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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-
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IV
V
VI
II
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-
Barrett, N. E., Grandison, A. S. & Lewis, M. J. 1999 Contribution of the lactoperoxidase system to the
keeping quality of pasteurised milk. Journal of Dairy Research 66 7380
Bjorck, L., Rosen, C. G., Marshall, V. & Reiter, B. 1975 Antibacterial activity of the lactoperoxidase system
in milk against pseudomonads and other Gram-negative bacteria. Applied Microbiology 30 199204
El Agamy, E. S. I., Ruppanner, R., Ismail, A., Champagne, C. P. & Assaf, R. 1992 Antibacterial and antiviral
activity of camel milk protective proteins. Journal of Dairy Research 59 169175
Kumar, S. & Mathur, B. N. 1989 Preservation of raw bualo milk through activation of LP-system. Part I.
Under farm conditions. Indian Journal of Dairy Science 42 339341
Marshall, V., Cole, W. M., Bramley, A. J. 1986 Inuence of the lactoperoxidase system on susceptibility of the
udder to Streptococcus uberis infection. Journal of Dairy Research 53 507514
Medina, M., Gaya, P. & Nunez, M. 1989 The lactoperoxidase system in ewes milk: levels of lactoperoxidase
and thiocyanate. Letters in Applied Microbiology 8 147149
Pruitt, K. M. & Kamau, D. N. 1994 Quantitative analysis of lactoperoxidase system components and of the
eects of the activated system on bacterial growth and survival. In Indigenous antimicrobial agents of
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Reiter, B. 1985 The lactoperoxidase system in bovine milk. Journal of Immunology Series 27 123141
Reiter, B. & Harnulv, B. G. 1984 Lactoperoxidase antibacterial system: natural occurrence, biological
functions and practical applications. Journal of Food Protection 47 724732
Ridley, S. C. & Shalo, P. L. 1990 Farm application of lactoperoxidase treatment and evaporative cooling for
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Saad de Schoos, S., Oliver, G. & Fernandez, F. M. 1999 Relationships between lactoperoxidase system
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Sokal, R. R. & Rohlf, J. F. 1981 Biometry. The principles and practice of statistics in biological research, 2nd
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Lactoperoxidase and thiocyanate in milk 409
Thakar, R. P. & Dave, J. M. 1986 Application of the activated lactoperoxidase-thiocyanate-hydrogen
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Zapico, P., Gaya, P., Nunez, M. & Medina, M. 1990 Lactoperoxidase and thiocyanate contents of goats milk
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Zapico, P., Gaya, P., Nunez, M., Medina, M. & De-Paz, M. 1991 Inuence of breed, animal and days of
lactation on lactoperoxidase system components in goat milk. Journal of Dairy Science 74 783787