LACTULOSE PREPARATION USING FOOD-SAFE REAGENTS by ANNE
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. All 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 initial lactose was converted to lactulose via primarily the Lobry de Bruyn and Alberda van Ill 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 vii
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
13 Figure 5. Schemes used in the L27(3 ) design for the 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
13 Figure 7. Schemes used in the Taguchi design L^7(3 ) 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 13 permeate using an L27(3 ) fractional factorial design 81
Figure 17 The influence of sodium phosphate concentration a, b interacting with lactose and citric acid concentrations on lactulose yield of heat 13 treated whey permeate using an L27(3 ) design 83
Figure 18, The influence of citric acid / sodium citrate concentration on lactulose yield during heat treatment of whey permeate using combined 13 results of L27(3 ) designs #1 and #2 85
Figure 19, The influence of citric acid concentration a, b interacting with lactose and sodium phosphate concentrations on lactulose yield during heat 13 treatment of whey permeate using an L27(3 design 86
Figure 20, The influence of lactose concentration on lactulose yield during heat treatment of 13 whey permeate using L27(3 ) design #1 88
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 13 LJ2?77 (3 ) design 89 Figure 22 The influence of temperature on lactulose yield during heat treatment of whey permeate using the 13 combined results of L27(3 ) designs #1 and #2 91
Figure 23 The influence of pH and sugar concentration on a, b the cold precipitation of a lactulose: lactose 13 solution using an L27(3 ) design 97
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, 13 orthogonal array L27(3 ) 24
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 13 L27(3 ) design 3 0
13 Table 7. Heat processing of the two L27(3 ) 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 13 solution using an L27(3 ) design 41
Table 10. Standard solutions containing varying ratios of five carbohydrates prepared for standard curves of all 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 X
Table 14. The absorbance of aqueous carbohydrate solutions and the TLC solvent by spectrophotometer between 200 and 400 nm 68
Table 15. Proximate analysis, based on % total solids, of whey permeate at different stages of lactulose manufacture and purification 75
Table 16. Design factor combinations and results 13 summary of the L27(3 ) design #1 77
Table 17. Design factor combinations and results 13 summary of the L27(3 ) design #2 78
13 Table 18. Analysis of variance [Taguchi L27(3 ) ] design #1 and #2, obtained from 27 experiments of heat processed demineralized whey permeate 80
13 Table 19. Results of the L27(3 ) design, assaying lactose and lactulose in both decant and precipitate portions after cold precipitation of a 28:72 lactulose:lactose solution 93
13 Table 20. Analysis of variance of the L27(3 ) design of cold precipitation in a lactulose:lactose solution 95
Table 21. Results of 5 replicates of a second cycle of cold precipitation in a lactulose: lactose solution 98
Table 22. Rf values of carbohydrate standards and various samples using thin-layer chromatography on a derivatized silica plate . 105 xi
ACKNOWLEDGEMENTS
The author would like to thank Dr. Durance for his encouragement, advice and support throughout this thesis. Also to
Canadian Inovatech Inc., Abbotsford, BC for their interest in the study and financial support. She also extends sincere appreciation to the other members of the supervisory committee, Dr. Vanderstoep,
Dr. Li-Chan, and Dr. Nakai, for their input, and to Mr. S. Yee for technical assistance during the course of this study. 1
1. INTRODUCTION
Lactulose, a synthetic isomer of lactose, usually sold as the
major constituent in a mixed carbohydrate syrup, has strong
worldwide markets. As a pharmaceutical it is used in the treatment
of chronic constipation and portal systemic encephalopathy. As an
ingredient in health foods and infant formulae it is a digestive
aid, acting as an energy source for beneficial bacteria in the
colon.
Purified lactose is efficiently converted to lactulose by
alkaline enolization using sodium borate and triethylamine as
catalysts. Lactulose yields of 86% have been reported using these
catalysts (Hicks et al., 1984). Since neither sodium borate nor triethylamine are food-safe, both must be removed after processing.
Lactulose is also produced in small quantities during thermal processing of evaporated and UHT milks. Its yield reaches only
approximately 4% in these products. Still, studies have indicated that altering thermal processing conditions and milk composition can influence lactulose production (Andrews, 1989; Martinez-Castro
& Olano, 1980). Some studies have further suggested that using purified lactose as the substrate for lactulose production is not necessary (Hicks et al., 1984). Whey, a waste product of cheese manufacture, may prove a more economical substrate for lactulose production.
There was no published research found in which the conditions of thermal processing were manipulated to maximize lactulose yield in whey using only food-safe reagents. In the present study, a process for lactulose production was investigated using food-safe reagents and using the lactose in deproteinized whey as the substrate. The goal was to produce a colourless, high-lactulose,
low-lactose mixed carbohydrate preparation. The main objectives of
this thesis were to:
1. Evaluate eight thermal processing factors in influencing
lactulose yield using a fractional factorial experimental design
-Temperature of heat treatment -Duration of heat treatment -Whey permeate demineralization -pH -NaOH concentration -Lactose concentration -Added sodium citrate/citric acid concentration -Added sodium phosphate concentration
2. Evaluate four cold precipitation factors in influencing the preferential precipitation of lactose over lactulose, again
utilizing a fractional factorial experimental design
-Cooling rate -Final temperature -pH -Carbohydrate concentration
3. Investigate further purification processes of the lactulose
sugar mixture, including demineralization by ion exchange and decolourization by activated charcoal.
4. Identify the changes occurring in sample composition as the whey permeate was heat-treated and purified. Numerous components of the whey permeate were measured at various stages of processing. 3
2. LITERATURE REVIEW
2.1 LACTULOSE
2.la Physical properties of Lactulose
Lactulose (4-0-Beta-D-galactopyranosyl-D-fructose) is a synthetic disaccharide of molar mass 342.30 g/mol and a melting point of 169°C. It is a white, crystalline, sweet tasting powder.
The disaccharide is composed of a galactose moiety linked to a fructose moiety by a 1 to 4 beta-glycosidic linkage. There are five possible isomers of lactulose: the fructose moiety in the form of an alpha or beta pyranose, an alpha or beta furanose, or lactulose in an acyclic form. Using current methods of lactulose production three isomers predominate: the beta furanose (Figure 1) , the beta pyranose, and the alpha furanose at a ratio of 0.74 5,
0.155, and 0.100 respectively (Jeffrey et al., 1983).
2.lb Lactulose in digestion
Lactulose was first synthesized from lactose in 1929 by
Montgomery and Hudson (1930), but it was not until 1957 that the significance of lactulose as a potential aid in digestion was recognized (Petuely, 1957). Unlike many other carbohydrates such as lactose, lactulose is not hydrolyzed by digestive enzymes in the small intestine to be absorbed as monosaccharides into the bloodstream. Instead, it is transported to the large intestine where it is hydrolyzed by resident saccharolytic microflora. Using lactulose as an energy source, some gram positive bacteria such as
Bifidobacteria species produce lactic and acetic acids, lowering pH and raising osmotic pressure. These in turn soften intestinal contents and may prevent the proliferation of some gram negative Figure 1 The beta-furanose isomer of free lactulose .
Of 5 possible isomers of free lactulose beta-furanose isomer (74.5% of total) beta-pyranose isomer (15.5% of total) alpha-furanose isomer (10.0% of total) alpha-pyranose isomer acyclic isomer
(Jeffrey et al., 1983) 5
putrefactive and pathogenic species such as Clostridium,
Bacteroides. Salmonella. Shigella. and Escherichia coli (Haenel,
1970; Mata et al., 1969a; Mata et al., 1969b; Rose & Gyorgy, 1955).
2.1c Lactulose in medical treatment
Lactulose is used in the treatment of chronic constipation and
in the prevention and treatment of portal systemic encephalopathy.
It is available as an impure syrup containing about 50% (w/w)
lactulose with lesser amounts of lactose, other sugars and acid.
Commercial syrups such as "Duphalac" and "Laevolac" are similar to
the product described in Table 1.
In the treatment of chronic constipation, the primary actions
of lactulose are:
1. the acidification of the colon's contents by organic acid
production during bacterial hydrolysis of lactulose,
2. the increase of osmotic pressure, and
3. a laxative effect.
The dosage range for chronic constipation is 15 to 45 mL of
lactulose syrup daily depending on the severity of the illness
(Avery et al., 1972).
Portal systemic encephalopathy (PSE), a complication of advanced hepatic cirrhosis, is caused by ammonia and other nitrogenous substances acting as toxins in the brain. Although its action is not yet completely understood, lactulose may decrease the production, decrease the absorption, and/or increase the excretion of ammonia in the digestive tract (Kosman, 1976). The dosage range for PSE is 3 0 to 50 mL of lactulose syrup three times daily, or Table 1 Composition of a typical lactulose syrup.1
Contents g / 100 mL
Lactulose 63 - 70 Lactose 4 - 8 Galactose 8 - 13 Other sugars 4 - 8 Orange flavours 2 Citric acid 0.16
General dosage: Chronic constipation- 15 - 45 mL/day Portal systemic encephalopathy- 90 - 150 mL/day
(Avery et al., 1972) i 7
enough to lower faeces pH from a normal 7 to a pH of 5.0 to 5.5
(Avery et al., 1972).
The toxicity of lactulose taken orally was reported to be
similar to sucrose when tested using rats (Okumura & Gomi, 1973).
When acute toxicity of lactulose was tested on adult baboons, the
animals survived a total dose of 37 mL lactulose syrup/kg body
weight, close to the volume of their stomachs. Subacute levels of
20 mL/kg body weight daily for six months showed no ill effects in
young baboons apart from a decreased growth rate (Avery et al.,
1972) . Still, an overdose of this potent laxative can lead to
diarrhoea and severe dehydration.
2.Id Commercial products
Lactulose is a component of various dairy products, either
added as an ingredient or occurring during the heat treatment of
the food. It is often added to those dairy products specifically
marketed as therapeutic digestive aids. The popularity of these
specialty products containing lactulose (and/or Bifidobacteria) is
currently centred in Japan and Europe, and nominally in the United
States.
Infant formulae have contained lactulose, at least in one
brand, since 1960. It was included to ensure that bottle-fed
infants had a similar intestinal microflora, with a high
Bifidobacteria faecal count, as breast-fed infants (Mizota &
Tamura, 1987). For most infant formulae, the heat treatment during processing produces the lactulose in the product. Spray dried and ultra-high temperature (UHT) formulae contain very little
lactulose, while in-container sterilized formulae can contain anywhere from 55 to 469 mg/100 mL of lactulose (Beach & Menzies, 8
1983) . Some other dairy foods, especially sterilized milks, can contain lactulose at relatively high levels. Studies of Beach and
Menzies (1983) and Hendrickse et al. (1977) suggested the high end of the lactulose range found in some in-container sterilized milks and infant formulae approached a level high enough to have a laxative effect. Table 2 details the lactulose content of various dairy foods.
2.2 WHEY
Whey is the fluid obtained by separating the coagulum from milk or cream. It is generally produced either as a sweet or an acid whey. Sweet whey, with a pH above 5.5, is produced during the manufacture of cheese or rennet casein. Acid whey is produced during the manufacture of cottage cheese, lactic casein, or mineral acid casein (Sienkiewicz & Riedel, 1990a) . The disaccharide beta- lactose (4-0-FJ-D-galactopyranose) , is a major constituent of whey along with proteins, minerals, acid, and water. Table 3 provides a breakdown of whey's typical components.
Whey is a waste product of the cheese industry produced at a rate ranging between 7.5 kg whey/kg soft cheese and 11.3 kg whey/kg hard cheese. It is used in the production of a wide range of products including lactose, whey powder, lactalbumin, animal feeds, and fermentation media (Sienkiewicz & Riedel, 1990a,b).
Often, whey protein is isolated from whey to be sold as a commercial ingredient. Through a process of ultrafiltration (UF) , whey can be separated by membrane filtration into two portions.
The permeate consists mainly of lactose and salts (Table 3). The portion remaining on the membrane contains the majority of protein, Table 2 Lactulose contents of some commercial products.
Product Lactulose (g/100 g) (mg/100 mL)
"BF-T" Infant formula1 0.5 "Hounyu" milk powder1 0.3 (>3xl07)3 "Sawayaka" sour milk1 4 (>lxl08)3 Pasteurized milk2 4-15 Sterilized milk2 80 -200 Powdered milk reconstituted2 2.5-30 UHT milk2 10 - 30
1 (Mizota et al., 1987)
2 (Andrews, 1989) 3 Bifidobacterium addition (/g) Table 3 Composition of whey and ultrafiltration whey permeate from cheddar cheese.
Constituent Permeate Whey
Total solids (%) 5. 7 6. 7 Total nitrogen (mg/g) 0. 26 1. 30 Protein (%) 0. 01 0. 60 Protein removal (%) 98. 4 Non protein nitrogen (mg/g) 0. 24 0. 34 Ash (%) 0. 50 0. 52 Lactose (%) 4. 9 5. 0 Lactic acid (%) 0. 14 0. 14 PH 6. 1 6. 1
(Hargrove et al., 1976) 11 including lactalbumin and lactoglobulin (Hargrove et al., 1976).
Still, the supply of whey far exceeds the demand. Excess whey is commonly disposed of by spreading on land or treated as waste water. The high biological oxygen demand (BOD) value of whey
(approximately 40 g 02/L) makes its disposal an environmental dilemma (Sienkiewicz & Riedel, 1990c).
2.3 LACTULOSE AS AN INTERMEDIATE IN THE DEGRADATION OF LACTOSE
Lactulose occurs as an intermediate in the degradation of lactose. There are two methods by which lactose in milk or whey is degraded via free or bound lactulose:
1. Lobry de Bruyn and Alberda van Ekenstein transformation, and
2. the interaction of amino acids with lactose in the Maillard reaction and subsequent Amadori rearrangement.
One reported study heated milk for 2 0 minutes at a temperature of 110°C-150°C. Eighty percent of the degraded lactose followed the LA transformation and 20% followed the Maillard reaction (Berg
& van Boekel, 1994).
2.3a Lobry de Bruyn and Alberda van Ekenstein transformation
The Lobry de Bruyn and Alberda van Ekenstein (LA) transformation occurs mostly under alkaline conditions and is the dominant reaction for this study. This reaction is not specific to lactose but describes more than 50 similar reactions, the common feature being an enediol intermediate (Davidson, 1967). Most occur in the presence of a base catalyst.
Common to nearly all recent descriptions of the LA transformation of lactose is the degradation of lactose to 12 lactulose, (compounds 1 to 4a) in Figure 2. Under the influence of a base catalyst, the glucose moiety of lactose is isomerized to fructose via an intermediate enolization. Beyond these initial reactions, studies have not always agreed upon pathways or reaction products.
Figure 2 further illustrates the possible degradative reaction routes of lactulose in milk according to Berg and van Boekel
(1994). In their study, the main reaction products attributed to the LA transformation of milk or model milk solutions - heated to between 100°C and 150°C and assayed by HPLC - were lactulose
(compound 4a), galactose (compounds 6a,b), formic acid (compounds
9a,b), and various C5 and C6 compounds. The formic acid produced was suggested as an explanation for much of the decrease in pH during heating. The C5 compounds were believed to be formed along with the formic acid. Possible C5 compounds included 2-deoxyribose
(compound 10), 3-deoxypentulose (compound 16) and/or furfurol
(compound 17) . Small amounts of furfural and hydroxymethylfurfural
(compound 8) were also measured. Reaction intermediate C6 compounds such as deoxyosones (compounds 7,13) were thought to be formed but were not identified. It was suggested that the main route for lactose degradation during LA transformation was through
4-deoxyosone (compound 13) which was not considered an intermediate during Maillard/Amadori reactions (Ames, 1992). Berg and van
Boekel also stressed that these were not simple reaction routes; reactions were occurring simultaneously and products could be formed through different routes.
In Berg and van Boekel's study (1994), epilactose, tagatose, and isosaccharinic acids were not detected. The small amount of 13
3 5 7 HC=0 HC-OH HC=0 HC=0 I II I I >HC=0 HC-OH C-OH C-OH c=o CH20H I I II I HO-CH - HO-CH —3 CH CH2 I * Hydroxymethylfurfural I ^ HC-O-Gal "OH HC-O-Gal Gal H?"OH HC-O-Gal I I 6a HC-OH HC-OH HC-O HC-OH I HC-OH Formic acid I 10 I HC-OH I I I CH20H CH2 CH20H CH20H 9a CH20H I + HC-OH I It It ^> 4b HC-OH 4a HC=0 I CH20H 1 CH20H 14b HC-OH HO-CH 2-Deoxyribose I I 0 I HC-OH c=o II HC-OH I I HO-CH Isosaccharinic 1 HO-CH HO-CH I Acid I HO-C-CH20H I Gal-C-OH I HC-O-Gal 9b HO-C-Gal I I CH2 I HC-OH I Formic acid HC-OH CH I HC-OH I I CH20H I CH20H CH20H CH20H CH20H Epilactose Lactose Lactulose Furfurol 16 It \ CH20H 13 I CH20H c=o 11 I I 12 c=o 14 15 CH2 CH20H CH20H I I I c=o HC-OH HC=0 HC-OH I II C-OH c=o I I I CH2 C-OH HC-OH / CH20H II I I I 1 HO-C C-OH > 1 HC-OH c=0 3-Deoxypentulose I II c=o I I ^— c=1 o HC-O-Gal CH CH20H CH2 CH2 I Gal I HC-OH | 1 HC-OH 6b I HC-OH HC-OH I CH20H i I CH20H CH20H CH20H
Figure 2 Possible degradative reaction routes of lactose in milk; gal refers to galactosyl or galactose. (Berg and van Boekel, 1994) 14 epilactose thought possibly produced may have had its HPLC peak obscured by another carbohydrate. Tagatose was detected only in model solutions which contained additional galactose.
Epilactose (compound 4b) has been described as a reaction product of lactose degradation in other studies. In one, it was detected by GLC after degradation of lactulose (Olano & Martinez-
Castro, 1981). Berg and van Boekel (1994) considered 1,2-enediol as the intermediate (compound 3) . In a study by Olano et al.
(1989), using a buffered 5% lactose solution (pH 6.6) heated at
120°C for 20 minutes and a GLC assay, 77.2 mg/100 mL of epilactose were formed (in conjunction with 474.2 mg/100 mL of produced lactulose). In another study, epilactose was detected by GLC, but not at temperatures below 120°C (Martinez-Castro et al., 1986).
D-Tagatose is a product of D-galactose degradation along with formic acid, whether the lactose reaction follows the LA transformation or the Maillard reaction / Amadori rearrangement route. Tagatose can then be further degraded to trioses, then oxidized to acids. While Berg and van Boekel (1994) did not detect tagatose using HPLC, Troyano et al. (1992) found 13 mg/L in milk heated at 120°C for 20 minutes using GLC.
Isosaccharinic acids have been described as lactose degradation products and often as the major organic acids produced
(Olano & Martinez-Castro, 1981). Corbett and Kenner (1953) found beta-isosaccharinic acid and tagatose as degradative products of alkali treated lactose; this acid was also detected by Carubelli
(1966). Isosaccharinic acids are thought to occur through the beta-elimination degradation of lactulose (compounds 4a to 14b, in
Figure 2. Apart from formic and isosaccharinic acids, other acids, 15 such as lactic and acetic acids have also been included as reaction products (Gould, 1945; Keeney et al., 1950).
2.3b MaiHard reaction and Amadori rearrangement
The Maillard reaction occurs in milk by the elimination of a water molecule, joining the carbonyl group of lactose to an amino acid. The amino acid is often protein-bound and is usually lysine.
This pairing forms an amino glycoside (again often protein-bound), named lactosyl-lysine. Amadori rearrangement transforms lactosyl- lysine to its isomer lactulosyl-lysine, Figure 3 (Andrews, 1986).
While early reports suggested that this reaction could produce free lactulose upon further degradation, it is now confirmed that this does not occur (Berg and van Boekel, 1994). Free lactulose has been produced in the laboratory from Amadori rearrangement reactions but these have only occurred under specific conditions such as treating lactose with p-toluene and hydrochloric acid
(Adachi & Patton, 1961) .
The main reaction products of heated milk and model milk solutions following these reactions, according to the Berg and van
Boekel study (1994), were lactulosyl-lysine, galactose, formic acid, hydroxymethylfurfural, and other Maillard products. They
^suggested that the formation of galactose; formic acid, and C5 and
C6 compounds from lactulose degradation occurred whether initiated by the LA transformation or the Maillard reaction (Figure 2). 16
RNH2+ RNH RNH + RNH RNH I I II I I - CH - CH HC HC CH2 I I I HC-OH Ii HC-OH +H- HC-OH C-OH c=o I I I HO-CH HO-CH HO-CH HO-CH HO-CH I I I HO-C-Gal HO-C-Gal HO-C-Gal HO-C-Gal HO-C-Gal I I I CH CH HC-OH HC-OH HC-OH I I I I CH20H CH20H CH20H CH20H CH20H
Lactosyl-amino Lactulosyl-amino compound compound
Figure 3 Amadori rearrangement of lactosyl-amino product of the Maillard reaction in milk to form a lactulosyl-amino compound.
(Andrews, 1986) 17
2.4 LACTULOSE CONVERSION
2 . 4a Overview of conversion methods
Lactulose was first synthesized from lactose in 1929 in an alkaline solution of lime. Less than 20% of the lactose was converted to lactulose (Hicks & Parrish, 1980). Since then, lactulose conversion from lactose has been catalyzed using aluminates, magnesium oxide, ion exchange resins, and beta- galactosidases, among others, with varying degrees of success.
Using calcium hydroxide as a catalyst produced a preparation with lactulose comprising 15% of total sugar; using sodium aluminate produced a 73% lactulose mixture (Parrish et al., 1980). Using beta-galactosidase as a catalyst, only 8% of lactose was converted to lactulose (Vaheri & Kauppinen, 1978). The combination of borate and triethylamine has one of the highest conversion rates.
2.4b Borate and triethylamine catalysts
Borate is an efficient catalyst because it complexes with lactulose, shifting the equilibrium established during isomerization in favour of lactulose and prevents degradative side- reactions. Using borate, a lactulose yield of 70-80% has been achieved. However, it is impractical to use borate alone as borate: sugar ratios of 50:1 are needed to reach these high yields
(Mendicino, 1960). Triethylamine is a tertiary amine added to raise pH. Alone, it can produce lactulose yields of 32% but with high alkaline degradation (Parrish, 1970). Combining the two and using a boric acid lactose ratio of 1:1, high lactulose yields can be achieved with low monosaccharide production and few degradative side-reactions. The rate of lactulose conversion increases with 18 basicity from the addition of triethylamine (Hicks & Parrish,
1980). Both boric acid and triethylamine are toxic and must be removed, but can be recovered. Boric acid can be recovered using methanol; triethylamine can be removed by heating the solution and cold trapping the evaporation (Hicks & Parrish, 1980).
A study of isomerization of lactose using boric acid and triethylamine at 70°C for several hours at pH 11 produced a lactulose yield of 86%. The final product contained the following sugars: lactulose at 86.8% of total carbohydrates, 8.1% lactose,
4.1% galactose, and 1.0% tagatose (Hicks et al., 1984).
2.5 FACTORS AFFECTING LACTULOSE CONVERSION IN HEAT PROCESSING
2.5a The influence of temperature and heating time
Temperature and heating time have been tested in combination and have been shown in several studies to influence the production of lactulose. Geir and Klostermeyer (1983) measured lactulose using an enzymatic assay after heating skimmed milks at various temperatures - 60, 70, 80, and 90° C - at five time intervals between 5 and 3 0 minutes. There was a significant increase in yield at higher temperatures, though no sample exceeded 14 mg lactulose/100 mL. Increasing heating time also increased yield at higher temperatures. At 90°C, lactulose content rose from approximately 3 mg/100 mL after 5 minutes to 13 mg/100 mL after 25 minutes.
In testing the influence of milk dilution, sodium citrate concentration, and sodium phosphate concentration, five different time/temperature treatment combinations were tested (Andrews &
Prasad, 1987). These included 130°C for 2 and 4 minutes, 110°C for 19
3 and 15 minutes, and 90° C for 120 minutes. Although the manipulation of citrate and phosphate addition and milk dilution affected the results, temperature and heating time did appear to influence lactulose production. Heat treatments of 130°C for 2 minutes and 110° C for 3 minutes consistently produced the least lactulose of the combinations tested. The difference among the other three time/temperature combinations were dependent on the other variables. Generally increasing lactulose occurred with 90° C for 120 min, 110° C for 15 min, then 130° for 4 min (Andrews &
Prasad, 1987) . These results suggest that the increase from 2 to
4 minutes at 130° C and from 3 to 15 minutes at •110° C had a positive influence on lactulose production. As well, in light of the displayed influence of heating time, one could argue that the increase in lactulose at 110°C for 3 minutes to 130°C for 2 minutes was due to the influence of the higher temperature.
2.5b The influence of pH
The LA transformation occurs most commonly in alkaline solutions. The pH of a solution has been shown to be an influential factor in lactulose production. In commercial production of lactulose using lactose solutions, pH is raised substantially. A pH of 11 was described by Hicks et al. (1984) as an effective pH for lactulose production using boric acid and triethylamine.
Several studies have detailed the influence of pH. Although restricted to a pH range near that of milk, it was found that an increase in pH from 6.6 to 7.0 in casein-lactose solutions, heated at 12 0°C for 2 hours, corresponded to an increase in lactulose 20 production (Adachi, 1959) . A study by Martinez-Castro and Olano
(1980) found similar results as the pH of milk was raised from 6.0 to 7.5 and heated at 120°C for 15 minutes. At pH 6.6 there was a
1.6% lactulose yield, while at 7.5 there was a 13.7% yield.
Lactose solutions heated at 40° C for 96 hours at pHs of 9,10,10.5, and 11 using boric acid and triethylamine as catalysts, gave increasing lactulose yields of 6%, 32%, 74%, and 83% respectively
(Hicks & Parrish, 1980). • .
2.5c The influence of citrate and phosphate
Both phosphate and citrate are naturally present in milk, each at approximately 0.01 M (Florence et al, 1985; Jenness & Patton,
1959). The addition of citrate or phosphate buffers to milk has been associated with an increase in lactulose yield upon heat treatment. In one study, milk was tested after various time/temperature treatments (Andrews & Prasad, 1987). The most effective time/temperature combination (130°C for 4 minutes) produced lactulose at approximately 45 mg/100 mL without any buffer addition. After including 10 mL of 0.4 M of citrate buffer to 100 mL milk, lactulose reached approximately 200 mg/100 mL.
Adding 10 mL of 0.4 M phosphate buffer increased lactulose to 125 mg/100 mL. These authors proposed that both phosphate and citrate acted as ampholytes and catalyzed the LA reaction.
2.5d The influence of lactose concentration
In a 1989 study, lactulose production was measured in heat treated milk at normal and double lactose concentrations. The results suggested that lactulose production was a first order 21 reaction with respect to initial lactose concentration (Andrews,
1989). While the lactulose content rose in response to the increase in lactose content, the lactulose yield - the ratio of lactulose produced to initial lactose - remained the same. Similar results were found using whey UF permeate and a boric acid / triethylamine catalyzed reaction. Lactulose yields in whey permeates with lactose concentrations of 8.8 and 17.7 g/100 mL were equal at the end of the reaction (3 hours) . The more concentrated sample, however, had a somewhat slower reaction rate (Hicks et al.,
1984) .
2.5e The influence of demineralization
The presence of normal whey UF permeate components, including salts, did not interfere with lactulose yield, according to Hicks et al. (1984) . In their study, whey and a purified lactose solution (whey ultrafiltrate which had been deionized and decolourized) were isomerized with a boric acid catalyst and compared. It was argued by the authors that using whey UF permeate instead of purified lactose would eliminate an energy-intensive refining step. However, in a study by Andrews (1989), it was found that 50 ppm calcium as calcium chloride added to milk, depressed lactulose production in heated milk. Here, Andrews proposed that the added calcium was complexing with the citrate and associating with the phosphate found naturally in the milk. This reduced the amount of citrate and phosphate available to catalyze the LA reaction. 22
2.5f Other influences on lactulose yield
Other components of milk or processing conditions have been studied as to their effect on lactulose yield. A few of the more important findings - fat content, protein content and milk storage
- are summarized below.
Geir and Klostermeyer (198 3) found that a fat content of between 0% and 3.5% had no influence on lactulose yield, when testing milk samples at various temperatures and heating times.
Protein concentration was observed to be inversely related to lactulose production in milk by Andrews (1989). After studying lactulose yield in concentrated milks, he suggested that protein increased the formation rate of Maillard browning, forming condensation products of available lactose and lactulose.
The effect of storage was observed in UHT and sterilized milks, in the same study (Andrews, 1989). Storage for 6 months in darkness caused the lactulose content of some milks to rise and others to fall, with a mean increase of 12%. Since the lactulose contents of some samples declined, it appeared to the author that lactulose was produced and degraded during storage. Storage at different temperatures was studied recently by Jimenez-Perez et al.
(1992) . UHT milk samples were stored for 90 days at different temperatures. The study showed a significant increase in lactulose during storage, especially between 4 0-50°C.
2.6 FRACTIONAL FACTORIAL EXPERIMENTAL DESIGN
In testing the influence of a single factor on any given result, in this case lactulose yield, the number of experiments equals the number of levels at which the factor is tested. The 23 difficulty occurs when more than one factor is tested, since it is well known that one factor can influence another. With two or more factors, the number of experiments increases exponentially with the number of factors, as each factor level must be tested at each level of all other factors.
Fractional factorial designs reduce the number of experiments by selecting only portions of a full factorial design, yet still providing similar information. . Interactions among factors not identified in the design were assumed to not be,significant. In the 1970s and 1980s, Taguchi devised approximately 20 orthogonal arrays. These arrays are multifactor experimental designs constructed from Graeco-Latin square designs. Orthogonal arrays can be used as long as factor responses show linear behaviour.
Arrays commonly occur in different combinations of from 3 to 40 factors, 2 to 5 levels, with various factor interactions and up to
169 experimental trials (Taguchi, 1987) . One example of an
13 orthogonal array is the L273 (L for latin, 27 trials, 3 levels and
13 factors), in Table 4. Interactions of two factors are shown by a connecting line. The columns of Table 4 represent factor levels and the rows are the experimental trials. Often, orthogonal arrays are used to test the significance of many factors at a few levels as a screening for subsequent optimization. An array with more levels and interactions and fewer factors is useful on its own to show factor and interaction trends. However, these designs are often not as useful for detailed optimization as are other more flexible computerized designs due in part to the restrictive nature of assigned levels. 24
Table 4 One example of Taguchi's experimental 13 design, orthogonal array L27(3 ) .
Factor Exp't No. ABCDEFGHIJKLM
1 1111111111111 2 1111222222222 3 1111 3 33333333 4 1222111222333 5 1222222333111 6 1222333111222 7 1333111333222 8 1333222111333 9 1333333 2 22111 10 2123123123123 11 2123231231231 12 2123312312312 13 2231123231 3 12 14 2 2312 3 1312123 15 2231312123231 16 2312123312231 17 2312231123312 18 23123122311 2 3 19 31321321321 3 2 20 3132 213213213 21 3132321321321 22 3213132213321 23 3213213321132 24 3213321132213 25 3 3 2 113 2 3 2 12 13 26 3321213132321 27 3321321213132
A 25
2.7 PURIFICATION OF LACTULOSE
2.7a Overview of lactulose purification methods
The separation of lactulose from lactose has proved to be a difficult task. Lactose and lactulose are similar sugars with the same molecular weight. Many methods have been used in the purification of lactulose: ion exchange to remove aldoses (Adachi
& Patton, 1961), oxidation of lactose to lactobionic acid and its precipitation as a calcium salt (Visser & van den Bos, 1988) , oxidation of aldoses with bromine, then precipitation using silver sulphate and hydrogen sulphide (Montgomery, 1962), and the reversible binding of lactulose to calcium hydroxide (Adachi,
1969), to name a few.
2.7b Cold temperature precipitation of lactose
Lactose consists of glucose and galactose moieties connected by a 1 to 4 beta-galactosidic linkage. Lactose is present as two isomers (alpha and beta) which describe the conformation of the glucose moiety of the disaccharide.
The ratio of beta to alpha lactose in an aqueous solution increases as temperature increases, the beta isomer being the only stable isomer above 93.5°C (Short, 1978). During cold temperature precipitation, the alpha isomer will precipitate first. The beta form will then convert to the alpha form as precipitation continues. Therefore, lactose crystals occur generally in the alpha monohydrate form. There are two rate determining steps in this precipitation, the crystallization of the alpha isomer and the conversion of the beta to the alpha isomer. Which is the limiting step in a given precipitation depends on temperature and other conditions (Zadow, 1984). Table 5 details the solubilities lactose and lactulose at various temperatures. At temperatures, lactulose is more soluble in water than lactose. 27
Table 5 Solubilities of lactose and lactulose in water at various temperatures.
Temperature Lactose1 Lactulose2 (°C) (% w/w) (%w/w)
15 14.43 76.4 30 19.88 81 60 36.87 80 51.12 90 > 86
(Visser, 1982) (Oosten, 1967) 28
3. MATERIALS AND METHODS
Figure 4 illustrates the final process of lactulose conversion and partial purification. It provides an overview to the methods described here.
3.1 RAW MATERIALS
A combination of cheddar and mozzarella wheys were supplied courtesy of Canadian Inovatech Inc., Abbotsford, BC. The whey mixture was previously deproteinized by ultrafiltration at that facility, concentrated to a total solids of approximately 5%, then frozen.
Sugar solutions used in this study included alpha-lactose monohydrate, lactulose, D-glucose, D-fructose, D-galactose, and
D-tagatose (Sigma Chemical Co., Mississauga, ON).
3.2 THE CONVERSION OF LACTOSE TO LACTULOSE
3.2a Sample preparation
The influences of processing and compositional factors on lactulose yield in deproteinized whey permeate were evaluated using
13 two L27(3 ) fractional factorial experimental designs. The factors of each design, together with their assigned levels are shown in
Table 6, the experimental schemes in Figure 5.
The 27 samples for the first experimental design were prepared as follows.
1. Eight litres of frozen ultrafiltrate whey permeate were thawed and the calcium content reduced, as described in Section 3.4d. The decalcified whey permeate was then demineralized by ion-exchange,
Section 3.4e, and concentrated using a roto-evaporator at 40°C. 29
WHEY PERMEATE Heat
NaOH Calcium B DECALCIFIED WHEY
Heat \ ( MODIFIED WHEY ) Discard Precipitate Citrate Water
NaOH
RETORTED WHEY PRECIPITATED PREPARATION NaOH. 1st PPT DECANT (lon--exchange e
Water Water Heat Heat DEIONIZED PREPARATION
^Charcoal Q
DECOLOURIZED PREPARATION
Figure 4 Overall process for the conversion of lactose to lactulose and partial purification of lactulose. 30
Table 6 Condition factors and their assigned levels of heat processed deproteinized whey investigated for influence on lactulose yield 13 using an LJ2?77(3 ) design.
Experimental design #1 Level
Factor pH 9.0 10, 12, Lactose Concentration 40 79 115 (mg/mL) Heating Temperature (°C) 90 110 130 Heating Duration (min) 5 20 90 Citric Acid (mM) 40 70 100 Disodium Phosphate (mM) 40 70 100
Experimental design #2 Level
Factor
NaOH Concentration (mM) 18 50 100 Purification1 DPW DCW LS Heating Temperature (°C) 105 115 125 Heating Duration (min) 10 40 80 Sodium Citrate (mM) 50 70 90 Disodium Phosphate (mM) 0 50 100
Purification of the whey: DPW = Deproteinized whey. DCW = Deproteinized whey, calcium removed. LS = Lactose monohydrate solution. 31
Lactose1 / Purification2
C,D/\ F,G
M •
Phosphate Citrate Time Temp. pH1 / NaOH2 Error
Experimental design #1 Experimental design #2
13 Figure 5 Schemes used in the L'2?77 (3'• ) design for the conversion of lactose to lactulose during heat treatment of whey permeate. 2. Citric acid and disodium phosphate were added to 15 mL graduated test tubes. Amounts were calculated to provide the molar level selected in the experimental design in a 10 mL final sample volume.
3. A portion of the concentrated demineralized permeate was diluted to three 100 mL volumes of 5, 10, and 15% total solids.
The lactose concentrations were assayed for use in the calculation of lactulose yield. Volumes of 8.0 mL of these diluted permeates were added to the test tubes containing citric acid and disodium phosphate according to the design.
4. The pH of the samples was raised with 5.0 M sodium hydroxide
(NaOH) according to the design and then diluted with distilled
deionized water (ddH20) to a final sample volume of 10 mL.
5. Heat processing utilized an agitating water bath for the 90°C treatments and a steam retort for the 110°C and 130°C treatments.
Section 3.2b details the heating process.
6. Each of the 27 samples was diluted if necessary and assayed for fructose and lactulose in duplicate using the enzymatic assays described in Section 3.5bl.
The samples of the second experimental design were prepared somewhat differently.
1. Two litres of whey permeate were thawed and the calcium reduced in one of the litres. Both litre volumes, the whey permeate and decalcified permeate, were concentrated by vacuum roto-evaporation and then diluted to both equal 89 mg/mL lactose.
An aqueous solution of lactose monohydrate was prepared of equal lactose content. Volumes of 8.2 mL of each solution were added to nine 15 mL graduated test tubes. 2. Stock aqueous solutions of 0.91 M disodium phosphate and 2.3 M sodium citrate were prepared. Amounts of 0, 0.55, and 1.1 mL of the disodium phosphate solution (0, 50, and 100 mM in 10 mL whey, respectively), and 0.22, 0.31, and 0.40 mL of the sodium citrate solution (50, 70, and 90 mM in 10 mL whey respectively) were added to the appropriate test tubes.
3. Selected volumes of 5.0 M NaOH were added to the test tubes according to the design. To each test tube was then added 1.8 mL
ddH20 less the combined amount of disodium phosphate, sodium citrate and NaOH. This brought each sample to a final volume of 10 mL and a lactose concentration of 73 mg/mL.
4. Retorting the samples is described in Section 3.2b.
5. Each of the 27 samples was diluted if necessary and assayed in duplicate for lactulose.
3.2b Heat processing
Samples (4.5 mL) were placed in 10 mL serum bottles ("400" brand, 25 X 54 mm diameter X height, 13 mm i.d. mouth, Wheaton
Inc., Millville NJ) with aluminum seals crimped over rubber flange- style stoppers. Heating time using a waterbath (Magniwhirl constant temperature bath, Blue M Electric Co., Blue Island IL) was defined as the time between the samples entering the waterbath and exiting to an ice bath for cooling.
For the higher temperature treatments using a retort (Model
500W, FMC Corp., Central Engineering Laboratory, Santa Clara CA), heating time was defined as the time between the shutting of the vent and the opening of the cold water inlet. Come up and cool down times of the heating vessels and samples are shown in Table 7.
Three samples were prepared with a copper and constantan 34
13 Table 7 Heat processing of the two L27(3 ) experimental designs for the conversion of lactose to lactulose.
Temperature1 Pressure2 Come Up Time Cool Down Time Vessel3 Sample of Sample to 80°C (°C) (lbs/inch2) (sec) (sec)
90 0.0 128 8 105 2.9 50 85 20 110 6.1 65 105 35 115 9.8 70 135 40 125 18.9 85 160 55 130 24.6 90 185 75
90° C samples were heated by water bath, higher temperature samples by retort. Gauge pressure was based on a sea level altitude.
Retort come up time measured from the closing of the retort vent to the retort interior reaching the set temperature. 35
Teflon coated wire thermocouple, (Omega Tech. Co. Stamford CT) ,
inside serum bottles filled with 4.5 mL of ddH20. The
thermocouple's Teflon coating was stripped, and the wire tips
soldered together. The wire tip was bent inward to keep the
soldered tip in the centre of the liquid, and the separated wires were folded over the mouth and flattened against the outside of the
flask. The rubber stopper was placed over the wires and the cover
crimped overtop. Two exposed 7 cm needle thermocouples (O.F.
Ecklund Inc., Fort Myers, FL) were placed in the waterbath or
retort to monitor the temperature of the heating medium. The
thermocouples were connected to a Field Logger (DT 100F Data Taker,
Data Electronics [AUST] Ltd., Rowville Australia) and personal
computer which tracked temperatures at set 5 or 10 second intervals
using the "Decipher" program (Data Electronics).
3.2c Calculation of lactulose yield
Experimental samples were assayed for sugars as described in
Section 3.5b. Percent lactulose yield was then calculated from
lactulose concentration:
% lactulose yield = (lactulose concentration mg/mL)(100%) (initial lactose concentration mg/mL)
(Equation 1)
This assay measured lactulose indirectly as fructose.
Therefore, free fructose in solution was also included in the
lactulose measurement. In experiment design #1, fructose was
assayed separately to be subtracted from the measured lactulose
amount. The following calculation would then provide a more 36
accurate lactulose measurement.
lactulose mg/mL = (lactulose mg/mL) (fructose mcf/mL) X (342.32 q/mol) (180.16 g/mol) where 342.32 g/mol molecular mass of lactulose 180.16 g/mol molecular mass of fructose
(Equation 2)
3.2d Statistics for fractional factorial designs
For each fractional factorial experimental design, the mean
was calculated from duplicate assays for each of the 27
experiments. These data were then analyzed by analysis of variance
(ANOVA) to determine if the factors and/or factor interactions
tested had significant influence on lactulose yield. A statistical
computer program "PTaguchi.BAS" designed by Nakai et al. (1994),
for the fractional factorial designs of Taguchi, was used to
simplify calculations.
Graphing factors and factor interactions used the mean of the
9 experiment samples for each factor level. Confidence limits
followed the equation:
1/2 1/2 X ± (t.0.05(2),DF e / n ) X (sse/DFe)
where t,0.05(2),DF e two-tailed t value at the degrees of freedom of the error at P=0.05. n = number of samples. sse = sums of squares of the error. DFe = degrees of freedom of the error.
(Equation 3)
3.3 CONTINUOUS FLOW CONVERSION OF LACTOSE TO LACTULOSE
3.3a Design of heat exchanger
A continuous flow heat exchanger was developed to produce the 37 heat treated whey permeate in larger amounts than was achieved using samples of 4.5 mL in the batch process. A heat exchanger was constructed using 1/4 inch outside diameter copper piping. The
13.4 m pipe was wound into a 13 cm diameter coil and placed in an oil bath and ice bath in sequence. A peristaltic pump (Minipuls 3,
Mandel, Guelph, ON) was used to circulate the whey through the pipe in a closed-loop system. The copper pipe had a volume of 167 mL of water at 110°C.
3.3b Determining heating times
A shorter copper coil heat exchanger was prepared to estimate the come up and cool down times of the whey permeate (Figure 6).
Needle thermocouples (described in Section 3.2b) were inserted along the copper piping in the oil bath at up to 5 points: 54, 110,
167, 224, and 280 cm (an initial 14 cm of piping remained outside the bath). The thermocouples were held in the piping by 5 cm long brass compression tee joints (ULN 365, Master Plumber, Vancouver,
BC) (Figure 6). Three thermocouples were rotated among the five tee joints; the tee joints were tightly capped if empty. One other tee joint connected the pipe to the inlet tubing. Using this construction, the thermocouples tracked the heating of the whey permeate in the oil bath or, when the piping was reversed, tracked the cooling of the permeate in the ice bath.
3.4 PURIFICATION
3.4a Materials
Two solutions were used in the purification studies:
1. lactose monohydrate:lactulose aqueous solutions of various 38
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. 39 ratios, and
2. decalcified UF whey permeate, heat treated using the conditions detailed in Table 8.
3.4b Fractional factorial design for cold precipitation
3.4bl Sample preparation
13 An L27(3 ) fractional factorial design was used to evaluate four factors on the preferential precipitation of lactose over lactulose in cooled, concentrated sugar solutions (Table 9, the experimental scheme in Figure 7) .
The 27 samples were prepared as follows.
1. A 200 mL aqueous solution containing 45.48 g lactose monohydrate and 16.80 g lactulose (0.39 lactulose/lactose) was prepared to simulate heat treated whey permeate. The solution was concentrated, removing 54 mL water through a roto-evaporator. Of the remaining solution, 28.5 mL were removed for the low sugar concentration samples. The remaining solution was concentrated to a mid sugar concentration solution by removing 3 0 mL water. Again,
28.5 mL were removed for samples. The solution was finally concentrated, removing 11.5 mL of water for the high sugar concentration solution. Each solution was then assayed for lactose and lactulose content.
2. Each of the three 28.5 mL solutions of differing sugar concentrations was separated into three 25 mL screw cap test tubes,
9.5 mL volumes each. To these was added enough 5.0 M NaOH to reach the pH assigned in the experimental design. The samples were
diluted with ddH20 so that all samples totalled 9.5 mL plus a
combined 0.2 0 mL of NaOH and ddH20. At this stage there were nine 40
Table 8 Heat processing conditions for lactulose production from whey selected using Taguchi's fractional factorial designs.
Conditions Level pH 10.5 - 11.0 Phosphate concentration 0 mM Sodium citrate concentration 50 mM Lactose concentration >70 mg/mL Purification decalcified UF whey permeate Heating temperature 110 °C Heating time 10 min 41
Table 9 Condition factors and their assigned levels of a cold precipitated lactulose: lactose 13 solution using a L27?7(3' ) design.
Level
Factor
7.0 9.0 10.7 Sugar concentration (%) 29 39 52 Temperature decrease (C°/hr) 3 5 7 Final temperature (°C) 7 12 20
Sugar concentration measured after separation. 29 ± 2 39 ± 2 52 ± 3 42
13 Figure 7 Schemes used in the Taguchi L27(3 ) design for the preferential cold precipitation of lactose, following the design of Table 4. 43 samples, each with a unique pH and sugar concentration combination.
3. Each of the nine solutions was further divided into three 3 mL volumes. Each sample was placed in an agitating water bath set at
60° C and 100 rpm. Each sample was then cooled following the factor levels of the experimental design. At 35°C, all samples were seeded with 2 mg lactose monohydrate. Samples were further cooled to their assigned final temperature by dropping waterbath temperature every 15 minutes, at which time they were moved to a refrigerated incubator and held for 24 hours. All the samples were cooled one at a time using three similar agitating waterbaths and
3 refrigerated incubators set at 7, 12, and 20°C (Forma-Scientific
Inc., Marietta, OH).
4. The decant layer was removed by pipette and diluted to 25 mL in a volumetric flask. The precipitate was resolubilized with warm
ddH20 and agitation, and then diluted to 2 5 mL.
3. 4b2 Calculation of lactulose yield and sugar ratio
Measured lactulose and lactose concentrations (mg/mL) for the decant and precipitate portions of each sample were adjusted based on the standard curves of Section 3.5b4 and then adjusted for volume: lactulose/lactose (mg/mL) = [carbohydrate (mg/mL)1(25 mL) (3.0 mL) where: carbohydrate = sugar adjusted for standard (mg/mL) curve. 25 mL = volume after dilution 3.0 mL = initial sample volume
(Equation 4)
Percent lactulose yield and sugar ratio were calculated using the following formulae: 44
%lactulose yield = _ (decant lactulose mg/mL) X 100%
{(decant lactulose mg/mL) + (precipitate lactulose mg/mL)}
(Equation 5)
%lactose yield = _ (decant lactose mg/mL) X 100% ._
{(decant lactose mg/mL) + (precipitate lactose mg/mL)}
(Equation 6) sugar ratio = decant lactulose (mg/mL) decant lactose (mg/mL) (Equation 7)
3.4c The second cycle of cold precipitation
Decant and precipitate portions from the first cycle were collected and adjusted to the ratios 50:50 and 14:86 lactulose:lactose respectively to simulate the sugar ratio of decanted and precipitated portions after a precipitation cycle.
After the pH was raised to 10.5 with 5.0 M NaOH, each portion was concentrated using a roto-evaporator to a point at which flakes began to form at a temperature of 60° C. This occurred at approximately 500 mg/mL in the decant portion and 3 50 mg/mL in the precipitate portion. Volumes of 2.0 mL of the adjusted decant were added to each of five screw cap 5 mL graduated test tubes. To five more test tubes were added 2.0 mL of the adjusted precipitate. All samples were cooled from 60° C at a rate of 5C° per hour. There was no lactose seeding of these samples. All samples were held at 20° C for 48 hours, at which time each was decanted, and the decant and precipitate portions resolubilized in separate 10 mL volumetric cylinders. Samples were assayed for lactose and lactulose in the
same manner as after the first precipitation cycle. 45
3.4d Calcium phosphate removal
After receiving the frozen, concentrated whey permeate from the supplier, it was thawed and heat treated to precipitate calcium salts. The pH of the whey permeate was raised to 6.8 with 5.0 M
NaOH and heated in a half jacket steam kettle. The permeate was held at a temperature of 85°C for 30 minutes. After it had cooled and the calcium phosphate settled, the permeate was decanted and the precipitate discarded (Durance Se Cross, 1992) .
3.4e Demineralization
Demineralizing by ion-exchange was used on two occasions in this study. Amberlite IR12 0, a strong acid cation exchange resin, and Duolite A368, a strong base anion exchange resin, were used in sequence for demineralization (Rohm and Haas Inc., Philadelphia,
PA) (Durance Se Cross, 1992) .
1. For most of this study, whey permeate was first decalcified, then heat-processed and cold-precipitated to synthesize and purify lactulose. As a final treatment, the lactulose mixture was demineralized by ion-exchange to remove natural and added salts as well as colour.
The lactulose mixture was pumped downward through 4 mL of
Amberlite IR120 then upwards through 3.8 mL of Duolite A368 at a flowrate of 0.3 mL/min using a peristaltic pump (Econo-column pump,
Bio-Rad Inc., Mississauga, ON). Flex-columns (Mandel) held the resin, each having a 20 micron porous polyethylene bed support and a 1.0 cm inside diameter. Tygon tubing (R-3 606, Cole-Parmer
Instruments Co., Vernon Hills, IL) size 14, inside diameter of 1.6 mm was used throughout. Various volumes of samples at differing 46 solids were demineralized on separate occasions with regeneration after each use. The pH of the mixture after flowing through each column was checked regularly for resin exhaustion.
Regeneration for each resin column was required after use and before the first use. The cation column was regenerated upward
with 6 mL of 1.0 M HC1 and 3 0 mL ddH20 at a flowrate of 0.9 mL/min.
The anion column was regenerated downward with 6 mL of 1.0 M NaOH
and 3 0 mL ddH20 at the same flowrate.
For the newly purchased anion exchange resin Duolite A368, an initial exhaustion/regeneration treatment consisted of three cycles
of 1.1 M HC1 and 1.0 M NaOH, then complete rinsing with ddH20. A previous study, however, suggested further treatment to minimize the odour and taste of the resin which can linger in the demineralized permeate. The resin was soaked in a 50% aqueous solution of methanol for one hour, then one hour in pure methanol, then a 50% solution again for an hour. After thorough rinsing in
ddH20, the resin was heated and held at 95°C until no resin odour was detected, approximately 35 hours.
2. The whey permeate in the first experimental fractional factorial design was initially demineralized by decalcification and ion-exchange to remove natural salts before any other treatment.
This practice was not continued.
The process was similar to the post-treatment demineralization but on a larger scale. Eight litres of thawed deproteinized whey at approximately 5% total solids were demineralized, using larger columns (jacketed Moduline medium-pressure 44 cm diameter columns,
Amicon Division W.R. Grace & Co., Danver, MA) packed with 400 mL of
Amberlite IR120 and 340 mL Duolite A368, a stronger pump 47
(Masterflex 7523-10, Cole-Parmer) and a flow rate of 35 mL/min.
Again the pH of the permeate after using each column was checked regularly for resin exhaustion. One cation resin bed volume could
effectively decationize 16 bed volumes of 5% decalcified whey permeate before exhaustion, and one bed volume of anion resin could
deanionize 15 bed volumes of 5% whey permeate.
The cation bed was regenerated upward with 750 mL of a 1.0 M
HC1 solution at a flowrate of 2 5 mL/min, and then rinsed with 4 L
of ddH20 at a flowrate of 50 mL/min. The anion exchange bed was
regenerated downward with 600 mL of 1.0 M NaOH at a flowrate of 25
mL/min. The bed was then rinsed with 3.25 L of ddH20 at a flowrate
of 50 mL/min.
3.4f Decolourization
While the Duolite A368 partially decolourized the heat treated
purified whey permeate, activated charcoal was used to further
remove the golden to amber colour. The same Flex-column, tubing,
and Bio-rad pump as was used for the post-treatment
demineralization was employed with the charcoal. A 9.5 mL bed
volume of activated charcoal was used for the small amounts of high
solids whey permeate with a downward flowrate of approximately 0.2
mL/min. Large volumes of ddH20 rinsed the sample through the
charcoal. Fresh activated charcoal was used for each
decolourization sample.
3.5 PROXIMATE ANALYSIS
Various assays were performed on the whey permeate at
different stages of processing to test for major compositional 48
changes taking place. These tests included measurements of total
solids, ash, nitrogen, protein, pH, titratable acid, lactose,
lactulose, glucose, fructose, and galactose. The whey permeate was
continually diluted and concentrated during processing so most test
results were based on the total solids of each sample.
3.5a Sampling throughout the process
The proximate analysis samples were all taken from one
processing batch, tracking decalcified whey permeate through
retorting and precipitation to a demineralized and decolourized
mixed carbohydrate solution. In this way, the same sample was
tested at each processing stage and volumes were controlled and
recorded.
3.5b Enzymatic Carbohydrate assays
3.5bl Quantitative assays for lactulose, lactose, glucose, and fructose.
The enzymatic assays used for the quantification of lactulose,
lactose, glucose, and fructose in a mixed carbohydrate solution
followed closely the lactulose assay method of Andrews (1984), but
with the following modifications.
1. The beta-galactosidase from Aspergillus oryzae was used in the
assay. Consequently, the pH of Buffer I was reduced from 7.5 to
5.0, the optimum pH for "Fungal Lactase 100,000" for hydrolysis as
outlined by the supplier.
2. Due to the higher concentration of carbohydrate in samples in
the present study, the amount of sample added to the cuvette for measurement was diluted ten-fold. 49
3. The method was adapted to include the measurement of lactose, fructose and glucose.
4. Fructose and lactulose were assayed in conjunction with glucose and lactose respectively.
The carbohydrates were assayed using the following buffers and solutions:
Buffer I - A 0.13 M citric acid - 0.31 M Na2HP04 0.33 mL (Mcllvaine) buffer of pH 5.0, containing
0.0041 M MgS04.7H20 and 0.046 M sodium azide as preservative.
Buffer II - A 0.75 M triethanolamine hydrochloride (Sigma) 1. 0 mL and 0.010 M magnesium sulphate buffer adjusted to pH 7.6 with 5.0 M NaOH.
Buffer III Buffer II diluted five-fold 0.4 0 mL
Enzyme Lactose and lactulose assays: a suspension of 0.10 mL "Fungal Lactase 100,000" beta-galactosidase from Aspergillus oryzae (Solvay Enzymes Inc., Elkhart, IN) of activity 230 units/mg (Section 3.5b2).
0.10 mL Glucose and fructose assays: ddH20
Oxidation Solution Lactulose and fructose assays: an aqueous solution 0.05 mL of 0.003 g glucose oxidase from Aspergillus niger grade II and 0.15 mL catalase (Boehringer Mannheim International Ltd., Laval, PQ)
0.05 mL Lactose and glucose assays Lactose/ lactulose assay
Glucose/ fructose assay: ddH20
ATP - An aqueous solution of 0.082 6 M disodium 0.10 mL Adenosine 5'-tetrahydrogen triphosphate (Boehringer Mannheim) and 0.47 M sodium carbonate
NADP - An aqueous solution of 0.013 M Beta- 0.10 mL nicotinamide adenine dinucleotide phosphate (Sigma)
HK/G6PD - A hexokinase and glucose-6-phosphate-dehydrogenase 0.02 mL solution (Boehringer Mannheim)
PI - A phosphoglucose isomerase solution (Boehringer 0.004 mL Mannheim) 50
1. To 0.50 mL of mixed carbohydrate sample was added Buffer I,
Enzyme, and ddH20 to 5 mL total volume and held 15 - 17 hours at
4 5°C in a gently agitating waterbath. Lactose and lactulose were hydrolyzed to glucose/ galactose and fructose/ galactose
respectively.
2. To the mixture was added Oxidation Solution, Buffer III, and
0.2 0 mL of 0.3 3 M NaOH. Two drops of silicone antifoam reduced
foaming during two hours of continued strong aeration at 45° C.
Aeration was accomplished by pumping air, driven by an air
compressor via thin tubing, to the bottom of the liquid-filled test
tubes. Then, 0.10 mL of the NaOH was added and the solution was
further incubated at 45° C for 15 minutes. Immersing for 20 minutes
in a boiling water bath denatured all added enzymes.
3. The solution was diluted to 10 mL and filtered (#42, Whatman
Ltd. , Maidstone, UK) . Into a cuvette (UV grade methacrylate,
Fisher Scientific, Ottawa, ON) were added 0.10 mL filtrate, 1.9 mL
ddH20, Buffer II, ATP, and NADP. The sample absorbance was measured
(Al, in Equation 8) at 340 nm on a UV visible spectrophotometer
(Shimadzu Scientific Instruments, Columbia, MD). To this was added
HK/G6PD, and incubated at room temperature for 15 minutes. Fructose
and glucose were phosphorylated to fructose-6-phosphate and
glucose-6-phosphate respectively. The oxidation of the latter was
coupled with a reduction of NADP which correlated to an increase in
extinction at the wavelength 340 nm (A2, in Equation 8). PI was
added with a further 2 0-minute incubation. The fructose-6-
phosphate isomerized to glucose-6-phosphate, and the latter
oxidized with residual G6PD, again causing a reduction in NADP (A3,
in Equation 8). 51
The difference in extinctions at 340 nm was proportional to
the amount of carbohydrate present in the mixture using the
following equation.
Carbohydrate mg/mL = (AE)(V)(MM)(D) 1000(1)(v)(e) where: AE = absorbance difference (A2 - Al) lactose and glucose assay (A3 - A2) lactulose and fructose assay V = total volume in cuvette, 3.22 mL MM = molecular mass of lactulose and lactose, 342.31 g/mole. molecular mass of glucose and fructose, 180.16 g/mole. D = dilution of sample, 20 1 = optical light path of cuvette, 1 cm v = volume of sample in cuvette = 0.1 mL e = molar extinction coefficient of reduced NADP at 340 nm, 6.31/(mmol)(cm)
(Equation 8)
In this assay lactulose was hydrolyzed to fructose and
galactose. Lactulose content was calculated stoichiometrically
from the measurement of fructose. Free fructose in the sample was
assayed separately and then subtracted (as lactulose) from the measured lactulose. A similar calculation was done for the lactose
assay. Lactose content was calculated stoichiometrically from
glucose, and then free glucose was subtracted.
3.5b2 Activity of Beta-galactosidase
The activity of the beta-galactosidase from Aspergillus oryzae was estimated using an artificial substrate, o-nitrophenyl-beta-D-
galactopyranoside (ONPG), instead of lactose (Dobrogosz, 1981).
Beta-galactosidase catalyses the hydrolysis of ONPG in the
following reaction:
ONPG + H20 —> galactose + o-nitrophenol 52
While ONPG is colourless, o-nitrophenol is yellow in alkaline
solution and can be measured spectrophotometrically.
A volume of 4.1 mL of 0.050 M sodium phosphate buffer, pH 7.5, was mixed with 0.2 0 mL of 0.03 2 M reduced glutathione (Sigma
Chemical Co.), and the mixture brought to 30°C. Enzyme was added
in a 0.20 mL volume, and 0.50 mL of pre-incubated 0.010 M ONPG was
then added. After exactly 15 minutes the reaction was stopped by
adding 1.0 mL of Na2C03 and the absorbance at 42 0 nm was measured.
A standard curve was prepared relating absorbance at 42 0 nm to
increasing o-nitrophenol concentrations, Figure 8. Conditions were
identical to the samples' assay except for the substitution of
ddH20 for ONPG (0.05 mL ddH20 less the amount of added o-nitro•
phenol) Linear regression was calculated and tested using ANOVA, in
the manner detailed in Section 35b4.
After the absorbances of test samples had been adjusted
against the standard curve, the following equation estimated enzyme
activity.
1 unit of enzyme activity1 = The amount of enzyme which produced 1 micromole of o-nitrophenol/hour
1 Enzyme activity is defined under the conditions specific to this assay. (Equation 9)
The beta-galactosidase used in this study was tested on two
occasions, at the beginning and end of two years of use. For each
test the enzyme was assayed in triplicate.
The two enzyme sample results were then tested using a two-
tailed variance ratio test to determine whether the samples came
from the same sample population using the following calculation: 53
Mean from 3 replicate samples. Error bars = ± 1 standard deviation. Regression curve: y = (7.6)x + 0.047 r2 = 0.992 n = 18
Figure 8 The standard curve of the absorbance of o-nitrophenol at 420 nm to determine the activity of the beta-galactosidase "Lactase 100,000" using ONPG hydrolysis. 54
2 2 F = s , / s 2
where s2 = sample variance (Equation 10)
3.5b3 Quantitative assay for galactose
Galactose was measured in mixed carbohydrate solutions using the Lactose/D-Galactose UV-method test kit (Boehringer Mannheim, cat no. 176303). D-galactose was oxidized by nicotinamide adenine dinucleotide (NAD) to galactonic acid in the presence of beta- galactose dehydrogenase. The spectrophotometer was used at wavelength 340 nm, measuring the sample before (Al) and after (A2,
in Equation 11) the addition of the beta-galactose dehydrogenase.
The amount of NADH formed was stoichiometric with the amount of galactose present, using the formula: galactose mg/mL = (AE)(V)(MM)(D) 1000(1)(v)(e) where: AE = absorbance difference, (A2-A1) V = total volume in cuvette, 3.30 mL MM = Molecular mass of galactose, 180.16 g/mole D = dilution of sample, 10 1 = optical light path of cuvette, 1 cm v = volume of sample in cuvette, 0.1 mL e = molar extinction coefficient of reduced NAD at 340 nm, 6.31 L/(mmole)(cm)
(Equation 11)
3.5b4 Standard curves for carbohydrate assays
Linear regression standard curves were prepared for all
carbohydrates using the described enzymatic assays. Aqueous
solutions of carbohydrate mixtures containing varying levels of the
five sugars were prepared to predict sugar concentrations in the
lactulose preparation at different stages of the study. Table 10 55
Table 10 Standard solutions containing varying ratios of five carbohydrates prepared for standard curves of all enzymatic assays.
Standard Aqueous Solutions B C D E Carbohydrate g/ 2 5 mL (% of sugar1)
lactose monohydrate2 1.8 3.4 1.0 2.9 2.4 (50) (93) (27) (80) (66) lactulose2 1.8 0.11 2.4 0. 63 1.0 (50) (3.0) (68) (18) (29) galactose2 0.018 0.11 0. 035 0.070 (0.50) (3.0) (1.0) (2.0) glucose 0.052 0. 035 0. 018 0. 070 (1.5) (1.0) (0.50) (2.0)
fructose 0.070 0.035 0. 018 0. 052 (2.0) (1.0) (0.50) (1.5)
Lactose monohydrate calculated as lactose Assayed carbohydrates at a 10/1 dilution 56
lists the five mixed carbohydrate solutions used in each standard
curve. All five solutions along with ddH20 were assayed in triplicate and sampled undiluted for glucose and fructose, and at
a 10 fold dilution for lactose, lactulose, and galactose.
The standard curve of glucose is shown in Figure 9. The means were calculated from replicate samples and error was defined as
± 1 standard deviation. The coefficient of determination, r2, measured the strength of the straight-line relationship by providing the proportion of the total variation in Y accounted for
by the fitted regression.
r2 = regression sums of squares total sums of squares
(Equation 12)
ANOVA was used to test the significance of the linearity and
slope of the regressional curve for each carbohydrate assay, Table
11 for glucose. The null hypotheses were defined as the curves
being linear and the slopes being equal to zero. The standard
curves for galactose and lactose, along with summaries of ANOVA
results are illustrated in Figures 10 and 11.
The standard curve of lactulose is shown in Figure 12, the
ANOVA results in Table 12. Regression curves for lactulose assayed
alone and in conjunction with lactose were tested for the
significance of linearity and slope. Then the two lactulose
regression curves were compared for significant differences in
slope and elevation using the following equations. The two fructose
regression curves were tested and compared in the same manner,
Figure 13 and Table 13. 57
0.00 0.50 1.00 1.50 2.00 2.50 3.00
Added glucose (mg/mL)
V
Experimental Data1 Regression Curve2
Mean from 3 replicate samples. Error bars = ± 1 standard deviation. Regression curve: y = (1.1)x - 0.0070 r2 = 0.998
Figure 9 The standard curve of glucose using an enzymatic spectrophotometric assay. 58
Table 11 Analysis of variance testing the significance of slope and linearity of the regression curve calculated for the glucose standard curve.
3 Source of Variation Degrees of Mean Square F-value Freedom (x 10"3)
Total 14 1180 Linear Regression1 1 16500 7600 Residual 13 2 .1 6 Deviations from 3 1. 33 0.55n.s, Linearity2 Within Groups 10 2.41
1 Testing the hypothesis that the slope of the regression curve is zero.
2 Testing the hypothesis that the regression curve is linear.
3 F = Q 07 r0.01(1)1,13
Fr = 3 71 0.05(1)3,10 Significant at p 0.01. n.s. not significant at p > 0.05. 0.50
—i—i—i—i—i—i—i—i—i—i—i i i i i i i i i i i i i i i *i 0-00 0.10 0.20 0.30 0.40 0.50
Added galactose (mg/mL) V
Experimental Data1 Regression Curve2
Mean from 3 replicate samples. Error bars = ± 1 standard deviation. Regression curve: y = (0.96)x + 0.00090 r2 = 0.997 n = 15 The regression curve was linear (p>0.05). The slope of the regression curve was not zero (p^O.Ol).
Figure 10 The standard curve of galactose using an enzymatic spectrophotometric assay. 60
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Added lactose (mg/mL) v Experimental Data1 Regression Curve2
Lactose calculated from lactose monohydrate added. Measured lactose less added glucose. Mean from 3 replicate samples. Error bars = ± 1 standard deviation.
Regression curve: y = (1.0)x - 0.059 r2 = 0.998 n = 18 The regression curve was linear (p>0.05). The slope of the regression curve was not zero (p<0.01).
Figure 11 The standard curve of lactose using an enzymatic spectrophotometric assay. 61
Measured lactulose less added fructose. Mean from 3 replicate samples. Error bars = ± 1 standard deviation.
Regression curve: y = (0.92)x + 0.034 r2 = 0.997
Figure 12 The standard curve of lactulose using an enzymatic spectrophotometric assay. t
62
Table 12 Analysis of varianc-e 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.
Source of Variation Degrees of Mean Square F-value Freedom (x 10"3)
A Lactulose assayed alone:
Total 17 10700 Linear Regression1 1 182000 22000** Residual 16 8.27 Deviations from 4 4 . 50 0.47n.s. Linearity2 Within Groups 12 9.52
B Lactulose assayed in conjunction with lactose:
Total 17 10400 Linear Regression1 1 175000 2900** Residual 16 60. 6 Deviations from 4 69 . 8 1.2n.s. Linearity2 Within Groups 12 57 . 6
Slope comparison3: t = 0.96 n.s. Elevation comparison4: t = 0.69 n.s.
C Lactulose assays combined5:
Total 35 10200 Linear Regression1 1 357000 11000** Residual 3 4 33 . 8 Deviations from 4 Linearity2 54.5 1.8n.s. Within Groups 3 0 31.1
1 Testing the hypothesis that the slope of the regression curve is zero. 2 Testing the hypothesis that the regression curve is linear. 3 Testing the hypothesis that the slopes of both regression curves are equal. 4 Testing the hypothesis that the elevations of both regression curves are equal. 5 Combined results of assays A and B. Significant at p ^ 0.01. n.s. not significant at p > 0.05. 63
Mean from 3 replicate samples. Error bars = ± 1 standard deviation. Regression curve: y = (1.0)x + 0.025 r2 = 0.997
Figure 13 The standard curve of fructose using an enzymatic spectrophotometric assay. 64
Table 13 Analysis of variance testing the slope and linearity of the regression curve calculated for the fructose standard curves and a t-test comparison of the slope and elevation of the curves.
Source of Variation Degrees of Mean Square F-value Freedom (xlO"3)
A Fructose assayed alone:
Total 14 1110 ** Linear Regression1 1 15600 9300 Residual 13 1. 68 Deviations from 3 0.70 0.35n.s. Linearity2 Within Groups 10 1. 98
B Fructose assayed in conjunction with glucose:
Total 14 1080 Linear Regression1 1 15100 3000** Residual 13 5. 11 Deviations from 3 7 . 63 1.8n.s. Linearity2 Within Groups 10 4.35
Slope comparison3: t = 0.82 n.s. Elevation comparison4: t = 0.94 n.s.
C Fructose assays combined :
Total 29 1060 Linear Regression1 1 30600 9400** Residual 28 3.27 Deviations from 3 5.24 1.7n Linearity2 Within Groups 25 3 . 04
1 Testing the hypothesis that the slope of the regression curve is zero. 2 Testing the hypothesis that the regression curve is linear. 3 Testing the hypothesis that the slopes of both regression curves A and B are equal. 4 Testing the hypothesis that the elevations of both regression curves A and B are equal. 5 Combined results of assays A and B. Significant at p 0.01. n.s. not significant at p > 0.05. 65
Slopes:
t = . (b1 - bJ ^ ^ 2 2 1/2 [(S Y:X)p/(2x2)1 + (S^y:X)p/(2x )2] where: b = 2xy /£x2
2 2 2 2 2 2 2 (S y.x)p = {fXY , ~ (sxy) 1/^x 1l + fy 2 - (£xy) 2/g x 2H
[(DF, - 2) + (DF2 - 2)]
Accept that the slopes are equal if t > tQ05(2 ) v where:
v = [(DF, - 2) + (DF2 - 2)]
(Equation 13) Elevations:
t = LLi,^i2) - b(x1_J^g2)i , 2 1/2 HS^.xJJl/n, + l/n2 + (X, - X2) /Ac} where:
2 2 2 2 2 2 (S Y:X)c = {f (^y )1+(£,y )2l - r f^xy) ,+ fcSxy)2 1 / [ (£x ),+ (Sx ) 21
n, + n2 - 3
2 2 Ac = (fix ), + (£x )2
Accept that the elevations are equal if t > t 05(2) v
(Equation 14)
3.5c Thin layer chromatography assay of carbohydrates
3.5cl Qualitative assay of carbohydrates
Thin layer chromatography (TLC) using aminopropyl-bonded silica plates was used to separate and identify sugars following the method of Doner et al. (1984).
Silica plates (Fisher Scientific Redi Plate Silica Gel G, 250 microns, calcium bonded) were first derivatized. 2 0 cm x 2 0 cm plates were immersed in a TLC developing tank with a 1.0% 3-amino- propyl triethoxysilane (3APTS) dry hexane mixture for 15 minutes.
The hexane was initially dried overnight over calcium chloride in a desiccator. The plates were then placed horizontally, covered in 66 dry hexane for 15 minutes to rinse off excess 3APTS. Plates were dried at 60° C (> 30 in. Hg) in a vacuum oven (Model #5850-5
National Appliance Co., Portland OR) for a further 15 minutes.
Aqueous solutions of 2 0% lactose, lactulose, glucose, galactose, fructose, and tagatose and a combination of 8% of all sugars were used as references to identify sugars of experimental samples. Standards (0.05 or 0.10 uL) were spotted at the plate's baseline. Concentrated whey permeate samples of 0.10 or 0.15 uL were spotted on the same plate.
Plates were irrigated with a 70:30 acetonitrile:ddH20 mixture in a TLC developing tank. Spots were made visible by spraying with a 6:4:3 tert-butanol:ethanol:sulphuric acid mixture and heated at
180°C for 5 minutes.
Rf factors were calculated and compared to Rf factors of the standard sugars for identification. The Rf factor is defined as the rate of movement of the leading edge of the solute zone divided by the rate of movement of the leading edge of the solvent zone.
In any given run, however, the distance travelled is proportional to the rate of movement. The Rf factor for this study then, was calculated as follows.
Rf = distance the solute moved distance the solvent moved where: The distance the solute moved is measured as the distance between the origin and the leading edge of the visible spot. The distance the solvent moved is measured as the distance between the origin and the mean of the solvent front line measured at several points.
(Equation 15)
3.5c2 Tagatose identification by TLC and spectrophotometry
A 70:30 acetonitrile:ddH20 solution and aqueous solutions of 67 lactose, lactulose, fructose, glucose, galactose, and tagatose were scanned on a spectrophotometer within the range of 200 to 400 nm, to determine the maximum absorbance wavelength of each (Table 14).
Silica plates were derivatized with 3APTS. A 20% tagatose aqueous solution and a reference mixture of 8% aqueous solutions of tagatose, glucose, and fructose were spotted at the baseline of one side of the 20 cm x 20 cm plate for spot visualization. Then, tagatose aqueous solutions of 0, 5.0, 10, 30, 50, 80, 100, and 150 mg/mL and concentrated process samples were spotted along the remaining baseline, all at 0.10 uL.
Plates were irrigated with 70:30 acetonitrile: ddH20 for 3 0 minutes. The plates were then protected, exposing only the two selected spots to the detection spray. Keeping the remaining spots protected, a hot air drier heated the sprayed area until the spots were visible.
Using a scalpel and the visible spots as a guide, tagatose standards and samples were scraped from the plate. As tagatose, glucose, and fructose all had absorbance peaks at 256 nm, the combination standard was used as a guide to ensure that only tagatose was removed. The samples were then mixed well with 1.0
mL ddH20, filtered (Whatman #541) , and rinsed with 3 mL ddH20 into a visible/UV cuvette.
A standard curve relating tagatose concentration and absorbance at 256.0 nm was prepared (Figure 14). The tagatose contents of process samples were then estimated based on absorbance at 2 56.0 and the standard curve. 68
Table 14 The absorbance of aqueous carbohydrate solutions and the TLC solvent by spectrophotometer between 2 00 and 4 00 nm.
Carbohydrate Peak Absorbance Wavelength (nm) Solution
Acetonitrile:ddH20 2 3 3.0
Lactose 255.8 Lactulose 255.8 Glucose 256.0 Fructose 256.0 Tagatose 256.0 Galactose 256.4 69
Regression Curve: y = (.00072)x + 0.018 r2 = 0.986 n = 10
Figure 14 The standard curve of the absorbance of tagatose concentrations at 256 nm. 70
3.5d Determination of nitrogen
3.5dl Determination of total nitrogen
A micro-Kjeldahl method following the study of Concon and
Soltess (1973) was used to determine the total nitrogen content at various steps in the process. As described in that study, samples
(from 0.50 g to 5.00 g) were mixed with a salt-catalyst mixture of potassium sulphate and mercuric oxide. Then the sample was digested with sulphuric acid and hydrogen peroxide until the protein was converted to ammonium sulphate. A chemical colourimetric method was used to determine ammonia content in the sample, using the "Technicon Autoanalyzer II", (Technicon
Instruments Co., New York, NY). The ammonia was adjusted for an internal standard, then the nitrogen was calculated and converted to a percentage (w/w).
Due to excessive foaming of the high carbohydrate sample in the current study, one change was made to the 19 73 procedure.
Digestion flasks of 100 mL were used instead of the standard 3 0 mL flasks for some samples. As well, the process took approximately double the 10 minute digestion time suggested in the method.
3.5d2 Determination of protein
An estimate of total protein was determined using the Bio-Rad
Protein Assay, micro-assay procedure. This dye-binding assay measured the colour change of Coomassie Brilliant Blue G-250 at a wavelength of 595 nm on spectrophotometer in response to protein concentration. Samples were diluted if necessary, dye was added, and after one hour the samples' absorbances were measured at 595 nm. Aqueous sugar solutions of similar concentration were used as 71 sample blanks. Blanks and a standard curve were prepared fresh for each testing batch. The commercially prepared standard for the
standard curve was a bovine gamma globulin protein (Bio-Rad Chem.)
and was prepared and tested at five concentrations (Figure 15).
3.5e Determination of total solids and ash
Total solids and ash were determined in sequence. Samples of
from 1 to 5 grams of whey (depending on sample concentration) were
weighed into pre-ashed, pre-weighed crucibles. The samples were
first concentrated at 60°C for 6 hours and then held in a vacuum
oven at 60°C for a further 15 hours, under > 3 0 in. Hg of pressure.
The portion of the initial sample which remained after vacuum
heating was calculated to be total solids.
These same samples were then transferred to a muffle furnace
(Box Type Muffle furnace, Blue M Electric Co.) for 15 hours at a
temperature of 425°C to 450°C. The remaining white residue after
heating was considered ash. Dark spots in the residue were
dampened with a small amount of ddH20 and ashed for a further 15
hours.
3.5f Determination of pH and titratable acidity
The pH of samples was determined using a Corning pH meter,
model 220, calibrated against standard pH solutions.
Titratable acidity (AOAC, 1990) was tested in both the
deproteinized whey and the retorted whey. Lactic acid in the whey
permeate was estimated using 0.10 M NaOH and phenolthalein as an
indicator. A conversion of 1.0 mL 0.10 M NaOH equalling
0.0090 g lactic acid was used for calculation. 72
Regression Curve: y = (0.0088)x + 0.072 r2 = 0.963 n = 19
Figure 15 The standard curve of the absorbance of bovine gamma globulin protein at 595 nm for the Bio-Rad Protein Assay. 73
To provide an estimation of the amount of acid produced during the heat treatment of lactose conversion, a different indicator and calculation were used. The pH of the permeate sample before heat treatment was 10.5; after heat treatment the sample pH dropped to
7.15. Berg and van Boekel (1994) suggested that the majority of acid produced during lactose degradation was formic acid; it was assumed for calculation that this was the only acid produced. A volume of 1.0 M NaOH was added to the post-heated permeate sample to bring the pH back up to 10.5. The NaOH used to raise the pH would be proportional to an estimate of the amount of formic acid produced. Using the stoichiometry of the reaction of formic acid and NaOH to produce sodium formate, the amount of formic acid reacting with the added NaOH was calculated. 74
4. RESULTS AND DISCUSSION
4.1 OVERVIEW OF PROCESS
This study describes a sequence of steps producing a high lactulose, mixed carbohydrate syrup from deproteinized whey.
Figure 4 (p.29) illustrates the overall process in its final stage.
In summary, the deproteinized whey was first decalcified, then heat treated under specific conditions to convert some of the lactose to lactulose, partially purified by cold precipitation, and finally deionized, decolourized, and concentrated.
Analyses of the lactulose preparation at the various steps in the process tracked the major compositional changes which occurred.
The results recorded in Table 15 were gathered after the fractional factorial experiments of conversion and purification, using the selected process conditions and levels of each study. Table 15 provides the best results of the lactulose preparation process in its final stage. Results were based on the total solids of each sample as their dilutions varied greatly. While this adjustment made comparisons possible, some comparisons were still difficult.
Minerals and sugars were added and removed from the preparation at various times, altering the amount of the solids.
Whey UF permeate (sample A in Table 15) is similar to the UF cheddar permeate of Table 3 (p.10) when the concentrations are adjusted to reflect concentrations based on the total solids. The lactic acid content of the UF permeate used in this study is somewhat lower than the sample in Table 3, and the nitrogen as well as protein content is substantially higher. The difference in protein is probably due to a difference in ultrafiltration effectiveness between the two samples. 75
Table 15 Proximate analysis, based on % total solids, of whey permeate at different stages of lactulose manufacture and purification.
Stages Of the process1 A B C D E F G
Solids (%) (w/v) 5. 20 11.8 9 .77 10.2 9. 55 4. 16 2 .5 8
Relative Solids Weight2 100 104 . 4 48 . 9 41. 3 34. 1
Ash (%)3 8. 04 7.68 12 .9 12.5 11. 6 1. 67 1. 82
Nitrogen (%) (w/w)3 0. 61 0. 61 0. 17 Protein (%)3 0. 48 0.44 0.32 0. 094 PH 6. 1 6.5 10 .5 7.2 5. 0
Titratable Acid4 (%) 0. 089 1.6
Lactose (% w/v)3 83 78 72 54 22 24 26 Lactulose (% w/v)3 0. 55 0.89 5 .4 19 55 58 59 Glucose (% w/v)3 0. 68 0.75 0 .70 0.53 0. 58 0. 77 1. 0 Fructose (% w/v)3 0. 092 0.13 0 .38 0.44 0. 40 0. 64 0. 81 Galactose (% w/v)3 2 .2 2.4 2 . 3 5.2 5. 1 4. 7 5. 0
Sample A: Deproteinized whey Figure 4 B: Decalcified whey C: Modified whey D: Retorted whey E: Purified lactulose preparation F: Deionized lactulose preparation G: Decolourized lactulose preparation 2 Relative solids weights based on sample volumes. 3 Calculated based on total solids of the sample, section 3.5e. 4 Titratable acid calculated as lactic acid for sample A and formic acid for sample D, section 3.5f. 76
The final product, sample G - decolourized lactulose preparation, is quite different in composition from the lactulose syrups detailed in Table 1 (p.6). In that typical commercial lactulose syrup, lactulose is the dominant sugar, up to 70% (w/v).
Lactose, galactose and other sugars are present in much lesser amounts, less than 13% (w/v) each. The final lactulose preparation of the current study was not concentrated to a syrup so straight comparison is impossible. However, of the total solids of sample
G, just under 60% was lactulose and 2 6% was lactose. Sample G had much less lactulose and substantially more lactose than commercial syrups.
4.2 THE CONVERSION OF LACTOSE TO LACTULOSE
The influence of eight factors - pH, NaOH, concentrations of sodium phosphate, citrate, and lactose, substrate purification, heating temperature, and heating time - on lactulose yield during heat treatment of whey permeate was investigated using two Taguchi
13 designs (L2?3 ) (Figure 5) . The 27 trials for each design are described along with results in Tables 16 and 17. The enzymatic assay for lactulose described in Section 3.5bl includes fructose in its measurement of lactulose. For this reason fructose was assayed separately in the first experimental design, to be subtracted from the measured lactulose. This provided a more accurate measurement of lactulose. As the fructose levels were very low in the high end lactulose yield samples, (less than 0.2% of measured lactulose was fructose) and testing was costly, fructose testing was not continued for design #2. To eliminate the discrepancy between the two designs, reported results of both include free fructose in the 77
Table 16 Design factor combinations and results 13 summary of the L27(3 ) design #1.
Trial Temp.' time lactose phosphate citric PH lactulose no. acid yield (°C) (min) (mg/mL) (mM) (mM) (%)
1 90 5 40 40 40 9.0 4. 0 3.8 2 110 20 40 40 70 10. 5 28 28 3 130 90 40 40 100 12.0 4. 1 4.1 4 110 20 40 70 40 12.0 7. 8 7.6 5 130 90 40 70 70 9.0 15 15 6 90 5 40 70 100 10. 5 18 18 7 130 90 40 100 40 10. 5 12 12 8 90 5 40 100 70 12.0 8. 9 9.5 9 110 20 40 100 100 9.0 27 28 10 130 20 79 40 40 10.5 29 29 11 90 90 79 40 70 12.0 16 16 12 110 5 79 40 100 9.0 18 18 13 90 90 79 70 40 9.0 23 21 14 110 5 79 70 70 10.5 26 25 15 130 20 79 70 100 12.0 8. 7 9.9 16 110 5 79 100 40 12.0 13 13 17 130 20 79 100 70 9.0 23 25 18 90 90 79 100 100 10.5 25 26 19 110 90 115 40 40 12.0 18 17 20 130 5 115 40 70 9.0 27 27 21 90 20 115 40 100 10.5 20 20 22 130 5 115 70 40 10.5 30 30 23 90 20 115 70 70 12.0 18 18 24 110 90 115 70 100 9.0 27 26 25 90 20 115 100 40 9.0 11 11 26 110 90 115 100 70 10.5 28 26 27 130 5 115 100 100 12.0 12 12
Each sample experiment was performed in duplicate, A and B. % lactulose was based on lactulose formed from original lactose. 78
Table 17 Design factor combinations and results 13 summary of the L27(3 ) design #2.
Trial Temp. Time Purif ication1 Phosphate Citrate NaOH lactulos no. (°C) (min) (mM) (mM) (mM) (%)
1 105 10 DPW 00 50 50 26 27 2 115 40 DPW 00 70 100 20 19 3 125 80 DPW 00 90 18 26 26 4 115 40 DPW 50 50 18 25 25 5 125 80 DPW 50 70 50 18 17 6 105 10 DPW 50 90 100 19 18 7 125 80 DPW 100 50 100 10 9 8 105 10 DPW 100 70 18 25 25 9 115 40 DCW 100 90 50 21 21 10 125 40 DCW 00 50 100 20 20 11 105 80 DCW 00 70 18 24 25 12 115 10 DCW 00 90 50 28 29 13 105 80 DCW 50 50 50 27 28 14 115 10 DCW 50 70 100 23 22 15 125 40 DCW 50 90 18 26 26 16 115 10 DCW 100 50 18 25 25 17 125 40 DCW 100 70 50 21 20 18 105 80 DCW 100 90 100 19 18 19 115 80 LS 00 50 18 24 24 20 125 10 LS 00 70 50 29 30 21 105 40 LS 00 90 100 24 23 22 125 10 LS 50 50 100 24 25 23 105 40 LS 50 70 18 18 17 24 115 80 LS 50 90 50 25 25 25 105 40 LS 100 50 50 27 27 26 115 80 LS 100 70 100 17 17 27 125 10 LS 100 90 18 28 28
DPW = deproteinized whey permeate DCW = decalcified DPW LS = lactose solution
Each sample experiment was performed in duplicate, A and B. % Lactulose based on 73 mg/mL initial lactose. 79 lactulose concentration.
Heat processing caused major changes to the adjusted whey permeate, as can be seen in Table 15. Lactulose and galactose contents increased while lactose content decreased. Lactose was converted to lactulose which in turn was degraded to galactose as detailed in Figure 2 (p. 13). A substantial drop in pH from 10.5 to
7.15 was caused by the production of acids, possibly formic and/or isosaccharinic acid.
The maximum lactulose yield was approximately 3 0% of the initial lactose content, and the treatment produced a liquid of golden amber colour. This conversion far exceeded reported studies manipulating lactulose conversion in milk using heat processing and pH adjustment. As described in Section 2.5, Andrews (1989) found a maximum of approximately 2 00 mg/100 mL lactulose in heated milk by individually adjusting lactose concentration, pH, addition of citrate and phosphate, heating temperature, or heating duration.
This corresponds to approximately a 4% lactulose yield. The larger yield in the present study was certainly due in part to the high pH of whey treatment, a pH that is not practical with milk. Still, the lactulose yield achieved in the present study clearly did not reach the 87% conversion level of one borate and triethylamine method (Hicks & Parrish, 1980) .
4.2a The influence of pH and NaOH concentration
In the fractional factorial design #1, pH at three levels -
9.0, 10.5, and 12.0 - were shown to significantly influence
lactulose yield (p<0.01) using ANOVA (Table 18). This is shown in
Figure 16a, with higher yield occurring at the mid pH level of 80
13 Table 18 Analysis of variance [Taguchi L27(3 ) ] , design #1 and #2, obtained from 27 experiments of heat processed demineralized whey permeate.
Source of variation DF1 Mean Square F -ratio2
Design #1
Lactose concentration (Lac) 2 127 45 ** Phosphate concentration (Phos) 2 3 .97 0 .56 n.s.3 Citric acid concentration (Cit) 2 54 . 8 19 ** PH 2 336 120 ** Heating temperature 2 62.2 22 ** Heating duration 2 9.60 3 .4 n.s. Lac X Phos 4 30.5 11 * Phos X Cit 4 51.2 18 ** Cit X Lac 4 32 . 9 12 *
Error 4 2.84
Design #2
Purification (Pur) 2 22 . 3 2 .4 n.s. Phosphate concentration (Phos) 2 27.6 3 .0 n.s. Sodium citrate concentration (SCit) 2 14.3 1 .6 n.s. NaOH concentration 2 88 . 2 9 .6* Heating duration 2 43.7 4 .7 n.s. Pur X Phos 4 9.24 1 .0 n.s. Phos X SCit 4 12.3 1 .3 n.s. SCit X Pur 4 3.51 0 .4 n.s.
Error 4 9.23
Degrees of freedom _0.05(1)2,4 6.94 ^0.01(1)2,4 18 . 0 ^0.05(1)4,4 6.39 ?0.01(1)4,4 16.0 * significant at p ^ 0.05 significant at p ^ 0.01 ** not significant at p > 0.05 n. s 3 The mean square for phosphate concentration was <1.0 and was incorporated into the error sums of squares. Figure 16a 81
T3 CD > CD CO O
o CO
PH Figure 16b 30
25 h
20 h 5 CD >- CD 15 h CO O «—• O 10 CO
5 h
J I L I I I I I I I I I I I I I I 1_ 0 10 20 30 40 50 60 70 80 90 100 110 120
NaOH Concentration (mM)
— V— • —A — Design #2 samples Design #2 pH's Design #1 samples
Figure 16 a,b The influence of pH and NaOH concentration on lactulose yield during heat treatment 13 of whey permeate using an L27(3 ) fractional factorial design. 82
10.5. In design #2, the NaOH concentration factor was substituted for pH. The 2 7 trial results of design #2 were incorporated into the pH figure by using the pHs measured for each sample. These data points, and those in design #1, suggested that a pH of 10.5 to
11.0 led to the highest lactulose yield. A pH of 11.0 was also found by Martinez-Castro and Olano (1980) and Hicks et al., (1984) to correspond to a higher lactulose yield when utilizing boric acid and triethylamine.
NaOH concentration tested by ANOVA in design #2 was found to be significant (p<0.05) at the levels 18, 50, and 100 mM, Table 18.
The lower NaOH concentrations corresponded to a higher lactulose yield, Figure 16b.
The set of conditions used in further study included a pH at between 10.5 and 11.0, set by the addition of NaOH, (Table 8, p.40) .
4.2b The influence of sodium phosphate concentration
Sodium phosphate concentration showed no significant influence
(p>0.05), at the levels of 40, 70 and 100 mM in design #1, nor at the levels of 0, 50 and 100 mM in design #2, Table 18.
Sodium phosphate concentration was shown to be significant in influencing lactulose yield in design #1 as it interacted with lactose (p^0.05) and citric acid concentration (p^O.01), Table 18.
The level of phosphate influenced the effect that lactose concentration had on lactulose yield. At the important higher levels of lactose concentration, the low and mid-phosphate concentrations corresponded to a higher lactulose yield, Figure
17a. In Figure 17b, sodium phosphate concentration influenced the Figure 17a 83 30 i
25 h T ? T 20 -A 1 -vT 1 (D 15 h CO o u 10 h CO
5 h
'l I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 'I I I l' 30 40 50 60 70 80 90 100 110 120
Lactose Concentration (mg/mL)
Figure 17b
30 40 50 60 70 80 90 100 110 120
Citric Acid Concentration (mM)
-A— -•- -V- 40 mM phosphate 70 mM phosphate 100 mM phosphate
Figure 17 a,b The influence of sodium phosphate concentration interacting with lactose and citric acid concentrations on lactulose yield of heat 13 treated whey permeate using an L27(3 ) design. 84 ability of citric acid to increase lactulose yield. At the important mid level of citric acid, it was the lowest phosphate level which corresponded to the highest lactulose yield. In design
#2, with modified levels, no interactions between sodium phosphate and sodium citrate or lactose were found.
The study of Andrews and Prasad detailed in Section 2.5c determined that sodium phosphate was influential in increasing lactulose yield in heat treated milk and it was suggested that it acted as a catalyst in the LA reaction. In that study, sodium phosphate was tested along with (but independent of) citric acid and the latter proved a better catalyst (Andrews & Prasad, 1987).
In the current study, sodium phosphate addition was not included in further study due to its questionable effectiveness and higher cost.
4.2c The influence of citrate concentration
In design #1, citric acid at 40, 70, and 100 mM levels showed a significant influence (p<0.01) on lactulose yield, (Table 18).
These results are in line with those of Andrews and Prasad (1987), who found that citric acid influenced lactulose yield in heat treated milk. In design #2, citric acid was replaced with sodium citrate, more suited for higher pH solutions. The citrate levels were narrowed to 50, 70, and 90 mM and there was no significant influence (p>0.05). Figure 18 illustrates the results of both designs.
Citric acid displayed a significant interaction with lactose and phosphate concentrations in Table 18. In Figure 19a, the mid level of citric acid and the highest lactose concentration led to 85
CD > CD CO o o CO
30 40 50 60 70 80 90 100 110 120
Citrate Concentration (mM)
- •-
Design #1 samples Design #2 samples
Figure 18 The influence of citric acid / sodium citrate concentration on lactulose yield during heat treatment of whey permeate using combined 13 results of L27(3 ) designs #1 and #2. Figure 19a 86
2 g> >- CD CO o
o CO
30 40 50 60 70 80 90 100 110 120
Lactose Concentration (mg/mL) Figure 19b
2
u CO
30 40 50 60 70 80 90 100 110 120
Phosphate Concentration (mM)
— A — • •• • - v- 40 mM citric acid 70 mM citric acid 100 mM citric acid
Figure 19 a,b The influence of citric acid concentration interacting with lactose and sodium phosphate concentrations on lactulose yield during heat treatment of whey permeate using 13 an L27(3 ) design. 87 the highest lactulose yield. In Figure 19b, the highest lactulose yield resulted from the combination of no phosphate and again the mid-level of citric acid. There was no significant interaction
(p>0.05) between sodium citrate and sodium phosphate concentration or lactose concentration in design #2, Table 18.
Sodium citrate was added at a level of 50 mM in further study. While there was no significant difference shown among 50,
70, and 90 mM, the lowest was selected as the most economical.
4.2d The influence of lactose concentration and purification
In experimental design #1, lactose concentration was determined to significantly influence lactulose formation (p$0.05)
(Table 18 and Figure 20) . The lower level of 40 mg/mL lactose corresponded to a substantially lower lactulose yield than the higher levels of 79 and 115 mg/mL which produced similar results in yield. Andrews (198 9) found that at levels of normal lactose concentrations (approximately 50 mg/mL) and higher, there was a first order reaction in regard to lactulose yield. Higher concentrations of lactose also increased lactulose yield as they interacted with phosphate and citric acid concentrations as shown in Figures 21a,b.
The rate of purification of the substrate was studied in experimental design #2. When deproteinized whey, deproteinized decalcified whey, and a lactose solution were compared, there was no significant difference (Table 18).
The set of conditions selected for further study, Table 8, included decalcified UF whey permeate of lactose concentration greater than 7 0 mg/mL. Further purification of the whey through 88
Figure 20 The influence of lactose concentration on lactulose yield during heat treatment of 13 whey permeate using L27(3 ) design #1. Figure 21a 89
2 CD > CD co O O CO
30 40 50 60 70 80 90 100 110 120
Phosphate Concentration (mM) Figure 21b 30
25
20 h 2 CD > 15 (D CO o o 10 CO
i i i i I i • I i i i i I i i i i I i i i i I i i i i I i i i i I i i i i I i i i i 30 40 50 60 70 80 90 100 110 120
Citric Acid Concentration (mM)
—A— ••• -V- 40 mg/mL lactose 79 mg/mL lactose 115 mg/mL lactose
Figure 21 a,b The influence of lactose concentration interacting with sodium phosphate and citric acid concentrations on lactulose yield during heat treatment of whey permeate 13 using an L27(3 ) design. 90 demineralization prior to heat treatment was not considered advantageous based on the results of this study and on minimizing cost. The demineralization step after heat treatment and purification removed added as well as natural salts.
4.2e The influence of temperature and time of heat treatment
Temperature and time of heat treatment at the chosen levels played a lesser role in the production of lactulose than was anticipated. While there was a significant difference (p^O.Ol) in the three widespread levels of temperature 90, 110, and 130°C in design #1, there was no difference when the levels were narrowed to
105, 115, and 125°C, (Table 18 and Figure 22). There may have been less conversion to lactulose at 90° C and a higher amount of lactulose degradation at 130°C. It is also possible that a temperature as high as 13 0°C might have shown a higher lactulose yield at a time shorter than 5 minutes.
Heating time showed no significant difference (p>0.05) in design #1 nor in #2. At a range of 5 to 9 0 minutes, there was surprisingly little response in lactulose yield.
4.2f Continuous flow heat exchanger
Using the continuous flow heat exchanger described in Section
3.3, acceptable lactulose conversion rates were noted at a flow rate of 6.8 mL/min. At this flowrate, a 2.3:1 ratio lactose:lactulose solution was produced. At flow rates below 6 mL/min, some of the sugar solution burned onto the interior surface of the piping.
A thermocouple was inserted into the copper piping 177 cm 91
30
25 h
20 h
CD >- CD 15 h oo o
o 10 h CO
'i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i t i i i i i i i r 85 90 95 100 105 110 115 120 125 130 135
Temperature (°C)
- CD-
Design #1 samples Design #2 samples
Figure 22 The influence of temperature on lactulose yield during heat treatment of whey permeate using 13 the combined results of L27(3 ) designs #1 and #2. 92 after entering the oil, roughly 15% of the distance the whey travelled. The whey temperature was within 2C° of the surrounding oil, even when the flowrate was more than doubled, to 15 mL/min.
Another thermocouple, placed at the mid-point of the cooling section of the pipe, monitored the whey permeate in the ice bath.
Here, the permeate temperature was always less than 6°C, even when the flowrate increased to over 30 mL/min.
4.3 PURIFICATION
4.3a Fractional factorial design for cold precipitation
13 A fractional factorial design L27(3 ) was used to test the influence of 4 factors - pH, concentration, the rate of temperature decrease, and the final temperature - on the precipitation of lactose and lactulose in a concentrated and cooled sugar solution.
The substrate for this purification study was a sugar solution which simulated the sugar ratio of the deproteinized decalcified whey after heat treatment outlined in Table 8. The 27 sample solutions of the factorial design were precipitated according to the experimental plan. The samples were then decanted and the precipitate and decant portions were assayed for lactose and lactulose (Table 19). Percent lactulose yield (the percentage of total lactulose which was not precipitated), percent lactose yield
(the percentage of total lactose which was not precipitated) and sugar ratio (the lactulose to lactose ratio in the decant portion) were calculated using equations 5, 6, and 7 respectively.
Different calculations of the data provided a different perspective on the precipitation: while a high lactulose yield is desirable, a 100% yield is achieved with no precipitation whatsoever. Some 93
13 Table 19 Results of the L27(3 ) design, assaying lactose and lactulose in both decant and precipitate portions after cold precipitation of a 28:72 lactulose: lactose solution.
Sample Lactulose Yield1 Lactose Yield2 Lactulose/Lactose3 (%) (%) unprecipitated 0.39 1 96 89 0.42 2 90 61 0. 62 3 83 39 0.95 4 92 59 0. 63 5 90 39 0.91 6 92 88 0.47 7 87 40 0.93 8 96 94 0.53 9 96 87 0.53 10 83 64 0.57 11 77 30 1.1 12 94 92 0.44 13 76 32 0.97 14 98 85 0. 49 15 91 69 0.56 16 100 96 0. 50 17 94 82 0.54 18 87 51 0.80 19 72 27 1.1 20 98 91 0.43 21 92 66 0.59 22 97 93 0.44 23 97 67 0. 57 24 80 41 0.81 25 96 77 0.57 26 87 43 0.92 27 95 97 0. 50
Lactulose (mg/mL) of decant . Lactulose (mg/mL) of decant + Lactulose (mg/mL) of precipitate
Lactose (mg/mL) of decant ._ Lactose (mg/mL) of decant + Lactose (mg/mL) of precipitate
Lactulose (mg/mL) of decant Lactose (mg/mL) of decant 94 precipitation of lactulose inevitably occurs as lactose is precipitated. On the other hand, sugar ratio alone cannot provide information on the lactulose lost in the process.
The results of the experiment included a wide variety of yield and ratio combinations. Lactulose yields ranged from 77% to
100%. This process did not aggressively precipitate lactulose and in all cases preferentially precipitated lactose over lactulose.
The sugar ratios of the decant portion ranged from 0.42 to 1.1, showing a marked increase in the lactulose to lactose ratio from the initial 0.3 9 ratio of the heat treated permeate.
The results of this study certainly illustrate the differences in solubilities between lactulose and lactose at low temperatures, as detailed in Table 5. Samples 3, 5, 7, 11, 13, 19, and 26, deemed by this author to have the most promising results, had on average a lactulose yield of 82% and a 1:1 lactulose: lactose ratio. This ratio is not as desirable as those found in commercial lactulose mixtures, such as those described in Table 1 with a 63-
70:4-8 lactulose:lactose ratio. There was also a substantial loss of lactulose as well. Using the same seven samples as above, there was on average an 18% loss of lactulose through purification. This corresponded to a 64% loss of lactose.
An ANOVA was used to test the significance of the factors and factor interactions on lactulose yield and sugar ratio (Table 20).
The pH and sugar concentration of the solution were found to be significant (p^0.05). The final temperature of cooling and the rate at which the solutions were cooled did not significantly influence precipitation at the chosen levels (p>0.05). The influence of pH and sugar concentration on precipitation is shown 95
13 Table 20 Analysis of variance of the L?7(3 ) design of cold precipitation in a lactulose:lactose solution.
Source of Variation DF Mean Square F-value1'2
ANOVA % lactulose yield
Temperature drop 2 12.6 1.4 n.s. pH 2 81.9 8.8 ** Final temperature 2 24.3 2.6 n.s. Concentration 2 469 51 **
Error 10 9 .28
ANOVA lactulose:lactose ratio
Temperature drop 2 0.00320 0.01 n.s.2 pH 2 0.00460 0.77 n.s. Final temperature 2 0.00770 1.9 n.s. Concentration 2 0.546 140 **
Error 22 0.00397
4. 10 n0.05(1)2,10 ^0.01(1)2,10 7, 56 ;V 05(1)4,10 3 ,4 8 ^0.01(1)4,10 5, 99 V05(1)2,22 3 .4 4 ^0.01(1)2,22 5, 72 * significant at p < 0.05 ** significant at p ^ 0.01 n.s. not significant at p > 0.05
2 The F-values of indicated factors and all interactions were below 1.0 and were incorporated into the error sums of squares. 96 in Figures 23a,b. Both lactulose yield and lactulose:lactose ratio are shown. While a pH of 7, 9, or 11 had no significant effect on the lactulose:lactose ratio, higher pH values increased lactulose yield. Increasing sugar concentration had a dual effect, increasing the lactulose:lactose ratio but also decreasing lactulose yield.
4.3b The second cycle of cold precipitation
The precipitate and decant portions of the first precipitation cycle were adjusted to either 14:86 or 50:50 lactulose:lactose ratio, raised in pH to 10.5, reconcentrated and cold-precipitated a second time. Five 2.0 mL replicate samples of each portion were precipitated a second time, separated, and assayed, (Table 21).
After the second cycle of precipitation, when the decant from the first cycle was used, a lactulose yield of approximately 78% was achieved. The lactulose:lactose ratio was 3.4:1. This means that after a second precipitation cycle, the majority of the sugar is lactulose. After the first precipitation cycle, lactulose and lactose contents were about equal.
When the precipitate portion of the first cycle was used for a second cycle, a much lower lactulose yield of approximately 60% occurred. The lactulose:lactose ratio was approximately 0.66:1, similar to many of the 27 samples from the first cycle.
The efficiency of two precipitations can be calculated theoretically, for example using sample #11 and the mean result of the second cycle. After two precipitation cycles, a substantial
40% of the initial lactulose would be lost during processing.
However, with this process it is possible that the lactulose lost Figure 23a 97 100 1.20
1.00 o CO k_ H 0.80 CD CO TJ O ' cc o CO CD H 0.60 CO CD o co o o H 0.40 CO o CO H 0.20
0.00
Figure 23b 100 1.20
H 1.00 o CO V— H 0.80 CD CO 32 o CD o CD H 0.60 CO CO CD o CO o o H 0.40 CO o CO H 0.20
0.00 25 30 35 40 45 50
Sugar concentration (%)
-A— -•- Lactulose yield (%) Lactulose/lactose ratio
Figure 23 a,b The influence of pH and sugar concentration on the cold precipitation of a lactulose: 13 lactose solution using an L27(3 ) design. 98
Table 21 Results of 5 replicates of a second cycle of cold precipitation in a lactulose: lactose solution.
Sample Lactulose Yield Lactulose/ (%) Lactose ratio Mean ± SD (range)
Decanted portion of the first cycle 50:50 lactulose: lactose 78 ± 8.0 3.4 ± 0.37 (67 - 88) (3.0 - 3.9)
Precipitated portion of first cycle 14:86 lactulose: lactose 60 ± 8.9 0.66 ± 0.18 (51 - 72) (0.49 - 0.90) 99
could be recovered through re-cycling of the precipitate portions.
In Table 15, the lactulose: lactose ratio of Sample E - the
purified lactulose preparation, was 55:22 (or 2.5:1). This is
lower than the 3.4:1 ratio achieved using a lactulose/lactose
solution. This may be due to the natural and added salts in the
whey permeate interfering with precipitation.
4.3c Demineralization and decolourization
Ion-exchange (Section 3.4e) was used to remove natural and
added salts. It can be seen in Table 15 that the ion-exchange was
successful in reducing the ash content of the permeate from an
initial 8.04% (of total solids) and post-purified level of 11.6% to
a level of 1.67% after demineralizing. The ion-exchange did adsorb
some of the carbohydrates as was calculated from the volumes before
and after ion-exchange. There was 6.1% of the initial total solids
lost during ion-exchange. There did not appear to be any
preferential adsorption as the ratio of carbohydrates are similar
before and after demineralization.
The amber colour of the lactulose preparation caused by
nonenzymatic browning during heating was significantly reduced by
activated charcoal (Section 3.4f) and to a small degree by anion
exchange. When the preparation was concentrated, however, a yellow tinge became apparent (Figure 24), which would certainly become more distinct if the preparation was further concentrated to a
syrup. More importantly, when the volumes of the whey permeate before and after decolourization were taken into account, there was a 30% reduction in total solids. Again, the ratio of the carbohydrates remained unchanged. 100
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). 101
4.4 PROXIMATE ANALYSIS
4.4a Carbohydrate standard curves using enzymatic assays
Standard curves were developed for glucose, galactose, lactose, lactulose, and fructose using the five standard solutions detailed in Table 10.
In the standard curve for glucose (Figure 9) , the significances of the linearity and slope of the regressional curve were tested using ANOVA (Table 11) . Similar results are shown for galactose and lactose, Figures 10 and 11. For all three carbohydrate standard curves, the r2 value was 0.997 or greater, the regression curve was linear, and in all, significant positive slopes existed.
Fructose and lactulose were assayed in a somewhat different manner: they were either assayed alone or in conjunction with glucose and lactose respectively (Section 3.5b5). The assay used for these carbohydrates was originally used for quantifying lactulose only. In the method of Hicks et al. (1984), the authors stated that glucose would interfere with the enzymatic assay of fructose. In the current study, lactulose and fructose were assayed with and without removing glucose. If there proved no difference in measuring lactulose or fructose with or without the removal of glucose, then fructose could be assayed with glucose and lactulose could be assayed with lactose with a considerable reduction in cost.
Each regressional curve was first tested separately for slope and linearity. The slope and elevation of the two regression assay curves for each sugar were then compared by a t-test for lactulose and fructose (Tables 12 and 13) . There was no significant 102 difference resulting from glucose removal for either the fructose or lactulose assay. This means that there was no influence on fructose measurement from the glucose present in the samples tested in this study.
The data for each sugar were combined and their regressional curves tested by ANOVA. Standard curves are depicted in Figures 12 and 13, ANOVA results of the combined curves are summarized in
Tables 12 and 13, for lactulose and fructose respectively.
4.4b Activity of beta-galactosidase
The activity of a galactosidase enzyme, its ability to hydrolyze lactose or lactulose, may lessen during conditions of storage. It was important to test the activity of the fungal enzyme at the start and the end of experimentation. The activity of the beta-galactosidase enzyme from the Aspergillus oryzae fungus was estimated to be 23 0 activity units/mg enzyme. One unit of enzyme activity is defined as the amount that produces 1 micromole of o-nitrophenol per hour under the specific conditions described in Section 3.5b2. Using ONPG and following the method in that section, a standard curve was prepared measuring the absorbance of increasing concentrations of the hydrolysed product, o-nitrophenol at 420 nm (Figure 8). The regression curve of the enzyme, tested using ANOVA, was linear at a significance of p .£0.01, and had a significant positive slope. The corresponding r2 value was 0.992.
The beta-galactosidase was used in experimentation for 22 months; the enzyme's activity was assayed in triplicate at the beginning and end of experimentation. Using a two-tailed variance ratio test (Section 3.5b2), there was no significant difference 103 between the variance of the sample sets, concluding they were sampled from the same population. It is suggested then that the activity of the beta-galactosidase did not significantly change during the study.
4.4c Thin-layer chromatography qualitative assay of carbohydrates
Thin-layer chromatography (TLC) was used to determine if significant amounts of any unexpected carbohydrates were present at any stage of the process. Derivatized silica plates, following the method in Section 3.5cl, were spotted with carbohydrate standards, standard mixtures, and process samples. An example of a plate spotted with standard carbohydrates and samples is shown in Figure
25 with corresponding Rf values in Table 22. Doner et al. (1984), used the same TLC method and tested many of the same sugars. Their
Rf findings of lactose (0.187) and lactulose (0.215), were very similar to the values obtained in this study, (0.18 and 0.21-0.22) for lactose and lactulose. The Rf values of the monosaccharides obtained in this study were not consistent with those of Doner et al., although they did occur in the same order. Doner et al., found tagatose to have an Rf of 0.425, fructose 0.406, and glucose
0.352. In this study, tagatose was 0.40, fructose 0.36-0.37, and glucose 0.30.
The deproteinized whey sample, assayed undiluted at 24.9% total solids, had a single strong Rf value of 0.18, similar to the lactose standard. In Table 15, enzyme assays showed the deproteinized whey sample contained levels of lactulose, glucose, and fructose below 0.7% (w/v); none were positively detected by this TLC method. Galactose was not detected either, although shown 104
Standards: #1 1 uL. 20% glucose 0.05 uL #12 2 1 uL. 20% galactose 0.05 uL 13 3 1 uL. 20% fructose 0.05 uL 14 4 1 uL. 20% lactose 0.05 uL 15 5 1 UL. 20% lactulose 0.05 uL 16 6 1 uL. 20% tagatose 0.05 uL 17 7 1 UL. .8% all sugars 0.05 uL 18 8 1 uL. ,10% all sugars 0.05 uL 19 no galactose
Samples; #9 0.15 uL. Decalcified whey....0.1 uL - #20 (24.9% TS) 10 0.15 uL. Precipitated 0.1 uL - 21 preparation (10.4%) 11 0.15 uL. Final preparation...0.1 uL - 22 (23.3% TS)
Figure 25 Thin-layer chromatography on silica plates shows standard sugar solutions at varying concentrations and whey at different stages of process.1 105
Table 22 Rf values of carbohydrate standards and various samples using thin-layer chromatography on a derivatized silica plate.
1 Sample Sample Sample Description Rf (0.10 uL) (0.05 uL) (0.15 uL)
1 glucose 0.28 12 0.32 2 galactose streaked 13 from origin 3 fructose 0.36 14 0.37 4 lactose 0.18 15 0.18 5 lactulose 0.21 16 0.22 6 tagatose 0.40 17 0.40 7 all sugars 0.17-0.36 18 0.17-0.36 8 all less gal 0.17-0.22& 0.30-0.38 19 0.17-0.21& 0.30-0.38 20 deprot. whey 0.18 9 0.18 21 retorted 0.14-0.19 10 purified whey 0.12-0.19 22 decolourized 0.12-0.19 11 purified whey 0.06-0.13& 0.18-0.19
The mean measurement of the solvent front was 14.5 cm. 106 by the enzyme assay to be present in the sample at 2.2% (w/v).
Using this TLC method, galactose is difficult to identify because it does not move up the solvent front, as described in Section
3.5cl.
The lactose preparation after precipitation (10.4% total solids) tested undiluted had overlapping spots from approximately
0.12 to 0.19 Rf value. The dominant carbohydrates according to the enzymatic assay (Table 15) were lactose and lactulose. In the TLC sample, there was no distinct spot at the lactulose Rf of 0.21.
Similar spotting occurred in the decolourized lactulose preparation, at 23.3% total solids, with an Rf range of 0.12 to
0.19 for the lower volume sample. The low end of the spots did not correspond with an identified carbohydrate. Doner et al. (1984), provided the Rf values for many sugars using this TLC method. Few tested sugars possessed an Rf value lower than lactose, but included melibiose, gentiobiose, raffinose, melezitose, maltotriose, and stachyose, none of which have been mentioned in literature involving lactose reactions in milk or whey.
4.4d Tagatose identification by TLC and spectrophotometer
No sample tested showed any spot formation near the tagatose standard's Rf value of 0.40. If tagatose was formed by the heat treatment, the amount was too low to be detected by this method.
A spectrophotometric method (Section3.5c2) was employed in an effort to identify tagatose in a process sample. The standard curve of tagatose using an absorbance of 256 nm, Figure 14, was not significant in its linearity. Dropping the three lowest tagatose concentrations (0, 5, and 10 mg/mL) produced a regression curve 107 which was significant in its linearity and slope. The three process samples tested, decalcified whey permeate at 24.9% total solids, purified whey permeate at 10.4% total solids, and decolourized whey permeate at 23.3% total solids, did not reach 30 mg/mL even though highly concentrated. It is therefore still unknown whether tagatose was produced by the heat treatment and in what amount. It can be said that the amount produced, if any, was likely to be under 3 0 mg/mL in these concentrated samples. This coincides with the findings of Berg and van Boekel (1994) who found no tagatose production in heated milk using HPLC and Troyano et al.
(1992) who found only 13 mg/L using GLC.
4.4e Determination of total nitrogen and protein
A micro-Kjeldahl method was used to determine total nitrogen present in the whey permeate at different stages of the treatment process, (Section 3.5dl). Protein was determined using the Bio-Rad
Protein assay described in Section 3.5d2. The regression curve of the standard curve, Figure 15, was tested using ANOVA.
Total nitrogen and protein, as listed in Table 15, were found to be 0.61% and 0.48% (w/w of total solids) respectively in the deproteinized whey permeate Sample A. In Table 3 (Hargrove et al.,
1976), whey from Cheddar cheese without deproteinization was described as having 1.9% (calculated as w/w of total solids) total nitrogen and 9.0% protein. After ultrafiltration, there was 0.4 6% total nitrogen and 0.18% protein. The UF whey permeate used in the present study contained somewhat higher levels of nitrogen and protein than the example of Hargrove.
In the decalcified whey permeate Sample B, there was 0.61% 108 total nitrogen and 0.44% protein (w/w of total solids). The process of decalcifying the whey permeate did not alter the nitrogen or protein content. In the final product, decolourized purified heat treated lactulose preparation - Sample G, there was 0.17% nitrogen and 0.094% protein (w/w of total solids) . It is presumed that much of the total nitrogen and protein was adsorbed onto the ion- exchange and activated charcoal columns. 109
5. CONCLUSIONS
A high lactulose, mixed carbohydrate preparation was produced without the use of toxic catalysts. The final syrup contained 57%
lactulose, 26% lactose, 5.0% galactose, 1.0% glucose, and 0.81%
fructose, based on total solids. Using TLC, there was no tagatose
detected, and there may have been an unidentified carbohydrate.
The final syrup was not as pure as commercial products, where
lactulose content can be ten-fold greater than lactose.
Manipulating conditions of thermal processing increased
conversion of lactose to lactulose. Taguchi's fractional factorial
experimental design aided in selection of conditions such that
approximately 3 0% of initial lactose was converted to free
lactulose via the Lobry de Bruyn and Alberda van Ekenstein
transformation. From eight initial factors - pH, NaOH
concentration, lactose concentration, purification, heating
temperature, heating time, citrate concentration, and sodium
citrate concentration - and two experimental designs, a set of
conditions was chosen for the heat treatment of whey permeate. In
the preferred method, decalcified UF whey permeate at a pH of
between 10.5 and 11.0 including 50 mM sodium citrate at a lactose
concentration of >70 mg/mL was heat treated at 110° C for 10
minutes.
Partial purification of lactulose was accomplished by
concentrating a sugar solution similar to heat treated whey
permeate and cooling, preferentially precipitating lactose over
lactulose. After one cooling cycle, there was on average a
lactulose yield of 82% and a 1:1 lactulose: lactose ratio.
Taguchi's fractional factorial design was again used determining 110 that pH and sugar concentration each had a significant effect on lactulose yield and the lactulose:lactose ratio in the precipitation decant. The selected conditions for cold precipitation based on these results was to maintain a pH of 10.5, concentrate to 50% lactose and lower the temperature by 5C°/hour from 65°C to 2 0°C, holding for 2 4 hours. 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 40% of the initial lactulose through two precipitation cycles.
Ion-exchange columns removed the majority of the natural and added salts from the lactulose preparations. Activated charcoal was not recommended as it removed most of the brown colour of the permeate but also removed 30% of the total solids.
This heat treatment process did not prove as efficient as current methods of lactose conversion to lactulose, such as using borate and triethylamine. However, the substrate, whey permeate, is a waste-product and a disposal dilemma for producers.
Efficiency in conversion may not be as crucial to a manufacturer as the cost of the process, adherence to local regulations, and customer acceptance. Conversion efficiency may also be overcome with a more effective purification process to remove or hydrolyze excess lactose, thus improving the sugar ratio of the final syrup. Ill
6. ABBREVIATIONS
ANOVA, analysis of variance
3-APTS, 3-aminopropyl triethoxysilane
ATP, adenosine 5'-tetrahydrogen triphosphate
CUT, come-up time
ddH20, deionized distilled water
GLC, gas-liquid chromatography
G6PD, glucose-6-phosphate-dehydrogenase
HK, hexokinase
HPLC, high-pressure liquid chromatography
LA, Lobry de Bruyn van Ekenstein
MW, molecular weight
NAD, nicotinamide-adenine dinucleotide
NADP, nicotinamide-adenine dinucleotide phosphate
ONPG, o-nitrophenyl-beta-D-galactopyranoside
PI, phosphoglucose isomerase
PSE, portal systemic encephalopathy
UF, ultrafiltration
UHT, ultra-high temperature 112
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