2 Lactose
2.1 Introduction
Lactose is the principal carbohydrate in the milks of all mammals; non-
mammalian sources are very rare. Milk contains only trace amounts of
other sugars, including glucose (50 mg l-’), fructose, glucosamine, galac-
tosamine, neuraminic acid and neutral and acidic oligosaccharides.
The concentration of lactose in milk varies widely between species (Table
2.1). The lactose content of cows’ milk varies with the breed of cow,
individuality factors, udder infection and especially stage of lactation. The
concentration of lactose decreases progressively and significantly during
lactation (Figure 2.1); this behaviour contrasts with the lactational trends
for lipids and proteins, which, after decreasing during early lactation,
increase strongly during the second half of lactation. Mastitis causes an
increased level of NaCl in milk and depresses the secretion of lactose.
Lactose, along with sodium, potassium and chloride ions, plays a major role
in maintaining the osmotic pressure in the mammary system. Thus, any
increase or decrease in lactose content (a secreted constituent, i.e. formed
within the mammary gland) is compensated for by an increase or decrease
in the soluble salt (excreted) constituents. This osmotic relationship partly
explains why certain milks with a high lactose content have a low ash
content and vice versa (Table 2.2).
Similarly, there is an inverse relationship between the concentration of
lactose and chloride, which is the basis of Koestler’s chloride-lactose test
Table 2.1 Concentration (%) of lactose in the milks of selected species
Species Lactose Species Lactose Species Lactose
California sea lion
Hooded seal
Black bear
Dolphin
Echidna
Blue whale
Rabbit
Red deer
Grey seal
Rat (Norwegian)
0.0
0.0
0.4
0.6
0.9
1.3
2.1
2.6
2.6
2.6
Mouse (house)
Guinea-pig
Dog (domestic)
Sika deer
Goat
Elephant (Indian)
cow
Sheep
Water buffalo
3.0
3.0
3.1
3.4
4.1
4.7
4.8
4.8
4.8
Cat (domestic)
Pig
Horse
Chimpanzee
Rhesus monkey
Human
Donkey
Zebra
Green monkey
4.8
5.5
6.2
7.0
7.0
7.0
7.4
7.4
10.2
22 DAIRY CHEMISTRY AND BIOCHEMISTRY
5
3
0 10 20 30 40 50 60
Week
Figure 2.1 Changes in the concentrations of fat (A), protein (0) and lactose (0) in milk during
lactation.
Table 2.2 Average concentration (%) of lactose and ash in the
milks of some mammals
Species Water Lactose Ash
Human 87.4 6.9 0.21
cow 87.2 4.9 0.70
Goat 87.0 4.2 0.86
Camel 87.6 3.26 0.70
Mare 89.0 6.14 0.51
Reindeer 63.3 2.5 1.40
for abnormal milk:
YO Chloride x 100
Koestler number =
YO Lactose
A Koestler number less than 2 indicates normal milk while a value greater
than 3 is considered abnormal.
Lactose plays an important role in milk and milk products:
products;
0 it is an essential constituent in the production of fermented dairy
LACTOSE 23
0 it contributes to the nutritive value of milk and its products; however,
many non-Europeans have limited or zero ability to digest lactose in
adulthood, leading to a syndrome known as lactose intolerance;
0 it affects the texture of certain concentrated and frozen products;
0 it is involved in heat-induced changes in the colour and flavour of highly
heated milk products.
2.2 Chemical and physical properties of lactose
2.2.1 Structure of lactose
Lactose is a disaccharide consisting of galactose and glucose, linked by a
pl-4 glycosidic bond (Figure 2.2). Its systematic name is j3-0-D-galac-
topyranosyl-( 1 -4)-ol-~-glucopyranose (a-lactose) or P-0-D-galactopyranosyl-
(1-4)-P-~-glucopyranose (p-lactose). The hemiacetal group of the glucose
moiety is potentially free (i.e. lactose is a reducing sugar) and may exist as an
a- or p-anomer. In the structural formula of the a-form, the hydroxyl group
on the C, of glucose is cis to the hydroxyl group at C, (oriented downward).
2.2.2 Biosynrhesis of lactose
Lactose is essentially unique to mammary secretions. It is synthesized from
glucose absorbed from blood. One molecule of glucose is isomerized to
UDP-galactose via the four-enzyme Leloir pathway (Figure 2.3). UDP-Gal
is then linked to another molecule of glucose in a reaction catalysed by the
enzyme, lactose synthetase, a two-component enzyme. Component A is a
non-specific galactosyl transferase which transfers the galactose from UDP-
Gal to a number of acceptors. In the presence of the B component, which
is the whey protein, a-lactalbumin, the transferase becomes highly specific
for glucose (its K, decreases 1000-fold), leading to the synthesis of lactose.
Thus, r-lactalbumin is an enzyme modifier and its concentration in the milk
of several species is directly related to the concentration of lactose in those
milks; the milks of some marine mammals contain neither a-lactalbumin nor
lactose.
The presumed significance of this control mechanism is to enable
mammals to terminate the synthesis of lactose when necessary, i.e. to
regulate and control osmotic pressure when there is an influx of NaC1, e.g.
during mastitis or in late lactation (lactose and NaCl are major determi-
nants of the osmotic pressure of milk, which is isotonic with blood, the
osmotic pressure of which is essentially constant). The ability to control
osmotic pressure is sufficiently important to justify an elaborate control
mechanism and the ‘wastage’ of the enzyme modifier.
DAIRY CHEMISTRY AND BIOCHEMISTRY
H B
H-C-OH
HO-C-H
HO-C-H
H-C
I
H-C
' CHzOH
I
CHzOH
-$
OH
O-&D-CPLPetopyrPnaPyl~i~)-@-D-Glucopy~naPe : @.Lactose
4 OH OH [xy n
n2
3
0
HO 3
H
HO
OH
H
Figure 2.2 Structural formulae of a- and p-lactose. (a) Fischer projection, (b) Haworth
projection and (c) conformational formula.
LACTOSE 25
Glucose- 1 -phoSPhE
UDP gliiccisr-4-rpinier.osr
gnlncros~llrr~~l~?.\:fr,.cl.vr
*LACTOSE
cr-I~/ctnlDu/ttil?
Glucose
Figure 2.3 Pathway for lactose synthesis.
2.2.3 Lactose equilibrium in solution
The configuration around the C, of glucose (i.e. the anomeric C) is not
stable and can readily change (mutarotate) from the x- to the /?-form and
vice versa when the sugar is in solution as a consequence of the fact that the
hemiacetal form is in equilibrium with the open chain aldehyde form which
can be converted into either of the two isomeric forms (Figure 2.2).
When either isomer is dissolved in water, there is a gradual change from
one form to the other until equilibrium is established, i.e. mutarotation.
These changes may be followed by measuring the change in optical rotation
with time until, at equilibrium, the specific rotation is + 55.4".
The composition of the mixture at equilibrium may be calculated as
follows:
Specific rotation [NIP
a-form + 89.4"
p-form + 35.0"
Equilibrium mixture + 55.4"
Let equilibrium mixture = 100
Let x% of the lactose be in the cr-form
Then (100 - x)% is the p-form
26 DAIRY CHEMISTRY AND BIOCHEMISTRY
oL I
I I I
2 4 6 8
PH
Figure 2.4 Effect of pH on the rate of mutarotation of lactose.
At equilibrium:
89.4~ + 35(100 - X) = 55.4 x 100
x = 37.3
100-x = 62.7
Thus, the equilibrium mixture at 20°C is composed of 62.7% 8- and
37.3% a-lactose. The equilibrium constant, P/a, is 1.68 at 20°C. The
proportion of lactose in the @-form increases as the temperature is increased
and the equilibrium constant consequently decreases. The equilibrium
constant is not influenced by pH, but the rate of mutarotation is dependent
on both temperature and pH. The change from m- to p-lactose is 51.1, 17.5
and 3.4% complete at 25, 15 and O"C, respectively, in 1 h and is almost
instantaneous at about 75°C.
The rate of mutarotation is slowest at pH 5.0, increasing rapidly at more
acid or alkaline values; equilibrium is established in a few minutes at pH 9.0
(Figure 2.4).
LACTOSE 27
2.2.4 Signgcance of mutarotation
The a- and 8-forms of lactose differ with respect to:
0 solubility;
0 crystal shape and size;
0 hydration of crystal form - hygroscopicity;
0 specific rotation;
0 sweetness.
Many of these characteristics are discussed in the following sections.
2.2.5 Solubility of lactose
The solubility characteristics of the a- and /?-isomers are distinctly different.
When a-lactose is added in excess to water at 20°C, about 7 g per 100 g
water dissolve immediately. Some a-lactose mutarotates to the 8 anomer to
establish the equilibrium ratio 62.78 : 37.3~; therefore, the solution becomes
unsaturated with respect to a and more a-lactose dissolves. These two
processes (mutarotation and solubilization of a-lactose) continue until two
criteria are met: - 7 g a-lactose in solution and a P/a ratio of 1.6 : 1.0. Since
the P/sc ratio at equilibrium is about 1.6 at 20"C, the final solubility is
7 g + (1.6 x 7) g = 18.2 g per 100 g water.
When /-lactose is dissolved in water, the initial solubility is -50g per
100 g water at 20°C. Some /?-lactose mutarotates to a to establish a ratio of
1.6: 1. At equilibrium, the solution would contain 30.8 g /? and 19.2 g
a/100 ml; therefore, the solution is supersaturated with a-lactose, some of
which crystallizes, upsetting the equilibrium and leading to further mutaro-
tation of /? -+ a. These two events, i.e. crystallization of a-lactose and
mutarotation of 8, continue until the same two criteria are met, i.e. -7g
a-lactose in solution and a P/a ratio of 1.6: 1. Again, the final solubility is
- 18.2 g lactose per 100 g water. Since 8-lactose is much more soluble than
a and mutarotation is slow, it is possible to form more highly concentrated
solutions by dissolving /?- rather than a-lactose. In either case, the final
solubility is the same.
The solubility of lactose as a function of temperature is summarized in
Figure 2.5. The solubility of a-lactose is more temperature dependent than
that of /?-lactose and the solubility curves intersect at 93.5"C. A solution at
60°C contains approximately 59g lactose per lOOg water. Suppose that a
50% solution of lactose (- 30 g p- and 20 g a-) at 60°C is cooled to 15°C.
At this temperature, the solution can contain only 7 g a-lactose or a total of
18.2 g per 100 g water at equilibrium. Therefore, lactose will crystallize very
slowly out of solution as irregularly sized crystals which may give rise to a
sandy, gritty texture.
Solubility,
g
anhydrous
lactose
I100
g
water
--*-
sggggsggg
LACTOSE
200
100
29
-
2.1
- 1
Figure 2.6 Initial solubility of a-lactose and b-lactose, final solubility at equilibrium (line l),
and supersaturation by a factor 1.6 and 2.1 (r-lactose excluding water of crystallization).
(Modified from Walstra and Jenness, 1984.)
Spontaneous crystallization can occur in the labile area without the
addition of seeding material.
The rate of nucleation is slow at low levels of supersaturation and in
highly supersaturated solutions owing to the high viscosity of the solution.
The stability of a lactose 'glass' is due to the low probability of nuclei
forming at very high concentrations.
Once a sufficient number of nuclei have formed, crystal growth occurs at
rate influenced by:
degree of supersaturation;
surface area available for deposition;
viscosity ;
agitation;
temperature;
mutarotation, which is slow at low temperatures.
?-Hydrate. cc-Lactose crystallizes as a monohydrate containing 5% water
of crystallization and can be prepared by concentrating aqueous lactose
solutions to supersaturation and allowing crystallization to occur below
30 DAIRY CHEMISTRY AND BIOCHEMISTRY
Figure 2.7 The most common crystal form of a-lactose hydrate.
93.5"C. The a-hydrate is the stable solid form at ambient temperatures and
in the presence of small amounts of water below 93.5"C, all other forms
change to it. The a-monohydrate has a specific rotation in water at 20°C of
+89.4". It is soluble only to the extent of 7g per 1OOg water at 20°C. It
forms a number of crystal shapes, depending on the conditions of crystalli-
zation; the most common type when fully developed is tomahawk-shaped
(Figure 2.7). Crystals are hard and dissolve slowly. In the mouth, crystals
less than 10 pm are undetectable, but above 16 pm they feel gritty or 'sandy'
and at 30pm, a definite gritty texture is perceptible. The term 'sandy' or
sandiness is used to describe the defect in condensed milk, ice-cream or
processed cheese spreads where, due to poor manufacturing techniques,
large lactose crystals are formed.
a-Anhydrous. Anhydrous a-lactose may be prepared by dehydrating
a-hydrate in V~CUO at temperatures between 65 and 93.5"C; it is stable only
in the absence of moisture.
B-Anhydride. Since /%lactose is less soluble than the a-isomer above
93.5"C, the crystals formed from aqueous solutions at temperatures above
93.5"C are p-lactose; these are anhydrous and have a specific rotation of 35".
/%Lactose is sweeter than a-lactose, but is not appreciably sweeter than the
equilibrium mixture of a- and p-lactose normally found in solution.
LACTOSE 31
Table 2.3 Some physical properties of the two common forms of lactose (modified from Jenness
and Patton, 1959)
Property a-H ydrate 8- Anhydride
Melting point" ("C) 202 252
Solubility in water (g 100 rn1-l) at 20°C I 50
Specific gravity (20°C) 1.54 1.59
Specific heat 0.299 0.285
Heat of combustion (kJ mol-') 5687 5946
"Decomposes; values vary with rate of heating, ti-hydrate loses water at 120°C.
bValues on anhydrous basis, both forms mutarotate to f55.4".
Specific rotaltionb [a]:' + 89.4" +35"
Some properties of c(- and !-lactose are summarized in Table 2.3. Mixed
a/! crystals, e.g. asp3, can be formed under certain conditions. The relation-
ship between the different crystalline forms of lactose is shown in Figure 2.8.
Lactose glass. When a lactose solution is dried rapidly, viscosity increases
so quickly that crystallization is impossible. A noncrystalline form is
produced containing a- and !-forms in the ratio at which they exist in
solution. Lactose in spray-dried milk exists as a concentrated syrup or
amorphous glass which is stable if protected from air, but is very hygro-
scopic and absorbs water rapidly from the atmosphere, becoming sticky.
2.2.7 Problems related to lactose crystallization
The tendency of lactose to form supersaturated solutions that do not
crystallize readily causes problems in many dairy products unless adequate
controls are exercised. The problems are due primarily to the formation of
large crystals, which cause sandiness, or to the formation of a lactose glass,
which leads to hygroscopicity and caking (Figure 2.9).
Dried milk and whey. Lactose is the major component of dried milk
products: whole-milk powder, skim-milk powder and whey powder contain
c. 30, 50 and 70% lactose, respectively. Protein, fat and air are dispersed in
a continuous phase of amorphous solid lactose. Consequently, the behav-
iour of lactose has a major impact on the properties of dried milk products.
In freshly made powder, lactose is in an amorphous state with an a/!
ratio of 1 : 1.6. This amorphous lactose glass is a highly concentrated syrup
since there is not sufficient time during drying for crystallization to proceed
normally. The glass has a low vapour pressure and is hygroscopic, taking
up moisture very rapidly when exposed to the atmosphere. On the uptake
of moisture, dilution of the lactose occurs and the molecules acquire
sufficient mobility and space to arrange themselves into crystals of a-lactose
32
LACTOSE IN SOLUTION
a-b
[Pl/[al= 1.64-0.0027T
DAIRY CHEMISTRY AND BIOCHEMISTRY
L
.-
Amorphous Lactose
V
a-Hydrate
(lactose. I.H,O)
Conipound crystal
asp3 (anhydrous)
[ p] / [a] = 1.25
I
T = IOW, presence
1
01 \vay
LI I
Anhydrous a
unstable
-1
Water uptake, T < !USo
I
stable (S)
TI 150", presence
01 water vapour
Dissolve, T c 93.5'
.......................
Silpersatiiration in ethanol
Figure 2.8 Modifications of lactose (T temperature in 'C) (from Walstra and Jenness, 1984).
monohydrate. These crystals are small, usually with dimensions of less than
1 pm. Crevices and cracks exist along the edges of the crystals, into which
other components are expelled. In these spaces, favourable conditions exist
for the coagulation of casein because of the close packing of the micelles and
the destabilizing action of concentrated salt systems. The fat globule
membrane may be damaged by mechanical action, and Maillard browning,
involving lactose and amino groups of protein, proceeds rapidly when
crystallization has occurred.
LACTOSE 33
Rapid drying Concentrated lactose syrup
MILK,WHEY,
PERMEATE
* “LACTOSE GLASS”
(Non-crystalline)
a-HYDRATE
Cryslallizatioir
4 MOLECULAR MOBILITY
CAKING OF MILK AND
WHEY POWDERS
AGGREGATES OF
CRYSTALS
Figure 2.9 Formation and crystallization of lactose glass.
Crystallization of lactose in dried milk particles causes ‘caking’ of the
powder into a hard mass. If a considerable portion of lactose in the freshly
dried product is in the crystalline state, caking of the powder on contact
with water is prevented, thereby improving the dispersibility of the powder.
Lactose crystallization is achieved by rehydrating freshly dried powder to
c. 10% water and redrying it, or by removing partly dried powder from the
drier and completing drying in a fluidized bed dryer. This process is used
commercially for the production of ‘instantized’ milk powders. Clustering of
the particles into loose, spongy aggregates occurs; these agglomerates are
readily wettable and dispersible. They exhibit good capillary action and
water readily penetrates the particles, allowing them to sink and disperse,
whereas the particles in non-instantized powder float due to their low
density which contributes to their inability to overcome surface tension.
Also, because of the small size of the particles in conventional spray-dried
powders, close packing results in the formation of inadequate space for
capillary action between the particles, thereby preventing uniform wetting.
As a result, large masses of material are wetted on the outside, forming a
barrier of highly concentrated product which prevents internal wetting and
results in large undispersed lumps. This problem is overcome by agglomer-
ation and, in this respect, lactose crystallization is important since it
facilitates the formation of large, sponge-like aggregates.
The state of lactose has a major effect on the properties of spray-dried
whey powder manufactured by conventional methods, i.e. preheating, con-
densing to about 50% total solids and drying to less than 4% water. The
powder is dusty and very hygroscopic, and when exposed to ambient air it
34 DAIRY CHEMISTRY AND BIOCHEMISTRY
has a pronounced tendency to cake owing to its very high lactose content
( - 70%).
Problems arising from the crystallization of lactose in milk and whey
powders may also be avoided or controlled by pre-crystallizing the lactose.
Essentially, this involves adding finely divided lactose powder which acts as
nuclei on which the supersaturated lactose crystallizes. Addition of 0.5 kg of
finely ground lactose to the amount of concentrated product (whole milk,
skim milk or whey) containing 1 tonne of lactose will induce the formation
of c. lo6 crystals ml- l, about 95% of which will have dimensions less than
10pm and 100% less than 15 pm, i.e. too small to cause textural defects.
Diagrams of spray dryers with instantizers are shown in Figures 2.10 and
2.11.
Feed-[ I /Hot air
Cyclone separators
(?/
II I1
Crystalization belt
b Vibrofluidizer
Hammer mill
Product out
Figure 2.10 Schematic representation of a low temperature drying plant for whey (modified
from Hynd, 1980).
LACTOSE 35
Figure 2.11 Schematic representation of a straight through drying plant for whey (modified
from Hynd, 1980).
Thermoplasticity of lactose. Unless certain precautions are taken during
the drying of whey or other solutions containing high concentrations of
lactose, the hot, semi-dry powder may adhere to the metal surfaces of the
dryer, forming deposits. This phenomenon is referred to as thermoplasticity.
The principal factors influencing the temperature at which thermoplasticity
occurs (‘sticking temperature’) are the concentrations of lactic acid, amor-
phous lactose and moisture in the whey powder.
Increasing the concentration of lactic acid from 0 to 16% causes a linear
decrease in sticking temperature (Figure 2.12). The degree of pre-crystalliza-
tion of lactose affects sticking temperature: a product containing 45%
pre-crystallized lactose has a sticking temperature of 60°C while the same
product with 80% pre-crystallization sticks at 78°C (Figure 2.12). Pre-
crystallization of the concentrate feed to the dryer thus permits considerably
higher feed concentrations and drying temperatures.
36 DAIRY CHEMISTRY AND BIOCHEMISTRY
Crystalline lactose (%)
45 55 65 75
1 I I
3
0 4 8 12 16
Lactic acid added (%)
Figure 2.12 Effect of added lactic acid (- - - -) and degree of lactose crystallization (-) on the
sticking temperature of whey powder (1.5-3.5% moisture).
In practice, the most easily controlled factor is the moisture content of
the whey powder, which is determined by the outlet temperature of the dryer
(to, Figure 2.13). However, as a result of evaporative cooling, the tempera-
ture of the particles in the dryer is lower than the outlet temperature (tp,
Figure 2.13) and the difference between to and t, increases with increasing
moisture content. The sticking temperature for a given whey powder
decreases with increasing moisture content (ts, Figure 2.13) and where the
two curves (t, and t,) intersect (point TPC, Figure 2.13) is the maximum
product moisture content at which the dryer can be operated without
product sticking during drying. The corresponding point on the outlet
temperature curve (TOC) represents the maximum dryer outlet temperature
which may be used without causing sticking.
Sweetened condensed milk. Crystallization of lactose occurs in sweetened
condensed milk (SCM) and crystal size must be controlled if a product with
a desirable texture is to be produced. As it comes from the evaporators,
SCM is almost saturated with lactose. When cooled to 15-20°C, 40-60% of
the lactose eventually crystallizes as a-lactose hydrate. There are 40-47
parts of lactose per 100 parts of water in SCM, consisting of about 40% a-
and 60% /?-lactose (ex-evaporator). To obtain a smooth texture, crystals
with dimensions of less than 10 pm are desirable. The optimum temperature
LACTOSE 37
110 -
100 -
h
90-
* :
* E 80-
E
?L
2
3
.-
‘ 70-
60
0 1 2 3 4 5
Powder moisture (%)
Figure 2.13 Influence of moisture content on the temperature of powder in a spray dryer (t,),
dryer outlet temperature (to) and sticking temperature (fJ The minimum product tempera-
tured required to avoid problems with sticking is at TPC with the corresponding dryer outlet
temperature TOC. (Modified from Hynd, 1980.)
for crystallization is 26-36°C. Pulverized @-lactose, or preferably lactose
‘glass’, is used as seed. Continuous vacuum cooling, combined with seeding,
gives the best product.
Ice-cream. Crystallization of lactose in ice-cream causes a sandy texture. In
freshly hardened ice-cream, the equilibrium mixture of a- and p-lactose is in
the ‘glass’ state and is stable as long as the temperature remains low and
constant. During the freezing of ice-cream, the lactose solution passes
through the labile zone so rapidly and at such a low temperature that
limited lactose crystallization occurs.
If ice-cream is warmed or the temperature fluctuates, some ice will melt,
and an infinite variety of lactose concentrations will emerge, some of which
will be in the labile zone where spontaneous crystallization occurs while
others will be in the metastable zone where crystallization can occur if
suitable nuclei, e.g. lactose crystals, are present. At the low temperature,
crystallization pressure is low and extensive crystallization usually does not
occur. However, the nuclei formed act as seed for further crystallization
38 DAIRY CHEMISTRY AND BIOCHEMISTRY
when the opportunity arises and they tend to grow slowly with time,
eventually causing a sandy texture. The defect is controlled by limiting the
milk solids content or by using /?-galactosidase to hydrolyse lactose.
Other frozen dairy products. Although milk may become frozen inadver-
tently, freezing is not a common commercial practice. However, concen-
trated or unconcentrated milks are sometimes frozen commercially, e.g. to
supply remote locations (as an alternative to dried or UHT milk), to store
sheep's or goats' milk, production of which is seasonal, or human milk for
infant feeding in emergencies (milk banks).
As will be discussed in Chapter 3, freezing damages the milk fat globule
membrane, resulting in the release of 'free fat'. The casein system is also
destabilized due to a decrease in pH and an increase in Ca2+ concentration,
both caused by the precipitation of soluble CaH2P0, and/or Ca,HPO, as
Ca,(PO,),, with the release of H + (Chapter 5); precipitation of Ca,(PO,),
occurs on freezing because pure water crystallizes, causing an increase in
soluble calcium phosphate with which milk is already saturated. Crystalli-
zation of lactose as a-hydrate during frozen storage aggravates the problem
by reducing the amount of solvent water available.
In frozen milk products, lactose crystallization causes instability of the
casein system. On freezing, supersaturated solutions of lactose are formed:
e.g. in concentrated milk at -8"C, 25% of the water is unfrozen and
contains 80 g lactose per 100 g, whereas the solubility of lactose at - 8°C is
only about 7%. During storage at low temperatures, lactose crystallizes
slowly as a monohydrate and consequently the amount of free water in the
product is reduced.
The formation of supersaturated lactose solutions inhibits freezing, and
consequently stabilizes the concentration of solutes in solution. However,
when lactose crystallizes, water freezes and the concentration of other
solutes increases markedly (Table 2.4).
Table 2.4 Comparison of ultrafiltrate from liquid and frozen skim milk
Ultrafiltrate of liquid portion of
Constituent Ultrafiltrate of skim milk frozen concentrated milk
PH
6.1 5.8
Chloride (mM) 34.9 459
Citrate (mM) 8.0 89
Phosphate (mM) 10.5 84
Sodium (mM) 19.7 218
Potassium (mM) 38.5 393
Calcium (mM) 9.1 59
Po
8. 3 C. 1
Protein
flocculation-
%P EL
volume
of
precipitate,
ml
Ern fur
W W
40 DAIRY CHEMISTRY AND BIOCHEMISTRY
It /
W"
1
ii
7
Srrd
,
Figure 2.15 Schematic representation of plant for the manufacture of crude and refined lactose,
from sweet whey.
only about 420000 tonnes of lactose are produced annually, i.e. only about
7% of that potentially available.
Production of lactose essentially involves concentrating whey or ultrafil-
tration permeate by vacuum concentration, crystallization of lactose from
the concentrate, recovery of the crystals by centrifugation and drying of the
crystals (Figure 2.15). The first-crop crystals are usually contaminated with
riboflavin and are therefore yellowish; a higher grade, and hence more
LACTOSE 41
Table 2.5 Some typical physical and chemical data for various grades of lactose" (from
Nickerson, 1974)
Analysis Fermentation Crude Edible USP
Lactose (YO) 98.0 98.4 99.0 99.85
Moisture, non-hydrate (YO) 0.35 0.3 0.5 0.1
Protein (YO) 1 .o 0.8 0.1 0.01
Ash (%) 0.45 0.40 0.2 0.03
Lipid (YO) 0.2 0.1 0.1 0.001
Acidity, as lactic acid (YO) 0.4 0.4 0.06 0.04
Specific rotation [%]i5
b b 52.4" 52.4"
"USP, US Pharmacopoeia grade.
bNot normally determined.
Table 2.6 Food applications of lactose
Humanized baby foods
Instantizingifree-flowing agent in foods
Confectionery products
Determineralized whey powder or lactose
Agglomeration due to lactose crystallization
Improves functionality of shortenings
Anticaking agent at high relative humidity
Certain types of icing
Maillard browning, if desired
Accentuates other flavours (chocolate)
Flavour adsorbant
Flavour volatiles
Flavour enhancement
Sauces, pickles, salad dressings, pie fillings
Table 2.7 Relative sweetness of sugars (approx. concentration, YO,
required to give equivalent sweetness) (from Nickerson, 1974)
Sucrose Glucose Fructose Lactose
0.5
1 .o
2.0
2.0
2.0
5.0
5.0
5.0
10.0
10.0
15.0
15.0
20.0
0.9
1.8
3.6
3.8
3.2
8.3
8.3
7.2
13.9
12.7
17.2
20.0
21.8
0.4
0.8
1.7
-
4.2
4.6
4.5
8.6
8.7
12.8
13.0
16.7
1.9
3.5
6.5
6.5
6.0
15.7
14.9
13.1
25.9
20.7
27.8
34.6
33.3
42 DAIRY CHEMISTRY AND BIOCHEMISTRY
Table 2.8 Relative humectancy of sucrose, glucose and lactose
(% moisture absorbed at 20°C)
Relative humidity
60% 100%
lh 9 days 25 days
Sugar Humectancy
Lactose 0.54 1.23 1.38
Glucose 0.29 9.00 47.14
Sucrose 0.04 0.03 18.35
valuable, lactose is produced by redissolving and recrystallizing the crude
lactose (Table 2.5). Lactose may also be recovered by precipitation with
Ca(OH),, especially in the presence of ethanol, methanol or acetone.
Lactose has several applications in food products (Table 2.6), the most
important of which is probably in the manufacture of humanized infant
formulae. It is used also as a diluent for the tableting of drugs in the
pharmaceutical industry (which requires high-quality, expensive lactose)
and as the base for plastics.
Among sugars, lactose has a low level of sweetness (Table 2.7), which is
generally a disadvantage but is advantageous in certain applications. When
properly crystallized, lactose has low hygroscopicity (Table 2.Q which
makes it an attractive sugar for use in icings for confectionary products.
2.4 Derivatives of lactose
Although the demand for lactose has been high in recent years, it is unlikely
that a profitable market exists for all the lactose potentially available. Since
the disposal of whey or UF permeate by dumping into waterways is no
longer permitted, profitable, or at least inexpensive, ways of utilizing lactose
have been sought for several years. For many years, the most promising of
these was considered to be hydrolysis to glucose and galactose, but other
modifications are attracting increasing attention.
2.4.1 Enzymatic mod$cation of lactose
Lactose may be hydrolysed to glucose and galactose by enzymes (p-
galactosidases, commonly called lactase) or by acids. Commercial sources of
8-galactosidase are moulds (especially Aspergillus spp.), the enzymes from
which have acid pH optima, and yeasts (Kluyveromyces spp.) which produce
enzymes with neutral pH optima. P-Galactosidases were considered to have
LACTOSE 43
considerable commercial potential as a solution to the ‘whey problem’ and
for the treatment of lactose intolerance (section 2.6.1). The very extensive
literature on various aspects of P-galactosidases and on their application in
free or immobilized form has been reviewed by Mahoney (1997). Techno-
logical problems in the production of glucose-galactose syrups have been
overcome but the process is not commercially successful. Glucose-galactose
syrups are not economically competitive with glucose or glucose-fructose
syrups produced by hydrolysis of maize starch, unless the latter are heavily
taxed. As discussed in section 2.6.1, an estimated 70% of the adult human
population have inadequate intestinal P-galactosidase activity and are
therefore lactose intolerant; the problem is particularly acute among Asians
and Africans. Pre-hydrolysis of lactose was considered to offer the potential
to develop new markets for dairy products in those countries. Various
protocols are available: addition of P-galactosidase to milk in the home,
pre-treatment at the factory with free or immobilized enzyme or aseptic
addition of sterilized free P-galactosidase to UHT milk, which appears to be
particularly successful. However, the method is not used widely and it is now
considered that the treatment of milk with P-galactosidase will be commer-
cially successful only in niche markets.
Glucose-galactose syrups are about three times sweeter than lactose
(70% as sweet as sucrose) and hence lactose-hydrolysed milk could be used
in the production of ice-cream, yoghurt or other sweetened dairy products,
permitting the use of less sucrose and reducing caloric content. However,
such applications have not been commercially successful.
The glucose moiety can be isomerized to fructose by the well-established
glucose isomerization process to yield a galactose-glucose-fructose syrup
with increased sweetness. Another possible variation would involve the
isomerization of lactose to lactulose (galactose-fructose) which can be
hydrolysed to galactose and fructose by some P-galactosidases.
8-Galactosidase has transferase as well as hydrolase activity and pro-
duces oligosaccharides (galacto-oligosaccharides, Figure 2.16) which are
later hydrolysed (Figure 2.17). This property may be a disadvantage since
the oligosaccharides are not digestible by humans and reach the large
intestine where they are fermented by bacteria, leading to the same problem
caused by lactose. However, they stimulate the growth of BiJidobacteriurn
spp. in the lower intestine; a product (oligonate, 6’-galactosyl lactose) is
produced commercially by the Yokult Company in Japan for addition to
infant formulae. Some galacto-oligosaccharides have interesting functional
properties and may find commercial applications.
2.4.2 Chemical modifications
Lactulose. Lactulose is an epimer of lactose in which the glucose moiety is
isomerized to fructose (Figure 2.18). The sugar does not occur naturally and
44 DAIRY CHEMISTRY AND BIOCHEMISTRY
Gal (1 4 2) Glu
Gal (1 + 3) Glu
Gal (1 + 6) Glu
(Allolactose)
I
Transglgcosylation
1
Gal (1 + 3) Gal
t
Tetrasaccliaricles
Hexasaccharides
Figure 2.16 Possible reaction products from the action of 8-galactosidase on lactose (from
Smart, 1993).
LACTOSE 45
so
Lo
8
- 60
2
Lo
cd
0
cr
0
0
Y
Y
40
*
8
E
fi 20
4
0
0 1 2 3 4
Time (hours)
Figure 2.17 Production of oligosaccharides during the hydrolysis of lactose by 8-galactosidase;
0, lactose; ., monosaccharides; 0, glucose: A, oligosaccharides; 0, galactose (modified from
Mahoney, 1997).
was first synthesized by Montgomery and Hudson in 1930. It can be
produced under mild alkaline conditions via the Lobry de Bruyn-Alberda
van Ekenstein reaction and at a low yield as a by-product of p-galactosidase
action on lactose. It is produced on heating milk to sterilizing conditions
and is a commonly used index of the severity of the heat treatment to which
milk has been subjected, e.g. to differentiate in-container sterilized milk from
UHT (ultra-high temperature) milk (Figure 2.19); it is not present in raw or
HTST (high temperature short time) pasteurized milk.
Lactulose is sweeter than lactose and 48-62% as sweet as sucrose. It is
not metabolized by oral bacteria and hence is not cariogenic. It is not
hydrolysed by intestinal 8-galactosidase and hence reaches the large intes-
tine where it can be metabolized by lactic acid bacteria, including Bifidobac-
terium spp. and serves as a bifidus factor. For this reason, lactulose has
attracted considerable attention as a means of modifying the intestinal
microflora, reducing intestinal pH and preventing the growth of undesirable
putrefactive bacteria (Figures 2.20-2.22). It is now commonly added to
infant formulae to simulate the bifidogenic properties of human milk -
apparently, 20000 tonnes annum-' are now used for this and similar
applications. Lactulose is also reported to suppress the growth of certain
tumour cells (Figure 2.23).
H %wAH20H CHzOH 5
Ho~~o~, HO CH,OH
7
OH
4 4
H 7
HO OH 0 H -0 H
OH
H H
L
Pyranose form - Furanose form
Figure 2.18 Chemical structure of lactulose.
LACTOSE
47
0 I0 2LI 30 40 SO 60 70 XO 90 1(X) 110 120
Lactulose concentration (mg 100 m1-l)
Figure 2.19 Concentration of lactulose in heated milk products (modified from Andrews, 1989).
LACTULOSE
Oral intake
-
Non-absorption and migration Not carogenic
to large intestine
Utilization by Bi@iobacterium
and increme In bifldobacteria
Favourable change of
intestinal microflora
,
n
Production of organic acids
I 1 I and lowering of intestinal pH 1
Ensuring intes- Lessening burdens
tinal function to hepatic function
Suppression of
inlestinal putrefactive bacteria
Suppression of production of
harmful subsun&
Viumin synthesis
Stimulation of
immune response
Figure 2.20 Significance of lactulose in health (modified from Tamura et al., 1993).
48 DAIRY CHEMISTRY AND BIOCHEMISTRY
Figure 2.21 Effect of lactulose on the intestinal microflora of 2-month-old infants (modified
from Tamura et al., 1993).
Lactulose is usually used as a 50% syrup but a crystalline trihydrate,
which has very low hygroscopicity, is now available.
Lactitol. Lactitol (4-O-~-~-galactopyranosyl-~-sorbitol), is a synthetic
sugar alcohol produced on reduction of lactose, usually using Raney nickel.
It can be crystallized as a mono- or di-hydrate. Lactitol is not metabolized
by higher animals; it is relatively sweet and hence has potential as a
non-nutritive sweetener. It is claimed that lactitol reduces the absorption of
sucrose, blood and liver cholesterol levels and to be anticariogenic. It has
applications in low-calorie foods ('jams, marmalade, chocolate, baked
goods); it is non-hygroscopic and can be used to coat moisture-sensitive
foods, e.g. sweets.
It can be esterified with one or more fatty acids (Figure 2.24) to yield a
family of food emulsifiers, analogous to the sorbitans produced from
sorbitol.
Lactobionic acid. This derivative is produced by oxidation of the free
carbonyl group of lactose (Figure 2.25), chemically (Pt, Pd or Bi), electro-
lytically, enzymatically or by fermentation. Its lactone crystallizes readily.
Lactobionic acid has found only limited application; its lactone could be
used as an acidogen but it is probably not cost-competitive with gluconic
acid-h-lactone. It is used in preservation solutions for organs prior to
transplants.
Refore intake During intake After intake
Figure 2.22 Increase in Ui/irlo/~ric,rc,,iir,,i spp. by administration of lactillox to healthy :idults (modified from Tamtila ('I (I/.. 1993)
50 DAIRY CHEMISTRY AND BIOCHEMISTRY
30
h
E
E
v
1
L
x 20
5
c)
h
0
L
4
c) 4
E
.o 10
W
0 10 20 30
Days after treatment
Figure 2.23 Effect of different doses of whole peptidoglycan (WPG) from Bifdobacterium
infantis on the growth rate of Meth A tumour. Mice were inoculated subcutaneously with a
mixture of lo5 Meth A cells and 0 (U), 10 (A), 20 (A), 25 (O), 50 (0) or 100 (0) pg of WPG.
(Modified from Tamura et al., 1993.)
Lactosyl urea. Urea can serve as a cheap source of nitrogen for cattle but
its use is limited because NH, is released too quickly, leading to toxic levels
of NH, in the blood. Reaction of urea with lactose yields lactosyl urea
(Figure 2.26), from which NH, is released more slowly.
2.4.3 Fermentation products
Lactose is readily fermented by lactic acid bacteria, especially Lactococcus
spp. and Lactobacillus spp., to lactic acid, and by some species of yeast, e.g.
Kluyveromyces spp., to ethanol (Figure 2.27). Lactic acid may be used as a
food acidulant, as a component in the manufacture of plastics, or converted
to ammonium lactate as a source of nitrogen for animal nutrition. It can be
converted to propionic acid, which has many food applications, by
Propionibacterium spp. Potable ethanol is being produced commercially
from lactose in whey or UF permeate. The ethanol may also be used for
industrial purposes or as a fuel but is probably not cost-competitive with
ethanol produced by fermentation of sucrose or chemically. The ethanol
may also be oxidized to acetic acid. The mother liquor remaining from the
production of lactic acid or ethanol may be subjected to anaerobic digestion
with the production of methane (CH,) for use as a fuel; several such plants
are in commercial use.
LACTOSE 51
H
CHIOH
no-c-H
I
0
H-C-OH
I
HO-C-H
I
I
H-C-OH
n-c
no-c-H
CHzOH
I
I
CHzOH
PH
Hme HO 0
HO
OH
H H
H
Lactitol, 4-O-~-D-galactopyranosyl-D-sorbitol
,600c 1 C1SH31COOH
NaOH,
0
CH20- C-CISHJI
H II
no-c-H
I
0
H-C-OH
L-II,""
H-Lon 1
HO-C-H '7 I '
I
I
CHZOH
HO-C-H
I
I
CHzOH
n-c
OH
0
Hm ey-C-cl&l I1
0
no
OH
HO
H H
H
Lactitol monoester
Figure 2.24 Structure of lactitol and its conversion to lactyl palmitate.
52 DAIRY CHEMISTRY AND BIOCHEMISTRY
CH,OH
Lactose
t
H-C-OH
I
HO-C-H
I
I
I
H-C
CHzOH
CH20H
Lactobionic acid
~H~OH CH,OH
Lactobionic acid-&lactone
Figure 2.25 Structure of lactobionic acid and its &lactone.
Lactose can also be used as a substrate for Xanthomonas campestris in
the production of xanthan gum (Figure 2.28) which has several food and
industrial applications.
All the fermentation-based modifications of lactose are probably not
really economical because lactose is not cost-competitive with alternative
LACTOSE 53
FH20H CH2OH
Lactosyl urea
Figure 2.26 Structure of lactosyl urea.
LACTOSE
0
II //”
c\
OH
H3C-C-
Pyruvic acid
J
2CH3CH2COOH + CHACOOII
propionic acid arctic acid
+ CO2 + H20 OH
H ‘ “ONH,
I //”
H3C-C-
Animoniuni lar ta te
(animal feed)
HH
II
H-C-C-OH II
HH
Ethanol
(potable or industrial)
t
70
H-C-C’
‘OH
Acetic acid
(vinegar)
Figure 2.27 Fermentation products from lactose.
54 DAIRY CHEMISTRY AND BIOCHEMISTRY
Figure 2.28 Repeating unit of xanthan gum.
fermentation substrates, especially sucrose in molasses or glucose produced
from starch. Except in special circumstances, the processes can be regarded
as the cheapest method of whey disposal.
2.5 Lactose and the Maillard reaction
As a reducing sugar, lactose can participate in the Maillard reaction, leading
to non-enzymatic browning. The Maillard reaction involves interaction
between a carbonyl (in this case, lactose) and an amino group (in foods,
principally the E-NH, group of lysine in proteins) to form a glycosamine
(lactosamine) (Figure 2.29). The glycosamine may undergo an Amadori
rearrangement to form a l-amino-2-keto sugar (Amadori compound) (Fig-
ure 2.30). The reaction is base-catalysed and is probably first order. While
the Maillard reaction has desirable consequences in many foods, e.g. coffee,
bread crust, toast, french fried potato products, its consequences in milk
products are negative, e.g. brown colour, off-flavours, slight loss of nutritive
value (lysine), loss of solubility in milk powders (although it appears to
LACTOSE 55
D-Glucopyranose
HO 0 OH HR
OH
Glycosylaiiiine
Figure 2.29 Formation of glycosylamine, the initial step in Maillard browning.
prevent or retard age-gelation in UHT milk products). Maillard reaction
products (MRPs) have antioxidant properties; the production of MRPs may
be a small-volume outlet for lactose.
The Amadori compound may be degraded via either of two pathways,
depending on pH, to a variety of active alcohol, carbonyl and dicarbonyl
compounds and ultimately to brown-coloured polymers called melanoidins
(Figure 2.31). Many of the intermediates are (off-) flavoured. The dicar-
bonyls can react with amino acids via the Strecker degradation pathway
(Figure 2.32) to yield another family of highly flavoured compounds.
56
1
DAIRY CHEMISTRY AND BIOCHEMISTRY
I
CHPOH
HO HpHR - L
Glycosylamine
C-NHR
c=o
I
L
.
HvNR
C
I
C-
I-
t-
\ JNHR
II
C-OH
1-Amiiio-2-kcto sugar
Figure 2.30 Arnadori rearrangement of a glycosylamine.
2.6 Nutritional aspects of lactose
Since the milks of most mammals contain lactose, it is reasonable to assume
that it or its constituent monosaccharides have some nutritional signifi-
cance. The secretion of a disaccharide rather than a monosaccharide in milk
is advantageous since twice as much energy can be provided for a given
osmotic pressure. Galactose may be important because it or its derivatives,
e.g. galactosamine, are constituents of several glycoproteins and glycolipids,
which are important constituents of cell membranes; young mammals have
a limited capacity to synthesize galactose.
Lactose appears to promote the absorption of calcium but this is
probably due to a nonspecific increase in intestinal osmotic pressure, an
effect common to many sugars and other carbohydrates, rather than a
specific effect of lactose.
However, lactose has two major nutritionally undesirable consequences
- lactose intolerance and galactosaemia. Lactose intolerance is caused by
an insufficiency of intestinal P-galactosidase - lactose is not completely
I
I
HO-T-H
\
1
I
HO-3-H
.lW
aiqiuy
HO-3-H
‘H3
I II I
o=j O’H HO-5 HO-5
I I II
0=3H )*N=H3 )N-H~
58 DAIRY CHEMISTRY AND BIOCHEMISTRY
2,3-butadione L-valine 3-amino-2-hutanone methjlpropanal
J-anuno-2.butnnone 3-amino-2.butanone letramethylpyrazine
Figure 2.32 Strecker degradation of L-valine by reaction with 2,3-butadione.
hydrolysed, or not hydrolysed at all, in the small intestine and, since
disaccharides are not absorbed, it passes into the large intestine where it
causes an influx of water, causing diarrhoea, and is fermented by intestinal
micro-organisms, causing cramping and flatulence.
2.6.1 Lactose intolerance
A small proportion of babies are born with a deficiency of P-galactosidase
(inborn error of metabolism) and are unable to digest lactose from birth. In
normal infants (and other neonatal mammals), the specific activity of
intestinal P-galactosidase increases to a maximum at parturition (Figure
2.33), although total activity continues to increase for some time postpartum
due to increasing intestinal area. However, in late childhood, total activity
decreases and, in an estimated 70% of the world's population, decreases to
a level which causes lactose intolerance among adults. Only northern
Europeans and a few African tribes, e.g. Fulami, can consume milk with
impunity; the inability to consume lactose appears to be the normal pattern
in humans and other species, and the ability of northern Europeans to do
so presumably reflects positive selective pressure for the ability to consume
milk as a source of calcium (better bone development).
Lactose intolerance can be diagnosed by (1) jujunal biopsy, with assay
for P-galactosidase, or (2) administration of an oral dose of lactose followed
by monitoring blood glucose levels or pulmonary hydrogen levels. A test
dose of 50 g lactose in water (equivalent to 1 litre of milk) is normally
administered to a fasting patient; the dose is rather excessive and gastric
8 c < E 5 El 5' L cn P, tl a f. i; &.
"2
pl 3 a 5.
p-Galadosidase
activity
(pmol
glucose
releasedl
mg
proteinllo
min)
2
N
r b 8 !4
60
0
0 r(
.
DAIRY CHEMISTRY AND BIOCHEMISTRY
'Tolerant'
50 fi lactiise perorally
'lntolerdnt'
0 20 4 0 60 80 100
Minutes
Figure 2.34 Examples of the 'lactose intolerance' test.
Enzyme
Enzyme
filter
MI ver
Liquid milk packaging
Further processing 1-1
Figure 2.35 (a) Scheme for manufacture of low-lactose milk using a 'high' level of soluble
8-galactosidase. (b) Scheme for the manufacture of low-lactose milk by addition of a low level
of soluble P-galactosidase to UHT-sterilized milk. (Redrawn from Mahoney, 1997.)
0 treatment with exogenous P-galactosidase, either domestically by the
consumer or the dairy factory, using free or immobilized enzyme; several
protocols for treatment have been developed (Figure 2.35).
Lactose-hydrolysed milks are technologically successful and commer-
cially available but have not led to large increases in the consumption of
LACTOSE 61
milk in countries where lactose intolerance is widespread, presumably due
to cultural and economic factors. However, there are niche markets for such
products.
2.6.2 Galactosaemia
This is caused by the inability to metabolize galactose due to a hereditary
deficiency of galactokinase or galactose-1-phosphate (Gal-1-P) : uridyl trans-
ferase (Figure 2.36). Lack of the former enzyme leads to the accumulation of
galactose which is metabolized via other pathways, leading, among other
products, to galactitol which accumulates in the lens of the eye, causing
cataract in 10-20 years (in humans) if consumption of galactose-containing
foods (milk, legumes) is continued. The incidence is about 1 : 40000. The
Galactose
f
Gal-1-P
UDP-1Clu
Glu-1-P
(e.g., chrondroitin sulphate)
1
I
Gd-1 -P-uridyl fransferase
UDP-Gal - Biopolymers
UDFGal-rpirnernse
UDP-Glu - Glycogen
Glu-1-p - Glycolysis
Figure 2.36 Pathways for the metabolism of galactose.
62 DAIRY CHEMISTRY AND BIOCHEMISTRY
lack of Gal-1-P : uridyl transferase leads to the accumulation of Gal and
Gal-1-P. The latter interferes with the synthesis of glycoproteins and
glycolipids (important for membranes, e.g. in the brain) and results in
irreversible mental retardation within 2-3 months if the consumption of
galactose-containing foods is continued. The incidence of this disease, often
called 'classical galactosaemia', is about 1 in 60 000.
The ability to metabolize galactose decreases on ageing (after 70 years),
leading to cataract; perhaps this, together with the fact that mammals
normally encounter lactose only while suckling, explains why many people
lose the ability to utilize lactose at the end of childhood.
2.7 Determination of lactose concentration
Lactose may be quantified by methods based on one of five principles:
1. polarimetry;
2. oxidation-reduction titration;
3. colorimetry;
4. chromatography;
5. enzymatically.
2.7.1 Polarimetry
The specific rotation, [a]?, of lactose in solution at equilibrium is + 55.4"
expressed on an anhydrous basis (+52.6" on a monohydrate basis). The
specific rotation is defined as the optical rotation of a solution containing
1 gml-' in a 1 dm polarimeter tube; it is affected by temperature (20°C is
usually used; indicated by superscript) and wavelength (usually the sodium
D line (589.3 nm) is used; indicated by subscript).
where a is the measured optical rotation; 1, the light path in dm; and c, the
concentration as g m1-I. It is usually expressed as:
where c is in g perlOOml.
The milk sample must first be defatted and deproteinated, usually by
treatment with mercuric nitrate (Hg(NO,),). In calculating the concentra-
tion of lactose, a correction should be used for the concentration of fat and
protein in the precipitate.
LACTOSE 63
2.7.2 Oxidation and reduction titration
Lactose is a reducing sugar, i.e. it is capable of reducing appropriate
oxidizing agents, two of which are usually used, i.e. alkaline copper sulphate
(CuSO, in sodium potassium tartrate; Fehling’s solution) or chloroamine-T
(2.1).
HNC I
I
o=s=o
I
0 I
kH,
Chloroamine-T
For analysis by titration with Fehling’s solution, the sample is treated
with lead acetate to precipitate protein and fat, filtered, and the filtrate
titrated with alkaline CuSO,, while heating. The reactions involved are
summarized in Figure 2.37.
Cu,O precipitates and may be recovered by filtration and weighed; the
concentration of lactose can then be calculated since the oxidation of one
mole of lactose (360 g) yields one mole of Cu,O (143 g). However, it is more
convenient to add an excess of a standard solution of CuSO, to the
lactose-containing solution. The solution is cooled and the excess CuSO,
determined by reaction with KI and titrating the liberated I, with standard
sodium thiosulphate (Na,S,O,) using starch as an indicator.
2CuS0, + 4KI -, CuI, + 2K,SO, + I,
1, + 2Na,S,03 --+ 2NaI + Na2S,06
The end point in the Fehling’s is not sharp and the redox determination
of lactose is now usually performed using chloramine-T rather than CuSO,
as oxidizing agent.
The reactions involved are as follows:
CH,C,H,SO,NClH + H,O + KI (excess)
* CH,H,H,SO,NH, + HCl + KIO (K hypoiodate)
KIO + lactose (- CHO) -+ KI + lactobionic acid ( - COOH)
KI + KIO -, 2KOH + I,
The I, is titrated with standard Na,S,O, (sodium thiosulphate):
I, + 2Na,S,O, --* 2NaI + Na,S,O,
64 DAIRY CHEMISTRY AND BIOCHEMISTRY
CU”
0
\c/H
I
HO-C-H
H
II
I
C-OH
H-C-OH
I
I
I
H-LOH
- Galactose -C-H
Alkali
Galactose -C-H
I HO-C-H
HO-C-H
CH2OH
CHaOH
I
LaCtW enediol
COOH
I
I
I
Red I
I
HO-C-H
H-C-OH
Heat
CuzO - CuOH Cu’ + Galactose -C-H
HO-C-H
CHIOH
Figure 2.37 Oxidation of lactose by alkaline copper sulphate (Fehling’s reagent).
One millilitre of 0.04 N thiosulphate is equivalent to 0.0072 g lactose
monohydrate or 0.0064 g anhydrous lactose.
The sample is deproteinized and defatted using phosphotungstic acid.
2.7.3 Colorimetric methods
Reducing sugars, including lactose, react on boiling with phenol (2.2) or
anthrone (2.3) in strongly acidic solution (70%, v/v, H,SO,) to give a
coloured solution.
0
P”
2.2 0 2.3
LACTOSE 65
The complex with anthrone absorbs maximally at 625 nm. The concen-
tration of lactose is determined from a standard curve prepared using a
range of lactose concentrations.
The method is very sensitive but must be performed under precisely
controlled conditions.
2.7.4 Chromaiographic methods
While lactose may be determined by gas liquid chromatography, high
performance liquid chromatography (HPLC), using a refractive index de-
tector, is now usually used.
2.7.5 Enzymatic methods
Enzymatic methods are very sensitive but are rather expensive, especially for
a small number of samples.
Lactose is first hydrolysed by 8-galactosidase to glucose and galactose.
The glucose may be quantified using:
1. glucose oxidase using a platinum electrode, or the H,O, generated may
2. glucose-6-phosphate dehydrogenase (G-6-P-DH)
be quantified by using a peroxidase and a suitable dye acceptor; or
Hexokinase
D-Glucose + ATP - Glucose-6-P + ADP
G-6-P-DH, NADP'
Gluconate-6-P + NADPH + Ht
The concentration of NADPH produced may be quantified by measuring
the increase in absorbance at 334, 340 or 365 nm.
Alternatively, the galactose produced may be quantified using galactose
dehydrogenase (Gal-DH):
D-galactose + NAD' - Galactonic acid + NADH + H+
Gal-DH
The NADH produced may be quantified by measuring the increase in
absorbance at 334, 340 or 365 nm.
References
Andrews, G. (1989) Lactulose in heated milk, in Heat-Induced Changes in Milk, (ed. P.F. Fox),
Horton, B.S. (1993) Economics of marketing lactose and lactose by-products in a global trading
Hynd, J. (1980) Drying of whey. J. Soc. Dairy Technol., 33, 52-4.
Bulletin 238, International Dairy Federation, Brussels, pp. 45-52.
environment, in Bulletin 289, International Dairy Federation, Brussels, pp. 7-9.
66 DAIRY CHEMISTRY AND BIOCHEMISTRY
Jenness, R. and Patton, S. (1959) Lactose, in Principles of Dairy Chemistry, John Wiley and
Sons, NY, pp. 73-100.
Mahoney, R.R. (1997) Lactose: enzymatic modification, in AdFanced Dairy Chemistry, Vol. 3:
Lactose, Water, Salts and Wtamins, 2nd edn (ed. P.F. Fox), Chapman & Hall, London, pp.
Nickerson, T.A. (1974) Lactose, in Fundamentals of Dairy Chemistry, (eds B.H. Webb, A.H.
Johnson and J.A. Alford), AVI Publishing, Westport, CT, pp. 273-324.
Smart, J.B. (1993) Transferase reactions of P-galactosidases - New product opportunities, in
Lactose Hydrolysis, Bulletin 239. International Dairy Federation, Brussels, pp. 16-22.
Tamura, Y., Mizota, T., Shimamura, S. and Tomita, M. (1993) Lactulose and its application
to food and pharmaceutical industries, in Lactose Hydrolysis, Bulletin 239, International
Dairy Federation, Brussels, pp. 43-53.
Tumerman, L.. Fram, H. and Comely, K.W. (1954) The effect of lactose crystallization on
protein stability in frozen concentrated milk. J. Dairy Sci., 37, 830-9.
Walstra, P. and Jenness, R. (1984) Dairy Chemistry and Physics, John Wiley and Sons, New
York.
77-125.
Suggested reading
Fox, P.F. (ed.) (1985) Developments in Dairy Chemistry, Vol. 3: Lactose and Minor Constituents,
Fox, P.F. (ed.) (1997) Advanced Dairy Chemistry, Vol. 2 Lactose, Water, Salts and Vitamins,
Holsinger, V.H. (1988) Lactose, in Fundamentals of Dairy Chemistry, (ed. N.P. Wong), Van
IDF (1989) Monograph on heat-induced changes in milk, Bulletin 238, International Dairy
IDF (1993) Proceedings ofthe IDF Workshop on Lactose Hydrolysis, Bulletin 289, International
Jenness, R. and Patton, S. (1959) Lactose, in Principles of Dairy Chemistry, John Wiley and
Labuza, T.P., Reineccius, G.A., Monnier, V.M. et a/. (eds) (1994) Maillard Reactions in
Nickerson, T.A. (1965) Lactose, in Fundamentals of Dairy Chemistry, (eds B.H. Webb and A.H.
Nickerson, T.A. (1974) Lactose, in Fundamentals of Dairy Chemistry, (eds B.H. Webb, A.H.
Walstra, P. and Jenness, R. (1984) Dairy Chemistry and Physics, John Wiley and Sons, New
Yang, S.T. and Silva, E.M. (1995) Novel products and new technologies for use of a familiar
Elsevier Applied Science Publishers, London.
Chapman & Hall, London.
Nostrand Reinhold, New York, pp. 279-342.
Federation, Brussels.
Dairy Federation, Brussels.
Son, New York, pp. 73-100.
Chemistry, Food and Health, Royal Society of Chemistry, Cambridge.
Johnson), AVI Publishing, Westport, CT, pp. 224-60.
Johnson and J.A. Alford), AVI Publishing, Westport, CT, pp. 273-324.
York.
carbohydrate, milk lactose. J. Dairy Sci., 78, 2541-62.
