Determination of Bacterial ATP Levels in Raw Milk: Selectivity of Non-Bacterial ATP Hydrolysis

Abstract

The selective destruction of non-bacterial ATP and subsequent determination of bacterial ATP using the ATP bioluminescent technique was investigated. Treatments to release ATP from somatic cells and hydrolyze free ATP also significantly reduced the ATP content of Enterobacter cloacae in skim and raw milk. The reduction can mainly be ascribed to apyrase (an ATPase) affecting the ATP content of intact bacteria. Somatic cell treatments failed to completely eliminate non-bacterial ATP. Although treatment with a somatic releasing reagent, EDTA and apyrase, resulted in a 96% reduction in the ATP content of raw milk, the remaining non-bacterial ATP was still considerably more than found in the bacterial component of raw milk studied here. Until reagents are available to selectively destroy all non-bacterial ATP without affecting the bacterial ATP content, the bioluminescent technique will have limited application in determination of the bacterial quality of raw milk.

Improved bioluminescent assay of somatic cell counts in raw milk

“…Strong acids, ionic detergents or organic solvents are applied to destroy bacterial cells. However, it was shown before that Triton X-100 does not destroy lymphocytes completely (Lundin, 1984) but partially damages some bacterial species, for example, Pseudomonas (Theron et al 1986). Therefore, to select the most appropriate ATP-releasing agent for somatic cells of milk, we compared the action of several neutral detergents, including Triton X-100, both on bacteria and somatic cells of milk (Table 1).…”

Comparaison de trois méthodes de dosage de l'ATP par bioluminescence pour l'estimation de la qualité bactériologique du lait cru

“…Il a alors conclu qu'une forte concentration en apyrase altérait l'ATP bactérien, et qu'un temps de digestion trop court ne permettait pas d'éliminer totalement l'ATP d'origine somatique. De plus, l'altération de l'ATP bactérien semble dépendre des souches microbiennes présentes dans l'échantillon (Theron et al, 1986a, Langeveld et Van der Waals, 1988.…”

“…L'estimation du niveau de pollution microbienne du lait cru par dosage d'ATP rencontre quelques difficultés principalement liées à la présence de cellules somatiques et de micelles de caséines qui contiennent aussi de l'ATP (Bossuyt, 1978;Richardson et al, 1980;Theron et al, 1986a). L'ATP d'origine somatique représente 96% de l'ATP total du lait cru, et son élimination est très difficile, voire même impossible sans altérer "ATP d'origine bactérienne (Theron et al, 1986a).…”