Preservation of raw milk by activation of the natural lactoperoxidase systems

Food Control, Vol. 7, No. 3, pp. 149-152, 1996 Copyright 0 1996 Elsevier Science Ltd ELSEVIER

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Preservation of raw milk by activation of the natural lactoperoxidase systems M.S. Haddadin,+ S.A. Ibrahim+ and R.K. Robinson** The preservation of raw ovine, bovine and caprine milks by activation of their natural lactoperoxidase (LP) systems was investigated. Milk samples with different concentrations of thiocyanate ions (SCN-) ranging from 1.5 to 150 mgll and hydrogen peroxide (Hz03 ranging from 10 to 100 mgll were stored at 4’, 22” and 30°C. Changes in titratable acidity, total colony counts and coliform counts of the milk samples were followed during storage. No significant diflerences were observed between samples with different concentrations of the added reagents, and this indicated that concentrations of 15 mgll (SCN-) and 10 mgll (Hz03 would be adequate for preserving milks of diflerent mammals - at least at 4°C. At higher temperatures, the effectiveness of the LP systems was, as monitored by the increases in acidity, of much shorter duration. Copyright 0 1996 Elsevier Science Ltd.

INTRODUCTION The lactoperoxidase (LP) system is a naturally occurring, antimicrobial mechanism found in raw milk. It can exert a bacteriostatic effect on both Grampositive and Gram-negative bacteria, including those psychrotrophic bacteria which decrease the shelf-life of liquid milk at 4°C (Bjorck, 1978; Marshall et al., 1986; Kamau et al., 1990; Wolfson and Sumner, 1994). The preservative action of the LP system in bovine milk has been well established (Reiter et al., 1976), and Pruitt and Reiter (1985) reported that activation of the system depends on the concentrations of two reactants, thiocyanate (SCN-) and hydrogen peroxide (H,O,). In particular, the LP system has the ability to catalyse the oxidation of thiocyanate by hydrogen peroxide with the produc+Department of Nutrition and Food Technology, University of Jordan, Amman, Jordan. *Department of Food Science and Technology, The University of Reading, Whiteknights, PO Box 226, Reading RG6 2AP, UK. *To whom correspondence should be addressed.

tion of the antibacterial hypothiocyanite (OSCN-) and other intermediates (Modi et al., 1991). These which can be further oxidized to compounds, end-products that are harmless to humans, have the ability to reduce bacterial growth by damaging the cell membranes and inhibiting the activity of many cytoplasmic enzymes. The normal concentrations of lactoperoxidase in bovine and ovine milks are reported to be above the minimum necessary for antibacterial activity, and hence the limiting factors are thiocyanate and hydrogen peroxide (Bjorck, 1978; Medina et al., 1989). In some circumstances, thiocyanate is present in bovine milk at levels sufficient to support an antimicrobial effect, eg up to 15 mg/l (Reiter, 1985) but, in ovine milk, the level can decline to 0.4 mg/l (Medina et al., 1989). Therefore, if the concentrations of SCN- and H,O, could be standardized, the milk of the important domestic mammals should show enhanced shelf-life even, perhaps, at ambient temperatures. However, while there are some reports on the effect of different concentrations of sodium thiocyanate/hydrogen peroxide on the activity of the Food Control 1996 Volume

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Preservation of raw milk: M.S. Haddadin et al.

LP system at different temperatures (Ewais et al., 1985), most of these studies have dealt with bovine milk. The purpose of this present study was, therefore, to evaluate the ability of the LP system to extend the shelf-life of ovine and caprine milks at refrigerated and ambient temperatures, using different concentrations of thiocyanate ions/hydrogen peroxide. The behaviour of bovine milk with the same levels of reactants was included for purposes of comparison.

MATERIALS AND METHODS The experiment was repeated four times (each in duplicate), and samples of fresh ovine, caprine and bovine milk were collected as required from the University of Jordan farm. Each bulk sample was kept on ice during transport to the laboratory, and was always used within 24 h of collection. More rapid usage of the milks would have been desirable, but logistical problems meant that some time delay was inevitable; in any event, the storage period would have only served to lower the bacteriological quality of milk. At the start of each experiment, the three milks were divided into portions (450 ml) in sterile, screwcap, &run bottles. The LP system of the samples was then activated by adding different amounts of sodium thiocyanate (Fisons, Loughborough, Leicestershire, UK) and sodium percarbonate (Fluka Chemie AG, CH-9470, Switzerland) - the latter acted as a source of hydrogen peroxide - to give four different concentrations of SCN- and H,O, in the ratios of 15: 10, 75 : 50, 125 : 75 and 150: 100. The control samples of raw milk received no treatment. After the addition of the different amounts of sodium thiocyanate/percarbonate, the samples were thoroughly mixed and duplicate bottles were stored at each of three temperatures. Samples at simulated ambient temperatures (30°C or room temperature, 22°C) were incubated in water baths, and a further pair of samples was kept in a refrigerator at 4°C. After 0, 3, 6, 9, 12, 15 and 18 h at 30°C or room temperature (22°C) portions (20 ml) were withdrawn from each bottle of milk for analysis; from samples ‘lbble 1 Effect of different concentrations milks stored at 4°C for up to 6 days

stored at 4°C portions were withdrawn once every 24 h over a period of 6 days. Bacteriological and chemical analyses

For measurement of the titratable acidity, a portion (10 ml) of each sample was mixed with 10 ml of distilled water and titrated with sodium hydroxide (N/9) to an end-point of pH 8.5 using an analyticalgrade pH meter (Hana Instrument Model HI 8416, Limena, Italy). The volume of sodium hydroxide dispensed was divided by ten to give the titratable acidity as percentage lactic acid. The bacteriological analyses were applied only to batches stored at 4”C, and these milks were tested for total colony counts and coliform counts on receipt from the farm, and then over six days storage. Duplicate sub-samples (1 ml) of the three milks were diluted in a series of peptone solutions (9 ml, 0.1%) down to lo+, and 1 ml aliquots from the appropriate dilutions (1O-*-1O-4 for coliforms and 10-5-10-7 for the total counts) were transferred to Petri dishes (9 cm). Yeast milk agar (Unipath Ltd, Basingstoke, UK) was employed for the total colony counts as recommended by BSI (1968), and the plates were incubated at 30°C for 72 hours; coliforms were enumerated on Violet Red Bile Agar (Unipath Ltd, Basingstoke, UK) after incubation at 37°C for 48 hours. Statistical analysis

All data were analysed using the General Linear Models of the Statistical Analysis System (SAS, 1988) to determine the significance between treatment means.

RESULTS When the different milks were stored at 4”C, the acidities of the treated samples of bovine milk were almost unchanged over four days (P ~0.01) and, even after six days, the increases were, at least in practical terms, negligible (see Table I). Thus, it is widely accepted that the maximum acidity in bovine milk for cheesemaking, for example, should be 0.16% lactic

of SCN- and H,O, (mg/l) on the developed acidity (% lactic acid) of ovine, bovine and caprine

Control

15:lO

75:50

150: 100*

Time/ratios (d)

(0)

(B)

(C)

(0)

(B)

(C)

(0)

(B)

(C)

(0)

(B)

(C)

0

0.18

0.14

0.13

0.17

0.14

0.12

0.17

0.13

0.12

0.17

0.12

0.12

:

0.20 0.22

0.16 0.14

0.15 0.14

0.17 0.18

0.14

0.11

0.18 0.20

0.14

0.12

0.18 0.17

0.12

0.12

: 5 6

0.25 0.29 0.30 0.40

0.19 0.22 0.24 0.30

0.17 0.20 0.28 0.33

0.20 0.22 0.23 0.26

0.14 0.15 0.15 0.16

0.15 0.17 0.19 0.24

0.20 0.22 0.22 0.27

0.14 0.15 0.16 0.16

0.13 0.14 0.16 0.18

0.20 0.22 0.22 0.22

0.13 0.13 0.14

0.13 0.15 0.16 0.19

(0) =ovine milk; (B) = bovine milk; (C) = caprine milk. *The results for 125 : 75 were similar. Least square means: standard error = f 0.05, replications = 4.

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acid (Tamime, 1993), so that even the lowest concentrations of thiocyanate and hydrogen peroxide were sufficient to keep the milk in an acceptable state for processing for six days. By contrast, the untreated samples showed an increase in titratable acidity after only two days (P
of raw milk: MS. Haddadin

et a/.

combination was disappointing. The more so as, according to Medina et al. (1989), sheep milk should contain sufficient lactoperoxidase to produce a bacteriostatic effect and the colony counts were little different from the caprine milk. Whether or not the level of catalase in the ovine milk was higher than in the caprine milk was not established, but a catalasemediated loss of hydrogen peroxide might explain, at least in part, the contrasted results. However, given that high bacterial counts in sheep milk are not uncommon (Nunez et al., 1984) activation of the LP system clearly cannot conceal poor microbiological quality (Bjorck et al., 1979). At 22°C the untreated samples of bovine milk became unacceptable after 3 hours, and the rate of deterioration accelerated dramatically after 6 hours (see Table 3). However, activation of the LP system increased the shelf-life of milk samples up to 18 hours (P ~0.01). At 30°C the acidity of the treated bovine milks had increased little after 6 hours, whereas the acidity of the untreated samples became totally unacceptable (P < 0.05). Indeed, even after 18 hours at 30°C all the treated samples of cow milk would have been usable for processing, while the control had an acidity of 0.66% lactic acid. Valdez et al. (1988) found that the natural LP system was inhibitory against microorganisms for up to 8 hours in samples of bovine milk stored at 30°C but it is clear

Table 2 Effect of different concentrations of SCN- and HzO, (mgil) on the bacteriological quality of ovine, bovine and caprine milks stored at 4°C; all counts as colony-forming units/ml of milk Time of storage, 6 days Ratios

Zero

Control

15:lO

75:50

100 : 150*

5.3 1.2 x 10” 4.1 x 10H

50.0 10.6 x 10” 8.5 x 10”

9.4 1.0 x 10’ 3.1 x 10’

3.5 x 10” 6.5 10’ 8.5 x 10’

8.5 x 10’ 8.2 10” 2.0 x 10’

2.9 x lo” 2.2 10’ 3.5 x 10”

8.5 x 10’ 5.1 10” 3.2 x 10’

1.5x 10’ 1.9 9.5 x IO’

1.8 x 10’ 1.9 2.5 x 10”

2.5 x lo3 7.5 102 4.0 x l(Y

Total colony count [:I (C) Coliform count IB9’ (C)

(0) = ovine milk; (B) = bovine milk; (C) = caprine milk. *The results for 125 : 75 were similar. Least square means: standard error = k 0.13, replications = 4.

Table 3 Effect of different concentrations of SCN and H,O, (mg/l) on the developed acidity (% lactic acid) of ovine, bovine and caprine milks stored at ambient temperature (22°C) for up to 18 hours Control

150: 100*

75:50

15:lO

Time/ratios (d)

(0)

(B)

(C)

(0)

(B)

(C)

(0)

(B)

(C)

(0)

(B)

(C)

0 3 6 9 12 15 18

0.18 0.20 0.27 0.38 0.45 0.50 0.60

0.14 0.15 0.24 0.44 0.53 0.60 0.62

0.12 0.16 0.22 0.30 0.38 0.45 0.52

0.18 0.18 0.18 0.21 0.20 0.35 0.38

0.12 0.12 0.13 0.14 0.14 0.15 0.16

0.12 0.12 0.13 0.14 0.14 0.16 0.25

0.18 0.18 0.19 0.19 0.20 0.21 0.38

0.13 0.13 0.14 0.14 0.15 0.16 0.17

0.12 0.12 0.13 0.16 0.17 0.18 0.30

0.18 0.19 0.21 0.21 0.25 0.35 0.38

0.14 0.14 0.15 0.15 0.15 0.15 0.16

0.12 0.12 0.13 0.16 0.18 0.18 0.26

(0) = ovine milk; (B) = bovine milk, (C) = caprine milk. *The results for 125: 75 were similar. Least square means: standard error = + 0.07, replications = 4.

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that raising the levels of thiocyanate and hydrogen peroxide can double the span of the bacteriostatic effect. The acidity of the treated samples of caprine milk held at both room temperature and 30°C was stable for 9-12 hours even at the lowest concentrations of added reactants, while the acidity of the control was above 0.30% lactic acid. After 15 hours, the acidity of the treated caprine samples (both temperatures) was just on the borderline of acceptable, but the stability declined sharply thereafter. Once again, it was the ovine milk that derived least benefit from activation of its LP system, with 6-9 hours being the maximum period for comparative pH stability. Obviously these values are an improvement over the controls, but it would appear that the practical benefits of adding thiocyanate / peroxide to high count ovine milks are likely to be small in the absence of refrigeration. Overall, the results show that, by activation of the LP system in raw milk, it is possible to store ovine, bovine and caprine milks at 4°C for several days. High concentrations of sodium thiocyanate and hydrogen peroxide were only marginally more effective than the minimum levels tested, and hence a level of thiocyanate ions of 15 mg/l and H,O, of 10 mg/l of milk should be sufficient to delay the risk of spoilage prior to processing; at this level, the potential health risk of the thiocyanate to humans should be negligible (Bjorck et al., 1979). At higher temperatures of storage, the major advantage was observed with bovine milk, and any serious increase in acidity was inhibited for around 18 hours. With caprine and ovine milks, the high initial cell counts of the test samples tended to nullify the preservative effect of the LP system at ambient temperatures, but even so, acid development was suppressed for several hours vis-a-vis the control samples; 4°C and a 15: 10 (mgll) ratio of thiocyanate : hydrogen peroxide are recommended for retention of the quality in raw ovine and caprine milks. The effect of the system on the nutritional value of the milk merits further study but, even if there is an associated destruction of vitamins for example, the loss may be preferable to wasting entire batches of milk in regions where supplies are already limited.

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REFERENCES Bjork, L. (1978) Antibacterial effect of the lactoperoxidase system on psychrotrophic bacteria in milk. J. Dairy Res. 45, 109-118 Bjork, L., Claesson, 0. and Schulthess, W.M. (1979) The lactoperoxidase/thiocyanate/drogen peroxide system as a temporary preservative for raw milk in developing countries. Milchwissenschafi 34,726-729 BSI (1968) British Standard London.

4285. British Standards

Institute,

Ewais, S.M., Hefnawy, SLA. and Abd El-Salam, M.H. (1985) Utilization of lactoperoxidase system in preservation of raw milk under local conditions. EgyptianJ. Dairy Sci. 13, l-7 Kamau, D.N., Doores, S. and Pruitt, K.M. (1990) Enhanced thermal destruction of Listeriu monocytogenes and Srccphylococcus aureus by lactoperoxidase system. Appl. Environ. Microbiol. 56, 2711-2716 Marshall, V.M.E., Cole, W.M. and Bramley, A.J. (1986) Influence of the lactoperoxidase system on susceptibility of the udder to Streptococcus uberis infection. J. Dairy Res. 53,507-514 Medina, M., Gaya, R and Nunez, M. (1989) The lactoperoxidase system in ewe’s milk: levels of lactoperoxidase and thiocyanate. Let. Appl. Microbial. 8, 147-149 Modi, S., Deodhar, S.S., Behere, D.V. and Mitra, S. (1991) Lactoperoxidase-catalysed oxidation of thiocyanate by hydrogen peroxide. Biochemistry30, 118-124 Nunez, J.A., Chavarri, EJ. and Nunez, M. (1984) Psychrotrophic bacterial flora of raw ewe’s milk with particular reference to Gram-negative rods. J. Appl. Bacterial. 57, 23-29 Pruitt, K.M. and Reiter, B. (1985) Biochemistry of peroxidase svstem: antimicrobial effects. in K.M. Pruitt and J. Tenovuo (kds), The Lactoperoxidase System, 143-178, Marcel Dekker Inc, New York, USA Reiter, B., Marshall, M.E., Bjorck, L. and Rosen, L.G. (1976) Nonspecific bactericidal activity of the lactoperoxidase-thiocyanate-hydrogen peroxide system of milk against Escherichia coli and some Gram-negative pathogens. Infect. Immune. 13, 800-807 Reiter, B. (1985) Protective proteins Federation Bull. 191,2-35

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Statistical Analysis System (SAS) (1988) User’s Guide in Statistics. SAS Institute Inc, Cary, NC, USA Tamime, A.Y. (1993) Modern cheesemaking: hard cheeses, in Modem Dairy Technology, 49-220, R.K. Robinson (ed), Chapman & Hall, London Valdez, G.E, Bibi, W. and Bachmann, M.R. (1988) Antibacterial effect of the LP system on the activity of thermophilic starter culture. Milchwissenschaft43, 350-352 Wolfson, L.M. and Sumner, S.S. (1994) Antibacterial activity of the lactoperoxidase system against Salmonella typhimurium in trypticase soy broth in the presence and absence of a heat treatment. J. Food Prot. 57, 365-368