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INFORMATION TO USERS This material was producad from a microfilm copy of the original document. While the moit advanced technological meant to photograph and reproduce thii document have been used, the quality it heavily dependent upon the quality of the original submitted. The following explanation of techniques is provided to help ou understand markings or patterns which may appear on this reproduction. 1. The sign or "target" for pages apparently lacking from the document photographed is "Missing Page(s)". If it was possible to ob tain the mining page(s) or section, they are spliced into the film along w ith: adjacent pages, This may have necessitated cutting thru an image and dupli eating adjacent pages to insure you complete continuity. 2. When an image on the film is obliterated with a large round black mark, it is an indication that the photographer suspected that the copy may have moved during exposure and thus cause a blurred image, fou will find a good image of the page in the adjacent frame. 3. When a map, drawing or chart, etc., was part of the material being photographed the photographer followed a definite method in "sectioning” the material. It is customary to begin photoi ig at the upper left hand comer of a large dieet and to continue photoi ig from left to right in equal sections with a small overlap. If necessary, sectioning is continued agein — beginning below the first row and continuing on until complete. 4. The majority of users indicate that the textual content is of greatest value, however, a somewhat higher quality reproduction could be made from "photographs" if essential to the understanding of the dissertation. Silver prints of "photographs" may be ordered at additional charge by writing the Order Department, giving the catalog number, titls, author and specific pages you wish reproduced. 5. PLEASE NOTE: Some pages may have indistinct print. Filmed received. Xorox Univerelty Microfilms S00 North Zoot>Road Ann Aibor, MicNgan 48106 KEHAGIAS, Christos Haralapos* 1945- COMMERCIAL CASEIN AS A SOURCE OF EDIBLE SIALIC ACID AND A GROWTH PROMOTING FACTOR FOR LACTOBACILLUS BIFIDUS VARIANT PENNSVCVWnttJS'. The Ohio State U niversity* Ph.D., 1976 Food Technology Xerox University Microfilms # Ann Arbor, Michigan 4B106 © 1976 CHRISTOS HARALAPOS KEHAGIAS ALL RIGHTS RESERVED COMMERCIAL CASEIN AS A SOURCE OF EDIBLE SIALIC ACID AND A GROWTH PROMOTING FACTOR FOR LACTOBACILLUS BIFIDUS VARIANT PENNSYLVANICUS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of Tho Ohio State University By Christos II. Kehagias, B.Sc. ***** Tho Ohio State University 1976 Reading Committee: Approved By P .M .T , IlnnBcn E.M. Micolojcik T. Krlstoffersen Adviser Department of Food Science and Nutrition This investigation was supported in part by Public Health Service Research G rants, Numbers FD-4G2 and FD-117 from tho Food and Drug Ad­ ministration, Washington, D. C,, and by IIATCIi funds. Dedicated to My Family ACKNOWLEDGMENTS I would like to express my sincere appreciation to Dr. P. M. T, Hansen for his patience and encouragement throughout my graduate program, for his enthusiasm and guidance in the preparation of this dissertation. It has been an exciting experience for me to do research under his guidance. Dr. Hansen's affectionate concern for the students will always be remembered and will serve as a guide to mo in the fuluro. 1 would also liko to thank the Ilanscn family for their hospitality during my stay at Ohio State. Special appreciation is expressed to Dr. E. M. Mikolajcik and Dr, Y. C. Jao for their guidance with respect to microbiological studies. I am greatly indebted to Dr. T. Kristoffcrsen, Chairman, and to the Department of Food Science and Nutrition for giving mo the opportunity as a graduate research nssociato. iv VITA April 4, 1945 Born, Pteleos, Volos, Greece 1968 Diploma in Agriculture, Aristotelian Univer­ sity of Thessaloniki, Greece 1971-1972 (a) Certificate in Dairy Technology, Agri­ culture College, Wageningcn, Netherlands (b) lies ear ch with the Nether I and Institute for Dairy Research (NIZO), Edc, Netherlands 1972-1973 Employment by the Ministry of Agriculture, Research Section - Dairy Science Research Station, DIabata, Thessaloniki, Greece 1973 -1970 Graduate Research Associate, Department of Food Scicnco and Nutrition, The Ohio State University, Columbus, Ohio 1970 Ph.D., Tho Ohio Stato University, Columbus, Ohio PUBLICATIONS Kelmgins, C. and Radema, 1., 1973: "Storage of Buttor Oil Under Various Conditions," Ncih. Milk and Dairy J . , 27:379. Kehagias, C. and Hansen, P. M. T ., 1975: "Properties of Casein After Limited Acid Hydrolysis," J. Dairy Soi. , 58;798. (Presented at the American Dairy Science Assoc, Meeting, Juno 1975.) V FIELDS OF STUDY Major Field: Food Science and Nutrition Studios in Food Science; Professors J. L. Blaisdell, P. M, T. Hansen, W.J. Harper, R. V. Joscphson, T. Kristoffersen, and E.M. Mikol- ajcik Studies In Biochemistry: Professors E. L. Gross and J, F. Snell Studies in Chemistry: Professors D. Horton, M. H. Klapper, and Q. Vnn Winkle Studies in Meat Chemistry: Professor II, W. Ockerman Studies in Microbiology: Professors A.M. Ackormann and G. J. Bamvnrt Studies in Nutrition: Professor V.M. Vivian Studies in Statistics: Professor R. V, Skavaril vi TABLE OF CONTENTS Page Dedication Hi Acknowledgments iv Vita v Fields of Study vi Tablo of Contents vli List of Tables x List of Figures xi I. INTRODUCTION 1 II. LITERATURE REVIEW 3 A. Sialic Acids 3 1. Sialic Acids in Milk 5 a. Free (Dialy stable) Sialic Acids 6 b. Colloidal (Non-Dialystable) Forms 9 2. Isolation 13 a. Sinlyllnctoso (NANA-Lac) 14 b. Sialic Acids IS 3. Stability Toward Acid and Heat 17 4. Identification 20 5. Biosynthesis 23 6. Significance of Sinlic Acids 28 a. In Various Glycoproteins 28 b. In Casein 33 7. Nutritional Aspects 34 vii Page B. Growth Promoting Factors of Lactobacillus bifidus variant Pcnnsylvanicus 36 1. Low Molecular Weight Compounds 36 2. Iligli Molecular Weight Compounds 40 HI. SCOPE OF INVESTIGATION . 42 IV. EXPERIMENTAL PROCEDURES 43 A. Materials 43 B, Methods 43 1. Liberation, Isolation and Purification of Sialic Acid 44 2. Direct Ehrlich Test 46 3. Colorimetric Determination of Sialic Acid 46 4. Paper and Thin-Layer Chromatography (TLC) 48 6* Crystallization of Sialic Acid 50 6. Growth of L. bifidus 51 7. Total Sugar Determination 54 8. Qualitative Test for Fructose 54 0. llexosnmincs 54 10. N-Acetylhoxosamines 54 11. Biuret Tost 54 12. Phosphorus 54 13. Ultra centrifugation-Sedimentation Analysis 55 14. Ultrafiltration 55 15. Urea Starch Gel Electrophoresis 55 16. Rennet Sensitivity 66 17. Calcium Sensitivity 56 18. Solubility Studies 56 19. Viscosity Measurements 57 20. Flavor 57 V. EXPERIMENTAL RESULTS 58 A. Liberation, Isolation and Purification of Sialic Acid 58 from Casein 1. Casoin 61 2. Hydrolyzing Agent 66 3. Neutralizing Agent 66 4. Methods of Purification 69 viii Pago B. Identification and Crystallization of the Sialic Acid 70 1. Paper and Thin-Layer Chromatography 70 2. Crystallization 75 a. From Solution 76 b. By Solvent Evaporation 76 C. Characterization of the Casein Bifidus Factor (CBF) 82 1. Stimulation of L. bifidus by CBF and N-AcGluc 82 2. Stimulation of L. bifidus of Ultrafiltrates from CBF 83 3. Comparison of the Bifidus Activity of CBF and Casein 90 4. Chemical Composition 90 5. Physico-Chemicnl Properties 96 D. Properties of the Residual Sialic Acid-Free Casein 99 1. Yields of Residual Casein 102 2. Urea Starch Gel Electrophoresis 102 3. Sialic Acid and Phosphorus Content 110 4. Rennet and Calcium Sensitivity 111 5. Solubility Studies 112 6. Viscous Properties 121 7. Flavor 121 E. Simplified Production of a Crude Preparation 123 Containing Sialic Acid and Casoin Bifidus Fraction (SA/CBF) VI. DISCUSSION 127 VII. SUMMARY 132 BIBLIOGRAPHY 134 ix LIST OF TABLES Title Page 1 Nomenclature and Structure of Sialic Acids 4 2 Distribution of Sialic Acid in Milk 5 3 Oligosaccharides of Human Milk and Bovine 9 Colostrum Containing Sialic Acid 4 Sugar Content of Casein Fractions 10 5 Carbohydrate Composition of Sugar Fraction 11 6 Structure of the Polysaccharide Part of Cow 12 k -Caseinoglycopeptido 7 Sialic Acid Content of Bovine Whey Proteins 12 8 Ingredients of ATCC Medium 70 53 9 Chemical Composition of CBF 95 10 Effect of Hydrolysis on the Sialic Acid and 110 Phosphorus Contont of Casein 11 Rennet Sensitivity of Intact and Hydrolyzed Caseins 111 12 Viscosities of Hydrolyzed Casein 122 x LIST OF FIGURES Title • Structure of Hexasaccharide Isolated from Human 7 Milk and Containing Two NANA Residues Pyrrole Derivatives Arising from NANA Under the 18 Action of Acid Metabolic Pathways for the Interconversion of Amino 24 Sugars Metabolism and Biosynthesis of NANA 29 Liberation, Isolation and Purification of Sialic Acid 59 from Casein Elution Diagram of Dry Isolcctric Casein Hydrolysate 62 on DOWEX-l, X 8(2 x 23 cm) Column in Acetate Form Elution Diagram of Fresh Isolectrlc Casein Hydrolysate 64 on DOWEX-l, X 8 (2 x 23 cm) Column in Acetate Form Elution Diagram of Formic Acid Hydrolysate on 67 DOWEX-l, X 8 (2 x 23 cm) Column in Formate Form Thin-Layer Chromatography of the Isolated Sialic Acid 71 Paper Chromatography of the Isolated Sialic Aoid 73 Microphotographs of Crystalline Sialic Acids 76 Stimulation of L» bifidus by CBF and N-AcGluc 84 UV Spectrum of FHtor-Sterilizcd and Autoclaved CBF Fractions xi Figure Title Page 14 Growth Effect of Ultrafiltrate of CBF on L. bifidus 88 15 Comparison of Bifidus
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  • LACTULOSE SOLUTION USP - Lactulose Solution Hi-Tech Pharmacal Co., Inc

    LACTULOSE SOLUTION USP - Lactulose Solution Hi-Tech Pharmacal Co., Inc

    LACTULOSE SOLUTION USP - lactulose solution Hi-Tech Pharmacal Co., Inc. DESCRIPTION Lactulose is a synthetic disaccharide in solution form for oral administration. Each 15 mL of lactulose solution contains: 10 g lactulose (and less than 1.6 g galactose, less than 1.2 g lactose, and 1.2 g or less of other sugars). Lactulose solution contains potassium sorbate as an inactive ingredient. Lactulose is a colonic acidifier which promotes laxation. The chemical name for lactulose is 4-O-β-D-galactopyranosyl-D-fructofuranose. It has the following structural formula: The molecular weight is 342.30. It is freely soluble in water. CLINICAL PHARMACOLOGY Lactulose is poorly absorbed from the gastrointestinal tract and no enzyme capable of hydrolysis of this disaccharide is present in human gastrointestinal tissue. As a result, oral doses of lactulose reach the colon virtually unchanged. In the colon, lactulose is broken down primarily to lactic acid, and also to small amounts of formic and acetic acids, by the action of colonic bacteria, which results in an increase in osmotic pressure and slight acidification of the colonic contents. This in turn causes an increase in stool water content and softens the stool. Since lactulose does not exert its effect until it reaches the colon, and since transit time through the colon may be slow, 24 to 48 hours may be required to produce the desired bowel movement. Lactulose given orally to man and experimental animals resulted in only small amounts reaching the blood. Urinary excretion has been determined to be 3% or less and is essentially complete within 24 hours.