The care of the freshness of the fish has a batch of importance, as fish is the major of import point for the development of international trade and besides the earner of foreign exchange ( Venugopal, 2002 ) . The alterations in fish depend on the environment they are stored and besides the chemical reactions that undergo in them. The fish is the most mostly consumable nutrient so the attention of keeping its freshness is the most of import undertaking. Many devices and techniques are available to keep the fish fresh but all the procedures are clip devouring and extremely expensive. So the usage of biosensors came into execution. Biosensors detect the freshness of the fish by mensurating the sum of the biogenic aminoalkanes that are released after the decease of the fish as a consequence of their metabolic activities. The usage of Trimethylamine ( TMA ) biosensor is discussed in this subject. The biosensor is constructed by immobilising the FMO3 ( Flavin incorporating monooxygenase type 3 ) and analyzing it with the flow injection analysis ( FIA ) .
Freshness of fish is the basic belongings of fish that influences the quality of the fish ( Pedrosa etal, 1990 ) . Fish freshness biosensor is the device chiefly used to observe the freshness of the fish that is used for ingestion. It is really of import for the nutrient industrialists to gauge the freshness of fish. To gauge the freshness of fish different concentrations of bases ( Saito et al, 1959 ) , ammonia ( Ota et al, 1952 ) , aminoalkanes ( Karube et al, 1980 ) were estimated. A biosensor is the device that detects records and transmits the information related to a physiological alteration or the presence of assorted biological stuffs.
It is really hard to command the quality of fish due to the broad assortment of species, the age of fish, the action of hydrolytic enzymes and autolytic enzymes of the micro being on the fish and besides the home ground of the fish ( Venugopal, 2001 ) . The loss of the freshness of the fish chiefly depends on the intrinsic and extrinsic factors like the nature of the fish, its home ground, feeding wonts, H2O 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 stuff is determined by the bio catalytic membrane which converts the reactant to the merchandise. The bio accelerator nowadays in the biosensor must be extremely specific to analyze and the reaction should be independent of physical parametric quantities like the temperature and pH. The constituents of a biosensor include a bio accelerator, a transducer, an amplifier and an end product device. The transition of substrate into merchandise is done by the bio accelerator and the reaction is determined by the transducer. The transducer converts the reaction into an electric signal. This end product from the transducer is amplified by the amplifier and so visualized on the screen. The transducer plays a cardinal function in the biosensor which converts the biological stuff into an electrical signal.
Components of biosensor:
First component- biological component:
The biological elements like micro beings, tissues, cells, nucleic acids, cell organs, enzymes, receptors and anti organic structures are used. These biological constituents are used to adhere the mark molecule. These constituents must be specific and stable under storage conditions.
Second component- physico chemical transducer:
The transducer plays a critical function. It measures the physical alteration that occurs in the reaction at the biological constituent and so converts that energy into an electrical end product.
Third component- sensor:
The signals obtained from the transducer are passed into a micro processor. These signals get amplified at the micro processor and so the informations obtained is converted into concentration units and eventually stored in a information storage device.
Types of biosensors:
There are about five types of biosensors based on the rule of sensing. They are:
Optical and acoustic moving ridge biosensor.
The basic rules involved in these biosensors are: the calorimetric biosensors measure the alteration in the temperature when the substrate is converted into merchandise. It is most widely used type of biosensor. The potentiometric biosensors measure the alteration in distribution of charge. The amperometric biosensors detect the motion of negatrons between two electrodes.
The biosensors detect the freshness of the fish by supervising the sum of O consumed, alteration in the concentration of NADH, the sum of H peroxide formed, alteration in pH, temperature and besides the fluorescence.
Electro chemical transducers are the incorporate devices that give the quantitative analysis by utilizing the biological acknowledgment component ( Toth et al, 2001 ) . These are farther classified into impedimetric, potentimetric, conductimetric and the amperometry.
The conductimetric biosensors work on the basic rule of the conduction and the alterations in the conduction of the medium where the micro organisms metabolise the saccharides ( L.D. Mello et Al, 2002 ) .
The impedimetric biosensors work on the rule of electric resistance ( Gibson et al, 1992 ) . It chiefly involves in analyzing the quality of the nutrient and the pathogens responsible for the spoilage of nutrient ( Feng, 1992 ) . These biosensors are based on the microbic metamorphosis which decreases the electric resistance and increases the electrical capacity and conductance.
The potentiometric biosensors are based on the rule of potentiometry. In this, the concentrations of the species of the micro organisms that are alive are measured across the mention electrode. It is besides used to mensurate the alteration in the ion concentration of the sample and besides the alteration in pH. Here immobilisation of enzymes plays a critical function. 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 composite is measured ( L.D.Mello et al, 2002 ) . The enzyme based potentiometric biosensors have huge application in the field of industrial procedures monitoring and the quality control of the merchandises ( Taylor et al, 1991 and Renault et Al, 2000 ) .
The amperometric biosensors work on the rule of mensurating the current produced during the chemical reaction of the species. This is farther related to the species concentration in the given solution. Among all other biosensors the amperometric biosensors are more sensitive, fast and give accurate consequences. The first and best used amperometric biosensor is the Clark O 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 consequences due to their enzymatic reaction with the substrate. The amperometric transducers that are chiefly used for H peroxide monitoring are more sensitive than the 1s used for the sensing of O ingestion.
The optical biosensors are chiefly based on the rule of UV- Vis soaking up or fluorescence or chemiluminescence or coefficient of reflection or the refractile index that is caused due to the bio catalytic interaction with the mark analyte ( Seitz, 1988 ) .
Applications of biosensors:
Food and agricultural procedures
Detection of warfare agents
Food industry uses the biosensors for look intoing the quality control. They are chiefly used to find the contaminations. The biosensors must be cheap, dependable and robust to run under realistic conditions.
The fish freshness is a major portion during the canning of the fish and besides during its ingestion. Fish is one of the most extremely perishable nutrient merchandises ( F. Ozogul et Al, 2004 ) . The shelf life of fish is really low. This shelf life can be increased by altering the atmospheric conditions like the addition in C dioxide and lessening in O concentration. The chief cause for the harm of the freshness of fish is the presence of micro beings and their microbic activities on the fish. The fish that are fresh do non exhibit any smell. The fishy smell is developed after some clip of the crop of the fish. The fishy smell is formed due to the dislocation of proteins. This smell is an indicant that the fish is stored for longer periods. The bacterium occupy the topographic point in the gills, tummy liner and tegument of the fish and they cause the fish to break up.
The freshness of fish is by and large tested by seeing the coloring material of the gills, and besides the freshness of the graduated tables present on the tegument of the fish. The softening of the tegument is besides an indicant of the spoilage of fish ( Jeremy Hammond et Al, 2001 ) .
The freshness of fish can be determined by utilizing the biosensors. The freshness can be determined by mensurating the biogenic aminoalkanes such as putrescine, cadaverine, agmatine, tyramine, histamine, spermidine and spemine. These aminoalkanes are chiefly produced during the late phase of microbic decomposition of fish. The alteration in coloring material of the fish, its smell and the texture occur due to the microbic activity on the fish.
Biogenic aminoalkanes are the basic nitrogen-bearing compounds that are chiefly formed due to the amino acerb decarboxylation or by the amination and transamination of aldehydes and ketones. The biogenic aminoalkanes occur in a huge assortment of nutrients like meat, cheese, vino, piscary merchandises, beer and many fermented nutrients ( Stratton et al, 1991 ) . The biogenic aminoalkanes are the low molecular weight aliphatic and acyclic organic bases. These biogenic aminoalkanes are chiefly generated during microbic and carnal metamorphosiss. They influence the procedure of ordinance of organic structure temperature, nutrition consumption and besides the addition and lessening of blood force per unit area. They are omnipresent i.e they are present everyplace. Agmatine, spermine and spermidine are the secondary aminoalkanes that form nitrosamines. Putrescine, cadaverine and spermidine act as free extremist scavengers. These biogenic aminoalkanes are produced by the enzymes that are involved in microbic decarboxylation ( Stratton et al, 1991 ) . The biogenic aminoalkane Histamine is chiefly responsible for the poisoning. This poisoning can be increased with the other biogenic aminoalkanes like putrescine or cadaverine ( Stratton et al, 1991 ) .
Biogenic aminoalkanes are chiefly used to gauge the freshness of fish and besides the grade of spoilage. The sum of these aminoalkanes is really low in fresh fish and when the bacterial spoilage additions so the sum of these biogenic aminoalkanes additions ( Fernandez et al, 1987 ) . The spoilage of the fresh fish chiefly occurs due to the biogenic aminoalkane like Histamine.
Spoilage of fish:
The fish spoilage is loosely classified into two major types. They are: the bacterial spoilage, spoilage caused by the bacteriums and the autolytic spoilage, spoilage caused due to self-digestion.
Bacterial spoilage occurs chiefly due to the growing of a broad assortment of micro beings on the fish. This spoilage can be controlled by the methods like sterilisation and besides by the usage of bactericidal agents, which kill the bacterium. A healthy and fresh fish is impermeable to bacteria ass the tegument is integral. The bacteriums can non turn on the fresh fish due to the absence of foods on fresh fish. As there are no sufficient nutrients the bacteriums can non turn and multiply on the fresh fish ( Mukundan et al, 1986 ) .
Autolysis is the procedure in which the endogenous enzymes degrade the tegument and musculus of the fresh fish. Autolysis makes the tegument of fish permeable for the entry of bacteriums into the tegument and besides releases the free amino acids, sugars and fatty acids which are good foods for the growing and generation of the bacterium. The ratio of autolytic spoilage is less when compared to the microbic spoilage ( Mukundan et al, 1986 ) . A broad assortment of enzymes viz. the lipases, phosphorylases and the cathepsins are involved in autolytic spoilage of fish ( Mukundan et al, 1986 ) .
Drying the fish to cut down the H2O activity prevents the autolytic spoilage of fish in which the denaturation of enzymes occurs. The care of pH besides helps in forestalling the autolytic spoilage. The suited pH is 7 i.e. the impersonal pH ( Mukundan et al, 1986 ) .
( As given by V. Venugopal, 2002 and H.Okuma et Al, 1992 ) .
Many alterations occur after the catching of fish. They include the self-digestion and besides the physico chemical alterations. These alterations are followed by the biochemical reactions of the micro organisms that contaminate the fish and cause harm 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 besides stops and leads to the debasement of the bases, by enzymatic action, that are present in the musculus of the fish. The debasement eventually leads to the formation of Uric acid ( U ) . The stairss are-
ATP ADP AMP IMP HXR HX H U
The adenosine tri phosphate ( ATP ) is converted to adenosine 5’di phosphate ( ADP ) which is so converted to adenosine 5 ‘ glandular fever phosphate ( AMP ) . This AMP is subsequently converted to inosine 5 ‘ glandular fever phosphate ( IMP ) which forms inosine ( HXR ) . This inosine bit by bit converts to hypoxanthine ( HX ) , xanthine ( X ) and eventually signifiers uric acid ( U ) . These are a concatenation 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 eventually the uric acid formation occur easy as the enzymes are released from the micro organisms that are responsible for the spoilage of the fish. These enzymes are responsible for the oxidization procedure of hypoxanthine and xanthine ( Venugopal, 2002 ) .
The freshness of fish can be measured utilizing the K value. The K value is chiefly based on the debasement 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 suited for ingestion. The K value above 40 is non suited for ingestion and is considered to be spoiled. After 24 hours after decease of fish the ATP, ADP and AMP bit by bit disappears, so the expression to gauge the K value is somewhat modified as KI. ( Karube et al, 1984 ) .
KI = ( HXR ) + ( HX )
( IMP ) + ( HXR ) + ( HX )
A big assortment of nitrogen compounds like the ammonium hydroxide are accumulated on the fish due to the microbic spoilage. The accretion of ammonium hydroxide occurs due to the bacterial decarboxylases ( Liston et al, 1980 ) . Trimethylamineoxide ( TMAO ) is the compound that is found in broad assortment of fish. Due to the bacterial enzymatic action the TMAO is converted to trimethyl aminoalkane ( TMA ) . The enzyme that is involved in the transition of TMAO to TMA is trimethlyamine oxide recuctase. The visual aspect of these trimethyl compounds indicates the loss of the freshness of fish ( Gram and Huss, 1996 ) .
Microbial ecology of the fish:
Each and every nutrient merchandise exhibits alone vegetations. The alterations in the nutrient merchandises occur due to the fluctuations in temperature, pH, the ambiance in which they are preserved, and besides the alimentary composing ( Lone Gram and Paw Dalgaard, 2002 ) . The most common pathogens that are found in the piscary merchandises include: Staphylococcus aureus, Salmonella species, Bacillus Cereus, Clostridium botulinus, Escherichia coli, Camphylobacter jejuni, Yersinia enterocolitica and Vibrio parahaemolyticus. The spoilage of the fish due to these microbic pathogens occurs during their gaining control and their storage ( Venugopal, 2002 ) . The spoilage of the unpreserved fish occurs due to Gram negative fermentative bacteriums ( Gram et al, 2000 ) .
The degree of the TMAO formed in the fish which are stored for longer periods can be reduced to TMA with the microbic activity of many species like Shewanella putrefaciens and Vibrio species ( Fonnesbech Vogel B et Al, 1997 ) .
A batch of research is being done from the past 20 five cryings on mensurating the freshness of fish. Many scientists proposed different methods to mensurate the freshness of the fish. But all the methods are clip taking procedures and are besides of extremely expensive. HPLC techniques are most widely used techniques to mensurate the freshness of fish.
The usage of biosensors made the procedure of mensurating the freshness of fish small easier. To observe the quality of the nutrient by utilizing the biosensors is classified loosely into two types: enzyme detectors for the nutrient constituents and the immunosensors for the infective bacteriums ( Venugopal, 2002 ) .
Many analytical techniques were done in order to mensurate the freshness of fish. Some of them include the colorimetric techniques, aerophilic and anaerobiotic bacterial home base count, pH trials, SMO detectors and nervous webs. The colorimetric technique is besides known as the Dyer ‘s method. In this method the biogenic aminoalkanes that are produced by the fish during long storage are measured. The sample is taken and so 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 mention sample and the content of the aminoalkanes are determined.
The sum of bacteriums present in the fish musculus can be determined utilizing the bacterial home base count method. The figure of lactic acid bring forthing aerophilic bacteriums can be determined utilizing media that contains 10 % of tomato juice. To number the figure of anaerobiotic bacteriums present the home bases are placed in C dioxide rich environment and the growing is estimated ( J. Hammond et Al, 2002 ) .
The pH trial is done by utilizing the pH metre. Initially the pH metre is set with 4.0 and 7.0 pH so the pH metre is placed on the surface of the fish and the reading is noted. The pH scope of 6.2 – 6.6 indicates acidic pH and this indicates that the fish are fresh and if the pH bit by bit decreases to basic pH it indicates that the fish is spoiled and non suited for devouring.
In this manner a broad scope of techniques were developed for mensurating the freshness of fish. But among all these processes the best and accurate procedures is the usage of biosensors. Among these biosensors the best used is the amperometric biosensor.
Construction of a TMA biosensor:
A TMA biosensor for the measuring of the fish freshness is constructed with the aid of the immobilized membrane of FMO3 ( flavin incorporating monooxygenase type-3 ) to an country medium to fade out O electrode. This procedure is done by utilizing the nylon cyberspace and a silicon O- ring ( Mitsubayashi et al, 2000 ) . The TMA is catalysed to TMAO by the FMO3, with NADPH as the coenzyme. The immobilisation of the enzyme is done by blending the solution with polyvinyl intoxicant ( 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 so placed at 10a„? for 1 hr in dark and so exposed to fluorescent visible radiation for the exposure crossing of the polyvinyl solution. Thus the immobilisation of the enzyme into the dialysis membrane occurs. Now the FMO3 membrane was taken and placed at the dissolved O electrode and so covered with the aid of nylon cyberspace. Finally this detector was set flow injection analysis into the reaction cell.
The standard TMA solution is used to measure the biosensor. The flow injection analysis system is the system in which a potentiostat which is controlled by the computing machine is placed. The potency of the potentiostat is fixed to -600mV and a mention electrode Ag/ AgCl. The end product of the biosensor is monitored on the computing machine screen continuously and saved for farther analysis. The TMA solution is easy injected to the flow system with the aid of a syringe and the biosensor is evaluated.
In the same manner the fish sample is immobilized with the FMO3 biosensor and so the consequences are analysed. The fish musculus sample is collected and it is mashed and homogenized with the buffer solution and so subjected to centrifugation. The obtained supernatant is taken and the pellet is discarded. This supernatant is loaded to the flow system and therefore 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 O electrode ( Kohji Mitsubayashi et Al, 2004 ) .
The future work includes the development of the biosensors with little alterations in them. The detectors are designed in order to react to the TVB-N concentration when the sample of fish gets spoiled and unsuitable for ingestion. These biosensors are made in order to observe the coloring material alterations as in the pH paper. In order to mensurate the strength of the coloring material the LED and photodiode based scanner can be used. These scanners give more accurate values ( Liam Byrne et Al, 2002 ) .