LACTULOSE PREPARATION USING FOOD-SAFE REAGENTS by ANNE ALEXANDRA LAYTON B.Sc. (Agriculture), The University of British Columbia, 1992 THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Food Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA APRIL 1997 ©ANNE ALEXANDRA LAYTON, 1997 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT Lactulose is efficiently synthesized from lactose using catalysts such as boric acid and triethylamine. However, since neither catalyst is food-safe, both must be removed after processing. Lactulose is also produced inadvertently during heat treatment of dairy products, although in small quantity. Studies have indicated that altering the heat processing conditions can improve lactulose yield. A high lactulose, mixed carbohydrate preparation was produced without the use of toxic catalysts. Using two Taguchi's fractional factorial designs, eight factors were tested as to their influence on lactulose yield: pH, lactose, NaOH, citrate and phosphate concentrations, heating temperature and duration, and purification of the lactose substrate. In the first design, lactose concentration (at levels of 40, 79, and 155 mg/mL) , pH (9.0, 10.5, and 12.0), heating temperature (90, 110, and 130°C), citric acid concentration (40, 70, 100 mM) and in the second design, NaOH concentration (18, 50, and 100 mM) , was shown to significantly influence lactulose yield. A ll other factors did not significantly influence lactulose yield at the selected levels. The interactions of lactose, citrate, and phosphate concentrations of the first design also significantly influenced lactulose yield. The conditions selected for the conversion of lactose to lactulose was decalcified whey permeate at > 70 mg/mL lactose, a pH of 10.5-11.0, with an added 50 mM sodium citrate, was heat treated at 110°C for 10 minutes. Approximately 30% of i n i t i al lactose was converted to lactulose v ia primarily the Lobry de Bruyn and Alberda van I ll Ekenstein transformation. Again using a Taguchi design, four factors were tested to if they significantly influenced the preferential precipitation of lactose over lactulose in a cooled aqueous solution: pH, sugar concentration, temperature decrease, and final temperature. The pH of the mixed carbohydrate solution (at levels of 7.0, 9.0, and 10.7) and sugar concentration (29, 39, and 52%) both significantly influenced either the lactulose yield of precipitation or the sugar ratio in the decant. For further study, the lactulose preparation was concentrated to approximately 50% solids and pH 10.5, cooled from 65° C to 20° C at 5C°/hour, and held for 24 hours, preferentially precipitating lactose over lactulose. After one cooling cycle, there was a lactulose yield of approximately 82% and a 1:1 lactulose: lactose ratio. After a second precipitation of the decanted portion, there was a 78% lactulose yield and a 3.4:1 lactulose: lactose ratio. There was a total loss of about 4 0% of lactulose through the two precipitation cycles. Ion-exchange columns removed the majority of the natural and added salts from the lactulose preparations. Activated charcoal removed most of the brown colour of the preparation but also 3 0% of the solids. The final syrup contained 59% lactulose, 2 6% lactose, 5.0% galactose, 1.0% glucose, and 0.81% fructose, based on total solids. Carbohydrates were assayed using an enzymatic spectro-photometric method. An unidentified substance was detected using thin-layer chromatography of carbohydrates. iv TABLE OF CONTENTS Abstract ii Table of Contents iv List of Tables ix List of Figures v ii Acknowledgement xi 1. INTRODUCTION 1 2. LITERATURE REVIEW . . 3 2.1 LACTULOSE 3 2.la Physical properties of lactulose 3 2.lb Lactulose in digestion 3 2.Ic Lactulose in medical treatment 5 2.Id Commercial products 7 2.2 WHEY 8 2.3 LACTULOSE AS AN INTERMEDIATE IN THE DEGRADATION OF LACTOSE 11 2.3a Lobry de Bruyn and Alberda van Ekenstein transformation 11 2.3b Maillard reaction and Amadori rearrangement 15 2.4 LACTULOSE CONVERSION 17 2.4a Overview of conversion methods 17 2.4b Borate and triethylamine catalysts 17 2.5 FACTORS AFFECTING LACTULOSE CONVERSION IN HEAT PROCESSING 18 2.5a The influence of temperature and heating time 18 2.5b The influence of pH 19 2.5c The influence of citrate and phosphate . 2 0 2.5d The influence of lactose concentration . 20 2.5e The influence of demineralization 21 2.5f Other influences on lactulose yield .... 22 2.6 FRACTIONAL FACTORIAL EXPERIMENTAL DESIGN 22 2.7 PURIFICATION OF LACTULOSE 25 2.7a Overview of lactulose purification methods 25 2.7b Cold temperature precipitation of lactose 25 V 3. MATERIALS AND METHODS 28 3.1 RAW MATERIALS 2 8 3.2 THE CONVERSION OF LACTOSE TO LACTULOSE . 28 3.2a Sample preparation 28 3.2b Heat processing 33 3.2c Calculation of lactulose yield 35 3.2d Statistics for fractional factorial designs 36 3.3 CONTINUOUS FLOW CONVERSION OF LACTOSE TO LACTULOSE 3 6 3.3a Design of heat exchanger 3 6 3.3b Determining heating times 37 3.4 PURIFICATION 37 3.4a Materials 37 3.4b Fractional factorial design for cold precipitation 39 3.4bl Sample preparation 39 3.4b2 Calculation of lactulose yield and sugar ratio 43 3.4c The second cycle of cold precipitation . 44 3.4d Calcium phosphate removal 45 3.4e Demineralization 45 3.4f Decolourization 47 3.5 PROXIMATE ANALYSIS 47 3.5a Sampling throughout the process 48 3.5b Enzymatic carbohydrate assays 48 3.5bl Quantitative assays for lactulose, lactose, glucose, and fructose 48 3.5b2 Activity of beta-galactosidase 51 3.5b3 Quantitative assay for galactose 54 3.5b4 Standard curves for carbohydrate assays 54 3.5c Thin layer chromatography assay of carbohydrates 65 3.5cl Qualitative assay of carbohydrates 65 3.5c2 Tagatose identification by TLC and spectrophotometry 66 3.5d Determination of nitrogen 70 3.5dl Determination of total nitrogen 7 0 3.5d2 Determination of protein 70 3.5e Determination of total solids and ash .. 71 3.5f Determination of pH and titratable acidity 71 vi 4. RESULTS AND DISCUSSION 74 4.1 OVERVIEW OF PROCESS 74 4.2 THE CONVERSION OF LACTOSE TO LACTULOSE 7 6 4.2a The influence of pH and NaOH concentration . 79 4.2b The influence of sodium phosphate concentration 82 4.2c The influence of citrate concentration 84 4.2d The influence of lactose concentration and purification 87 4.2e The influence of temperature and time of heat treatment 90 4.2f Continuous flow heat exchanger 90 4.3 PURIFICATION 92 4.3a Fractional factorial design for cold precipitation 92 4.3b The second cycle of cold precipitation ..... 97 4.3c Demineralization and decolourization 99 4.4 PROXIMATE ANALYSIS 101 4.4a Carbohydrate standard curves using enzymatic assays 101 4.4b Activity of beta-galactosidase 102 4.4c Thin-layer chromatography qualitative assay of carbohydrates 103 4.4d Tagatose identification by TLC and spectrophotometer 106 4.4e Determination of total nitrogen and protein . 107 5. CONCLUSIONS 109 6. ABBREVIATIONS 111 7. REFERENCES 112 vii LIST OF FIGURES Figure 1. The beta-furanose isomer of free lactulose .... 4 Figure 2. Possible degradative reaction routes of lactose in milk; gal refers to galactosyl or galactose 13 Figure 3. Amadori rearrangement of lactosyl-amino product of the Maillard reaction in milk to form a lactulosyl-amino compound 16 Figure 4. Overall process for the conversion of lactose to lactulose and partial purification of lactulose 29 Figure 5. Schemes used in the L (313) design for the 27 conversion of lactose to lactulose during heat treatment of whey permeate 31 Figure 6. The heat exchanger with thermocouples determined come up and cool down times: top) the pressure tight system, bottom) the heating or cooling coil with thermocouples in sequence 38 Figure 7. Schemes used in the Taguchi design L^(313) 7 for the preferential cold precipitation of lactose, following the design on Table 4 42 Figure 8. The standard curve of the absorbance of O-nitrophenol at 42 0 nm to determine the activity of the beta-galactosidase "Lactase 100,000" using ONPG hydrolysis 53 Figure 9. The standard curve of glucose using an enzymatic spectrophotometric assay 57 Figure 10. The standard curve of galactose using an enzymatic spectrophotometric assay 59 Figure 11. The standard curve of lactose using an enzymatic spectrophotometric assay 60 Figure 12. The standard curve of lactulose using an enzymatic spectrophotometric assay 61 Figure 13. The standard curve of fructose using an enzymatic spectrophotometric assay 63 Figure 14. The standard curve of the absorbance of tagatose concentrations at 256 nm 69 viii Figure 15 The standard curve of the absorbance of bovine gamma globulin protein at 595 nm for the Bio- Rad Protein Assay 72 Figure 16 The influence of pH and NaOH concentration on a, b lactulose yield during heat treatment of whey permeate using an L (313) fractional factorial 27 design 81 Figure 17 The influence of sodium phosphate concentration a, b interacting with lactose and citric acid concentrations on lactulose yield of heat treated whey permeate using an L (313) design 83 27 Figure 18, The influence of citric acid / sodium citrate concentration on lactulose yield during heat treatment of whey permeate using combined results of L (313) designs #1 and #2 85 27 Figure 19, The influence of citric acid concentration a, b interacting with lactose and sodium phosphate concentrations on lactulose yield during heat treatment of whey permeate using an L (3 13 27 design 86 Figure 20, The influence of lactose concentration on lactulose yield during heat treatment of whey permeate using L (313) design #1 88 27 Figure 21. The influence of lactose concentration a,b interacting with sodium phosphate and citric acid concentrations on lactulose yield during heat treatment of whey permeate using an L (313) design 89 J2?77 Figure 22 The influence of temperature on lactulose yield during heat treatment of whey permeate using the combined results of L (313) designs #1 and #2 91 27 Figure 23 The influence of pH and sugar concentration on a, b the cold precipitation of a lactulose: lactose solution using an L (313) design 97 27 Figure 24 Lactulose preparation at three stages of processing, from left to right - decalcified UF whey permeate (24.7% TS), after precipitation (10.4% TS), and after deionization and decolourization (23.3% TS) .. 100 Figure 25. TLC on silica plates shows standard sugar solutions at varying concentrations and whey at different stages of process 104 ix LIST OF TABLES Table 1. Composition of a typical lactulose syrup 6 Table 2. Lactulose contents of some commercial products 9 Table 3. Composition of whey and ultrafiltration whey permeate from Cheddar cheese 10 Table 4. One example of Taguchi's experimental design, orthogonal array L (313) 24 27 Table 5. Solubilities of lactose and lactulose in water at various temperatures 27 Table 6. Condition factors and their assigned levels of heat processed deproteinized whey investigated for influence on lactulose yield using an L (313) design 3 0 27 Table 7. Heat processing of the two L (313) 27 experimental designs for the conversion of lactose to lactulose 34 Table 8. Heat processing conditions for lactulose production from whey selected using Taguchi's fractional factorial designs 40 Table 9. Condition factors and their assigned levels of a cold precipitated lactulose: lactose solution using an L (313) design 41 27 Table 10. Standard solutions containing varying ratios of five carbohydrates prepared for standard curves of a ll enzymatic assays 55 Table 11. Analysis of variance testing the significance of slope and linearity of the regressional curve calculated for the glucose standard curve 58 Table 12. Analysis of variance testing the significance of slope and linearity of the regression curve calculated for the lactulose standard curves with a t-test comparison of the slope and elevation of the curves 62 Table 13. Analysis of variance testing the significance of slope and linearity of the regression curve calculated for the fructose standard curves with a t-test comparison of the slope and elevation of the curves 64