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Resazurin reduction in milk Moyer, Rudy H.

Abstract

Methods were developed to quantitatively measure resazurin reduction and the reducing capacity of milk. The method for resazurin determination involved butanol extraction of the dye from milk and measurement of the optical density of extracts at 582 mμ and 615 mμ after saturation with sodium bicarbonate; that for reducing capacity was based on reaction of 2,6-dichloro-phenolindophenol with milk at its normal pH. For the latter determination excess indophenol was added and the quantity of dye remaining estimated spectrophotometrically at 660 mμ in butanol extracts saturated with sodium bicarbonate. This assay could be applied in the presence of resazurin or resorufin, since these compounds had negligible absorbance at 660 mμ. Both of these methods were reproducible in milk and their respective accuracies estimated at greater than 90 percent. The quantitative assay for resazurin was employed in order to study the behavior of resazurin in milk and the systems in fresh milk responsible for reduction of the dye. Results obtained on aging whole and skim milk were used to demonstrate that the resazurin reduction dealt with in the present investigation was due to the reducing systems of milk rather than to bacterial activity. These results showed that the reducing system was less stable and more sensitive to temperature of aging in skim than in whole milk. Measurement of rates of resazurin reduction by various fractions of normal milk showed that the major portion of the reducing ability of whole milk was associated with the cream. The aqueous phase from centrifuged warm gravity cream had a greater ability to reduce resazurin than did the whole milk from which the fractions were derived. The rate of resazurin reduction by milk decreased with incubation at 37°C, however, in the presence of sufficient numbers of bacteria, an acceleration of rate with incubation was noted. The point at which washed suspensions of added bacteria became significant in reduction was demonstrated as a change in slope of logarithmic plots of dye reduction rates. Resazurin was shown to have a destabilizing influence on the reducing capacity of milk. This Influence was catalytic and dependent on total concentration of dye; rate of inactivation being constant for a given dye concentration. Evidence was presented to show that the component of the reducing system that was inactivated was ascorbic acid. The influence of fractions, obtained from passage of resazurin through a silicic acid column, suggested that this catalytic effect was probably due to the dye as such, rather than to artifacts in the commercial dye preparation used. Examination of the reducing capacity of milk fractions before and after treatment with ascorbic acid oxidase indicated that resazurin reduction was brought about by that part of the reducing capacity that could not be accounted for as free ascorbic acid. In mastitic samples, this element of the reducing capacity was concentrated completely in the fat and centrifuged sediment. It was concluded from these investigations that the reducing system of milk existed as a measurable entity at any given time rather than as a continuous evolution of electrons from some slow enzymatic reaction. This system consisted of the measurable ascorbic acid of the milk, which occurred in the plasma, and some reducing agent bound to structural components of the cream and sediment. The measurable ascorbic acid accounted for approximately 80 percent of the reducing capacity but was concluded to have little influence on resazurin reduction. It was concluded that the bound reducing agent depended on structural elements in the milk for its ability to reduce resazurin, and that it lost this ability on dissociation from whatever particle it occurred on. It was postulated that this reducing agent was ascorbate and that it occurred bound to leucocytes and other cellular debris in the milk in situations analogous, to its reported occurrence in blood. Attempts to identify this reducing agent as ascorbate were unsuccessful in this investigation, but the techniques employed were probably inadequate.

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