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Determination of the extracellular lipases of Pseudomonas fluorescens spp. in skim milk with the β-naphthyl caprylate assay

Published online by Cambridge University Press:  01 June 2009

Robin C. McKellar  and
Hilaire Cholette
Robin C. McKellar
Affiliation:
Food Research Institute, Research Branch Agriculture Canada, Ottawa, Ontario K1A 0C6, Canada
Hilaire Cholette
Affiliation:
Food Research Institute, Research Branch Agriculture Canada, Ottawa, Ontario K1A 0C6, Canada
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Summary

A method based on the hydrolysis of β-naphthyl caprylate (β-NC) has been developed for quantitating extracellular lipase from Pseudomonas fluorescens. The assay was extremely sensitive to skim milk (SM); as little as 0·02 ml raw SM in a 2·0 ml reaction mixture resulted in an apparent loss of 50% of the lipase activity. Activity improved 3-fold when trypsin (50 μg/ml) was included in the reaction mixture. When super-simplex optimization was used to determine the optimum levels of β-NC, Na taurocholate (NaTC), SM/lipase mixture and trypsin for maximum activity, NaTC was found to be unnecessary for activity. Subsequent addition of 15 mM-NaTC resulted in 80% loss of activity. On the other hand, NaTC was required for native lipase activity in the presence of SM. Native lipase was completely inhibited by heating at 70 °C for 2 min, while B52 lipase retained 75% of its activity under the same conditions. The assay was able to detect lipase produced by Ps. fluorescens B52 in SM at 5 °C when the cell density exceeded 108 colony forming units/ml. The presence of butterfat (3°5%) in the SM assay inhibited B52 lipase by 97%. The β-NC assay gave results comparable to the tributyrin agar diffusion assay using cell-free extracts of ten strains of common dairy psychrotrophs. The results suggest that the β-NC assay may be useful for determining lipase activity in raw SM.

Type
Original Articles
Information
Journal of Dairy Research , Volume 53 , Issue 2 , May 1986 , pp. 301 - 312
Copyright
Copyright © Proprietors of Journal of Dairy Research 1986

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