Fundamental Food Microbiology, Third Edition - Fuad Fathir

318 FUNDAMENTAL FOOD MICROBIOLOGY
shelf life of dairy products. Some of the earlier methods, such as UV absorbance,
Folin–Ciocalteu reagent reaction, and gel diffusion assay, are probably not sensitive
enough for this purpose. Several new methods, such as the use of TNBS (trinitrobenzene
sulfonic acid) and fluorescamine reagents are quite sensitive and are being
tested to assay proteinases in milk. In the TNBS method, the reagent reacts with
free amino groups and, under the experimental conditions, develops color that can
be colorimetrically measured to determine the amount of free amino acids present
because of proteolysis. Fluorescamine reacts with amino acids to form fluorescent
compounds at pH 9.0 and can thus be fluorimetrically measured to determine protein
hydrolysis. Other methods, such as enzyme-linked immunosorbant assay (ELISA)
and luciferase inactivation assay are extremely sensitive and need further development
before they can be used reliably.
B. Heat-Stable Lipases in Milk
Because natural lipases are present in milk, the measurement of lipases produced
specifically by psychrotrophic bacteria creates some difficulties. 6 However, it can
be overcome by heating the milk, which destroys milk lipases but not the bacterial
heat-stable lipases. Assay methods that measure release of free fatty acids (FFAs)
due to hydrolysis of milk fat by the lipases can be titrated to determine the potential
of lipolysis of the lipases. As milk contains FFAs naturally, this method may not be
accurate. Methods in which esterases of chromogenic and fluorogenic compounds
react with lipases to produce color or fluorescent products have also been developed,
but they have limitations. Recently, a rapid and sensitive sandwich ELISA method
was tested to determine lipases of Pseudomonas spp. An antibody produced against
a Pse. fluorescens strain and linked to horseradish peroxidases reacted with lipases
from many strains of Pseudomonas spp. Reliability and sensitivity of this technique
are being studied.
V. CONCLUSION
Food spoilage causes not only economic loss but also unavailability of food, especially
in places of food shortage and with high population, such as in some developing
countries. To reduce both, efficient techniques need to predict storage potential
of foods, especially because the consumption of processed foods is going to increase.
The methods now used have limitations and are not highly efficient. New methods
that are now being studied could be more effective in the future. One of these methods
is the development of biosensors, which could detect important bacterial metabolites
at very low concentrations. Another method is to determine bacterial load rapidly
by PCR. However, this technique cannot differentiate between live and dead cells.
As with lactic acid bacteria at present, if in the future the genome sequences of
important spoilage bacteria become available, better and specific biosensors and
PCR techniques can be developed to detect spoilage potential of a food at an early
stage.