PREPARATION OF HYDROLYZED LACTOSE SYRUP FROM WHEY PERMEATE AND ITS FUNCTIONAL PROPERTIES IN WHITE PAN BREAD FORMULATION by OLUYEMI A. OGUNRINOLA 6.S. University of Illinois, 1983 A MASTER'S THESIS submitted in partial fulfillment of the requirements for the degree MASTER OF SCIENCE FOOD SCIENCE Department of Animal Sciences and Industry KANSAS STATE UNIVERSITY MANHATTAN, KANSAS 1986 Major Professor 1 ACKNOWLEDGEMENTS A112D7 23AZ31 The author wishes to express his gratitude to his major professor Dr. I.J. Jeon for his guidance, suggestion, constructive criticism, patience, and encouragement throughout this study. Thanks are also given to the other members of the author's supervisory committee, Dr. L.H. Harbers, Professor J.G. Ponte, Jr. and the chairman of the Food Science Graduate program, Dr. D.Y.C. Fung. Gratitude and thanks are also extended by the author to all staff members, friends, and colleagues who have helped made this a thorough and unique learning experience. Sincere appreciation is also extended to the Department of Animal Sciences and Industry for the opportunity the author has to be part of it. Thanks also are expressed to Professor H.A. Roberts for preparing the whey permeate used for this study as well as to the Grain Science Department staff members who assisted during the breadbaking phases of this study. Last but not the least, the author is grateful to his parents, brothers and sisters for their love, encouragement and financial supports. 1 TABLE CF GCNTENTS PAGE ACKrCMLEDCvENTS i i LI ST CF TABLES v i i LIST CF FIGURES viii INTRODUCTION 1 LITERATURE REVIEW 3 I. Whey Permeate: General Outlook 3 II. Whey Permeate Composition 4 A. Lactose 4 B. Minerals and Ash and other Components 7 III. Whey Permeate Problems 8 A. Production vs. Utilization 8 B. Disposal Problem 8 C. Technical Problem 9 D. Nutritional Problem 11 IV. Whey Permeate Processing 11 A. Obtaining whey permeate (IF Process) 13 B. Demi nera 1 i zat ion 13 C. Deco lor i zat ion 14 D. Concentration 14 V. Irrmobilized Enzyme 15 A. Matrix System 16 B. Type of Reactor 16 C. ivethod of Immobilization 17 i i i D. The Substrate 17 VI. Hydrolysis of Lactose 19 A. Beta-ga lactosidase 19 B. Oligosaccharide Formation 21 C. Determination of Lactose 22 VII. Uses of Hydrolyzed Lactose 23 A. Hydrolyzed Lactose in Baked Products 23 B. Use in Ice Cream 24 VIII. Sensory Evaluation Methods 25 MATERIALS AND ivETKDS 27 I. Inrmobi 1 i zed Enzyme System 27 A. The components of the IlvE System 27 1. Carrier 27 2. The Reactor System 27 3. The Enzyme 27 4. The Substrates 29 3. Inrmobi 1 izat ion of Fungal Lactase to the Carrier . 29 1. Carrier Preparation and Enzyme Attachment 29 II. Wiey Permeate Preparation and Hydrolysis 30 A. Hydrolysis 30 B. Preparation of Hydrolyzed Permeate into Variables . 30 1. Demi neral i zat ion 31 2. Decolori zation 31 3. Concentration 31 III. Bread Formulation and Production 33 A. Ingredient vVeighing and Mixing 33 iv B. Dough Fermentation 33 C. Dough Make-Up 39 1. Scaling 39 2. Rounding 39 3. Intermediate Proof 39 4. Moulding 39 5. Panning 39 6. Final Proof 39 D. Bread Baking 40 IV. Analytical Methods 40 A. pri 40 B. % Total Solids 41 C. Microbial Standard Plate Count 41 D. Minerals and Proteins 41 E. % Lactose Hydrolyzed 42 F. Fermentation 43 G. Specific Volume 43 H. Firmness Test 43 I. Residual Sugars 43 J. Bread Scoring 44 K. In-House Sensory Evaluation 44 L. Statistical Analysis 45 RESULTS AND DISCUSSION 46 I. Immobilized Enzyme System 46 A. IME System 46 B. Attachment of the Enzyme to the Glass Beads .... 46 II. Hydrolysis of vVhey Permeate and hydrolyzed Lactose Syrup v Preparations 47 1. vWiey Permeate Hydrolysis 47 2. Demi nera 1 i zat ion 48 3. Deco lor i zat ion 50 4. Concentration 50 5. Microbial Quality 51 III. Bread Formulation and Production 51 A. Dough Development and Breaornaking 51 B. Fermentation and Proofing 52 C. Bread Baking and Specific Volume 56 D. Bread Scores and Physical Characteristics 58 E. Residual Sugar Content 64 F. In-house Sensory Evaluation 70 CCNdUSICN 74 REFERENCES 76 A°PE>DIX 81 VI LIST OF TABLES Table Page 1A. Typical gross composition (%, wt/vol) and mineral composition (%, wt/vol) for ultrafiltration-derived deproteinated milk serum (DPMS) 5 IB. Typical compositions of whey permeate, skim milk permeate, and whole milk permeate 5 2. Relative sweetness and solubility of common saccharides 6 3. Baking equipment used during breadmaking with the hydrolyzed lactose syrups 4. The general baking formula used for the hydrolyzed lactose syrup breads 36 5 The amount of water added to each dough batch 37 6. pri and total soluble solids of the hydrolyzed lactose syrups ... 38 7A. Comparison of mineral content of demineralized and the undemineralized whey permeate 49 7B. Comparison of mineral content of the demineralized and the undemineralized hydrolyzed lactose syrups 49 8. Selected data on hydrolyzed lactose syrup and the control doughs and breads 57 9. Residual sugars in the hydrolyzed lactose syrup and control bread crumbs 69 10. Summary of the in-house evaluation on the hydrolyzed lactose syrup and the control breads 71 11. Proximate analysis of the hydrolyzed lactose syrup and the control breads 73 vii LIST OF FIGURES Figure Page 1. Structure of lactose (I), galactose (II), and glucose (III) 10 2. Frequency of lactose intolerance in percent of the population in some parts of the world 12 3A. Proposed mechanism of lactose hydrolysis by 0-galactosidases . 18 3B. Proposed mechanism of galactosyl transfer reaction by S-galactosidases 20 4. Bench scale unit used for lactose hydrolysis 28 5. hydrolyzed lactose syrup production and usage in bread mix . 32 6. Steps involved in bread production 34 7A. Fermentation of undemineralized HLS in doughs 54 76. Fermentation of demineralized HLS in doughs 55 8A. Firmness measurement in undemineralized HLS bread 1 - Day . 60 8B. Firmness measurement in demineralized HLS bread 1 - Day ... 61 9A. Firmness measurement in undemineralized HLS bread 3 - Day . 62 96. Firmness measurement in demineralized HLS bread 3 - Day ... 63 10. HPLC chromatograms of 1% standard sugar solution (A) and control breads (6) 65 J 11. HPLC chromatograms of demineralized 50 /o HLS (C) and undemineralized 75% HLS breads (D) 66 12. HPLC chromatograms of demineralized 75% HLS (E) and undemineralized 95% HLS breads (F) 67 13. HPLC chromatograms of demineralized 95% HLS (G) and demineralized 50% HLS breads (H) 68 viii INTRODUCTION The future of the dairy industry looks brighter on the account that it is able to maximize the utilization of its by-products. There are significant increases in the production of by-products of cheese and related products. However, underutilization of these by-products remains a major concern of the industry. The advent of high technology such as the ultrafiltration process (UF) and immobilized enzyme (I1V1E) has opened the opportunity for the dairy industry to produce quality food ingredients such as whey protein concentrate (vvPC) and hydrolyzed lactose syrups (HLS) from whey and whey permeates. These by-products are generally underutilized inspite of their higher potentials and fuctionalities. For example, researchers in recent years have made HLS from whey and whey permeates and have attempted to use it in ice cream and dairy drink mixes. However, the use of this functional ingredient in baked products has yet to be reported as far as we understand, although the bakery industry has been a strona. dairy ingredient user in the past. Therefore, the objectives of this study were to: 1. immobilize Q-galactosidase on a solid support, 2. use the immobilized enzyme to hydrolyze whey permeates, 3. classify and prepare the hydrolyzed permeate into variables, 4. process each of the variables by deionizing and decolorizing, 5. concentrate the variables to syrups, 6. use the hydrolyzed lactose syrups in white pan bread formulation, 7. evaluate the doughs and breads made from the hydrolyzed syrups and observe some specific physical and chemical characteristics, 8. conduct in-house sensory evaluation on the breads. vVhey permeate was obtained from the Kansas State University Dairy Processing Plant (Manhattan, KS) and used as the substrate for the enzymatic hydrolysis process using an immobilized enzyme system. The hydrolyzed whey permeates were divided into three variables of different degrees of lactose hydrolysis. Each variable was then classified as "demineralized" and "undemineralized" and processed accordingly. All the variables were decolorized and concentrated to syrups. The syrups were used in white pan bread formulation at 6% flour basis (700g flour) to substitute sucrose. Sucrose was used as the control. The breads were made in triplicate batches. REVIEW OF THE LITERATURE I. \A<hey Permeate: General Outlook vVhey permeate is the watery liquid by-product of ultrafiltration (UP) process of whey and /or milk. It is largely made up of lactose, ash and traces of non-protein nitrogenous materials (Chandan, 1982; hausser, 1984). Approximately 90% of whey is whey permeate with its contents estimated at about 85% lactose, 9% ash and 4% non-protein materials on a dry weight basis (Chandan, 1982). In 1983, American dairy farmers produced about 140 billion pounds of milk and approximately one-third of this was processed into cheese. This represents 5 billion pounds of cheese and 45 billion pounds of whey (Luksas, 1984). The UF process which fractionates the whey solids from whey, especially its protein, results in the production of whey permeate. Thus, it is known that most permeate on the market today comes from cheese manufacturing, especially from sweet cheeses such as Cheddar cheese. Precise data on whey permeate production is not easy to obtain at present time.