Journal of Dairy Research (2002) 69 401409.

# Proprietors of Journal of Dairy Research 2002

DOI: 10.1017\S0022029902005538 Printed in the United Kingdom
401
Variations of lactoperoxidase activity and thiocyanate content in
cows and goats milk throughout lactation
B. FLORENCE A. FONTEH, ALISTAIR S. GRANDISON*
. MICHAEL J. LEWIS
School of Food Biosciences, The University of Reading, Whiteknights campus,
P.O. Box 226, Reading RG6 6AP, UK
(Received 5 April 2001 and accepted for publication 12 November 2001)
Snn.v.. Lactoperoxidase activity and thiocyanate content were monitored in the
milks of seven individual cows and six goats during lactation. Lactoperoxidase
activity exhibited a cyclic pattern with alternating peaks and troughs throughout
lactation. Extremely large variations were observed between and within animals.
For example, lactoperoxidase activity ranged from 0n055n60 U\ml for one cow on
dierent sampling days. Variations between cows were also large, ranging from
0n985n1 U\ml on a specic sampling day. Mean lactoperoxidase activity in cows
ranged from 1n52n7 U\ml with an overall mean of 2n3p1n0 U\ml while the
thiocyanate concentration ranged from 6n010n2 g\ml with an overall average
of 8n5p5n1 g\ml. Lactoperoxidase activity means for goats ranged from
0n040n16 U\ml with a grand mean of 0n1p0n06 U\ml while the thiocyanate content
means ranged from 6n68n2 g\ml with a grand mean of 7n0p2n59 g\ml. There was
no correlation between lactoperoxidase activity and thiocyanate content in either
cows (R# l0n011) or goats (R# l0n015) milk. These experiments have revealed that
lactoperoxidase activity is aected by many factors including the individual
animals, species, feed and stage of lactation. Therefore the exogenous supply of
thiocyanate and hydrogen peroxide needed to activate the lactoperoxidase system
for raw milk preservation will vary in quantity depending on these factors.
K.vov-: Lactoperoxidase, cow, goat, lactation, thiocyanate.
The lactoperoxidase (LP) system, composed of thiocyanate, LP and hydrogen
peroxide, occurs naturally in milk (Reiter & Harnulv, 1984). When active, the LP-
system produces hypothiocyanite ions, a strong antimicrobial agent (Bjorck et al.
1975; Barrett et al. 1999). Thiocyanate and hydrogen peroxide occur naturally in
milk, but in insucient amounts (Reiter & Harnulv, 1984) for the LP system to be
fully operational. Several workers have demonstrated the ecacy of the system in
milk preservation when the concentrations of these substrates are increased by an
exogenous supply (Kumar & Mathur, 1989; Ridley & Shalo, 1990; El-Agamy et al.
1992). Quantities to be added would vary depending on how active the LP system
is naturally, which can in turn be estimated by monitoring variations in enzyme
* For correspondence; e-mail : A.S.Grandison!reading.ac.uk
402 F. A. Fo1n . o1nv-
activity and thiocyanate content in animals during lactation. Although some work
has been carried out with goats milk (Zapico et al. 1991; Saad de Schoos et al. 1999),
no detailed information on such variations in cows milk is available in the literature.
The objective of the present work was therefore to monitor variations in LP activity
and thiocyanate content in cows and goats milk throughout lactation. This may
serve as a basis for estimating how much exogenous material would be needed to
activate the LP system such that the shelf life of milk can be prolonged.
n.1vI.i- . n1no-
Milk samples
Seven Holstein-Friesian cows were selected from the dairy cows at the Centre for
Dairy Research at the University of Reading. Lactation number of the cows ranged
from second to ninth. All cows received identical treatment (feeding, housing, health
care, etc.) throughout the study period. Milk was sampled from these cows as soon
as possible after calving. Sampling was carried out in the evening for all cows
throughout the study period. Cows were sampled biweekly for the rst 2 months,
then weekly for the next 6 weeks, followed by sampling fortnightly for the next 6
months. Six goats were selected from a local farm and morning milk was sampled
every 2 weeks during a 5-month lactation period. In both cases (cows and goats),
milking was carried out by hand and the samples assayed for enzyme activity within
2 h of milking. Samples were frozen at k18 mC, thawed later at room temperature
and assayed for thiocyanate content.
Chemical analysis
Lactoperoxidase activity was determined (ve replicates per sample, - between
replicates was less than 5%) according to the modied method of Pruitt & Kamau
(1994). In this procedure, 0n1 ml milk was mixed with 2 ml 1 mn-2,2h azino-di-
3-ethylbenzthiazoline-6-sulphonic acid (ABTS) in phosphate buer (0n1 n, pH 6n7) in
a cuvette. Reaction was initiated by adding 1 ml 0n3 mn-hydrogen peroxide to the
mixture. The reaction was monitored for 1 min. and the absorbance recorded at
412 nm (A
%"#
). Results are expressed in units\ml. One unit (U) is dened as the
amount of enzyme which oxidises 1 mol ABTS\min. Thiocyanate content was
determined in duplicate according to Marshall et al. (1986) : milk samples were rst
deproteinised by mixing 2 ml trichloroacetic acid (200 g\l) with 4 ml milk.
Supernatant uid (1n5 ml) was mixed with 1n5 ml ferric nitrate dye and the A
%'!
measured immediately and compared with a standard curve.
Statistical analysis
Signicant dierences between means at 95% condence interval were es-
tablished using a Students t test.
v-i1- . I---Io
Lactoperoxidase activity in cows
Patterns of enzyme activity for individual cows are presented in Fig. 1. Cows A
and D were sampled for a shorter period of time because the former died while the
latter dried o much earlier than was expected.
Lactoperoxidase and thiocyanate in milk 403
6
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0 40 80 160 120 200 240 280 320
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Fig. 1. Variation in #, lactoperoxidase activity and $, thiocyanate content of milk from individual
cows (AG) throughout lactation.
4
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Table 1. Summary of lactoperoxidase activity and thiocyanate concentration during lactation in cows. Each cow was sampled n
number of times, maximum or minimum levels were recorded at d number of days after calving
(Mean valuesp-)
Lactoperoxidase activity (Units\ml) Thiocyanate concentration (g\ml)
Cow Maximum Minimum Mean Maximum Minimum Mean
A 5n41 0n03 1n50ap1n0 27n9 3n6 10n2cp5n2
dl39 dl25 nl22 dl11 dl3 nl22
B 5n33 0n55 2n65cp1n10 14n3 2n9 9n0cp4n1
dl96 dl54 nl36 dl47 dl1 nl36
C 4n43 0n18 2n30bp0n81 24n2 2n6 9n5cp5n7
dl77 dl63 nl37 dl59 dl18 nl37
D 5n56 0n06 2n72cp1n12 33n5 3n4 10n2cp5n4
dl195 dl28 nl40 dl21 dl3 nl40
E 5n33 0n24 2n65cp1n04 21n1 2n0 7n9bcp4n3
dl69 dl48 nl35 dl240 dl23 nl35
F 3n63 0n25 2n15bp0n74 15n4 1n8 7n0abp3n1
dl8 dl50 nl38 dl162 dl1 nl38
G 4n01 0n42 2n1bp0n86 10n9 2n4 6n0ap2n2
dl15 dl47 nl29 dl218 dl12 nl29
a,b,c Means bearing the same superscript letter(s) are not signicantly dierent.
Lactoperoxidase and thiocyanate in milk 405
Table 2. Winsorised means of lactoperoxidase activity (Units\ml) per lactation period
in cows
(Values are meansp-)
Early lactation Mid lactation
61150 d
Late lactation
150 d
Cow 030 d 3160 d (nl9) (n9)
(nl8) (nl8)
A 1n4p0n51 1n4p0n17 1n1p0n98
B 2n3p0n51 2n5p0n69 3n8p0n58 2n1p0n21
C 2n3p0n77 2n1p0n41 2n7p0n46 1n8p0n39
D 2n0p0n48 2n2p0n28 3n7p0n88 2n3p0n39
E 3n2p0n49 2n4p0n74 3n0p0n64 1n9p0n38
F 2n4p0n52 1n95p0n48 2n8p0n33 1n7p0n37
G 2n4p0n62 2n4p0n72 2n6p0n39 1n3p0n19
In all cows sampled, there were huge variations in LP activity throughout
lactation reecting a cyclic pattern of the enzyme activity with alternating peaks
and troughs. This pattern appeared to be more pronounced during the early stages
of lactation, then levelled o towards the end. No consistent trend in enzyme activity
throughout lactation was obvious from the data so far collected, in agreement with
the observations of Saad de Schoos et al. (1999). Variations in LP activity were
observed between cows as well as within each cow, with grand mean enzyme activity
of 2n3 U\ml for the entire population of cows (Table 1). Comparison of means of
individual cows revealed statistical dierences between them. For each cow, there
were signicant dierences in enzyme activity between most days of sampling.
Greatest variation within any single cow was observed in cow D where maximum
enzyme activity was recorded as 5n65 U\ml while the minimum was 0n064 U\ml.
Although cow F had the least variation between its maximumand minimum enzyme
activity values, the maximum value (3n6 U\ml) was nonetheless more than ten times
greater than the minimum value (0n25 U\ml). Such massive changes in enzyme
activity were common in all cows sampled. It is notable that the maximum and
minimumvalues recorded for each cowoccurred on very dierent lactation days, and
therefore dierent days of sampling\assaying.
A general trend emerged upon plotting the winsorised means (Sokal & Rohlf,
1981) per lactation period for each cow (Table 2) : for the majority of cows, LP
activity seemed to increase from the onset of lactation to peak at mid lactation, and
then decreased thereafter.
Thiocyanate content in cows
Although variations in thiocyanate content in each cow also appeared to present
an alternating pattern, these variations were not as dramatic as with LP activity.
Large variations were observed between cows and within each cow. The highest
content (33n5 g\ml) was obtained 11 d after cow D had calved. The lowest content
(1n8 g\ml) was recorded in cow F on the 1st day after calving. Highest overall mean
throughout lactation (10n2 g\ml) was found in both cows A and D while the lowest
mean (6n0 g\ml) was recorded in the milk from cow G. Levels ranging from 5 to
6 g\ml in bualo milk were reported by Thakar & Dave (1986). Reiter (1985),
however, found higher levels of 15 g\ml in the milk of cows on natural pastures
containing clover. The high values obtained in this study could be a result of the
feeding system administered to the cows. These cows calved between February and
March and were put out to graze on pastures 1 month later (April). For most cows,
406 F. A. Fo1n . o1nv-
0 50 100 150 200 250
0 50 100 150 200 250
0 50 100 150 200 250
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III
IV
V
VI
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I
Days after kidding
Fig. 2. Variation in #, lactoperoxidase activity and $, thiocyanate content in milk from individual
goats (IVI) during lactation.
Lactoperoxidase and thiocyanate in milk 407
Table 3. Summary of lactoperoxidase activity and thiocyanate concentration during
lactation in goats. Each goat was sampled n number of times, maximum or minimum
levels were recorded at d number of days after calving
(Mean valuesp-)
Lactoperoxidase activity (Units\ml) Thiocyanate concentration (g\ml)
Goats Maximum Minimum Mean Maximum Minimum Mean
I 0n17 0n01 0n06bp0n04 14n1 4n0 7n0ap2n7
dl45 dl144 nl14 dl158 dl89 nl14
II 0n25 0n10 0n16dp0n05 11n6 2n2 7n1ap2n7
dl84 dl56 & 103 nl12 dl128 dl70 nl12
III 0n16 0n05 0n11cp0n04 10n9 2n7 6n6ap2n5
dl38 dl137 nl12 dl151 dl110 nl12
IV 0n09 0n01 0n04ap0n00 16 3n3 7n5ap3n0
dl72 dl30 nl13 dl30 dl88 nl13
V 0n23 0n02 0n10cp0n07 9n9 3n3 7n1ap1n0
dl62 dl133 nl13 dl48 dl78 nl13
VI 0n19 0n02 0n13cdp0n07 14n4 4n1 8n2ap3n4
dl95 dl135 nl8 dl81 dl149 nl8
a,b,c,d Means bearing the same superscript letter(s) are not signicantly dierent.
the thiocyanate content was lowest at the onset of lactation. This was followed by
a gradual increase such that the highest concentrations were recorded 2 months after
calving, in contradiction to Saad de Schoos et al. (1999) who reported the thiocyanate
content to be highest during the 1st month of lactation. The grand mean calculated
for the whole population (nl237) was 8n5p5n1 g\ml. Statistical analysis (at 95%
condence interval) showed that the thiocyanate content in cows A, B, C and D were
signicantly higher than the other cows, but not signicantly dierent from each
other. There appeared to be no direct correlation (R# l0n011) between lactoperoxi-
dase activity and thiocyanate content in the milk of the cows sampled (nl237).
Lactoperoxidase activity in goats milk
Enzyme activity patterns in all the goats sampled are given in Fig. 2. LP activity
was much lower in goats milk than in cows milk. Table 3 shows that milk sampled
from goat II on day 84 had the highest enzyme activity (0n25 U\ml) whereas milk
sampled from goat IV on day 30 was the lowest in enzyme activity (0n01 U\ml).
Unfortunately, it was not possible to assay the milk from these goats during the 1st
month of lactation. Nevertheless, there was no denite trend in LP activity
throughout the assay period as was the case with cows milk. Overall mean was
0n1p0n06 U\ml. There were again signicant dierences between goats, with means
ranging from0n040n16 U\ml for goats IVand II respectively. Variations within each
goat were also quite remarkable, the greatest population standard deviation
(0n07 U\ml) was found in goat V samples. Medina et al. (1989) also reported
signicant dierences in LP activity between individuals and between weeks of
sampling. For most of the goats selected, the highest enzyme activity was recorded
in samples that were collected during mid lactation (5075 d), agreeing with the
observations made by Zapico et al. (1990).
Thiocyanate content in goats milk
Again, no distinct pattern in thiocyanate content was evident from the goats
sampled. The highest concentration (16 g\ml) was recorded in the milk sampled
408 F. A. Fo1n . o1nv-
from goat IV on day 30 after kidding while the lowest (2n7 g\ml) was from milk of
goat III, sampled on day 110 after kidding. Zapico et al. (1991) reported an
individual sample from goats milk with thiocyanate content as high as 15n85 g\ml.
Half of the goats in the present study had their highest thiocyanate content towards
the end of lactation (i.e. more than 100 d after kidding). This observation agrees with
that of Zapico et al. (1991). Means ranged from 6n6 g\ml (Goat III) to 8n2 g\ml
(Goat VI). Grand mean for the entire population (nl72) throughout the study
period was 7n0p2n59 g\ml. Zapico et al. (1991) reported a mean thiocyanate content
content of 8 g\ml for milk from the Verata breed of goat while Medina et al. (1989)
reported a mean of 10n3 g\ml for ewes milk. There were no signicant dierences
between the individual average thiocyanate content of the goats monitored.
In the present study, there were also large variations in thiocyanate content
within goats throughout the sampling period. The widest variation ranging from
4n114n4 g\ml was observed in samples collected fromgoat VI. Such wide variations
have been reported by other workers (Medina et al. 1989; Zapico et al. 1990). As with
cows milk, there appeared to be no correlation (R# l0n015) between LP activity and
thiocyanate content in goats milk.
These results reveal that extremely wide variations occur in LP activity and
thiocyanate content within and between cows throughout lactation as well as
between animal species. Less extreme variations would be expected to occur in bulk
milk although this was not monitored. Nevertheless, it would therefore be
inappropriate to prescribe a single dose of substrates to be added to milk in order to
activate its LP system.
These results also have implications for the keeping quality of pasteurised milk,
as it is believed that the LP system plays a signicant role in its preservation
(Barrett et al. 1999). Considering that present regulations require all pasteurised
milks to test positive for LP activity, these massive variations in raw milk could
result in false negative\positive tests in some pasteurised milks.
vv-
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