The maintenance of the freshness of the fish has a lot of importance, as fish is the major important item for the development of international trade and also the earner of foreign exchange (Venugopal, 2002). The changes in fish depend on the environment they are stored and also the chemical reactions that undergo in them. The fish is the most largely consumable food so the care of maintaining its freshness is the most important task. Many devices and techniques are available to maintain the fish fresh but all the processes are time consuming and highly expensive. So the use of biosensors came into implementation. Biosensors detect the freshness of the fish by measuring the amount of the biogenic amines that are released after the death of the fish as a result of their metabolic activities. The use of Trimethylamine (TMA) biosensor is discussed in this topic. The biosensor is constructed by immobilizing the FMO3 (Flavin containing monooxygenase type 3) and analysing it with the flow injection analysis (FIA).
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Freshness of fish is the basic property of fish that influences the quality of the fish (Pedrosa etal, 1990). Fish freshness biosensor is the device mainly used to detect the freshness of the fish that is used for consumption. It is very important for the food industrialists to estimate the freshness of fish. To estimate the freshness of fish different concentrations of nucleotides (Saito et al, 1959), ammonia (Ota et al, 1952), amines (Karube et al, 1980) were estimated. A biosensor is the device that detects records and transmits the information related to a physiological change or the presence of various biological materials.
It is very difficult to control the quality of fish due to the wide variety of species, the age of fish, the action of hydrolytic enzymes and autolytic enzymes of the micro organisms on the fish and also the habitat of the fish ( Venugopal, 2001). The loss of the freshness of the fish mainly depends on the intrinsic and extrinsic factors like the nature of the fish, its habitat, feeding habits, water temperature, its handling, methods of catching and the storage (Ashie et al, 1996).
The analytical device that converts the biological response into an electrical signal is called a biosensor. The response of the biological material is determined by the bio catalytic membrane which converts the reactant to the product. The bio catalyst present in the biosensor must be highly specific to analyse and the reaction should be independent of physical parameters like the temperature and pH. The components of a biosensor include a bio catalyst, a transducer, an amplifier and an output device. The conversion of substrate into product is done by the bio catalyst and the reaction is determined by the transducer. The transducer converts the reaction into an electric signal. This output from the transducer is amplified by the amplifier and then visualized on the screen. The transducer plays a key role in the biosensor which converts the biological material into an electrical signal.
Components of biosensor:
First component- biological element:
The biological elements like micro organisms, tissues, cells, nucleic acids, organelles, enzymes, receptors and anti bodies are used. These biological components are used to bind the target molecule. These components must be specific and stable under storage conditions.
Second component- physico chemical transducer:
The transducer plays a vital role. It measures the physical change that occurs in the reaction at the biological component and then converts that energy into an electrical output.
Third component- detector:
The signals obtained from the transducer are passed into a micro processor. These signals get amplified at the micro processor and then the data obtained is converted into concentration units and finally stored in a data storage device.
Types of biosensors:
There are almost five types of biosensors based on the principle of detection. They are:
Optical and acoustic wave biosensor.
The basic principles involved in these biosensors are: the calorimetric biosensors measure the change in the temperature when the substrate is converted into product. It is most widely used type of biosensor. The potentiometric biosensors measure the change in distribution of charge. The amperometric biosensors detect the movement of electrons between two electrodes.
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The biosensors detect the freshness of the fish by monitoring the amount of oxygen consumed, change in the concentration of NADH, the amount of hydrogen peroxide formed, change in pH, temperature and also the fluorescence.
Electro chemical transducers are the integrated devices that give the quantitative analysis by using the biological recognition element ( Toth et al, 2001). These are further classified into impedimetric, potentimetric, conductimetric and the amperometry.
The conductimetric biosensors work on the basic principle of the conductivity and the changes in the conductivity of the medium where the micro organisms metabolize the carbohydrates (L.D. Mello et al, 2002).
The impedimetric biosensors work on the principle of impedance (Gibson et al, 1992). It mainly involves in analysing the quality of the food and the pathogens responsible for the spoilage of food (Feng, 1992). These biosensors are based on the microbial metabolism which decreases the impedance and increases the capacitance and conductance.
The potentiometric biosensors are based on the principle of potentiometry. In this, the concentrations of the species of the micro organisms that are alive are measured across the reference electrode. It is also used to measure the change in the ion concentration of the sample and also the change in pH. Here immobilization of enzymes plays a vital role. The enzymes or the antibodies or the antigens are immobilized on the surface of the transducer as an electric signal and the obtained reaction in the formation of the antigen antibody complex is measured (L.D.Mello et al, 2002). The enzyme based potentiometric biosensors have vast application in the field of industrial processes monitoring and the quality control of the products (Taylor et al, 1991 and Renault et al, 2000).
The amperometric biosensors work on the principle of measuring the current produced during the chemical reaction of the species. This is further related to the species concentration in the given solution. Among all other biosensors the amperometric biosensors are more sensitive, fast and give accurate results. The first and best used amperometric biosensor is the Clark oxygen electrode which is used for the analysis of glucose in which the glucose oxidase enzyme is used (Updike and Hicks, 1967). The amperometric biosensors exhibit better results due to their enzymatic reaction with the substrate. The amperometric transducers that are mainly used for hydrogen peroxide monitoring are more sensitive than the ones used for the detection of oxygen consumption.
The optical biosensors are mainly based on the principle of UV- Vis absorption or fluorescence or chemiluminescence or reflectance or the refractive index that is caused due to the bio catalytic interaction with the target analyte (Seitz, 1988).
Applications of biosensors:
Food and agricultural processes
Detection of warfare agents
Food industry uses the biosensors for checking the quality control. They are mainly used to determine the contaminants. The biosensors must be inexpensive, reliable and robust to operate under realistic conditions.
The fish freshness is a major part during the canning of the fish and also during its consumption. Fish is one of the most highly perishable food products (F. Ozogul et al, 2004). The shelf life of fish is very low. This shelf life can be increased by changing the atmospheric conditions like the increase in carbon dioxide and decrease in oxygen concentration. The main cause for the damage of the freshness of fish is the presence of micro organisms and their microbial activities on the fish. The fish that are fresh do not exhibit any odour. The fishy odour is developed after some time of the harvest of the fish. The fishy odour is formed due to the breakdown of proteins. This odour is an indication that the fish is stored for longer periods. The bacteria occupy the place in the gills, stomach lining and skin of the fish and they cause the fish to decompose.
The freshness of fish is generally tested by seeing the colour of the gills, and also the freshness of the scales present on the skin of the fish. The softening of the skin is also an indication of the spoilage of fish (Jeremy Hammond et al, 2001).
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The freshness of fish can be determined by using the biosensors. The freshness can be determined by measuring the biogenic amines such as putrescine, cadaverine, agmatine, tyramine, histamine, spermidine and spemine. These amines are mainly produced during the late stage of microbial decomposition of fish. The change in colour of the fish, its odour and the texture occur due to the microbial activity on the fish.
Biogenic amines are the basic nitrogenous compounds that are mainly formed due to the amino acid decarboxylation or by the amination and transamination of aldehydes and ketones. The biogenic amines occur in a vast variety of foods like meat, cheese, wine, fishery products, beer and many fermented foods (Stratton et al, 1991). The biogenic amines are the low molecular weight aliphatic and acyclic organic bases. These biogenic amines are mainly generated during microbial and animal metabolisms. They influence the process of regulation of body temperature, nutrition uptake and also the increase and decrease of blood pressure. They are ubiquitous i.e they are present everywhere. Agmatine, spermine and spermidine are the secondary amines that form nitrosamines. Putrescine, cadaverine and spermidine act as free radical scavengers. These biogenic amines are produced by the enzymes that are involved in microbial decarboxylation (Stratton et al, 1991). The biogenic amine Histamine is mainly responsible for the intoxication. This intoxication can be increased with the other biogenic amines like putrescine or cadaverine (Stratton et al, 1991).
Biogenic amines are mainly used to estimate the freshness of fish and also the degree of spoilage. The amount of these amines is very low in fresh fish and when the bacterial spoilage increases then the amount of these biogenic amines increases (Fernandez et al, 1987). The spoilage of the fresh fish mainly occurs due to the biogenic amine like Histamine.
Spoilage of fish:
The fish spoilage is broadly classified into two major types. They are: the bacterial spoilage, spoilage caused by the bacteria and the autolytic spoilage, spoilage caused due to autolysis.
Bacterial spoilage occurs mainly due to the growth of a wide variety of micro organisms on the fish. This spoilage can be controlled by the methods like sterilization and also by the use of bactericidal agents, which kill the bacteria. A healthy and fresh fish is impermeable to bacteria ass the skin is intact. The bacteria cannot grow on the fresh fish due to the absence of nutrients on fresh fish. As there are no sufficient nutrients the bacteria cannot grow and multiply on the fresh fish (Mukundan et al, 1986).
Autolysis is the process in which the endogenous enzymes degrade the skin and muscle of the fresh fish. Autolysis makes the skin of fish permeable for the entry of bacteria into the skin and also releases the free amino acids, sugars and fatty acids which are good nutrients for the growth and multiplication of the bacteria. The ratio of autolytic spoilage is less when compared to the microbial spoilage (Mukundan et al, 1986). A wide variety of enzymes namely the lipases, phosphorylases and the cathepsins are involved in autolytic spoilage of fish (Mukundan et al, 1986).
Drying the fish to reduce the water activity prevents the autolytic spoilage of fish in which the denaturation of enzymes occurs. The maintenance of pH also helps in preventing the autolytic spoilage. The suitable pH is 7 i.e. the neutral pH (Mukundan et al, 1986).
(As given by V. Venugopal, 2002 and H.Okuma et al, 1992).
Many changes occur after the catching of fish. They include the autolysis and also the physico chemical changes. These changes are followed by the biochemical reactions of the micro organisms that contaminate the fish and cause damage to the freshness of the fish. The fish are said to be dead when, the respiratory system fails. After the failure of the respiratory system, adenosine triphosphate (ATP) bio synthesis also stops and leads to the degradation of the nucleotides, by enzymatic action, that are present in the muscle of the fish. The degradation finally leads to the formation of Uric acid (U). The steps are-
ATP ADP AMP IMP HXR HX H U
The adenosine tri phosphate (ATP) is converted to adenosine 5'di phosphate (ADP) which is then converted to adenosine 5' mono phosphate (AMP). This AMP is later converted to inosine 5' mono phosphate (IMP) which forms inosine (HXR). This inosine gradually converts to hypoxanthine (HX), xanthine (X) and finally forms uric acid (U). These are a chain of reactions. The formation of inosine from ATP occurs fast due to the reaction of the endogenous enzymes that are present inside the fish. But the formation of hypoxanthine and xanthine and finally the uric acid formation occur slowly as the enzymes are released from the micro organisms that are responsible for the spoilage of the fish. These enzymes are responsible for the oxidation process of hypoxanthine and xanthine (Venugopal, 2002).
The freshness of fish can be measured using the K value. The K value is mainly based on the degradation of the compounds. The K value is defined as-
K = HXR + HX
ATP + ADP + AMP + IMP + HXR + HX
The fish with a K value 20 is determined as fresh and is considered to be suitable for consumption. The K value above 40 is not suitable for consumption and is considered to be spoiled. After 24 hours after death of fish the ATP, ADP and AMP gradually disappears, so the formula to estimate the K value is slightly modified as KI. (Karube et al, 1984).
KI = (HXR) + (HX)
(IMP) + (HXR) + (HX)
A large variety of nitrogen compounds like the ammonia are accumulated on the fish due to the microbial spoilage. The accumulation of ammonia occurs due to the bacterial decarboxylases (Liston et al, 1980). Trimethylamineoxide (TMAO) is the compound that is found in wide variety of fish. Due to the bacterial enzymatic action the TMAO is converted to trimethyl amine (TMA). The enzyme that is involved in the conversion of TMAO to TMA is trimethlyamine oxide recuctase. The appearance of these trimethyl compounds indicates the loss of the freshness of fish (Gram and Huss, 1996).
Microbial ecology of the fish:
Each and every food product exhibits unique flora. The changes in the food products occur due to the variations in temperature, pH, the atmosphere in which they are preserved, and also the nutrient composition (Lone Gram and Paw Dalgaard, 2002). The most common pathogens that are found in the fishery products include: Staphylococcus aureus, Salmonella species, Bacillus cereus, Clostridium botulinum, Escherichia coli, Camphylobacter jejuni, Yersinia enterocolitica and Vibrio parahaemolyticus. The spoilage of the fish due to these microbial pathogens occurs during their capture and their storage (Venugopal, 2002). The spoilage of the unpreserved fish occurs due to Gram negative fermentative bacteria (Gram et al, 2000).
The level of the TMAO formed in the fish which are stored for longer periods can be reduced to TMA with the microbial activity of many species like Shewanella putrefaciens and Vibrio species (Fonnesbech Vogel B et al, 1997).
A lot of research is being done from the past twenty five tears on measuring the freshness of fish. Many scientists proposed different methods to measure the freshness of the fish. But all the methods are time taking processes and are also of highly expensive. HPLC techniques are most widely used techniques to measure the freshness of fish.
The use of biosensors made the process of measuring the freshness of fish little easier. To detect the quality of the food by using the biosensors is classified broadly into two types: enzyme sensors for the food components and the immunosensors for the pathogenic bacteria (Venugopal, 2002).
Many analytical techniques were done in order to measure the freshness of fish. Some of them include the colorimetric techniques, aerobic and anaerobic bacterial plate count, pH tests, SMO sensors and neural networks. The colorimetric technique is also known as the Dyer's method. In this method the biogenic amines that are produced by the fish during long storage are measured. The sample is taken and then acid is added to the sample and centrifuged. The supernatant is collected and the pellet is discarded. The obtained supernatant is analysed spectrophotometrically against the reference sample and the content of the amines are determined.
The amount of bacteria present in the fish muscle can be determined using the bacterial plate count method. The number of lactic acid producing aerobic bacteria can be determined using media that contains 10% of tomato juice. To count the number of anaerobic bacteria present the plates are placed in carbon dioxide rich environment and the growth is estimated (J. Hammond et al, 2002).
The pH test is done by using the pH meter. Initially the pH meter is set with 4.0 and 7.0 pH then the pH meter is placed on the surface of the fish and the reading is noted. The pH range of 6.2 - 6.6 indicates acidic pH and this indicates that the fish are fresh and if the pH gradually decreases to basic pH it indicates that the fish is spoiled and not suitable for consuming.
In this way a wide range of techniques were developed for measuring the freshness of fish. But among all these processes the best and accurate processes is the use of biosensors. Among these biosensors the best used is the amperometric biosensor.
Construction of a TMA biosensor:
A TMA biosensor for the measurement of the fish freshness is constructed with the help of the immobilized membrane of FMO3 (flavin containing monooxygenase type-3) to an area sensitive to dissolved oxygen electrode. This process is done by using the nylon net and a silicon O- ring (Mitsubayashi et al, 2000). The TMA is catalysed to TMAO by the FMO3, with NADPH as the coenzyme. The immobilization of the enzyme is done by mixing the solution with polyvinyl alcohol (Ichimura et al, 1984). The obtained solution is placed on the dialysis membrane and spread over it until it gets permeated (Mitsubayashi et al, 1994). The dialysis membrane is then placed at 10â„ƒ for 1 hour in dark and then exposed to fluorescent light for the photo crossing of the polyvinyl solution. Thus the immobilization of the enzyme into the dialysis membrane occurs. Now the FMO3 membrane was taken and placed at the dissolved oxygen electrode and then covered with the help of nylon net. Finally this sensor was set flow injection analysis into the reaction cell.
The standard TMA solution is used to evaluate the biosensor. The flow injection analysis system is the system in which a potentiostat which is controlled by the computer is placed. The potential of the potentiostat is fixed to -600mV and a reference electrode Ag/ AgCl. The output of the biosensor is monitored on the computer screen continuously and saved for further analysis. The TMA solution is slowly injected to the flow system with the help of a syringe and the biosensor is evaluated.
In the same way the fish sample is immobilized with the FMO3 biosensor and then the results are analysed. The fish muscle sample is collected and it is mashed and homogenized with the buffer solution and then subjected to centrifugation. The obtained supernatant is taken and the pellet is discarded. This supernatant is loaded to the flow system and thus the obtained values are noted and the freshness of the sample can be measured (Mitsubayashi et al, 2004).
Fig.1. Structure of the FMO3 immobilized biosensor with Clark-type oxygen electrode (Kohji Mitsubayashi et al, 2004).
The future work includes the development of the biosensors with slight modifications in them. The sensors are designed in order to respond to the TVB-N concentration when the sample of fish gets spoiled and unsuitable for consumption. These biosensors are made in order to detect the colour changes as in the pH paper. In order to measure the intensity of the colour the LED and photodiode based scanner can be used. These scanners give more accurate values (Liam Byrne et al, 2002).