LACTULOSE PREPARATION USING FOOD-SAFE REAGENTS by ANNE ALEXANDRA LAYTON B ...

128 Pages·2009·5.13 MB·English

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