. The coeffi cients of variation for 177, 218 and 246°C (350, 425 and 475°F) are less than 5% and less than 6% for 288°C (550°F). The heat penetration curves, where the mass average temp erature (MAT) is plotted against time, are given for all the different oven temperatures for enriched white bread in Fig ure 7, and for whole wheat bread in Figure 8. 47 TABLE 8 Thiamine retention of white and whole wheat bread baked at 4 different temperatures for several baking times (Exp.1). Nominal Enriched white Whole wheat oven temp (°C) Baking Thiamine Baking Thiamine time retention* time retention* (min) (%) (min) (%) 10 90.7 10 115.1 (325°F) 20 81.7 20 122.5 32 101.1 31 98.3 45 73.7 43 102. 3 191 7 97.2 7 131.8 (375°F) 13 98.8 13 108.5 20 95.4 20 120.8 27 97.8 27 79.7 218 4 94.5 4 93.6 (425°F) 8 80.5 8 98.5 13 82.3 13 104. 1 19 94.2 19 94.9 246 3 119.9 3 71.9 (475°F) 7 102.3 7 95.8 9 88.4 10 109.3 12 100. 6 13 104.7 *Values are means of 2 replicates. 48 TABLE 9 pH values for white and whole wheat dough and bread baked at 252°C (485°F) (nominal)' for 17 min (Experiment 2). pH* Enriched white Whole wheat Dough before proofing 5.35 6.03 Dough after proofing 4.93 5. 40 Baked bread 5.03 5.38 *Values are means of 2 replicates. 49 TABLE 10 Oven temperatures for enriched white and whole wheat bread baked in different loads at 4 nominal oven temperatures. Nominal Oven temperature ( C) oven temp (°C) Enriched white* Whole wheat* 1770 152.2 + 7.3 154.9 + 6.8 (350°F) 218 172.6 5.6 177.6 ± 6.9 (425°F) 246 189. 1 ± 8.3 189.5 + 6.3 (475°F) 288 215.8 + 12. 1 217-7 + 11.9 (550°F) *Values are means + standard deviations. 50 Figure 7. Heat penetration curves for enriched white bread baked at 4 nominal oven temperatures. 51 160 H 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 60 65 90 95 TIME (MIN) LEGENOi OVEN°TeF 350 e-B-e 425 475 550 Figure 8. Heat penetration curves for whole wheat bread baked at 4 nominal oven temperatures. 52 Figure 9 shows the heat penetration curves for the middle and crust of enriched white bread at 218°C (425°F) (nominal temperature). 4 Thiamine retention results. Table 11 summarizes the results of thiamine retention in enriched white and whole wheat bread at different baking times at 177, 218, 246 and 288°C (350, 425, 475 and 550°F (nominal oven temperatures). Variability in % thiamine re tention values among the 4 replicates as expressed by the coefficients of variation were 2.8 to 35.7% for enriched white.bread, and 3.7 to 23.4% for whole wheat bread. Vari ability of thiamine retention for whole wheat bread appears to be smaller than for enriched white bread. Figures 10, 11, 12 and 13 show the thiamine destruction curves for each different oven temperature comparing en riched white and whole wheat bread on the same graph. From these experimental data, it is evident that thiamine reten tion for both types of bread decreases with baking time for all baking temperatures. Since most of the data points seemed to form a straight line, regression equations were calculated from the data and the lines were drawn through the points and the slopes were calculated. The slopes with r values are given in Table 12. When the statistical hy pothesis Ho: the population regression is linear is tested (Zar,1974, p. 215), only the curve for the enriched white 53 Figure 9. Heat penetration curves for the middle and crust of enriched white bread baked at 218 C (425 F) (nominal). 54 TABLE 11 Thiamine retention of white and whole wheat bread baked at 4 different temperatures for several baking times (Exp.2). Nominal Enriched white Whole wheat oven _______ temp (°C) Baking Thiamine Baking Thiamine time retention** time retention** (min) (%) (min) (%) 175^ 15 72.3 + 1.7 16 78.0 ± 4.4 (350°F) 35 63-6 ± 6.6 40 66. 1 + 2.5* 55 53-3 ± 1.5 65 52.2 ± 1.9 92 51.6 ± 7.1 90 43.5 + 9.4 220 10 88.3 ± 31.5 10 92.9 ± 11.2 (425°F) 20 89.0 ± 18.3 25 71.0 ± 4.2 35 57.2 + 7.9 40 59.0 + 10.1 55 42. 3 + 3. 1 71 20.7 + 4.3 245 6 76.2 + 21.0 7 83-0 + 7.3 (475°F) 15 72.4 ± 16.2 20 70.2 + 7.2 25 71.7 ± 10.8 35 52.2 + 8.2* 40 56. 1 + 8.1 60 26.3 + 8.2 280 5 91.5 ± 12.4 6 83-0 ± 9.4 (550°F) 12 86.8 ± 10.3 15 67.0 + 5.7 22 65.3 + 7.0 22 60.4 + 14. 1 31 45.0 ± 7.8 31 35.3 ± 7.7 **Values are means + standard deviations for 4 replicates. *0nly 3 replicates were used. 55 bread at 177°C (350°F) deviates significantly from linearity o at the 5% probability level. Values of r are quite signif-2 icant, especially for the whole wheat bread. The lowest r value obtained was for enriched white bread at 246°C (475°F) (r2=0.7618). For all oven temperatures, the slopes for whole wheat bread are higher than the ones for enriched white bread. Only the curves for 177°C (350°F) did not show any signifi cant difference (see Appendix B). These results demonstrate that thiamine in whole wheat bread (a natural source) is less stable than thiamine in enriched white bread (a syn thetic form) . Thiamine destruction lines for all different oven temper atures for each type of bread were also drawn (see Figures 14 and 15). When comparing the slopes from each graph, the slope values increase as oven temperature increases, which means that thiamine become less stable as oven temperature rises. When each line is compared to each other (SNK test) at 5% level, 218 and 246°C (425 and 475°F) lines for en riched white and whole wheat bread do not show a significant difference (see Appendix B). Half-life values are given in Table 13. 56 Figure 10. Thiamine retention curves for enriched white and whole wheat bread baked at 177 C (350 F) (nominal). 57 Figure 11. Thiamine retention curves for enriched white and whole wheat bread baked at 218 C (425 F) (ncminal). TIME WIN) LEGEND i TYPE • o • ENR.MHIT * * • M. HHEBT Figure 12. Thiamine retention curves for enriched white and whole wheat bread baked at 246°C (475°F) (rcminal). 59 Figure 13. Thiamine retention curves for enriched white and whole wheat bread baked at 288 C (550 C) (nominal). 6© TABLE 12 Slope and r2 values of thiamine destruction curves for white and whole wheat bread baked at 177, 218, 246 and 288°C. Nominal oven Enriched white Whole wheat temp (°C) Slope (min-1) r2 Slope (min~n) r2 177„ (350°F) 0.003931 0.8956 0.004264 0 .9619 218 (425°F) 0.006545* 0.8576 0.008491* 0 .9333 246 (475°F) 0.006800** 0.7618 0.009475** 0 .9487 288 (550°F) 0.01039* 0.9327 0.01317* 0 .9187 *Difference between slopes in the same row is significant at 0.02<p<0.05. **Difference between slopes in the same row is significant at 0.01<p<0.02. 61 Figure 14. Thiamine retention curves for enriched white bread baked at 177, 218, 246 and 288 C (roriinal). 62 •i 11111111111 i i" "i i 0 5 10 15 20 25 30 35 40 45 SO 55 60 65 70 75 80 85 90 TIME (MIN) LEGEND i TEMP"F • • • 350 * * * 425 A A A 475 ODD 550 Figure 15. Thiamine retention curves for whole wheat bread baked at 177, 218, 246 and 288°C (nominal). 63 TABLE 13 Half-life values for thiamine in enriched white and whole wheat bread baked at 177, 218, 246 and 288°C (nominal). i 1/2 Nominal (min) oven temp (°C) Enriched White Whole wheat 177„ (350°F) 77 71 218 (425°F) 46 36 246 (475°F) 44 32 288 (550°F) 29 23 V DISCUSSION This study on kinetics of thiamine degradation in a phos phate buffer confirms again the work done by the many au thors mentioned earlier where thermal degradation of thia mine follows a first order reaction and adheres to the Arrhenius equation. The Ea value of 34.2 kcal/mole found in this experiment is slightly higher than values recorded by Feliciotti and Esselen (1957) and Mulley et al. (1975) in the same buffer system (see Table 1). A likely explanation for the higher value is that all of the destruction curves were forced through 100% retention, whereas these other workers did not correct their curves accordingly. When the present data were not forced through 100% an Ea value of 27.8 kcal/mole was obtained, which is closer to the values found by these authors. The pH experiment agrees with the findings of the re searchers cited earlier, where thiamine destruction is greater at higher pH. Furthermore, the results show a high er loss between 6.0 and 7.0, which is also found by Lincoln et al. (1944), Pace and Whitacre (1953) and Feliciotti and Esselen (1957). The pattern of the curve of log k versus pH in Figure 16 agrees fairly well with the ones of Farrer - 64 -65 (1945), Feliciotti and Esselen ( 1957) and Mulley e_t al. (1975b). The only obvious difference is that in this study there appears to be a small drop in the k value at pH 5.5 when compared to the trend of the 3 preceeding points. All the k and f1/2 values found in the model system are in the same range as those found in other studies (Table 1). When the results of different authors, using similar sys tems, are compared, the values obtained are not identical. These differences among k and 4 1/2 values of different au thors and this work may be due to experimental variation in the buffer system used, in the thiamine analysis procedure and/or in the considerations for the treatment of data (e.g. forcing slopes through 100% thiamine retention). The pH values of baked white bread in experiment 1 and 2 agree with the values given by Pomeranz and Shellenberger (1973) where pH of white bread crumb varies from pH 5.1 to 5.4. It is surprising to see that there is no loss of thiamine during normal baking of 450 g (1 lb) loaves of enriched white bread. This is especially interesting because 30 to 50 percent thiamine destruction was found in the 12g bread formula at normal baking and 30% in the slurry assay at 75 and 90 minutes (although 0% was found at 60 minutes). Cer tainly this 0% destruction in 1 lb loaves does not corre spond well with the 20% destruction results of other work ers, and is in sharp contrast to the 50% destruction result 66 0.31623 -| Figure 16. A review of pH/k curves for thiamine in buffered solutions. (1) from this work. (2) Mulley'et'al. 11979b) - phosphate buffer at 129 C. (3) Feliciotti and Esselen (1957) - phosphate buffer at 119°C. (4) Farrer (1945) - phosphate buffer at 100°C. (5) Farrer (1945) - phosphate-citric acid buffer at 100°C. 67 of Tabekhia and D'Appolonia (1979), where the same condi tions were used . One explanation for the 0% destruction is that yeast might have been active still during the initial period of baking. Because the mass of the bread is large, it takes a while for the middle of the bread to reach a temperature where the yeast is killed. Some thiamine would then be pro duced during this initial period, which could account for an error in the percent retention calculation. The reason why other workers obtained up to 20% destruction could be be cause the production of thiamine might vary with the variety of yeast, and the type they used produced less thiamine. More 450 g (1 lb) loaf experiments should be done at the normal bake and over bake stage to confirm these results. The 12 g bread formula was used instead of the 450 g (1 lb) loaves mainly because it was easier to handle. However, these breads are very small and the proportion of surface crust to volume is much higher compared to 450 g ( 1 lb) loaves. Because most of the thiamine destruction occurs in the crust, one would expect more destruction in these smaller loaves than in the 450 g (1 lb) loaves. This is well demonstrated by the 30 to 50 percent thiamine destruc tion found in the 12 g loaves at the normal bake time com pared to the 0% found in the 1 lb loaves here and the 20% average found by the other workers cited in Appendix A. 68 Because of the unsteady temperatures in the bread during baking, as shown by the heat penetration curves, it was not possible to calculate the kinetic rate constant (k) and the energy of activation (Ea) for thiamine destruction in bread by means of the steady state approach. Bread being a con duction heating food with changing temperatures, it is prob ably better analyzed by the a more complex non-steady state approach. This was not done for this work. However, exper imental results showed that log percent thiamine retention decreased at a constant rate with time. Thus these slopes made it possible to compare mathematically the difference between whole wheat and enriched white bread. When comparing the slopes for enriched white and whole wheat bread baked at different oven temperatures, the 218 and 246°C (425 and 475°F) slopes were found to be not sig nificantly different. This can be explained for the whole wheat by the fact that their MAT curves are similar for the two temperatures, therefore the same percent destruction would be expected. However, this explanation cannot be true for the enriched white bread since their MAT curves for 218 and 246°C (425 and 475°F) are quite different. However, the difference in oven temperature between these two nominal oven temperatures is only 16.5°C for enriched white and 11.9°C for whole wheat, whereas the difference between the other temperatures are much higher (see Table 10). This lat er observation is a more likely explanation for the non-sig-69 nificant difference between the slopes for 218 and 246°C (425 and 475°F). Slopes show a higher thiamine instability as oven temper ature increases. But because bread is baked in less time as oven temperature increases, during normal baking of bread there is not more destruction of thiamine at the higher temperature. For all curves, it appears that the MAT increases accord ing to a pattern approximated by two straight lines. First, the MAT increases very rapidly for 6 to 15 minutes, with slopes increasing as oven nominal temperature increases. Then, forming a second line, the MAT continues to increase at a constant rate, but slower than the first line. This rate also is greater with increasingly higher oven tempera tures. The 288°C (550°F) curve is the only curve where the experimental points would not fit well on this hypothetical 2nd straight line pattern. Also, when curves for enriched white and whole wheat bread are compared for the same oven temperature, they do not agree very well with each other, especially the curves at 246°C (475°F). MAT curves for en riched white bread are all steeper than the whole wheat bread curves. This is likely due to the different composi tion and moisture retention of the two types of bread, hav ing different heat penetration properties. Whole wheat bread would have a greater moisture retention, because of the high fiber content. Like for Figure 7 and 8, the two 70 straight lines pattern can be observed. Both curves show a rapid increase in temperature (being slighty higher for the crust) for the 12 first minutes- Afterwards, the curve for middle of the bread is almost horizontal (slighty positive slope), whereas for the crust, the temperature increases at a constant rate. This almost constant temperature for the middle of the bread is about 100°C on the graph. The temp erature data for all the other bread treatments also reveal that the middle temperature data follow a very similar pat tern with the temperature never exceeding 100 to 108°C. From the MAT formula and graphs, it is obvious that the crust temperature is the one that governs the MAT of the bread loaf. Because MAT curves were all lower for whole wheat bread compared to enriched white, less thiamine destruction should be expected. But in fact the destruction curves demonstrat ed more thiamine destruction for whole wheat bread. This observation on MAT curves reinforces the thiamine destruc tion data which showed that thiamine in whole wheat bread is more vulnerable to temperature than synthetic thiamine in enriched white bread. Two different theories could be proposed to explain the lower stability of thiamine to heat for whole wheat bread. The first is the pH effect. In this study, whole wheat bread had a slightly higher pH than enriched white bread, and it has been demonstrated that thiamine stability de-71 creases with an increase in pH. As a second theory there is the ash or inorganic constituents explanation proposed by Dawson and Martin (1942) and Farrer (1955), where higher thiamine losses are found with higher extractions of flour. Since k values are very similar between pH 5.0 and 5.5, higher thiamine losses in whole wheat are most probably caused by both higher pH and an increase in the inorganic constituents. This theory was also proposed by Farrer ( 1955). To take into consideration the pH effect only, other ex periments should be done using the same type of bread but varying the pH, for example, by adding an acid like vinegar to the bread, or using a sour-dough type bread. To verify the ash theory, different percent extraction flours with controlled pH could be used. VI CONCLUSION Thiamine destruction in phosphate buffer follows a first order reaction and the Arrhenius equation, where Ea was 34.2 kcal/mole. It increased as pH increased, with greater de struction at pH 6.0 and above. Kinetic data could not be obtained with bread system be cause of unsteady temperature change of the bread during baking. However, a linear relationship was obtained when logarithm of percent thiamine retention was plotted against time, showing higher destruction with time. When slopes were compared, the stability of thiamine decreased with higher oven temperatures, and thiamine in whole wheat bread (a natural source) was less stable than in enriched white bread (a synthetic source). This difference might be ex plained by higher pH and higher ash content in the whole wheat bread. Baking losses were found to be 30 to 50% for normal bak ing of 12g loaves. To interpret these results in terms of a normal loaf (450 g or 1 lb), one would expect to get less destruction in larger loaves of both types of bread, al though not necessarily 0%. - 72 _ LITERATURE CITED AACC. 1969. Methods of Analysis of the American Association of Cereal Chemists. St-Paul, MN. Adamson, A.W. 1979- A Textbook of Physical Chemistry, 2nd ed. Academic Press, New York, NY. p.543-AOAC. 1980. Official Methods of Analysis, 13th ed. Association of Official Analytical Chemists, Washington, DC. Association of Vitamin Chemists. 1966. Methods of Vitamin Assay, 3rd ed. Interscience Publishers, New York, NY. p.123-Aughey, E. and Daniel, E.P. 1940. Effect of cooking upon the thiamine content of foods. J. Nutrition 19: 285. Bendix, G.H., Heberlein, D.G., Ptak, L.R. and Clifcorn, L.E. 1951. Factors influencing the stability of thiamine during heat sterilization. Food Res. 16: 494. Booth, R.G. 1943. The thermal decomposition of aneurin and cocarboxylase at varying hydrogen ion concentrations. Biochem. J. 37: 518. Boudart, M. 1968. Kinetics of Chemical Processes. Prentice-Hall Inc., Englewood Cliffs, NJ. Capellos, C. and Bielski, B.H.J. 1972. Kinetic systems: Mathematical Description of Chemical Kinetics in Solution. John Wiley & Sons Inc., New York, NY. p.5. Coppock, J.B.M., Carpenter, B.R. and Knight, R.A. 1956. Cereal product fortification: The B vitamins with special reference to thiamine losses in baked products. J. Sci. Food Agric. 7: 457. Daniels, F. and Alberty, R.A. 1958. Physical Chemistry. Wiley-Interscience, New York, NY. p.318. Dawson, E.R. and Martin, G.W. 1941. Vitamin B.. Estimation in yeast and bread and stability during breadmaking. J. Soc. Chem. Ind. (Transactions). 60: 241. - 73 -74 Dawson, E.R. and Martin, G.W. 1942. Vitamin B.. Estimation in wheatmeal and brown bread and stability of different forms of vitamin B. during bread-baking. J. Soc. Chem. Ind. (Transactions;. 61: 13. Dwivedi, B.K. and Arnold, R.G. 1972. Chemistry of thiamine degradation. Mechanisms of thiamine degradation in a model system. J. Food Sci. 37: 886. Dwivedi, B.K. and Arnold, R.G. 1973- Chemistry of thiamine degradation in food products and model systems: A review. J. Agric. Food Chem. 21(1): 54. Farrer, K.T.H. 1941. The influence of pH on the destruction of aneurin at 100°C. Austr. Chem. Inst. J. Proc. 8: 113. Farrer, K.T.H. 1945- The thermal destruction of vitamin B^. 1. The influence of buffer salts on the rate of destruction of aneurin at 100°C. Biochem. J. 39: 128-132. Farrer, K.T.H. 1953- The thermal destruction of vitamin B1 in vegetables. Austr. J. Sci. 16: 62. Farrer, K.T.H. 1955. The thermal destruction of vitamin B^ in foods. Adv. in Food Res. 6: 257. Farrer, K.T.H. and Morrison, P.G. 1949- The thermal destruction of vitamin B.. 6. The effect of temperature and oxygen on the rate of destruction of aneurin. Austr. J. Exptl. Biol. Med. Sci. 27: 517. Feliciotti, E. and Esselen, W.B. 1957. Thermal destruction rates of thiamine in pureed meats and vegetables. Food Technol. 11 (Feb.): 77. Food and Nutrition Board. 1980. Recommended Dietary Allowances, 9th ed. National Research Council, National Academy of Sciences, Washington, DC. Gomori, G. 1955. Preparation of buffers for use in enzyme studies. In: Methods in Enzymology, Volume I. S.P. Colowick and N.O. Kaplan (Eds). Academic Press Inc., New York, NY. p. 143-Greenwood, D.A., Beadle, B.W. and Kraybill, H.R. 1943. Stability of thiamine to heat. II. Effect of meat-curing ingredients in aqueous solutions and in meat. J. Biol. Chem. 149: 349. Greenwood, D.A., Kraybill, H.R., Feaster, J.F. and Jackson, J.M. 1944. Vitamin retention in processed meat. Effect of thermal processing. Ind. Eng. Chem. 36: 922. 75 Harper, H.A. 1973. Precis de Biochimie. Les Presses de 1'Universite Laval, Quebec, Que. p.116. Hoffman, C, Schweitzer, T.R. and Dalby, G. 1940. The loss of thiamin in bread on baking and toasting. Cereal Chem. 17: 737. Holvey, D.N. 1972. The Merck Manual, 12th ed. Merck & Co., Inc., Rahway, NJ. p.1049-Lincoln, H., Hove, E.L. and Harrel, C.G. 1944. The loss of thiamine on cooking breakfast cereals. Cereal Chem. 21: 274. Lund, D.B. 1975. Effects of blanching, pasteurization, and sterilization on nutrients. In: Nutritional Evaluation of Food Processing, 2nd ed. R.S. Harris and E. Karmas (Eds). AVI Publishing Co., Inc., Westport, CT. p.210. Mcllvaine, T.C. 1921. A buffer solution for colorimetric comparison. J. B. C. 49: 183• Mulley, E.A., Stumbo, CR. and Hunting, W.M. 1975a. Kinetics of thiamine degradation by heat. A new method for studying reaction rates in model systems and food products at high temperatures. J. Food Sci. 40: 985. Mulley, E.A., Stumbo, CR. and Hunting, W.M. 1975b. Kinetics of thiamine degradation by heat. Effect of pH and form of the thiamine on its rate of destruction. J. Food Sci. 40: 989-Neal, R.A. and Sauberlich, H.E. 1973- Thiamin. In: Modern Nutrition in Health and Disease, 5th ed. R.S. Goodhart and M.E. Shils (Eds). Lea & Febiger, Philadelphia, PA. p.186. Pace, J.K. and Whitacre, J. 1953- Factors affecting retention of B vitamins in corn bread made with enriched meal. 1. The relation of pH to the retention of thiamine, riboflavin and niacin in corn bread. Food Res. 18: 231. Pomeranz, Y. and Shellenberger, J.A. 1973. Bread Science and Technology. AVI Publishing Co., Inc., Westport, CT. Rice, E.E. and Beuk, J.F. 1945. Reaction rates for the decomposition of' thiamine in pork. Food Res. 10: 99. Schultz, A.S., Atkin, L. and Frey, CN. 1942. The stability of vitamin B1 in the manufacture of bread. Cereal Chem. 19: 532. Sognefest, P. and Benjamin, H.A. 1944. Heating lag in TDT cans and tubes. Food Res. 9". 234. 76 Stumbo, CR. 1965. Thermobacteriology in Food Processing. Academic Press, New York, NY. p.322. Tabekhia, M.M. and D'Appolonia, B.L. 1979- Effects of processing steps and baking on thiamine, riboflavin, and niacin levels in conventional and continuous produced bread. Cereal Chem. 56(2): 79-Williams, R.R. 1938. The chemistry of thiamin (vitamin B.). J. Am. Med. Assoc. 110: 727-Zar, J.H. 1974. Biostatistical Analysis. Prentice-Hall, Inc., Englewood Cliffs, NJ. p. 198. Appendix A THIAMINE LOSSES IN BREAD Type of bread White Whole wheat Rye Regular_white High B- white Whole wheat Regular white High B. white Whole wheat Regular white High B1 white Whole wheat Regular white High B- white Whole wheat Melba toast Reg. white High B1 Whole wheat White White High B1 white Enriched white 85% extraction 95% extraction 100% extraction (stone ground) Hovis(wheat germ) Baking Baking % loss Reference time temperature (min) light toasting medium toasting heavy toasting almost 0 8 5 9 0 4, 12 0 0 10, 17 3 0 24,21 40 9 26 45 20 30 190-218 C 15 425°F 475°F 45-48 510-520UF 6-8 6-8 11 3-14 22 27 33 35 19 Morgan and Frederick (1935)*^ Hoffman et al (1940) Aughey and Daniel (1940) Harrel1et al. (1941)1 Dawson and Martin (1941) Dawson and Martin (1942) - 77 ~ 78 (continued) Type of bread Baking Baking % loss Reference time temperature (min) Enriched white (1 lb) Whole wheat (1 lb) High B1 white (1 lb) ' Commercial scale Enriched white High B. white Whole wheat 10 410°F 3 20 13 30 21 40 33 10 2 20 17 30 23 40 310 6 20 14 30 23 40 32 'commercial conditions' 21 (corresponding 22 to 30 min 26 labor atory bakes) Schultz et al (1942) White (1.25 lb) White (10 lb) White (4 lb) 35 44 80 75 80 440°F 360-440°F 360-475°F 17 14 21 19 24 Meckel and Anderson (1945) 1 British 85% extr British 80% extr South Africa 95% extr. Canned bread Good baking cond itions 16 15 29 15 Goldberg and Thorpe (1946) 1 Brenner.et al. (1948a)1 White Bread rolls (various) 30 40 50 15 20 25 400°F 17 20 26 7- 12 8- 16 12-22 Zaehringer and Personius (1949) 75% extraction (0.35% ashes) 85% extraction (0.49% ashes) 18.0 21.7 Farrer ( 1949)' 79 (continued) Type of bread Baking Baking % loss Reference time temperature (min) 98% extraction (0.97% ashes) 100% extraction (1.06% ashes) Wheat Rye Melba toast Loaves Muffins Sticks Wheat Rye White patent flour 80% extr.(unblea ched ,untreated) 80% extr.(with dough improver) 80% extr.(with chlorine dioxide) 100% extr. (no additives) American army type canned bread National bread 5mm thickness 9mm 12mm (all loaves were 1 Enriched white (1 lb) Conventional method 30 45 225-240°F 240-260°F 30 475°F Toasting lb loaves) 8 13 18 23 30 430°F 30.0 31.0 11- 20 15-25 20 15 21 34 12- 14 30 21 .0 17-3 20.7 19- 1 23-3 19.6 31.0 14.7 13.4 4 10 10 21 47 Pulkki et al. ( 1950) 1 Menger (1952)2 Pace and Whitacre ( 1953)' Bukin et al. ( 1954)^ Coppock et al. (1956) Tabekhia and D'Appolonia (1979)** 80 (continued) Type of bread Baking Baking % loss Reference time temperature (min) Continuous 8 430°F 8 Tabekhia and method 13 12 D'Appolonia 18 5 (1979)** 23 27 30 43 •Feeding tests. **Approximate values from their Fig. 1. ^Referenced in Farrer (1955). Referenced in Coppock et al. (1956). Appendix B STATISTICAL ANALYSIS - 12G LOAVES (EXPERIMENT 2) B. 1 T-TEST b1 - b2 Sb1-b2 350°F 475°F Enr. white W. wheat Enr. white W. wheat sx; 51756 55124 9944 19871 ZY^ 0.8931 1 .0419 0.6035 1.8804 ZXY 203.4679 235.0443 67-6186 188.2795 n 16 15 16 15 b 0.003931 . 0 .004264 0.006800 0 .009475 Residual SS 0.09323 0 .03973 0.1437 0 .09640 Residual DF (S^y.x)p 15 14 15 14 0. 004585 0. 008281 Sb^bp t exp 0. 0004144 0. 001118 -0 .8035 -2 .3931 V 29 29 t.05(2),29 +2 .045 +2 .045 Conclusion Bew=Bww (p=0. 2140) Bew^Bww (p=0. 0204) 425°F 550°F Enr. white W. wheat Enr. white W. wheat EY EXY n b Residual SS Residual DF (S y.x)p 19000 0. 9491 124.3641 16 0.006545 0.1351 15 29464 2.2763 250.1912 16 0.008491 0.1519 15 6120 0.7081 63-5770 16 0.01039 0.04765 15 6824 1.2881 89.8601 16 0.01317 0. 1048 15 0.009566 0.0050807 - 81 -82 (continued) M25°F 550°F Enr. white W. wheat Enr. white W. wheat Sb ..-bp 't ixp t .05(2),30 Conclusion 0.0009100 2.1384 30 ±2.042 Bew^Bww (ps0.0117) 0.001255 2.2154 30 ±2.0.42 Bew^Bww (p=0.01720) B.2 F-STATISTIC SSc - SSp k - 1 SSp DFp Enr. white W. wheat Res. SS Res. DF Res. SS Res. DF Regression 350 Regression 425 Regression 475 Regression 550 Pooled regression Common regression F experimental F.05(1),3,DFp Conclusion 0. 09323 15 0.03973 14 0. 1351 . 15 0.1519 15 0. 1437 15 0.0964 14 0. 04765 15 0.1048 15 0. 4197 60 0.3928 58 0. 7270 59 1.2502 57 14.6396 42.2034 2.76 b350*b425*b475*b550 (p<<0.0001) 2.77 b350*b425*b475*b550 (p<<0.0001) 83 B.3 SNK TEST q S.E. B.3•1 Enriched white bread See following page. B.3.2 Whole wheat bread See following page. B.4 DEVIATION FROM LINEARITY HQ: The population regression is linear. H.,: The population regression is not linear. MS deviation from linearity F MS within groups Example of anova table for whole wheat at 550°F: Source of variation SS DF MS Total Between groups Linear regression Deviation from linearity Within groups 1.288089 1.211828 1.183316 0.0285119 0.0762604 2 13 0.014256 0.0058662 F = 2.4302 F.05(1),2,13 = 3-89 conclusion: accept HQ (p=0.1269) Enriched white bread Comparison (1 vs 2) Difference 0>L - b2) S.E. q P q.05,60,p Probability P Conclusion 0.0007995 8.0792 4 3.737 p<0.001 b550f*b350 0.0008693 4.4266 3 3.399 0.01<p<0.005 b550^j425 0.0009609 3.7361 2 2.829 0.025<p<0.01 b550#3475 0.0006476 4.4304 3 3.399 0.01<p<0.005 b475?^350 0.0007320 0.3483 2 2.829 p>0.50 b475=b425 0.0005017 5.2105 2 2.829 p<0.001 b425^350 550 vs 350 550 vs 425 550 vs 475 475 vs 350 475 vs 425 425 vs 350 0.006459 0.003848 0.003590 0.002869 0.0002550 0.002614 Whole wheat bread 350 425 475 550 Comparison (1 vs 2) Difference (bl " b2> S.E. p q.05,58,p Probability P Conclusion 550 vs 350 0.008906 0.0007467 11.9267 4 3.737 p<0.001 550 vs 425 0.004679 0.0007817 5.9855 3 3.399 p<0.001 550 vs 475 0.003695 0.0008164 4.5257 2 2.829 0.001<p<.005 475 vs 350 0.005211 0.0004815 10.8230 3 3.399 p<0.001 475 vs 425 0.0009840 0.0005341 1.8428 2 2.829 O.l0<p<0;20 425 vs 350 0.004227 0.0004199 10.0650 2 2.829 p<0.001 b550#3350 b550#>425 b550#>475 b475#>350 b475-b425 b425#>350 350 425 475 550 85 Summary: F exp F.05(1),2,DFwi Conclusion p Enr.white 350 26. 050 3. 89 reject Enr.white 425 1. 395 3. 89 accept Enr.white 475 2. 908 3. 89 accept Enr.white 550 2. 107 3. 89 accept W.wheat 350 1. 432 3. 81 accept W.wheat 425 1. 027 3. 89 accept W.wheat 475 1 . 252 3. 81 accept p<<0.0001 0.2826 0.0904 0.1612 0.2769 0.3853 0.3207 Appendix C EXAMPLE OF CALCULATION FOR % THIAMINE RETENTION C.1 MODEL SYSTEM Example: Sample at 100°C after 4 hrs of heating. Photomultiplier reading '0' (no heating) 50.5 •0' blank 0.3 Standard 50.0 Standard blank 0.Sample 25.8 Sample blank 3 Thiamine yg/ml Sample reading - Sample blank reading 1 x Standard reading - Stand.blank reading 5 50.5 - 0.3 1 yg thiamine/ml '0' = x — = 0.2020 yg/ml ( 1) 50.0 - 0.3 5 25.8 - 0.3 1 yg thiamine/ml sample r x —= 0.1026 yg/ml (2) 50.0 - 0.3 5 (2) % thiamine retention = x 100 = 50.8% (1) C.2 BREAD SYSTEM Example: Whole wheat bread baked at 350°F for 65 min (2nd run). Photomultiplier reading Dough 93.0 Dough blank 1.8 Standard 85.85 Sample 63.- 86 -87 Sample blank 5.6 Sample weight: 9-0 g dry weight: 0.8506 g/g Dough weight: 10.0 g dry weight: 0.5906 g/g S.reading - S.blank read. 1 100 Thiam.yg/g sample = x — x Stan .read .-St.blank read. 5 dry sa mple wt (g) (63.5 - 5.6) 1 100 yg thiam./g sample = x x (4) (85.85-0.65) 5 (9 x 0.8506) = 1.7745 yg/g (3) (93.0 - 1.8) 1 100 yg thiam./g dough x x (85.58-0.65) 5 (10 x 0.5906) = 3-5950 yg/g (3) % thiamine retention = x 100 = 49.4% (4) Dry weight calculation: (dry sample wt + container wt) - container wt wet sample wt dry wt g/g 4.7082 - 1.2991 Duplicate 1 : = 0.8523 g/g (1) 4.0000 4.7004 - 1.3047 Duplicate 2 : = 0.8589 g/g (2) 4.0000 (1) + (2) Average dry wt g/g = = 0.8506 g/g 


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