1. Rich media are recommended to obtain good growth of the microorganisms.

2. Maximum esterase and lipase production is observed at the early stationary phase of growth.

3. Other methods to lyse cells can be used, such as lysozyme treatment with sonication or pressure techniques.

4. Include blanks containing substrate without enzyme, boiled enzyme, and enzyme alone.

5. This time is recommended for formation and stabilization of the color complex.

6. When the intracellular fraction is obtained by the procedure described in Subheading 3.2.2., color is observed in the blank containing substrate without enzyme. When the pressure technique is used, this blank has no color. This method is recommended for purified enzymes.

7. The best sensitivity is obtained with 0.25% triglyceride.

8. Observe the presence of lipase activity at 12, 24, 48, and 72 h.

9. To quantify, measure the clearing-zone size surrounding the wells and express in mm.

10. Activity can be expressed as diffusion units, defined as the minimum fold dilution of the enzyme preparation that failed to give a detectable zone of hydrolysis (7).

12. In this technique, the lipid substrate and the cells are constantly mixed, resulting in more direct contact between the substrate and the enzyme. However, in the agar-well assay technique, the substrate is essentially immobile, and the enzyme or cells must diffuse or spread within the agar to reach the substrate, a process that is relatively slow.

13. Extraction efficiency is not always high, depending on the nature of the solvents and the FFAs. Calibrations must be done. Measurements do not take the acid molecules into account.

14. Make a standard curve with butyric acid.

15. All assays are performed in duplicate.


1. McKay, A. M. (1993) Microbial carboxylic ester hydrolases (EC 3.1.1) in food biotechnology. Lett. Appl. Microbiol. 16, 1-6

2 Fox, P. F. and Stepaniak, L. (1993) Application of enzymes in cheese technology. Int. Dairy J. 3, 509-536.

3. Lee, S. Y. and Lee, B. H. (1990) Esterolytic and lipolytic activities of Lactobacillus casei subsp. casei LLG. J. Food Sci. 55, 119-122.

4 Jaeger, K. E., Ransac, S., Dijkstra, B. W., Colson, C., van Huevel, M., and Misset, O. (1994) Bacterial lipases. FEMS Microbiol. Rev. 15, 29-64.

5 Layer, P. and Keller, J. (1999) Pancreatic enzymes: secretion a nd luminal nutrient digestion in health and disease. J. Clin. Gastroenterol. 28, 3-10.

6. De Man, J. C., Rogosa, M., and Sharpe, M. E. (1960) A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23, 130-135.

7. Fox, P. and Stepaniak, L. (1983) Isolation and some properties of extracellular heat-stable lipases from Pseudomonas fluorescens strain AFT 36. J. Dairy Res. 50, 77-89.

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