Letters in Applied Microbiology 1989,8, 147-149 FLD/89/01

The lactoperoxidase system in ewes’ milk : levels of lactoperoxidase and thiocyanate

M A R G A R I T A M E D I N A , P I L A R GAYA & MANUEL NUREZ Departamento de Produccibn y Tecnologia de Alimentos, CIT-INIA, Apartado 8111, Madrid 28080, Spain

Received 5 January 1989 and accepted 17 January 1989

M E D I N A , M., GAYA, P. & NUREZ, M . 1989. The lactoperoxidase system in ewes’ milk : levels of lactoperoxidase and thiocyanate. Letters in Applied Microbiology 8, 147-149.

Ewes’ milk lactoperoxidase levels in the range of 0.142.38 units/ml were detected, with significant differences between individuals, herds and weeks of sampling. Thio- cyanate concentration ranged from 0.4 to 20.6 ppm, varying significantly between individuals and weeks of sampling.

The activity of the lactoperoxidase system (lac- toperoxidase/thiocyanatejhydrogen peroxide) in bovine milk and its preservative effect at ambient and refrigeration temperatures are well known (Oram & Reiter 1966; Bjorck et al. 1975, 1979; Reiter et al. 1976; Bjorck 1978).

Lactoperoxidase, usually present at levels of 10-30 pg/ml, constitutes about 1% of the whey proteins in bovine milk (Reiter 1985). Different methods of analysis employed render the com- parison of published data difficult. A mean value of 1.4 units/ml was reported for bovine milk by Stephens et al. (1979) using the sensitive method of Shindler et al. (1976).

Thiocyanate is widely distributed in animal tissues and secretions. Natural thiocyanate content of bovine milk varies between 1 and 10 ppm (Wood 1975).

The third component of the system, hydrogen peroxide, may be generated in milk by leuco- cytes and by lactic acid bacteria.

Ewes’ milk produced in Spain (245 million litres in 1986) is used for the manufacture of cheese, a large proportion of which is made from unpasteurized milk. Total viable counts of lo7 cfu/ml and psychrotroph counts of lo6 cfu/ml are common in ewes’ milk sampled on arrival at the dairy, due to unsatisfactory milking practices and to multiplication during farm storage and collection (Nuiiez et al. 1984).

No information on the lactoperoxidase system in ewes’ milk is available in the liter- ature. The aim of the present work was to deter- mine levels of lactoperoxidase and thiocyanate in milk from this species, in order to evaluate the feasibility of this system as a method of improving the bacteriological quality of ewes’ milk.

Materials and Methods

M I L K S A M P L E S

Milk from 18 Manchega breed ewes of three dif- ferent herds (six ewes/herd) was examined. Morning and evening milkings from individual animals were separately collected on three dif- ferent days during mid-lactation, one week elapsing between consecutive sampling days.

Samples were stored at 4°C and analysed within 24 h.

C H E M I C A L A N A L Y S I S

Lactoperoxidase activity was determined in duplicate after Marshall et al. (1986), by follow- ing the oxidation of ABTS (2,2’-azinodi-3-ethyI- benzthiazoline-6-sulphonic acid) (Sigma Chemi- cal Co., St Louis, MO, USA) at 412 nm using a Beckman DU7 spectrophotometer.

148 Margarita Medina et al. Table 1. Lactoperoxidase levels

(units/ml) in ewes’ milk

Samuling*

Table 2. Thiocyanate levels (ppm) in ewes’ milk

Sampling*

Herd 1 2 3 Herd 1 2 3

A 0.70t 0.66 0.61 B 0.78 0.78 0.66 C 0.85 1.05 0.87

* One week elapsed between

t Mean of morning and evening consecutive sampling days.

samples from six animals.

Thiocyanate content was determined in dupli- cate, after deproteinization with 20% (w/v) tri- chloroacetic acid, as the ferric complex formed with the ferric nitrate reagent, by measuring the absorbance at 460 nm (Bjorck et al. 1975).

S T A T I S T I C A L A N A L Y S I S

Analysis of variance and correlation on data obtained were performed by means of programs BMDOSV and BMDP8D (Department of Bio- mathematics, UCLA, Los Angeles, USA), respectively.

Results and Discussion

L A C T O P E R O X I D A S E

The lactoperoxidase content of ewes’ milk samples ranged from 0.14 to 2.38 units/ml, with a mean value of 0.77 units/ml (Table 1). The lac- toperoxidase level and milk production were not significantly correlated (r = 0.055). Analysis of variance detected significant differences between individuals (P < 04)01), herds (P < 0.001) and weeks of sampling (P < 0.001), but not between morning and evening milkings.

Differences in the lactoperoxidase content of bovine milk have been reported, with variations due to individual, breed and lactation stage. Lactoperoxidase level in bovine milk is also influenced by feeding and oestrogen concentra- tion (Reiter 1985). Lactoperoxidase activity in human milk ( 0 . 0 6 4 9 7 units/ml) is lower than in bovine milk (Reiter 1985). Peroxidative activ- ity in human milk seems to be exclusively derived from milk leucocytes, the enzyme thus being myeloperoxidase (Moldoveanu et al. 1982). The highest value for lactoperoxidase activity has been found in guinea pig milk, with a mean content of 22 units/ml (Stephens et al.

A 12.06t 6.98 11.93 B 10.06 10.20 12.02 c 10.04 8.28 11.13

* One week elapsed between con-

t Mean of morning and evening secutive sampling days.

samples from six animals.

1979). A level of 0.9 units/ml was reported for buffaloes’ milk (Harnulv & Kandasamy 1982).

T H I O C Y A N A T E

The mean thiocyanate content in ewes’ milk samples reached 10.3 ppm, with a maximum of 20.6 ppm and a minimum of 0.4 ppm (Table 2). Milk production and thiocyanate concentration were not correlated (r = -0.135). Significant differences in thiocyanate content were only observed between individuals (P < 0.001) and between weeks of sampling (P < 0.001).

Thiocyanate levels of 3 . 2 4 6 ppm were found in cows’ milk by Bjorck et al. (1979), whereas 5-6 ppm have been reported for buffaloes’ milk (Harnulv & Kandasamy 1982; Thakar & Dave 1986). Ewes’ milk thus seems to contain higher levels of thiocyanate than cows’ and buffaloes’ milk. However, cows on natural pastures con- taining clover give milk with up to 15 ppm of thiocyanate, particularly at the height of the summer (Reiter 1985).

Ewes’ milk lactoperoxidase activity compares well with values previously reported for milk from other species. As the limiting factors for activation of the lactoperoxidase system in milk are thiocyanate and hydrogen peroxide, addi- tion of thiocyanate to a level of 15 ppm and 8.5 ppm of hydrogen peroxide could be feasible, according to the results obtained by Bjorck et al. (1979), for ewes’ milk preservation in areas where refrigeration is not available and for improving the bacteriological quality of refriger- ated ewes’ milk.

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