Indicators of Microbial Food Spoilage ◾ 299
during storage. More emphasis needs to be given to develop suitable indicators to reduce the loss of
food by microbial spoilage. In the future, biosensors (Chapter 42) may be developed that could be
effective for indicating changes in specific metabolites by a group of bacteria with similar characteristics, which are considered important spoilage bacteria in a food group. 3 Commercially available
electric nose (EN) biosensors, such as SensorfreshQ TM , LibraNose, FreshSense, etc. have been used to
monitor microbial spoilage of fresh food, including meat, fish, and poultry. These devices detect volatile byproducts, such as H 2 S, NH 3 , CO 2 , diacetyl, and acetoin, resulting from microbial metabolism.
Molecular Methods in Spoilage Microbe Detection
Inherent drawbacks in conventional methods led to the application of modern molecular techniques in detection and identification of microorganisms or microbial community in food products. Because spoilage in products, such as raw meat, poultry, or meat products, are associated
with multiple organisms, molecular tools allow analysis of microbial community. Methods, such
as randomly amplified polymorphic DNA (RAPD)-PCR, real-time PCR, pulsed field gel electrophoresis (PFGE), rep-PCR, multilocus sequence typing (MLST), and whole genome sequencing,
have been used on various meat products 4 (see Chapter 42).
Assay of Heat-Stable enzymes
Heat-Stable Proteinases in Milk
Proteinases of some psychrotrophic bacteria, such as Pseudomonas fluorescens strain B52, even when
present as low as 1 ng/mL, in raw milk can reduce the acceptance quality of UHT-treated milk
during normal storage. 5,6 Because of this, it is very important that sensitive assay methods be used
in their estimation to predict the 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 trinitrobenzene sulfonic acid
(TNBS) 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.
Heat-Stable Lipases in Milk
Because natural lipases are present in milk, the measurement of lipases produced specifically by
psychrotrophic bacteria creates some difficulties. 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) as a result of the 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
during storage. More emphasis needs to be given to develop suitable indicators to reduce the loss of
food by microbial spoilage. In the future, biosensors (Chapter 42) may be developed that could be
effective for indicating changes in specific metabolites by a group of bacteria with similar characteristics, which are considered important spoilage bacteria in a food group. 3 Commercially available
electric nose (EN) biosensors, such as SensorfreshQ TM , LibraNose, FreshSense, etc. have been used to
monitor microbial spoilage of fresh food, including meat, fish, and poultry. These devices detect volatile byproducts, such as H 2 S, NH 3 , CO 2 , diacetyl, and acetoin, resulting from microbial metabolism.
Molecular Methods in Spoilage Microbe Detection
Inherent drawbacks in conventional methods led to the application of modern molecular techniques in detection and identification of microorganisms or microbial community in food products. Because spoilage in products, such as raw meat, poultry, or meat products, are associated
with multiple organisms, molecular tools allow analysis of microbial community. Methods, such
as randomly amplified polymorphic DNA (RAPD)-PCR, real-time PCR, pulsed field gel electrophoresis (PFGE), rep-PCR, multilocus sequence typing (MLST), and whole genome sequencing,
have been used on various meat products 4 (see Chapter 42).
Assay of Heat-Stable enzymes
Heat-Stable Proteinases in Milk
Proteinases of some psychrotrophic bacteria, such as Pseudomonas fluorescens strain B52, even when
present as low as 1 ng/mL, in raw milk can reduce the acceptance quality of UHT-treated milk
during normal storage. 5,6 Because of this, it is very important that sensitive assay methods be used
in their estimation to predict the 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 trinitrobenzene sulfonic acid
(TNBS) 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.
Heat-Stable Lipases in Milk
Because natural lipases are present in milk, the measurement of lipases produced specifically by
psychrotrophic bacteria creates some difficulties. 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) as a result of the 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