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Microbiological analysis is an important technique to assure the quality and safety of the food products for consumption. Conventional microbiological methods are commonly used for the identification and enumeration of harmful microorganisms in food products. However, these methods are time consuming and tedious (Vasavada, 1993). Furthermore, these methods cannot be used during the processing system of food products (Bell et al., 1994, Damez and Clerjon, 2008).
In modern food processing industries, rapid microbiological techniques are becoming an important tool over conventional methods because rapid techniques provide results more quickly than conventional methods as it require less time for the preparation and processing of samples and interpretation of the results. In addition, the use of rapid microbiological techniques increases the total number of samples which can be analysed per day which is tedious work to perform by conventional microbiological analysis (Vasavada, 1993).
Rapid microbiological methods are automated or semi automated methods such as impedance measurement, conductance analysis, bioluminescence, colorimetry, enzyme-linked immunosorbent assay (ELISA), flow cytometry, reflectometry, radiometry etc. which are now a days commonly used for the rapid detection of microorganisms during the processing system of food products (White, 1993, Vasavada, 1993).
Impedance measurement is an important transduction technique used in microbiological analysis of food products (Madrid et al., 1999). When micro organism grow in the culture medium, then its growth and metabolic activity transforms proteins, carbohydrates and fats into smaller, highly charged end products such as amino acids, lactate, acetate etc (Vasavada, 1993).
Furthermore, the metabolic activity causes changes in the chemical composition of culture media such as ionic content. The change in ionic content of culture medium further changes the conductivity of culture broth which can be monitored by the use of electrodes on an alternating current flowing through the medium and thus impedance can be measured (Swaminathan and Feng, 1994).
The measured impedance determines the microbial content in the medium (Madrid et al., 1999). Impedancimetry is also used for the detection of aerobic mesophiles, faecal and total coliforms (Orsi et al., 1997). Bactometer and Malthus system instruments are used to measure the impedance and conductance changes in the medium (Vasavada, 1993).
Impedancimetry technique has been approved by AOAC (Association of Official Analytical Chemists) for its use in the food borne pathogens such as Staphylococcus aureus, Clostridium Botulinum, Listeria monocytogenes, Yersinia enterocolitica and Campylobacter. It has also been experimentally observed that impedance technique can be used for the measurement of microbial contaminants in juices and beverages (Vanne et al., 1996).Furthermore, impedancimetry can be used as a powerful tool for the monitoring of various organisms such as total aerobic mesophiles ,faecal and coliform in dairy and ready to use vegetables (Orsi et al., 1997).
Impedancimetry technique can be used for the quality indication of meat as a non destructive method. As meat is electrically anisotropic and its electrical properties depends on the direction of the electrical field in the sample. It has been studied that the electrical properties can be measured by placing the electrodes in three different directions in meat. Furthermore, the impedance value of muscle reduces rapidly during the pre-rigor stage in pork and beef and is an indicator of ageing of meat (Lepetit et al., 2002).
It has been studied and observed that impedancimetry techniques can be used as a rapid method in the milk processing plant for measuring the total, mesophilic and psychrotropic counts in raw milk (R and M. K, 1983).
ATP- bioluminescence is another rapid method for estimating the populations of microorganisms in food sample as well as on the clean surfaces, utensils (T. R, 1985) and process equipments (Vanne et al., 1996). Furthermore, it is also helpful in maintaining hygiene in food processing plant (de Boer and Beumer, 1999).
ATP - bioluminescence is a rapid method which measures the number of bacteria presents in the sample by measuring the light produced by the stoichiometric conversion of ATP into photons of light (Griffiths, 1993). Microbial and food ingredients cell contains adenosine-5-triphosphate (ATP) which is calculated by using the luciferin- luciferace enzyme complex found in the tail of firefly Photinus pyrali (Figure 1) (de Boer and Beumer, 1999).
Figure 1: Representation of the firefly luciferase reaction (Griffiths, 1993)
The total light output of the sample is directly proportional to the amount of ATP present and can be measured by luminometer (de Boer and Beumer, 1999). It has been observed that ATP bioluminescence method can be used to detect as few as 1 x 104 cfu/ml of bacteria in food within 5 to 10 min (Griffiths, 1993).
ATP bioluminescence technique can be applied in dairy industry to assess the hygiene of food contact surface in various situations (Vilar et al., 2008). Furthermore, it has been claimed that ATP-bioluminescence technique can be used to detect the bacteria in milk up to the concentration of 1 x 104 within 5 to 10 minutes. This rapid analysis of bacteria in milk makes the technique useful as a rejection parameter for the incoming milk in milk processing industry. However, it has also been claimed that the technique can measure 24 samples within 1-2 hours without culturing (Griffiths, 1993).
ATP - bioluminescence technique can also be used in microbial analysis of food such as pork and beef carcasses (Vilar et al., 2008).Furthermore, the technique can be used as an index of bacterial activity and can also be used to detect the presence of antibiotic residue than the methods based on acid production in milk (J et al., 1993).
ATP - bioluminescence technique has a potential to be utilised as a tool for quality control of fruit juices. It has been analysed that the technique can be used during the processing operation at ambient condition for the measurement of active population of yeast and bacteria in fruit juices (T. R, 1985).
Flow cytometry method
Flow cytometry is another rapid technique which is used to measure the microbial populations and can be used for both qualitative and quantitative analysis of food (Comas-Riu and Rius, 2009, Gunasekera et al., 2000).It is extremely sensitive method and avoids the need for culturing procedures (Gunasekera et al., 2000).
Flow cytometric technique measures the microbial population of the sample by using the principle of light scattering and emission of fluorochrome molecules. A flow cytometer consists of several systems such as light source (laser light); series of lenses; optical filters and light detectors (photodiodes or photomultipliers).In its operation, the sample is injected into the centre of the sheath flow, forcing the cell to pass across the focussed light and the system detects the microbial population by measuring the signal produced by light detectors (Figure 2). It has been observed that the system can detect up to 100,000 cells (Comas-Riu and Rius, 2009).
Figure 2: Diagram of flow cytometry technique(Comas-Riu and Rius, 2009).
Flow cytometry technique can be applied in dairy industry for the measurement of total bacterial numbers. It has been observed that the sensitivity of the flow cytometry method is less than 104 total bacteria/ml of raw milk. Furthermore, the method takes 30 seconds ââ‚¬" 2 minutes for the measurement depending upon the number of bacteria present in the raw milk (Gunasekera et al., 2000).
Flow cytometry technique can be used as an indicator for the microbiological quality of milk (Comas-Riu and Rius, 2009). Furthermore, the technique has also been used for the detection of listeria monocytogenes in raw milk and salmonella spp. in processed milk (Gunasekera et al., 2000).
In addition of that, it has been also found that the flow cytometry technique is appropriate for the monitoring of the growth of bacteria in refrigerated raw milk , homogenized milk and flavoured milk during the shelf storage period (Gunasekera et al., 2000).
Enzyme linked immuno-sorbent assay (ELISA)
Enzyme linked immuno-sorbent assay is another rapid method for the analysis of mycotoxins in food products and has been used since a decade for the analysis of toxins. The technique works on the basis of interaction between antibody and antigen (Asensio et al., 2008). In its operation, the mycotoxin is firstly extracted from the ground sample with the help of solvent followed by the mixing of a portion of the sample extract and a conjugate of an enzyme coupled mycotoxin and then is added to the antibody-coated micro-titer wells.
Further any mycotoxin present in the sample extract or control standards is legitimated to compete for the antibody binding sites with the enzyme-conjugated mycotoxin. After washing the micro titer wells, addition of an enzyme substrate is done and the addition results in the development of blue colour. The colour intensity is inversely proportional to the concentration of mycotoxin in standard or sample.
Furthermore, a solution is then added to stop the enzyme reaction and the intensity of the solution colour in the microtiter wells is measured optically using an ELISA reader
with an absorbance filter level of 450 nm. The optical densities (OD) of the samples are then compared with theoptical densities of the standards followed by the determination of interpretative result (Zheng et al., 2006).
ELISA is used in the determination of authenticity of food such as meat, fish, milk products, fruit juices etc from adulteration (Asensio et al., 2008). Furthermore, ELISA is also broadly used as an effective screening tool in regulatory, residual and industrial laboratories (M. Vass et al., 2008) as it is the rapid technique for detecting the hidden allergens in food (Poms et al., 2004).
Radiometric technique is another rapid method for the analysis of microbial concentration in food products. Radiometry techniques works on the measurement of CO2 produced by microorganisms by the conversion of 14C-labeled substrates and the measurement of CO2 is done by the instrument named as Bactec (W. S et al., 1977, Durwood B et al., 1978).
Radiometric Technique is a screening method for determining whether the food is safe to consume or not within few hours as compared to the conventional methods such as standard plate count which takes 24 -48 for the analysis (W. S et al., 1977).
It has been experimentally observed that radiometric technique is equally effective to plate count technique for the measurement of microorganism and can be used in the microbiological analysis of cooked and frozen foods (Durwood B et al., 1978).
It is a rapid microbiological technique for the detection and enumeration of microbes such as coliform, yeast and mold. The technique works on the principle that the growth and metabolism of micro organisms in the sample changes the dye pigmentation, which can be measured directly by reflectance colorimetry (Vasavada, 1993, Gunasekera et al., 2000).
It has been experimentally observed that reflectance colorimetry can be used as a method to moniter the microbial growth and lipase activity in milk and dairy products (M. R. BLAKE et al., 1996).
Rapid microbiological methods such as impedance measurement, ATP-bioluminescence, colorimetry are becoming an important tool for the rapid analysis of microbial population and enzymatic growth during the processing of food products such as milk, dairy products, ready to eat vegetables, fish, beef, pork etc as these techniques provides accurate results within short time than conventional microbiological techniques and can be useful for maintaining the hygiene and safety for the production of food products for consumption.