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Peroxidases catalyse reactions in which hydrogen peroxide is reduced and a suitable electron donor is subsequently oxidised.  A wide variety of organic and inorganic substances can serve as electron donors, but substrate specificity varies between various peroxidases.

Most assays are based on the principle that the electron donor in oxidised form absorbs light and can be determined by a spectrophotometric assay.

Currently 2, 2’-azino-di-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) is accepted as the electron donor of choice for peroxidase assay.  ABTS is relatively stable in aqueous solution and there is no information, to the best of this author's knowledge, to indicate that it is hazardous to health.

The peroxidase assay using ABTS was originally developed by Shindler for the determination of peroxidase activity in cervical mucus.  However, work by Björck and others has shown that the method is suitable for determining LP activity in milk and milk products.

Detailed instructions for using ABTS in LP assay in milk and milk products have been described by Björck and Mullan (1993).

LP is a basic protein and therefore it can absorb to charged surfaces, particularly glass.  However, it retains its catalytic activity when absorbed to glass and this can cause difficulties in assaying the enzyme.  Glass cuvettes must be cleaned in acid between samples to remove any residual LP.  Alternatively, disposable cuvettes can be used.

Pruitt and his colleagues have also demonstrated that high concentrations of SCN (>0.5 mM) will interfere, giving lower LP activities due to simple competitive interference.  While it is unlikely that levels >0.5 mM will be encountered in milk it is advisable to measure SCN levels in unknown milk samples prior to critical LP analysis.

Björck and Mullan (1993) have also explained the need for excellent laboratory technique to minimise the coefficient of variation (CV) between samples.  Small differences in D A413/min values will have a significant effect on the results, for example a difference of 0.1 in DD A413/min will result in a 10% difference in activity (U/ml) of a solution of LP.

Mullan and Waterhouse (unpublished results) have found that the sensitivity of the ABTS method and the CV of the method can be significantly improved by using a pH of 6.0.  This modification also avoids some difficulties experienced with milk samples of low LP activity due to the precipitation of caseins in the pH 4.6 acetate buffer used in the original method.

Determination of biological activity or antimicrobial activity

Antimicrobial activity can be assessed by adding thiocyanate and hydrogen peroxide at the appropriate concentrations to a suitable medium containing LP and a designated strain of E. coli and determining the change in total viable count of the E. coli population.  Alternatively the effect on acid production by the E. coli can be measured.  The latter method is limited in that it cannot provide information on the level of bactericidal effect but it is simple and convenient to perform.  The relationship between total enzyme-protein concentration and antimicrobial activity can be obtained by diluting an enzyme preparation of known concentration in a quantitative manner and determining the concentration at which LP system activation is limiting

Methods were originally developed to enable the in vitro evaluation of the antimicrobial activity of calf milk replacers containing a LP system. There is no standard reference method and the evolution of the approach has been  described (Björck and Mullan, 1993).  Skim milk prepared from medium-heat skim milk powder, free from antibiotic residues and inhibitors of the LP system and containing no detectable LP activity, is reconstituted using distilled water to give 10% (w/v) milk solids.  A simple acidification assay can be performed using E. coli  NCDO 904. Tubes of milk (10 ml), inoculated with E. coli NCDO 904 to give 107 CFU/ml and supplemented with thiocyanate (0.25 mM) and hydrogen peroxide (0.25 mM), are incubated at 37°C for 6 h.  pH values at 6 h are subtracted from time 0 values and compared with controls containing 5 µg LP/ml and ± L- cysteine at an added concentration of 1 mM. The control without cysteine should give little or no change in pH whereas tubes containing 1 mM cysteine should show D pH values ranging from 0.25 to 0.4.

E. coli NCDO 2328 is much more sensitive to the LP system than strain NCDO 904. Consequently, the bactericidal effects of the LP system are studied most easily using NCDO 2328.  Media based on milk as discussed previously or 0.01 M phosphate buffer can be used. The method is essentially as described for the acidification assay except that viable calls of E. coli are enumerated using violet red bile agar (VRBA) at time 0 and after 2 h incubation as described by Extrand et al. (1985).

Antimicrobial effects can be determined in 0.01 M phosphate buffer using commercial LP preparations diluted to give LP activity equivalent to that given by enzyme concentrations between 0.05 and 0.5 µg/ml.  Optimal antimicrobial activity requires >0.5 µg LP/ml.

Specific details on factors affecting precision and accuracy have not been published in full but are known to include: strain of E. coli used, temperature of assay, incubation time and presence or absence of inhibitors of the LP system.  Controls incorporating a specific inhibitor of the LP system, for example L-cysteine, are required.


How to cite this article

Mullan, W.M.A. (2003) . [On-line]. Available from: https://www.dairyscience.info/index.php/exploitation-of-anti-microbial-proteins/98-lactoperoxidase-assay.html . Accessed: 26 September, 2016.

 

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