CAKE PRODUCTS PREPARED FROM SIMPLE SUGARS AND . A SACCHARIDE SIRUP AND THE CARIOGENIC ACTIVITY ' IN RATS: DIETARY INTAKE AND SALIVA PARAMETERS ' 7- A Dissertation for-the Degree Of Ph. 0.? l * MICHIGAN STATE UNIVERSITY v CHARLOTTE MAY THOMPSON ‘ ‘ . f j '1975 . ; I-w-‘r‘ .‘GLF'V'I’ ' LEBRAER III _ Inger; State Univers-it This is to certify that the thesis entitled “I CAKE PRODUCTS PREPARED FROM SIMPLE SUGARS AND A SACCHARIDE SIRUP AND THE CARIOGENIC ACTIVITY IN RATS: DIETARY INTAKE AND SALIVA PARAMETERS presented by Charlotte May Thompson has been accepted towards fulfillment of the requirements for Minna—degree in JumaxLNnnrition and Foods 4: I 0.4%42 752C%0¢cz4—A¢{/ Major professor Date /{/7//7C5’ 0-7 539 a IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII - ~‘%i % - £113.28 Mg- Qmw gWZag ABSTRACT CAKE PRODUCTS PREPARED FROM SIMPLE SUGARS AND A SACCHARIDE SIRUP AND THE CARIOGENIC ACTIVITY IN RATS; DIETARY INTAKE AND SALIVA PARAMETERS By Charlotte May Thompson Dental decay is a major health problem in the U.S. as indi- cated by the Ten-State Nutrition Survey (l968-1970). Dietary modi- fication becomes a practical approach to control of dental disease through alteration of dietary carbohydrate available as substrate for cariogenic oral microorganisms. Further investigation was con- sidered necessary to evaluate the cariogenic effect of a cake product containing one of the following simple sugars or saccharide sirup: (1) sucrose, superfine quality, (2) fructose, (3) glucose, (4) a high fructose corn sirup, and (5) a mixture of equal parts of crystalline fructose and glucose. The purpose of this study was to devel0p an acceptable cake product using monosaccharides for sucrose in a cake formula and to test the cariogenic effect of this product in a caries-susceptible animal model. Butter-type cakes containing fat were prepared by the solu- tHMi method of mixing and evaluated by both sensory and objective measurements. Eleven taste-panel members scored each of the cakes for grain, crumb color, silkiness, tenderness and flavor. Product Charlotte May Thompson quality was assessed using objective measurements for pH, batter viscosity, moisture loss, moisture content, relative cake volume, resistance to breaking (Allo-Kramer shear press) and color (Hunter Color Difference Meter). According to objective measurements of product quality, cakes made with monosaccharides were smaller, more easily broken and more moist than the cakes made with sucrose. When compared to 'other cakes, those which contained fructose were darkest in color as indicated by Hunter Color Difference Meter L or lightness values. As measured by aL or green-red values, fructose cakes were also the least green. These color differences suggest that increased brown- ing reactions occurred during baking in cakes prepared from fructose. The composite methods used for evaluation of cakes prepared with sucrose, fructose, high fructose corn sirup, and fructose/glucose were sufficiently high so that these products were all considered acceptable whereas cakes made with glucose were considered unacceptable. Multisurface caries were produced in Osborne-Mendel rats infected with Streptococcus mutans and fed diets containing cakes prepared from one of the simple sugars or saccharide sirup. The baked cakes were dried and used at a level of 70% in rations fed rats. These rations contained on a dry weight basis 32% of one of the simple sugars being tested, 14% fat and l2% protein. Groups of 10 male Osborne-Mendel rats were weaned at 18 days of age and given penicillin in the drinking water (0.5 g/lOO ml) for 2 days. The test diet and oral infection with S. mutans was then begun. At Charlotte May Thompson 116_ days of age, the animals were sacrificed by decapitation, the jaws cleaned of soft tissue, and the molar teeth scored for caries by the method of Keyes (J. Dent. Res. 32, 1088, 1958). The cari- ogenic effects of sugars incorporated into cakes were greater with sucrose, as compared with glucose, or with fructose, or with high fructose sirup or with a mixture of fructose and glucose. Techniques for collecting and measuring saliva were inves- tigated to evaluate the feasibility of following nutritional responses in humans associated with changes in dietary intake. Mixed saliva was collected 3 hours after the morning meal. Resting saliva samples (10 min samples) obtained from 7 subjects were analyzed for urea using the Hyland Phenate Hypochlorite method and for glucose using the enzyme glucose oxidase. For subjects who ate 2.3 to 4.9 g protein/100 kcal, the range for urea in saliva was 9.8 to 14.9 mg %. Increasing the protein level from 44 g/day to 115 g/day in one subject resulted in an increase in urea in the saliva from 22.1 to 26.1 mg%. The usual carbohydrate (g/lOO kcal) for the 7 subjects in the morning meal was 8.0 to 21.0 g and the glucose level in saliva ranged from O to 4.3 mg %. Ingestion of 9 g of carbohydrate (6 g sugar) just prior to the collection of saliva resulted in glucose values of 98.4 mg % as compared to 1.3 mg % salivary glucose when collection was done 1 hour after ingestion of the carbohydrate (10 g caramel), as tested in one subject. CAKE PRODUCTS PREPARED FROM SIMPLE SUGARS AND A SACCHARIDE SIRUP AND THE CARIOGENIC ACTIVITY IN RATS; DIETARY INTAKE AND SALIVA PARAMETERS By Charlotte May Thompson A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1975 ACKNOWLEDGMENTS Appreciation for the support of this research and thesis development is extended to my committee composed of Dr. Rachel Schemmel, Dr. Olaf Mickelsen, Dr. Willis Wood, Dr. Duane Ullrey and Dr. Kusum Patel. Special gratitude is extended to the following: Dr. Kaye Funk for providing the incentive and initiative to ensure an early and productive start for research activities. Dr. Rachel Larson and the staff of the Caries Prevention and Research Branch, National Institutes of Health, for providing cariogenic streptococci cultures, training for dental scoring and verification of caries values used in this study. Dr. J. William Thomas and Elaine Kibbey of the Dairy Science Department for going the extra mile to find solutions to problems. Family, friends and fellow graduate students who provided unfailing support and encouragement when spirits flagged. The National Institutes of Health Traineeship #GMO 1818, the Department of Food Science and Human Nutrition and the Agricultural Experiment Station for financial support as well as scholarship funds from MHEA, ADA, College of Human Ecology and the Graduate School. The Data Analysis Unit of the College of Human Ecology for statistical advice.‘ ii TABLE OF CONTENTS LIST OF TABLES . LIST OF FIGURES LIST OF APPENDICES INTRODUCTION BIBLIOGRAPHY PART I. REVIEW OF LITERATURE: DECAY OF ENAMEL IN MULTISURFACE CARIES CARIOUS PROCESS Experimental Animal Models . Carious Lesion in the Enamel CARIES-TEST CHALLENGE FOR MULTISURFACE LESIONS Bacterial Factors . Plaque Formation Acid Formation . . . . Microbial-Substrate Factors Host Factors SUMMARY BIBLIOGRAPHY PART II. USE OF MONOSACCHARIDES AND A SACCHARIDE SIRUP IN BUTTER-TYPE CAKES INTRODUCTION LITERATURE REVIEW . Overview: Sugars in Cake Products Functions of Sugar . Effect of Reducing Sugars Use of Liquid Sweeteners Page viii ix Properties of Sugars . Solubility Sweetness . Sugar Caramelization . . . Sugar-Amine Interactions of the Maillard Reaction Color changes during heating Flavor changes . . . Polymer formation . Relative Browning Rates . MATERIALS AND METHODS Preparation . Ingredients . Procedure Evaluation Objective Measurements Sensory Evaluations Analyses of Data RESULTS Objective Measurements Sensory Evaluations DISCUSSION SUMMARY BIBLIOGRAPHY PART III. CARIOGENICITY 0F MONOSACCHARIDES AND A SACCHARIDE SIRUP IN DIETS 0F RATS INTRODUCTION LITERATURE REVIEW . Cariogenicity of Foodstuffs . Carious Process and Factors Influencing It Cariogenic Streptococci in the Rat Use of Penicillin in Caries Research Nutrient Factors MATERIALS AND METHODS Experimental Design Infection of Rats . iv Scoring Carious Lesions . Preparation of Jaws . Values for Linear Area of Decay Analyses of Data . . . RESULTS Body Weights . . . Diet and Fluid Consumption . Dental Caries Incidence . Wet Tissue Weights DISCUSSION SUGGESTIONS FOR FURTHER STUDY . SUMMARY BIBLIOGRAPHY PART IV. DIETARY INTAKE AND SALIVA PARAMETERS INTRODUCTION LITERATURE REVIEW . Mixed Saliva Resting Flow Rate Collection . Flow Rate and Salivary Composition Properties of Saliva . . . pH Urea . . Specific Gravity Glucose Dietary Factors pH . Rate of Flow Carbohydrates METHODS RESULTS AND DISCUSSION Urea Glucose . Salivary Parameters and Dental Data . 104 106 111 112 112 113 113 114 114 115 116 116 117 117 118 119 120 122 122 125 Page SUMMARY . . . . . . . . . . . . . . . . . . 127 BIBLIOGRAPHY . . . . . . . . . . . . . . . . 128 APPENDICES . . . . . . . . . . . . . . . . . 131 vi LIST OF TABLES Table Page 1. Cake formula for one replication . . . . . . . . 46 2. Score-card used to evaluate cake samples . . . . . 50 3. Averages, standard deviations and statistical analysis of objective measurements of batter and baked butter-type cakes prepared with mono- saccharides and sucrose . . . . . . . . . . 53 4. Averages, standard deviations and statistical analysis of quality characteristics of butter-type cakes prepared with monosaccharides and sucrose . . . . 55 5. Frequency of flavor responses to interior portion and crust of butter- -type cakes by 11 panelists with 3 replications of each cake . . . . 58 6. Composition of cake rations . . . ._ . . . . . 8O 7. Percentage of carbohydrate, protein and fat in the partially dried cake rations . . . . . . . . 82 8. Means, standard deviations and statistical analyses in growth of Osborne-Mendel rats fed either a sucrose- or monosaccharide-containing cake diet for 13 weeks . 9O 9. Means, standard deviations and statistical analyses for dental caries incidence of male Osborne-Mendel rats fed either a sucrose- or monosaccharide- containing cake diet for 13 weeks . . . . . . 92 10. Correlation coefficients (r) and significance between caries and intakes and weight gain at 13 weeks in male Osborne-Mendel rats fed dried cake diets pre- pared with 5 different sweeteners . . . . . . . 94 11. Means, standard deviations and statistical analyses of wet tissue weights of male Osborne-Mendel rats fed either a sucrose- or monosaccharide-containing diet . 96 12. Dietary intake in the morning meal, selected salivary parameters, and dental information in 7 subjects con- suming either their regular food intake or experi- mental meals high in protein or carbohydrate . . . 123 vii LIST OF FIGURES Figure Page 1. Mean growth rates of male Osborne-Mendel rats fed diets containing 70% dried cake made from 1 of 5 sweeteners for 13 weeks . . . . . . . . . 89 viii LIST OF APPENDICES Appendix A. General Sensory Instructions B Photomicrographs C. Cariogenic Working Cultures D Dietary-Dental Data Sheet E. Saliva Collection and Measurements F. Salivary Urea Nitrogen Determination G. Enzymatic Determination of Glucose ix Page 132 135 138 140 142 145 147 INTRODUCTION Currently, dental decay is one of the few health problems that is of importance in both industrial and developing countries. The Ten—State Nutrition Survey (1968-1970) indicated that in areas of the U.S. the incidence of dental decay and the delivery of dental care was a major health problem especially among children. For persons 18 years of age and over, extensive dental decay (10 or more decayed, missing and filled teeth) was found in permanent teeth for 80% of the surveyed population. Values for edentulous persons were not included (Department Health, Education, and Welfare, 1972). Attention to human dietary habits has been suggested as a means for reducing the incidence of dental caries (Nizel, 1969). This suggestion is based on the finding that certain oral micro- organisms can synthesize extracellular polysaccharides from food debris which, in turn, become an integral part of the dental plaque. Formation of the latter is thought to be the initiating step in dental caries. Cariogenic bacteria enmeshed in the plaque are protected from easy dislodgment; they use the plaque material as substrate for continued cariogenic action. These polysaccharides are formed jn_vitrg_from sucrose but not from glucose or other monosaccharides (Newbrun, 1967). Dietary modification, through alteration of the oral environment, then becomes a practical approach to the control of dental disease (Berman, 1971; Miller, 1973; Hartles, 1970). Studies with human subjects have shown an association between the incidence of dental decay and sucrose consumption. The latter includes not only the amount of sucrose consumed but also the frequency with which it is eaten and the physical consistency of the food contain- ing this carbohydrate (Lilenthal et al., 1953; Gustafsson et al., 1954; Harris, 1963). The effects of a variety of simple sugars on dental caries have been demonstrated recently in hamsters by Campbell and Zinner (1970). They showed that a sucrose containing diet supported a more destructive carious process over a 100 day feeding period than did diets containing fructose, glucose, lac- tose or a 1:1 mixture of fructose and glucose. To alter the bacterial flora of the mouth to resemble that of caries-free individuals, Jay and co—workers (1959) recommended a series of low carbohydrate diets. Results from these dietary plans, which restricted carbohydrate to 100 g daily, showed that 70% of the individuals who had been caries-susceptible became caries-inactive. However, the diets were inconsistent with usual eating patterns and therefore inconvenient to maintain. Bibby (1961) has also recommended a dietary approach based on recognition of the need of altering the cariogenicity of foodstuffs. Befbre dietary advice for the reduction of dental caries will be accepted, the less cariogenic foods must be well received so that they can be readily incorporated into the individual's regular diet: Thus, the development of acceptable food substitutes using monosaccharides for sucrose in foods which commonly contain sucrose provides a realistic means for reducing dental caries. The purpose of this study was to develop an acceptable food product made from simple sugars and/or saccharide sirup and to test these in a caries-susceptible animal model. BIBLIOGRAPHY Berman, D. S. (1971) Dental caries--a review. Nutr. 25, 154-160. Bibby, B. G. (1961) Cariogenicity of foods. J. Amer. Med. Ass. 114, 316-321. Campbell, R. G. & Zinner, D. D. (1970) Effect of certain dietary sugars on hamster caries. J. Nutr. 100, 11-20. Department Health, Education, and Welfare (1972) Ten-state nutri- tion survey 1968-1970, DHEW Publication (HSM) 72-8131, Atlanta, Ga. Gustafsson, B. E., Quensel, C. E., Lanke, L. S., Lundqvist, C., Grahnen, H., Bonow, B. E. & Krasse, B. (1954) Effects of different levels of carbohydrate intake on caries activity in 436 individuals observed for five years. Acta Odontol. Scand. 11, 232-364. Harris, R. (1963) Biology of the children of Hopewood House, Bowral, Australia. Observations on dental-caries experience extending over five years (1957-1961). J. Dental Res. 42, 1387-1399. Hartles, R. L. (1970) Dietary modification as a means of the con- trol of dental caries. Roy. Soc. Health J. 90, 316—319. Jay, P., Beeuwkes, A. M. & MacDonald, H. B. (1959) Dietary Program for the Control of Dental Caries. The Overbeck Co., Ann Arbor, Mich. Lilienthal, B., Goldsworthy, N. E., Sullivan, H. R. & Cameron, D. A. (1953) The biology of the children of Hopewood House, Bowral, North South Wales: 1. Observations on dental caries extending over five years (1947-1952). Med. J. Aust. 1, 878-881. Miller, J. (1973) Prevention for the individual practitioner. Brit. Dental J. 134, 181-187. Newbrun, E. (1967) Sucrose, the arch criminal of dental caries. Odont. Rev. 18, 373-386. Nizel, A. E. (1969) Food habits and their modification for dental caries. Nutr. News 32 (FebruarYI, l-2. 4 PART I REVIEW OF LITERATURE: DECAY OF ENAMEL IN MULTISURFACE CARIES CARIOUS PROCESS Evaluation of the interdependency of diet and caries pro- duction is dependent upon the recognition of the multifactorial nature of the carious process. That is, dental decay is primarily an infectious process dependent upon interactions involving micro- flora, oral dietary substrate, and the resistance of the tooth (host factors) (Orland, 1954; Keyes, 1960). Caries do not develop with only one active factor but control can be effected by altering only one of these conditions (Keyes and Jordan, 1963). After eruption of the tooth, destructive carious activity begins on an external surface with selective penetration into the interior of the tooth. Formation of a carious lesion in the enamel involves two distinct steps: one of these is demineralization and the other is proteolytic breakdown and destruction of the organic matrix of the tooth structure (Sognnaes, 1955; Darling, 1963). Dietary factors play a role in both the location and extent of these lesions. Experimental Animal Models X-ray diffraction studies reveal that the mineral salt in teeth of rats resembles the hydroxyapatite of human enamel (Gilda, 1951). The calcium-phosphorus weight ratio expressed on an ash basis of human enamel is usually in the range of 2.0 to 2.2 (Leicester, 1949). The calcium-phosphorus ratio in the incisor teeth of the rat was calculated to be 1.71 (Matsuda, 1927) and in the enamel to'be 2.0 (McCluree_t_a_1_., 1966). In hamsters, Lobene and Burnett (1954) reported the calcium-phosphorus ratio of molars for males and females, respectively, was 1.87 and 1.45. Patterns of cavitation seen in humans are primarily located in the enamel of crevices (pits and fissures), or on root surfaces, or develop as multisurface cavitation (both sulci and smooth surface). McCollum et_gl, (1922) reported that the destruc- tion of molar crowns initiated in the occlusal sulci of white rats was similar to that found in man. The developmental pattern of enamel carious lesions from early enamel penetration to deep dentine destruction and cavitation was observed UJbe analogous u)human caries as evidenced by histopathological examination of ground and decal- cified sections of rat molars (Agnew gt_al,, 1932). Gross carious lesions in the molar teeth of the Syrian hamster have been described in sulci, occlusal areas and frequently near the cemento-enamel junction and extending around the cervical portion of the teeth (Arnold, 1942). Smooth-surface caries have also been reported in the albino rat (located on the buccal, lingual and proximal surfaces and also following the gingival line) (Stephan, 1951; McClure, 1952) and on the smooth lingual surfaces of the maxillary second molars of ham- sters (Sognnaes, 1948). A condition of periodontal disease and root surface cavi- tation (cementum caries) has been observed in the Syrian hamster and is associated with the gingival accumulation of food debris (King, 1950). K6nig (1965) has observed that early interproximal carious lesions in Sprague-Dawley rats start from the tip of the cusp whereas those lesions in Osborne-Mendel rats originate from the depth of the fissure similar to the pattern of attack seen in human lesions. With respect to distribution, the location of the proximal caries between the two strains of rats was similar. Carious Lesion in the Enamel The carious enamel lesion consists of demineralization with loss of calcified external dental tissue, obliteration of dentinal canals, and finally bacterial invasion of the underlying dentine and pulp structure. Experimental caries limited to enamel surfaces permits identification of zones of demineralization while avoiding the complications of proteolytic changes and bacterial invasion with loss of structure. Experimental carious lesions found on bucco-lingual, proximal, and occlusal surfaces correspond in several respects to common types of lesions found in molar teeth of humans (Johansen, 1963; Darling, 1963; Stepehan and Harris, 1955). The process of carious attack is a selective demineraliza- tion of certain preformed structural patterns within the dental hard tissues. Early dental caries are characterized by deep sub- surface demineralization (up to 1000 microns). This pattern may extend horizontally involving only the enamel surface area or may penetrate along enamel prisms or rods extending to the dentine- enamel junction (Sognnaes, 1963). These enamel prisms are composed of apatite crystals in a hydrated organic matrix which is principally protein. In mammalian teeth, the particular apatite present in the enamel is the calcium phosphate salt hydroxyapatite Ca10(PO4)6(OH)2. In acid media, the hydroxyapatite reacts with lactic acid at a pH of 5.2 to pro- duce tricalcium phosphate, calcium lactate and water. If the acidity goes below pH 5.2, tricalcium phosphate breaks down further to dicalcium phosphate, calcium lactate and water. These calcium phosphate compounds are more soluble than hydroxyapatite and the enamel surface becomes demineralized (Nizel, 1972). Hydroxyapatite is characterized by a specific crystalline configuration. In enamel structure, deve10ping apatite crystals take a spiraling path to the enamel surface forming the structure of the enamel prisms. The process of mineralization in the enamel involves the displacement of water by minerals and as the enamel matures the crystallites become more densely packed. During early caries development, an increase in sub-microscopic spaces within the enamel structure can be seen as opaque areas represent- ing decalcified lesions (Johansen, 1965). Within the body of the demineralized lesion, there is a differential decalcification of certain structures. The pattern of attack involves areas between adjacent enamel prisms; regions in which the crystallite orientation is discontinuous. The individual prisms then become more completely involved, often leaving rings of unaffected enamel, which correspond in size to the prisms (Darling, 1963). 10 A dominant feature of many lesions is demineralization of enamel sections longitudinal to the prisms (the striae of Retzius). These areas are formed due to normal periodic variation in calcifi- cation and represent fronts of incremental or normal growth found at an angle to the direction of enamel prisms. Enamel caries spread mainly along those striae of Retzius which are less calcified than neighboring enamel segments (Darling, 1961). Tooth destruction rapidly occurs once the enamel surface is demineralized. With disintegration of the organic matrix and collapse of the dentine and pulp, loss of tooth surface is seen with evidence of cavity formation (Sognnaes, 1963; Keyes and Jordan, 1963). CARIES-TEST CHALLENGE FOR MULTISURFACE LESIONS Experimental multisurfaCe caries (both sulci and smooth surface lesions) develop under specific dietobacterial conditions in vulnerable teeth (Keyes, 1968). Although a number of factors influence the course of the disease, the active carious process involves accumulation of microorganisms and subsequent acid produc- tion (Burnett and Scherp, 1962; Gibbons, 1964). A microbial and genetic caries-test challenge is essential in order to evaluate the dietary effect on the caries rate of multisurface enamel caries in erupted teeth. Bacterial Factors Bacterial activity is essential to the tooth decay process as shown by Orland gt_gl, (1954) in germ-free studies in the rat. Cariogenic organisms differ with respect to the type of carious lesions they produce (Stephan and Harris, 1955). However, rampant caries are found with organisms that form dental plaque and are also highly acidogenic (Gibbons, 1968). Cariogenic bacteria were first identified in rodents but have also been isolated from man (Zinner and Jablon, 1968). Cer- tain strains of enterococci and streptococci show specific cari- ogenic activity (Orland et_al,, 1955; Fitzgerald §t_al,, 1960). The majority of bacteria known to initiate caries are streptococci 11 12 (Gibbons, 1968). Specific anaerobic strains of bacteria of cari- ogenic importance have been designated as Streptococcus mutans (Clarke, 1924; Edwardsson, 1968; Guggenheim, 1968; and Krasse and Carlson, 1970). These strains of streptococci induce plaque forma- tion and lead to multisurface caries. Lactobacilli are often present in the oral flora but have not been shown to be associated with highly active carious disease (Fitzgerald, 1963). There are very few lactobacilli in the plaque but as many as 70% of the plaque microflora are streptococci (Bibby, 1938). Lactobacilli are aciduric and are metabolically active at pH 5.0 (Snyder, 1965) while streptococci have a terminal pH in glucose broth between 4.0 and 4.5 (Zinner and Jablon, 1968). How- ever, Burnett and Scherp (1968) have reported that many streptococci produce 3 to 6 times as much acid as lactobacilli. Proteolytic properties of microorganisms have not been related to the production of experimental dental caries in animals. Fitzgerald, Jordan and Stanley (1960) produced caries in germ-free rats with a single streptococci strain that was acidogenic but non- proteolytic. Lactobacilli are reported to have little proteolytic activity (Orland, 1959). Gibbons et_al, (1966) have suggested that characteristics of cariogenic microorganisms are the ability to accumulate carbohydrate and to form acid (Gibbons, 1964). In germ-free animals, cariogenic- plaque forming strains of streptococci typically form extracellular polysaccharides of the dextran type whereas non-cariogenic strains 13 produce large amounts of extracellular levans (Fitzgerald and Jordan, 1968). Cariogenic bacteria typically store intracellular poly- saccharide of the glycogen-amylopectin type (Gibbons and Socransky, 1962). Plaque Formation Localized sites of demineralization and cavitation have been ascribed to the activity of the adhering plaque of colonies of microorganisms (Black, 1886; Stephan, 1953). When exposed to sucrose solutions, these adhering plaques of dental bacteria have been observed to accumulate polysaccharide material (Manly, 1961). Dex- tran polymers located outside the bacterial cells within the plaque matrix form insoluble precipitates with proteins (Gibbons and Banghart, 1967) which provide the basis for the adhesive nature of dental plaque and favor initiation of smooth-surface lesions. Significantly more extracellular dextran is formed from sucrose than from equal amounts of fructose, glucose, maltose, lactose (Gibbons gt_al,, 1966) or fructose and glucose combined (Gibbons and Banghart, 1967). A sucrase enzyme system (Koepsell §t_al,, 1953; Hestrin et_al,, 1956) on the cell surface of oral bacteria located within the plaque matrix degrades the sucrose entering the plaque and both 1,6 linked dextrans (Gibbons and Banghart, 1967) and levans (Wood, 1964) are formed. Glycosyl transfer from sucrose is favored due to the high energy yield from hydrolysis (-66OO cal/mole) (Bernfeld, 1963). This extracellular synthesis of dextrans occurs by direct transfer 14 of the glucose unit to a growing polymer chain (Critchley §t_al,, 1967) with the release of free fructose. The high energy in the bond originally between the 2 anomeric carbons of sucrose, C-1 of glucose and C-2 of fructose, is maintained during the polymer syn- thesis (Newbrun, 1967). Extracellular levans are produced in plaque material follow- ing incubation with sucrose and Streptococcus salivarius (Wood, 1964); but not with fructose, glucose, or a mixture of fructose and glucose (Manly and Dain, 1961). Only small amounts of levans are accumulated in the plaque; the fructose molecules are readily soluble and can be lost by diffusion and therefore do not readily promote plaque formation (Gibbons and Banghart, 1967). Levans retained in the plaque may be metabolized by the microflora present to yield acid products; however, acid production has been shown to occur only at a very slow rate from these compounds (Manly, 1961; Wood, 1964). Acid Formation Acid production by cariogenic bacteria is sufficient to account for the decalcification phase of the carious process. Stephan (1940) showed that a pH (pH 5.0 to 5.5) sufficient to ’dissolve enamel was reached in plaque on enamel surfaces within 2 minutes after the mouth was rinsed with a 10% sucrose or glucose solution. Stephan (1944) found that plaque from caries-resistant subjects did accumulate less acid than plaque from caries-active subjects. 15 Acid formation occurs in the oral cavity as a result of microbial fermentation of carybohydrate. Sreebny and co-workers (1950) showed that removal of microorganisms from human saliva by filtration inhibits an increase in titratable acids that is found in whole saliva when these systems are incubated in a glucose solu— tion for 24 hours. Extended exposure of teeth to acid within the plaque is due to the continued production by cariogenic microflora of lactic acid from stored carbohydrate. Gibbons and Socransky (1962) showed that streptococci isolated from plaque of humans accumulated polysac- charide when exposed to glucose and formed lactic acid with loss of polysaccharide when the glucose source was removed. It is this prolonged depression of plaque pH during periods of exogenous carbohydrate depletion due to catabolism of intracellular poly- saccharide by cariogenic bacteria that contributes to a dietary role in the carious process (Stephan, 1940). These stored polysaccharides (amylopectin) are generally believed to be polymers of glucose and to function as energy reserves for the cariogenic bacteria (Gibbons and Socransky, 1962). These polymers resemble the starches of plants and the glycogen of mammals and are cleaved by a-1,4-glucan hydrolase (a-amylase) (Gibbons and Kapsimalis, 1963). Streptococci that are cariogenic are strong amylopectin producers (Fitzgerald and Jordan, 1968). Streptococci do not possess a cytochrome system (do not utilize oxygen) and obtain energy by metabolizing glucose to lactic or other acids (Hartles, 16 1954). However, Cowman et a1. (1974) have reported that aerobic conditions resulted in increased growth of a number of strains of S, mutans due to increased utilization of amino acids. Microbial-Substrate Factors Kite EILEQ: (1950) showed that food in the oral cavity is an essential nutrient source for bacteria associated with caries production. Weanling rats fed a caries-producing ration for 16-25 weeks by a stomach tube failed to develop caries. Compared to con- trol littermates fed the same diet ad libitum, the tube-fed rats showed normal growth and molar structure of teeth but no dental decay whereas control rats had an average of 5 molars that were decayed. B. F. Miller et_al, (1940) showed that dental plaque readily produced lactic acid from glucose, maltose and sucrose but less readily from lactose and starch. The role of dietary carbohydrate in the carious process is largely dependent in multisurface caries upon the availability of the carbohydrate substrate to the cari- ogenic microorganisms lodged in the plaque. Sucrose has a high energy yield (-6600 cal/mole) from hydrolysis and is capable of producing both intracellular and extracellular polymers which contribute to the carious process in the plaque matrix (Newbrun, 1967; Navia, 1970). When the free glucose molecule is the only carbohydrate substrate available to the cariogenic bacteria, stored amylopectin is formed (Gibbons and Socransky, 1962); but in the absence of a high energy bond the extracellular synthesis of dextran does not occur. 17 The fact that sucrose is generally consumed in large quantities in human diets and is readily fermented contributes to the cariogenic activity of this sugar. Haldi gt_al, (1953) observed that the cariogenicity of sucrose fed to rats was greatly reduced by dissolving sugar in water before ingestion. A purified diet free of sucrose was fed to each rat by stomach tube. One group of 13 animals was then fed 9 g of granulated sucrose that was placed in a food cup for oral ingestion; 2 out of 13 rats remained caries free. The second group of 13 rats was provided with measured amounts of 40% sugar solution to equalize the sucrose intake between groups; 7 out of 13 rats remained caries free. Dietary maltose and lactose do not favor establishment of dextran-synthesizing, plaque-forming streptococci. The glycosidic llink in these disaccharides is a hemiacetal between an aldehyde and hydroxyl carbon with a low free energy of hydrolysis; therefore, these compounds do not serve directly as a glycosyl donor in dextran synthesis (Bernfeld, 1963). However, maltose is readily fermented and this carbohydrate may favor acid production in areas where metabolically active microorganisms are already present (areas of plaque or areas of impactation). Large starch molecules do not readily serve as substrate for the carious process since they do not diffuse into the viscous plaque material (Critchley gt_gl,, 1967) and therefore do not come in contact with the surface of the tooth and the metabolically active bacteria. Acid production from starch'hsalso small. Small amounts of maltose may originate from the action of amylase on 18 the starch in the oral cavity; but it is readily neutralized by the saliva. Host Factors Caries occur on vulnerable surfaces of teeth of a susceptible host in the presence of colonies of characteristic groups of bac- teria. In the hamster, extensive coronal caries have been shown when animals were infected with streptococci and fed a fine particle, high sucrose caries-test diet (Fitzgerald and Keyes, 1960). Under the same conditions, marked smooth-surface caries are seen in the NIH Osborne-Mendel rat (Kfinig gt_al,, 1969). The use of a coarse- particle diet has been associated with the development of carious lesions limited to the occlusal surface (Stephan et_al,, 1952). In order to demonstrate that heredity is an important fac- tor in the development of dental caries, Hunt et_al, (1944) studied the extent of caries activity with inbred strains of rats. A high prevalence of carious lesions in the dental sulci were found in the caries-susceptible strain fed a coarse ground rice diet for 35 days (Hoppert et;al,, 1932) while under the same con- ditions the resistant strain remained free of caries. Substitu- tion of fine-ground rice for the coarse ground particles in the diet delayed the onset of molar sulci caries in these rats while differences between the strains remained consistent (Braunschneider et_al,, 1948; Hunt etgalg, 1955). The bacterial flora or salivary secretions in Hunt-Hoppert strains of rats have not been found to differ between caries—resistant and caries-susceptible animals 19 (Jay, 1948; Rosen §t_al,, 1955; Keller et_al:, 1954; Sreebny et_al,, 1956). Therefore, this could not account for the differences in caries susceptibility. Kifer and co-workers (1956) did note that the fissures of caries-resistant rats were narrower than those of the caries-susceptible rats. An important role in host susceptibility to caries "my be dietary-microflora interaction (Larson et_al,, 1967). A coarse particle diet low in sucrose (diet 585 with 25% cane sugar of Stephan et_al,, 1952) showed predominantly sulci caries in Hunt- Hoppert caries-susceptible rats (Larson §t_al,, 1968). This occurred regardless of whether or not the animals were infected with cariogenic microflora (exposure to caries active rats). Using the same experimental conditions, a different pattern of caries development in the Hunt-Hoppert rat was found when a fine particle-high sucrose diet (diet 2000 with 56% confectionary sugar of Keyes and Jordan, 1964) was substituted for the coarse ground- low sucrose ration described above. Uninfected animals again showed predominantly sulci activity with the caries-susceptible rats show- ing twice as many carious areas. However, infected rats showed the same total number of carious areas in both caries-susceptible and caries-resistant strains of rats; but 60% of the carious areas in the caries-susceptible rats were sulci lesions while 67% of the carious areas in the caries-resistant animals represented smooth- surface lesions. Thus, the site of caries development appears to reflect differences inherent in the host-species while total caries development is dependent upon a response of all tooth surfaces to 20 an appropriate cariogenic challenge of both microflora and dietary factors. Differences in caries activity among strains of rats have been attributed to a dietary response involving the pattern and frequency of food intake. Osborne-Mendel rats fed Diet 2000 ad libitum (K6nig‘gt_al,, 1969) had high caries activity correlated with a long, frequent and slow feeding period (average of 213 minutes eating time and 16 meals/day and 0.65 g/intake/minute). Under the same regimen, NIH Black rats had less caries activity and a feeding period that was shorter, less frequent but with a more rapid food intake (98 minutes eating time and 12 meals/day and 0.77 g intake/minute) as compared to the Osborne-Mendel rats. SUMMARY An approach to the study of dental caries prevention or con- trol requires recognition of the multifactorial (bacterial, dietary and host) aspects of the disease. In animal experiments, evaluation of dietary factors that modify the appearance of carious lesions include the composition and pattern of food intake and the residue effect in contact with teeth and the presence of cariogenic bacteria. Fully effective caries control can be achieved under laboratory conditions. This has been shown with tube feeding studies (Kite et_al,, 1950) and with germ-free studies with animals (Orland gt_al., 1954). Sucrose, in particular, has been shown to be cariogenic (Konig, 1968; Pigman, 1970). The pattern and extent of carious lesions reflect the inter- action of diet and microflora in the test animal. Rats fed a coarse cereal diet alone (Hoppert et_al,, 1932) or in combination with 25% cane sugar develop sulci (occlusal crevice) carious lesions with or without infection of cariogenic microorganisms. Diets high in sucrose (56-66%) in the absence of infection of the animals also primarily resulted in sulci lesions (Larson gt_al., 1967; 1968). Carious lesions on the smooth surfaces appear in rats on cariogenic diets containing 56% confectionary sugar (Larson and 21 22 6055, 1967) or 66% granulated sucrose (Larson et_al,, 1967) only in the presence of cariogenic bacteria. Smooth-surface lesions do not occur in rats fed a diet favoring only sulcal caries even in the presence of bacterial flora that favor multisurface lesions (Larson et al., 1967). BIBLIOGRAPHY Agnew, M. C., Agnew, R. G. & Tisdall, F. F. (1932) Experimental oral lesions in rats. J. Dental Res. 12, 449. Arnold, F. A., Jr. (1942) The production of carious lesions in the molar teeth of hamsters (C, auratus). 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(1960) The infectious and transmissible nature of experimental dental caries. Findings and implications. Arch. Oral Biol. 1, 304-320. Keyes, P. H. (1968) Research in dental caries. J. Amer. Dental ASS. 76, 1357—1373. Keyes, P. H. & Jordan, H. V. (1963) Factors influencing the initiation, transmission, and inhibition of dental caries. In: Mechanisms of Hard Tissue Destruction, pp. 261-283 (Sognnaes, R. F., ed.), A.A.A.S., Washington, D.C. 26 Keyes, P. H. & Jordan, H. V. (1964) Periodontal lesions in the Syrian hamster. III. Findings related to an infectious and transmissible component. Arch. Oral Biol. 9, 377-400. Kifer, P. E., Hunt, H. R., Hoppert, C. A. & Witkop, C. J. (1956) A comparison between the widths of the fissures of the lower molars of caries-resistant and caries-susceptible albino rats (Rattus norvegicus). J. Dental Res. 35, 620-629. King, J. D. (1950) Dental cavities in the golden hamster. Brit. MEd. J. 1, 876-877. Kite, O. W., Shaw, J. H. & Sognnaes, R. F. (1950) The prevention of experimental tooth decay by tube-feeding. J. Nutr. 42, 89-103. Kopesell, H. J. Tsuchiya, H. M., Hellman, N. N., Kazenko, A., Hoffman, C. A., Sharpe, E. S. & Jackson, R. W. (1953) Enzymatic synthesis of dextran. J. Biol. Chem. 200, 793-801. KUnig, K. G. (1965) Caries resistance in experimental animals. In: Caries Resistant Teeth, p. 113 (Wolstenholme, G. E. W. & O'Connor, M., ed.), Little, Brown and Co., Boston. Kdnig, K. G. (1968) Diet and caries: Cariogenic factors. Ala. J. Med. Sci. 5, 269-275. K6nig, K. G., Larson, R. H. & Guggenheim, B. (1969) A strain- specific eating pattern as a factor limiting the transmis- sibility of caries activity in rats. Arch. Oral Biol. 14, 91-103. Krasse, B. & Carlsson, J. (1970) Various types of streptococci and experimental caries in hamsters. Arch. Oral Biol. 15, 25-32. Larson, R. H. & Goss, B. J. (1967) Diet as a limiting factor in the transmissibility of caries activity between rats of dif- ferent strains. Arch. Oral Biol. 12, 1085-1093. Larson, R. H., Keyes, P. H. & Goss, B. J. (1968) Development of caries in the Hunt-Hoppert caries-susceptible and caries- resistant rats under different experimental conditions. J. Dental Res. 47, 704-709. Larson, R. H., Theilade, E. & Fitzgerald, R. J. (1967) The interaction of diet and microflora in experimental caries in the rat. Arch. Oral Biol. 12, 663-668. l—ericester, H. M. (1949) Biochemistry of the Teeth. C. V. Mosby Co., St. Louis, 306 pp. 27 Lobene, R. R. & Burnett, G. W. (1954) Studies of the composition of teeth. I. Chemical analysis for the principal inorganic constituents of the enamel and dentin from Syrian hamsters. J. Dental Res. 33, 487-496. Manly, R. S. (1961) Retention of carbohydrate from sugar solutions by salivary sediment (Abstract). J. Dental Res. 40, 3A. Manly, R. S. & Dain, J. A. (1961) Conversion of sucrose to poly- saccharide by oral samples (Abstract). J. Dental Res. 40, 747. Matsuda, Y. (1937) A biochemical study of tooth growth. J. Biol. Chem. 71, 437-444. McClure, F. J. (1952) Dental caries in rats fed a diet conUaining processed cereal foods and a low content of refined sugar. Science 116, 229-231. McClure, F. J., King, J. G., Derr, J. & Wick, A. L. (1966) Major components of the primary and secondary dentitionIrfminature and duroc swine fed normal vs. low phosphorus diets. Arch. Oral Biol. 11, 253-265. McCollum, E. V., Simmonds, N. & Kinney, E. M. (1922) The relation of nutrition to tooth development and tooth preservation. Johns Hopkins Hosp. Bull. 33, 202-215. Miller, B. J. Muntz, J. A. & Bradel, S. (1940) Decomposition of carbohydrate substrates by dental plaque material. J. Dental Res. 19, 473-478. Navia, J. M. (1970) Evaluation of nutritional and dietary factors that modify animal caries. J. Dental Res. 49, 1213-1227. Newbrun, E. (1967) Sucrose, the arch criminal of dental caries. Odontologisk Revy 18, 373-386. Nizel, A. E. (1972) Nutrition in Preventive Dentistry: Science and Practice, W. B. Saunders Co., Philadelphia, 506 pp. Orland, F. J. (1959) A review of dental research using germfree animals. Ann. N.Y. Acad. Sci. 78, 285-289. Orland, F. J., Blayney, J. R., Harrison, R. W., Reyniers, J. A., Trexler, P. C., Ervin, R. E., Gordon, H. A. & Wagner, M. (1955) Experimental caries in germfree rats inoculated with enterococci. J. Amer. Dental Ass. 50, 259-272. 28 Orland, F. J., Blayney, J. R., Harrison, R. W., Reyniers, J. A., Trexler, P. C., Wagner, M., Gordon, H. A. & Luckey, T. D. (1954) Use of the germfree animal technic in the study of experimental dental caries. J. Dental Res. 33, 147-174. Pigman, W. (1970) Carbohydrates, fats, and dental caries. In: Dietary Chemicals vs. Dental Caries, Advances in Chemistry Series No. 94, pp. 7-21 (Gould, R. F., ed.), Amer. Chem. Soc., Washington, D.C. Rosen, S., Benarde, M. A., Hunt, H. R. & Hoppert, C. A. (1955) Microbiological differences in the oral cavities of the Hunt-Hoppert caries-resistant and caries-susceptible rats. J. Dental Res. 34, 113-117. Snyder, M. L. (1965) The bacteriology of caries resistance. In: Caries Resistant Teeth, p. 245 (Wolstenholme, G. E. W. & O'Connor, M., eds.), Little, Brown and Co., Boston. Sognnaes, R. F. (1948) Caries-conducive effect of a purified diet when fed to rodents during tooth development. J. Amer. Dental Ass. 37, 676-692. Sognnaes, R. F. (1955) Effect of ingested sugars and other carbo- hydrates on the resistance of teeth to caries. J. Amer. Dental Ass. 51, 270-284. Sognnaes, R. F. (1963) Dental hard tissue destruction with special reference to idiopathic erosions. In: Mechanisms of Hard Tissue Destruction, pp. 91-153 (Sognnaes, R. F., ed.), A.A.A.S., Washington, D.C. Sreebny, L. M., Kirch, E. R. & Kesel, R. G. (1950) The location of the glycolytic enzymes in saliva. J. Dental Res. 29, 506-511. ‘ Sreebny, L. M., Meyer, J. & Bochem, E. (1956) Morphologic and enzymatic differences in caries-resistant and caries- susceptible albino rats. J. Amer. Dental Ass. 53, 9-13. Stephan, R. M. (1940) Changes in hydrogen-ion concentration on tooth surfaces and in carious lesions. J. Amer. Dental Ass. 27, 718-723. Stephan, R. M. (1944) Intra-oral hydrogen-ion concentrations asso- ciated with dental caries activity. J. Dental Res. 23, 257-266. Stephan, R. M. (1951) The development of caries on the buccal and lingual tooth surfaces of rats as well as proximal and fissure caries (Abstract). J. Dental Res. 30, 484. 29 Stephan, R. M. (1953) The dental plaque in relation to the ’ etiology of caries. Int. Dental J. 4, 180-195. Stephan, R. M., Fitzgerald, R. J., McClure, F. J., Harris, M. R. & Jordan, H. (1952) The comparative effects of penicillin, bacitracin, chloromycetin, aureomycin, and streptomycin on experimental dental caries and on certain oral bacteria in the rat. J. Dental Res. 31, 421-427. Stephan, R. M. & Harris, M. R. (1955) Location of experimental caries on different tooth surfaces in the Norway rat. In: Advances in Experimental Caries Research, pp. 47-65 (Sognnaes, R. F., ed.), A.A.A.S., Washington, D.C. Wood, J. M. (1964) Polysaccharide synthesis and utilization of dental plaque (Abstract). J. Dental Res. 43, 955. Zinner, D. 0. & Jablon, J. M. (1968) Human streptococcal strains in experimental caries. In: Art and Science of Dental Caries Research, pp. 87—109 (Harris, R. 5., ed.), Academic Press, New York. PART II USE OF MONOSACCHARIDES AND A SACCHARIDE SIRUP IN BUTTER-TYPE CAKES 3O INTRODUCTION Some humans seek sweetness in foods far beyond the need for relieving hunger, even to the detriment of health (Cabanac, 1971). Indeed, the sugar and sirup consumption in the U.S. increased from 25 kg per capita in 1899 to 54 kg in 1924 (Antar et_al,, 1964) to 57 kg in 1970 (Agricultural Economic Report, 1972). Although these values based on retail market supplies for sugar consumption have stabilized in excess of 50 kg per year for the past 45 years, the total simple sugars as a percent of total carbohydrate intake per day, has increased from 44% to 52% during the same time period; that is, from 1924 to 1970 while there was a concomitant decrease in starch consumed from grain products. The commercial availability of glucose sirups (Birch et;a1,, 1973) and high fructose corn sirups (Redfern and Hickenbottom, 1972) provide an economical and convenient source of monosaccharides as sweetening agents. Since consumer acceptance reflects sensory and quality characteristics of foods (Rubini, 1974) as well as avail- ability (Yudkin, 1967), changing nutritional intake should be easier if the new food is not excessively changed from the old. Therefore, before advocating dietary modifications in order to decrease the sucrose in the diet, it should be beneficial to first investigate the substitution of monosaccharides for sucrose in foods which contain this ingredient. The purpose of this study 31 32 was to investigate, both subjectively and objectively, quality char- acteristics of butter-type cakes prepared with fructose and/or glucose and to compare these characteristics with those of control cakes made with sucrose. LITERATURE REVIEW Overview: Sugars in Cake Products Functions of Sugar Structurally, the principal effects of sugar in flour mix- tures are to reduce gluten strength, increase tenderness and produce smaller, more uniform cell distribution. These effects on the physical pr0perties of baked products are partially due to the water binding capacity of sugar which in turn delays the hydration of proteins and starch. For this reason if the sugar is too high in relation to the liquid, a sunken center develops in cakes during baking (Kissel and Marshall, 1962). Competition of sugar for water prevents viscosity changes associated with starch gelatinization and leads to failure to develop characteristic cake structure. All sugars absorb moisture to a certain extent. Moisture is held in a batter or dough product as the solvent primarily for sugar. In plain cake formulas, sugar is found dissolved in the liquid phase (Lowe, 1955). Sugars not well dissolved can produce a sugary crust on cakes. Too much sugar can cause a sticky crust and a gummy texture. In a balanced cake formula, the absorptive ability of sugar contributes to the keeping quality of the product. The sweetness of cake is dependent upon the concentration and type of sugar used (Dahlberg and Penczek, 1941) and the effect of reactions occurring during product baking. A high sugar to 33 34 protein ratio contributes to flavor production by nonenzymatic browning reactions involving mainly reducing sugars with amines, amino acids, peptides and proteins (Hodge, 1966). High heat also causes flavor changes as sugars are decomposed and new compounds are formed (Meyer, 1960). Color changes also occur in baked products based on sugar content. Browning due to rearrangement of potential carbonyl groups from reducing sugars is accelerated by high temperature, low moisture content and an alkaline media. Reactions of ingredi- ents result in unsaturated polymers of varying composition which contribute color and flavor changes to the final food product. In general, the types of nonenzymatic browning reactions which occur during baking are caramelization of the sugar moiety and the sugar- amine interaction of the Maillard reaction. Effect of Reducing Sugars In starch—water-sugar systems, monosaccharides, when sub- stituted for disaccharides at the same concentration by weight, do not delay starch gelatinization of the same extent (Bean and Osman, 1959). Miller and Trimbo (1965) reported that replacement of sucrose with monosaccharides eliminated the surface dip in the center of cakes with a high sugar to water ratio (sugar levels of 140% based on the weight of flour and with water reduced to 80% of normal). However, excessive browning of crust and crumb of the glucose-containing cake was noted. Optimum flavor and eating quality was found in white and yellow plain cakes to be at pH 7.0 to 7.9 (Stamberg and Bailey, 35 1939). To minimize excessive browning when sweetening agents con- taining reducing sugars are substituted for sucrose in butter-type cakes, Miller gtgal: (1957) maintained the pH of the cake crumb at approximately 6.3 with the use of a leavening acid. However, accelerated loss of C02 was also found which resulted in decreased cake volume. Use of Liquid Sweeteners Invert sugar sirup, used at a 10% level in baked products, contributes to browning in the crust, fineness of grain, and soft- ness of texture (Anonymous, 1972). Honey, which contains 30% glucose, 30% fructose, and approximately 4% oligosaccharides (Siddiqui, 1970) has been used as an acceptable sweetening agent in cakes for up to 50% of the total sweetening; however, adjustments were made in the added liquid to account for the moisture content of the honey. In addition to water content, the use of honey in baked products makes it necessary to adjust the cake formula to take into account the free-acid content and distinctive flavor (Morgan, 1937). Corn sirup has been recommended at about 25% replacement level for sucrose (Nesetril, 1967). Corn sirup consists primarily of D-glucose, maltose, and d-1,4-linked oligosaccharides of D-glucose (Grenby and Leer, 1974). Corn sirups have been advocated for use in foods; their main advantage for automated mixing is in the reduction of ingredient and processing costs (Keeney, 1962). Although great strides have been made in production and analysis of 36 corn sirups, little is known of their actions in foods (Eopechino and Leeder, 1969). Corn and glucose sirups lack the necessary sweetness for total replacement for sucrose in food products (Red- fern and Hickenbottom, 1972). High fructose corn sirups (HFCS) contain more than 10% fructose (Wardrip, 1971) and are produced by enzymatic conversion of cornstarch to a sirup containing both glucose and fructose. ISOMEROSER 100 Brand High Fructose Corn Syrup (Clinton Corn Proces- sing Co., Clinton, Iowa) is prepared by the enzyme-isomerase and is composed of 42% fructose and 50% glucose on a dry weight basis. R solids, relative sweet- In water solutions containing 15% ISOMEROSE ness compared to sucrose is 100. The product is available commer- cially in a 71% solids solution. Microbiological stability and wholesomeness of the high fructose sirups have been reported to be comparable to invert sucrose sirups. No enzyme activity has been demonstrated in the refined fructose sirup. Furthermore, enzymes derived from strains of Streptomyces (Streptomyces olivochromogenes is used commercially as an enzyme source by CPC International Inc., Argo, Ill.) are nonpathogenic (K001 and Smith, 1972). Fructose-containing sirups have ready application in all foods which already utilize liquid sweeteners such as soft drinks or which require low sugar content such as yeast-leavened baked goods. Replacement levels as a total sweetener have been recom- nended for yellow and chocolate cakes at 40-50% of the sucrose 37 level and for chewy type cookies at 20-30% of the sucrose level (Redfern and Hickenbottom, 1972). Properties of Sugars Solubility In order of solubility, the sugars in pure form are ranked as follows: fructose, sucrose and glucose. At room temperature, about 4 parts of fructose are soluble in 1 part water and about 1 part glucose is soluble in 1 part water on a total weight basis (American Home Economics Association, 1971). This sugar-to-water ratio is basic to a consideration of substitution of monosaccharides into a cake formula. The quantity of solids which can be incorporated into a solution is dependent upon the solubility of the solute. Fructose/ glucose sirups can be prepared with a high concentration of solids since fructose has a very high solubility and glucose does not readily crystallize. Solubility curves of sucrose-invert sugar mixtures show that the percent of invert sugar increases to a peak solubility and then decreases. The proportion of invert sugar to sucrose gradually decreases from 75% at 40°C to about 60% at 10°C (Davis and Prince, 1955). The concentration of solute varies with the temperature of a solution. At 40°C, 70.4 9 sucrose will solubilize in 100 ml water (Handbook of Chemistry and Physics, 1959). A 78.9% fructose solution is present at 20°C (Jackson et_al,, 1926) while a 73.1% glucose solution is formed at approximately 55°C (Jackson and 38 Silsbee,l922). In baked products, the effects of differences in solubility are interdependent upon the hydration capacity of the other ingredients. The absorptive ability of sugar also prevents the drying of food products. Dittmar (1955) showed that sucrose and glucose W absorb less than 1% moisture below 60 to 65% humidity at 25°C whereas fructose and invert sugar hold 20% water in surface solution at 65% humidity. Lowe (1955) reported that products containing fructose do not dry out as rapidly as those made with sucrose. Sweetness Relative sweetness of sugars in water varies with the con- centration of the solution being tested. At recognition threshold levels (the lowest concentration at which sweetness is tasted under controlled conditions), 19 of 20 subjects detected sweetness in solutions containing sucrose, glucose, fructose and an equal mix- ture of fructose and glucose at the following % concentrations, respectively: 1.25, 1.75, 0.75 and 1.0% (Willaman, 1925). At 20% levels the relative sweetness of fructose is 119.8 and glucose is 91.7 when compared to a sucrose standard set at 100% (Dahlberg and Penczek, 1941). Pangborn (1965) has evaluated the intensity of apparent sweetness of aqueous solutions of fructose, glucose, lactose and sucrose in combinatiOn with acetic, lactic, tartaric and citric acids. Results showed that citric and acetic acids from among those used had the greatest sweetness depressing effect. Among the sugars, 39 the apparent sweetness of fructose decreased the most by acid and that of sucrose the least. This report emphasizes the need for further study before direct application can be made from simple *aqueous systems to more complex food systems. Sugar Caramelization Caramelization refers to the series of reactions that occur when sugars are heated to melting. The sugar decomposes, water is given off, and mixtures of aldehydes and ketones leading to brown colored compounds are formed. This reaction is accelerated in the presence of moisture and alkali. Products containing fructose and glucose are especially subject to caramelization changes. The melting point of fructose is approximately 104°C while sucrose melts at 186°C. Anhydrous glucose has a melting point at 146°C while that of the hydrate form nelts at 86°C (Handbook of Chemistry and Physics, 1973). Heating solutions of the common sugars (glucose, fructose, maltose and sucrose) yields caramels which are grossly similar (Ramaiah et_al,, 1957a,b). Greenwood et_al, (1961) and Bryce and Greenwood (1963) found that the 300°C pyrolysis volatiles from sucrose, maltose, glucose and starch as determined by gas chroma- tography, are identical. Hodge (1966) lists carbohydrate caramelization and dehydration products giving rise to bitter flavors as lactic aldehyde, furfuryl alcohol, 5-(hydroxymethyl)- 2-fura1dehyde and maltol. 4O Sugar-Amine Interactions of the Maillard Reaction Reducing sugars also undergo Maillard-type browning. In this case the unsaturated carbonyl compounds responsible for brown- ing are formed mainly as the result of sugar-amine interactions. Condensation between the carbonyl group of a reducing sugar and the free amino group of a protein to form a N-substituted glycosyl amine is an early step in the reaction. This carbonyl-amine con- densation is not dependent upon the presence of oxygen (Hodge, 1953). At moderate temperatures, this first stage of the reaction series is slow, incomplete, reversible to some extent and yields colorless products (Lea, 1950). During later stages of the reaction, rearrangement of the carbonyl-amino compound can occur with splitting off of the labile amine moiety and with dehydration and polymeriza- tion of the sugar moiety (Hodge, 1953). Under mild conditions (50°C, pH 5.5-6.0, low sugar:N ratio), Burton and McWeeny (1964) report that most of the browning occurs due to production of unsaturated carbonyl decomposition products followed by rapid reaction of these compounds with amino groups. The extent of browning is increased with long periods of heating (Burton et_al,, 1963b) and with increasing temperature (Barnes and Kaufman, 1947). Color changes during heating.--A key reaction for early color changes is the Amadori rearrangement of the condensation Product, an aldosylamine, to form a ketoseamine. This keto 41 intermediary compound leads to fragmentation, dehydration, and further condensation reactions and provides a basis for the auto- catalytic nature of the system (Hodge, 1953). Later browning is reported to be dependent upon the degree of unsaturation and the reactivity of the intermediates formed (Burton gt;al,, 1963b). Kato (1960) has suggested that in systems containing aldoses and amines the main cause of browning may be due to reactions between the amine and 2,3-enol compounds. Burton et_gl, (1963a) reported that saturated aldehydes and saturated and unsaturated a,B-ketones do not readily brown with glycine while the later appearance of highly colored products is accelerated with the use of o,B-unsaturated straight chain aldehydes. As compared to pH 4, the rate of browning at pH 6 has been shown by Barnes and Kaufman (1947) to double in simple glucose and glycine mixtures. Lea (1950) reported a rise in pH increased the rate of Maillard browning reaction of aldoses and amines approxi- mately linearly between pH 3 and pH 8. However, Cole (1967) found that this expected pH effect on reaction rate was not always seen between pH 6 and 7. In the presence of alkali, reducing sugars readily form 1,2-enol compounds (Reynolds, 1965). In nearly anhydrous condi- tions or in the presence of amines in high concentration, browning due to the formation of reductones is favored (Hodge, 1953); while at moisture contents of 20% of greater and with temperatures above 43°C, oxidative browning occurs (Reynolds, 1965). 42 Within acidic aqueous systems, color changes have been pri- marily attributed to dehydration of the sugar moiety and production of furfural compounds (Hodge, 1953). Reynolds (1965) reports that organic acids play a catalytic role in promoting decomposition of sugar molecules in mixtures of organic acids, sugars and amino acids. In these systems, the rates of formation of.ketoseamine and color changes increase with increasing solids content, and are still further increased in the presence of organic acids. Flavor changes.--Characteristic flavor changes develop as a result of the Strecker degradation of the d-amino acid to yield the aldehyde with one carbon less. This degradation occurs as a sec- ondary effect of the reaction between aldoses and amines and produces carbon dioxide, carbonyl compounds and free amines. The products are formed as a result of the reaction between amino acids and dicarbonyl compounds. Furthermore, these reactions occur through- out a wide range of pH and temperature conditions (Hodge, 1953; Reynolds, 1965). More than 80% of the carbon dioxide evolved from the reac- tion between aldoses and glycine has been reported to originate from the carboxyl group of glycine (Reynolds, 1965). However, Cole (1967) has shown that the limiting factor in the rate of carbon dioxide production is the type of sugar rather than the amino acid or pH. Barnes and Kaufman (1947) reacted single amino acid-sugar Pairs and found that development of flavor in this simple system 43 was specifically dependent upon the amino acid used. Herz and Shallenberger (1960) reported that, in general, reactions between glucose and 12 different amino acids led to a characteristic bitter reaction product as judged by a 6 member flavor panel. Exceptions were the development of an acid-bitter flavor when histidine was used and a sweet flavor when alanine was used. Polymer formation.--Fina1 stages of the Maillard browning system are marked by condensation of products formed as a result of the reaction between sugar and amino acids which yield brown polymers and co-polymers known as melanoidins (Hodge, 1953). The presence of brown color, formation of conjugated unsaturated carbonyl compounds, and production of carbon dioxide in sugar-amino acid systems are closely related chemically (Cole, 1967). Rooney et_al, (1967) found that the type of sugar used affected the rate and quantity of carbonyl produced while the type of amino acid used mainly affected the kind of aldehyde produced. Relative Browning Rates The major mechanism responsible for the chemical alteration of sugars during baking is the Maillard browning reaction since these sugar-amine reactions are autocatalytic and have a lower energy of activation than caramelization reactions (Kinsella, 1971). The rate of early browning associated with the use of carbohydrates increased in the following order: disaccharide, hexose and pentose (Lea, 1950). 44 Casey (1965) showed that the oxidant effect of fructose was twice as fast as that of glucose. Fructose, when reacted with 5 amino acids under controlled conditions (pH 6.5 for 30 minutes at 110°C), gave higher initial rates of production of low boiling volatiles than did glucose. Burton gt_al, (1963b) found that when fructose was reacted with glycine the rate of carbonyl development was initially rapid and the rate of increase of browning was approximately twice that seen in glucose-glycine systems. Others (Lea, 1950; Reynolds, 1965) have noted that while initial rates of reaction between amino acids and fructose are rapid, the extent of later browning may proceed more slowly with fructose than with glucose in buffered systems such as foods. Burton et_al, (1963b) also noted that the color density of glucose reacted with glycine did exceed the browning of fructose-glycine systems in later stages of browning. MATERIALS AND METHODS Butter—type cakes were prepared using 5 kinds of sweeteners. These sweeteners included (1) sucrose (control cake), (2) fructose, (3) glucose, (4) high fructose corn sirup and (5) fructose and glu- cose combined. The 4 latter sweeteners were substituted for the sucrose in the cake formula. Ingredients (Table 1) were combined by the solution method of mixing (Stateler, 1950) to permit adaptation of the recipe for use with saccharide sirups available on the market. The cake formula was scaled to produce 3 cakes at each mixing to provide sufficient samples for each evaluation. Each of the 5 vari- eties of cake was prepared in triplicate. Preparation Ingredients Sugars tested included a representative commercial high fructose corn sirup (HFCS) product1 as well as crystalline fructose and glucose alone and in a 1:1 ratio (fructose/glucose). Cakes made from sucrose produced a standard-type product and served as a basis against which the other products were evaluated. 1ISOMEROSER 100 Brand High Fructose Corn Syrup (HFCS) was generou51y provided by the Clinton Corn Processing Co., Clinton, ICN~a. This commercially available product contains 71% solids and IS composed of 35.5% glucose, 29.8% fructose and 5.7% other saccharides. 45 46 TABLE 1.--Cake formula for one replication.a Ingredients Amount ngcgqgfige Shortening, vegetable 195.0 g 43.3 Sugar,C superfine quality 473.0 9 105.0 Vanilla 5.0 ml 1.2 Eggs 225.0 g 50.0 Flour, cake 450.0 g (b) Baking powder, SAS-phosphate type 21.0 g 4.6 Salt 7.5 g 1.7 Milk solids, whole 60.0 g Water, distilledc 446.0 g 112.5d aBatter for three 8-inch layers. sensory and objective measurement S. These were used for bWeight of ingredients compared to weight of flour taken as equal to 100%. CFor cakes prepared with HFCS, 668 of sirup and 251 g of water were used as compared to sugar (473 g and water (446 g). dPercentage based on combined weight of milk solids and water. 47 Substitution of the simple sugars for sucrose in the cake fermula was based on an equal weight basis. The cakes were all prepared using the same proportion of ingredients with substitution of the sweeteners based on percent solids for the sirup product. Since the HFCS contained 71% solids and 29% water, each dry sugar was made up as a solution with distilled water and used in that form. This represented 45% of the water required for each cake formula. Although the relative sweetness of sugars varies with their concentration and interaction with other ingredients (Dahl- berg and Penczek, 1941), preliminary investigations demonstrated that this was the most fundamental way to modify recipes for the incorporation of the simple sugars in order to produce acceptable butter-type cakes. All ingredients except eggs were each taken from a common lot and each was weighed to the nearest gram. Sugars as well as cake flour, double-acting baking powder, and salt were weighed prior to baking and stored at room temperature. Just prior to mixing, hydrogenated vegetable shortening, fresh eggs, whole milk powder and distilled water were weighed. Sugar solutions were also prepared at this time. The whole milk powder was then combined with the previously weighed dry ingredients. Procedure The sugar solution was placed in a 5-quart bowl and blended with the fat and flour mixture using the whip attachment of a Kitchen Aid mixer, Model K5-A, set at speed 1 (56 rpm) for 2 minutes 48 and speed 2 (92 rpm) for 2 minutes. After scraping the bowl and beater, the eggs, remaining water and vanilla were added and mixed at speed 2 (92 rpm) for 1 minute and speed 4 (132 rpm) for 1 minute. For each of the 3 replications, 400 g of batter were placed in each of three 8-inch aluminum layer-cake pans prepared by cover- ing the oiled pan with oiled waxed paper. All cakes comprising a replication were baked simultaneously in a Hotpoint oven, Model HJ225, set and maintained at l77i2°C with a Versatronic controller. For each of the 3 replications of each kind of cake, cakes designated for Specific objective measurements and sensory evaluations were baked in a designated spot in the oven according to a preplanned schedule to negate any effects of oven position. Following baking for 28 minutes, cakes were cooled on wire racks for 15 minutes before removing from the pans and then cooled for 1 hour after removal from the pans. The cakes were then wrapped in plastic food wrap, placed on cardboard trays, labeled, placed in polyethylene bags and frozen at -23°C for later evaluation. This took place at a time more convenient for evaluation and was 2 weeks following baking. Evaluation Objective Measurements The Beckman Zeromatic pH meter was used to determine the pH of all cake batters as well as cake slurries. The latter was pre- pared from 1 g of baked cake and 60 m1 of distilled water at pH 6.8. Using the parameters described by Funk, Conklin and Zabik (1970) and 49 techniques outlined by Matthews and Dawson (1966), batter viscosity was determined with a Brookfield viscometer. Moisture loss during baking was calculated from the before- and after-baking weight with the latter determined following the first 15 minutes of cooling. Total moisture content was also determined by drying a 2 9 cake sample to constant weight in a vacuum oven set at 70°C (Association Official Agricultural Chemists, 1965). Relative cake volume index based on the height of a center slice of cake, tenderness as determined by the Allo-Kramer shear press, and measurements of crumb color using a Hunter Color Differ- ence Meter have been detailed by Funk gt_al, (1970). To follow browning changes caused by reducing sugars, color readings were made on the interior surface 3/4-inch from the bottom of the cake. Sensory Evaluations Cakes were scored for grain, crumb color, silkiness, tenderness, flavor of crust, and flavor of crumb using a 5-point scale as shown in Table 2. Ratings of 3 and 2 represented inter— mediate scores for those described in 4 and 1. Instructions explaining the score-card and descriptive terms used were available at each testing session (Appendix A). The taste panel consisted of 11 departmental faculty and graduate students. No Specific training was provided. 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Component Percentage Proteina 12.9 Fatb 14.2 Carbohydrate (32.5% sugar)C 66.6 Kilocalories/gd 4.35-4.42 aDried cake (70 g) supplied 1.96 g protein from cake flour, 0.9 g protein from whole milk solids, 1.68 g protein from egg; skim milk powder (15 g) supplied 5.3 g protein; dried yeast (4.0 g) supplied 1.9 g protein; alfalfa meal (3.0 g) supplied 0.5 9 pro- tein; and liver powder (1.0 g) supplied 0.7 g protein. (Agriculture Reseagch Service, Handbook No. 8, 1963; National Academy of Sciences, 1972. bDried cake (70 g) supplied 11.3 g fat from Crisco, 0.21 g fat from cake flour, 0.9 g fat from whole milk solids, 1.5 g fat from eggs; dried yeast (4.0 g) supplied 0.04 g fat; and liver pow- der (1.0 g) supplied 0.16 g fat. (Values obtained from Agriculture Reseagch Service, Handbook No. 8, 1963; National Academy of Sciences, 1972. cDried cake (70 g) supplied 27.5 g carbohydrate from sugar, 20.8 g carbohydrate from cake flour, 1.3 g carbohydrate from whole milk solids, 0.1 g carbohydrate from 699. 0.36 g carbohydrate from baking powder; added sugar (5 g) supplied 5 g carbohydrate; skim milk powder (15 g) supplied 7.7 g carbohydrate; dried yeast (4.0 g) supplied 1.6 g carbohydrate; alfalfa meal (3.0 g) supplied (2.1 g) carbo- hydrate; and liver powder (1.0 g) supplied 0.14 g carbohydrate. (Values obtained from Agriculture Research Service, Handbook No. 8, 1963; National Academy of Sciences, 1972.) dValues used for calculating kcal were 9, 4 and 4 for l g of protein, complex carbohydrate and fat, respectively. The energy value of monosaccharide is calculated as 3.68 kcal/g and sucrose is calculated as 3.87 kcal/g (Merrill and Watt, 1955). 83 and 29.6 for the rations-containing cake made with the mono- saccharides. The rats were housed by littermate-pairs in metal screen suspended cages. The environment was controlled with 12 hours of light of each 24 with a temperature maintained at 23i1°C. Diets and distilled water were available ag_libitum for a period of 15 weeks. Every week from age 20-111 days, rats were weighed and food and water intakes recorded. Food in porcelain cups was weighed and placed in each cage for each pair of rats. At the end of the week, the record of weekly food intake was determined by subtracting the weight of the cup and remaining contents from the original weight of the filled food cup. Values for food consumption were adjusted I for spillage by adding the spilled ration collected on a paper towel under each cage to the cup weight at the end of the week. Fluid intake was determined by weighing cylindrical glass water bottles equipped with ball—tipped drinking spouts when filled and subtracting from this weight the weight of bottle and spout at the end of a week. Minimum leakage was measured in 2 sample bottles over a period of a week. Leakage per day consisted of 0.2% (1.4 g/100 g water/7 days). At 116 days of age, the rats were sacrificed by decapita- tion. Food cups were removed at 7:45 A.M. Sacrificing occurred between 8:30 and 10:00 A.M.; animals were sacrificed on a random schedule following the same order used in assignment to diets. 84 Infection of Rats Rat litters were weaned at 18 days of age and were fed a grain diet (Schemmel gt_al,, 1972) and distilled water ag_libitum for 2 days. The distilled water contained sodium penicillin2 (0.5 g/100 ml) and was prepared fresh daily. Rats were weaned at an early age to permit maximum cariogenic challenge to tooth surfaces. Penicillin was used to reduce the normal oral flora in the rat. 0n the 20th day of life, each animal was weighed and those littermates weighing between 35-40 g were housed by pairs and the experimental ration assigned. Fresh drinking water was provided which consisted of distilled water without the added penicillin. This day without antibiotic was scheduled to permit loss of the penicillin from the gastrointestinal tract before infection of the animals was begun. Infection was carried out using a pure culture of Strepto- coccus mutans 6715-153 on animals of age 21 through 25 days. A drop (0.1 ml) of the 30-hour working culture (Appendix C) was placed in the mouth of each rat using a 1 ml syringe without needle on the let, 23rd and 25th day of age. Throughout this time period of days 21-25, drinking water was prepared fresh daily by adding the pure culture of S. mutans to distilled water (1/100 ml). The 2Penicillin-G (Benzylpenicillin) sodium salt, 25 million units, was obtained from Sigma Chemical Company, St. Louis, Missouri. 3Cariogenic streptococci cultures and instructions for maintenance were generously provided by Dr. Rachel H. Larson; Chief, Preventive Methods Development Section; Caries Prevention & Research Branch; NIDH. 85 remainder of the experimental period, the animals were provided with one of the experimental cake rations and distilled water ag_libitum (26-116 days of age). The animals were exposed to the total cariogenic challenge (diet plus infection) for 95 days (day 21-116). Preliminary experi- ments showed that 95 days was sufficient to permit accurate identi- fication of decalcified areas within each cake ration. Accurate evaluation of differences in caries activity is based on the number of individual lesions and the extent of each carious lesion. Within this time period, each experimental group of animals showed lesions large enough to be detected yet no group had decayed areas so exten- sive as to prevent identification of individual lesions. . . . 4 Scor1ng_Car1ous Les1ons Preparation of Jaws Animals were sacrificed by decapitation. The fur and skin were removed with the use of surgical scissors and then the heads were autoclaved at 121-123°C for 5 minutes to facilitate removal of soft tissue. Following these procedures, the skulls were then fur- ther cleaned by placing them in a 2% solution of ammonium hydroxide for 30 minutes. The jaws were then rinsed, dried and stored until scored. 4Dr. Rachel H. Larson, NIDH, generously provided the pro- cedures and training for dental scoring and verified the scores used in the present study. 86 Values for Linear Area of Decay Three molarsin each jaw quadrant were scored for caries by the method of Keyes (1958) to identify the location and the quanti- tative size of the lesions. Lesions evaluated were located at l of the following 4 sites on the tooth: buccal, lingual, proximal and sulci. I The incidence and extent of smooth-surface caries in unstained jaws were determined by examining moist jaws using a Bausch and Lomb dissecting microscope under low power magnification (20 X) while the teeth were rapidly dried under a stream of com- pressed air. The decalcified enamel dehydrates more rapidly than the sound enamel and appears as chalky white areas which can then be identified. The method of assigning values on the buccal or lingual surface involved judging by eye the extent of decay of enamel using an established (Keyes, 1958) linear scale of enamel units based on the size of the molar (the maximum number of buccal plus lingual linear units in the lower jaw quadrant is equal to 28). At proxi- mal sites, each surface is assigned a linear value of 1 unit (the maximum number of linear units assigned to proximal lesions for the 3 molars per jaw quadrant is equal to 4). Sulci lesions were evaluated after dyeing the jaws in a saturated solution of Kernechtrot B salt5 for 17 hours. The wet jaws were then held with a small straight hemostat beneath a slow 5Kernechtrot nuclear fast red dye distributed by Roboz Surgical Instrument Co., 810 18th Street, N.W., Washington, D.C.. 20006. 87 drip of water under magnification of 20 X. The molars of each jaw quadrant were sliced6 into equal halves at an angle parallel to the bucco-lingual surfaces. The method of assigning values for decalci- fied enamel on these exposed sulcal surfaces was based on the number of linear surfaces showing dye penetration. Maximum values for total linear sulci area in the lower jaw quadrant is equal to 14. Analyses of Data Means and standard deviations were determined for the groups of 10 rats on each of the 5 cake diets in the study. Extent of enamel caries for smooth surfaces (buccal plus lingual plus proximal) and for sulcal surfaces were evaluated for each rat; the summation of these linear values then represented the total linear area of enamel decay for each rat. Analysis of variance was performed by the 3600 Controlled Data Corporation (CDC) computer, Michigan State University. Significance of differences between mean values for caries was assessed by Dunnett's test (Dunnett, 1955) using sucrose as a control group. The Student's paired t-test (Sokal and Rohlf, 1969) was used for analyses of mean values for growth, intake and wet tis- sue weights. Correlation coefficients were determined between each caries location (smooth surfaces, sulci surfaces and total surfaces) and for cumulative l3-week food intake, fluid intake and weight gain for rats fed each of the cake rations. 6A steeldisc saw, 0.004 inch thickness and 0.75 inch in diameter was mounted on a mandrel held in a straight handpiece of a standard dental engine. RESULTS Body Weights Animals fed each of the 5 cake-containing rations increased in weight as they grew older (Figure 1). Mean live body weights at 111 days of age and body weight gains from 20 to 111 days of age are presented in Table 8. Rats fed the S ration gained a mean weight of 343:34 g and attained a final mean weight of 379:36 9 while rats fed diets which included fructose (FG, HFCS, and F alone) gained a mean range of 305i18 to 316:39 g and attained a final mean weight for the 3 groups of 345130 9, but differences were not significant. Diet and Fluid Consumption Rats fed the S cake diet consumed a mean of 109i9 g of diet per week over the 13-week feeding period from age 20 to 111 days (Table 8). Glucose and FG fed animals ate a mean of 114i9 and 111:5 g of diet per week, respectively, while rats fed F and HCS ate a mean of 105114 and 103:9 g diet per week, respectively. There were no significant differences in mean food intake among the dif- ferent dietary treatments. The mean intake of distilled water per week by rats fed either G, S, FG, F or HFCS cake rations was 265:58, 228:28, 220:11, 206:37 and 205:23 g, respectively (Table 8). The fluid intake of rats fed F and HFCS cake diets was significantly (P<<0.05) lower than the intake of rats fed G diets. 88 89 o Sucrose I Glucose IJFG o HFCS 400 «x- Fructose / l// ./ 300 //’ID / o E / ‘ 3% o p /-~a': ' 200. -” 1%» / no 3 / £3 100 "I. 3 7 10 (20 days) Week of Observation Figure 1.--Mean growth rates of male Osborne-Mendel rats fed diets containing 70% dried cake made from 1 of 5 sweeteners for 13 weeks. 13 90 .Amwm— .AAcom oco onomv Aumounu ooAAAa owoAAIozA A.AcoozumV w soA ooncA voAA AoA zoos AAA op ooAAoEoo AA some AA A new A AoA momma mxopcA oAzAA coozpon oocvoAAAo Amo.o VAV AcooAAAcmAA AAAAoAAmAAAAA A AA vocAo .AAvo co Axooz mAV mum Ao Azoo AAA\AAA\AAAAA> com: a .aoosm\mpoc oA mew; meosAo 6A A A; AA A ANN AN A AAN AA A AAN AA A AAN AN A ANN 3853 9.35 AA: AA A... AAAAA AAAAA AAAAAA AAAAA AAAAA E33 335 AS“. .A. A. AN A AAA AA A AAA AA A AAA AA A AAA AA A NAA Ate: A85 8.35 88 A.,. AA AAAA ANAAAA AAAAAA AAAANA AAAAAA AAV 338 .2 AN A AAA AA A AAA AA A AAA AA A AAA AA A AA AAV AAAAAA AAAA AA A A A A wmwmewmmwm \wmmwwhwm mom: omopootm omoovo Amocoom AAvoEAAAA AcoAAom oxoo ooA AAAA Amoco21oceoomo Ao gpzoA m .Axooz mA AoA Avo oxoo chcAAAcoouooAeogooomocoe Ao -voAoom A AAAAAA m :A AoAAAoco AAoAAAAAAAA oco AcoAAAA>oo oAAocopA .Acooz--.m AAqu 91 Dental Caries Incidence Means, standard deviations and statistical analyses of dental caries incidence are presented in Table 9. From among all treatments, rats fed the S-containing cake ration showed the greatest mean cariogenic effect in terms of the number of carious teeth per animal and the greatest loss of enamel surfaces. The mean number of carious teeth involved was 7.3:4.2. That is, on the average, in any one rat, 7 out of the 12 teeth scored were decayed. However, there was a wide range with one rat free of decay while 5 animals had 8 or more teeth showing some enamel decay; this wide range is reflected in the standard deviation of 4.2. Mean total enamel decay per animal was 20.0:14.8 linear units. A linear unit represents extent of decay out of a maximum arbitrary measure of total surface area of 172 linear units (Keyes, 1958). On enamel smooth surfaces alone, S fed rats showed 10.6t8.9 mean linear units of decalcified enamel from a possible area of 124linear units. An approximate equal area of decay (9.4:7.9 linear units) was located in the sulci. Rats fed G and FG cake rations had a mean of 4.5:2.3 and 4.1:2.3 carious teeth per animal, respectively. One hundred percent of the rats fed the G cake ration showed some carious enamel. For these rats, the mean total carious linear area was 8.8i5.4. This decay was approximately equally divided between smooth-surface areas (4.5:4.6 linear units) and sulci areas (4.3i5.4 linear units). Also, 100% of the rats fed FG rations showed 1 or more of the 12 molar teeth decayed. The mean linear area of total enamel involved .Ao.oAvAA moo v am AA AA A o A .AAAAA .AAAAAAAV A.A.AAA A +Ifl mews: m- 5 Ammo. 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