Most Popular Fish Product Consumed In United States Biology Essay

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According to (NASS-USDA, 2001), Catfishs (order Siluriformes) is now the fourth most popular fish product consumed in United States. In Malaysia, catfish is identified as one of the most-preferred marine fish for daily consumption that can be found in swamps, paddy fields and streams. There are reports on production of good quality fish oils from herring (Aidos et al., 2003) and catfish (Sathivel et al., 2003) by-products.

Much of the oil in catfish is in the viscera, which contain approximately 33 percent fats or lipids which could be converted into edible oil or biodiesel product. The oil extracted from fish, are rich in polyunsaturated fatty acids (PUFAs) especially those of the omega-3 family, mainly eicosapentaenoic acid (EPA; C20:5n-3) and docosahexaenoic acid (DHA; C22:6n-3). Although fish is a dietary source of n−3 fatty acids, fish do not synthesise them; they obtain them from the algae (microalgae in particular) or plankton in their diet (Falk-Petersen, 1998).

The importance of PUFAs in human health and nutrition is well recognised. Many clinical and epidemiologic studies have shown positive roles for n-3 PUFA in infant development, cancer, cardiovascular diseases and more recently, in various mental illnesses, including depression, attention-deficit hyperactivity disorder and dementia (Riediger et al., 2009). In addition, fish oil also helps to prevent brain aging and Alzheimer's disease (Kyle, 1999). The effect of long chain n-3 PUFA when treating asthma has been recently reviewed by Reisman et al. (2006), on bowel diseases (Razack and Seidner, 2007), psoriasis (Zulfakar et al., 2007) and in prevention some types of cancer, including colon, breast and prostate (Marchioli, 2001).

The emphasis on the importance of omega-3 long-chain PUFA has led to the commercial availability of purified fish oil supplements that are available in health food stores. The International Society for the study of Fatty Acids and Lipids (ISSFAL) recommended Adequate Intakes (AIs) of a minimum of 0.22 g/day for DHA and EPA combined, while the British Nutrition Foundation (BNF) has recommended a desirable population intake of 1.1 g (females) and 1.4 g (males) of EPA and DHA/day.

There are some extraction methods used to extract oil from fish in order to give better quality oil extraction and fine quality product at the end. The conventional fish processing methods for oil extraction including rendering process and hydrodistillation. These methods can contribute to the lost, denaturation, or decomposition of the precious nutritional fish oil. Hydrodistillation gives some disadvantages due to heat instability of the oils and loss of certain water-soluble components (Damjanovic, 2003). It is also involves substantial energy consumption to run long hours of extraction. The other conventional method of extracting oil from fish is by rendering, a process in which high heat is used to extract fat or oil mainly from animal tissues. This method used high temperature that can make the components of fish oil most susceptible to thermal degradation.

Consequently, there is an increasing demand today for new extraction technique with possibilities for automation, shorter extraction time and lower solvent consumption. Pressurised Liquid Extraction (PLE) technology and its applications have been developed and used by various researchers (Bautz, Polzer et al., 1998; Richter and Covino, 2000). It has also been applied for the analysis of food and biological samples (Schafer, 1998).

In this study, an experimental designed was planned and the experiment is performed with PLE which sample in the cell is pressurized with certain pressure, heated to desired temperature and sample is extracted statically for specific period time in order to obtain highest yield of extracted oil with the highest amount of the marker compound (EPA and DHA) in the extraction products. This PLE extraction using water and hexane as solvents and the obtained result will be compared with the extracted yield from conventional methods.

One of the major drawbacks of oil containing a high amount of omega-3 PUFA, such as fish oils, is their high susceptibility to oxidation, which involve the formation of toxic products as peroxides or volatile compounds relative to non-desirable off-flavours. Therefore, optimum processing, storage and packaging of fish oils are essential to preserve omega-3 PUFA from oxidation. The original of fish oil in liquid form make it have limited shelf-life and difficulty in storage because the temperature will affect the formation of peroxide value. This was reported by Pak (2005) that during storage at 4oC, peroxide values of several fish oils reached the acceptable limit of 8 meq/kg after 60 or 90 days and at -18 oC at least 150 days. This fact showed that the storage temperature had important effects on the storage stability of fish oil.

The use of antioxidants is one of the most common methods to prevent fish oil oxidation. However, this method still can't change the physical properties of fish oil. One technology for overcoming these problems and also reducing oxidation of omega-3 fatty acids is encapsulation of fish oil. Encapsulation or microencapsulation with a coating material has also been proposed as strategy to retard lipid auto-oxidation, improve the oil stability, prolong its shelf-life and control off-flavours (Matsuno and Adachi, 1993).

Cyclodextrin, derived from enzymatically modified starch molecules is used to encapsulate fish oil. The application of cyclodextrin in pharmaceutical, cosmetic and food industries is extensive because it is inexpensive and non-toxic. Szente and Szejtli (2004) reviewed the applications of cyclodextrin as a food ingredient in association with encapsulation technology.

In this study, β-cyclodextrin (β-CD) will be used as encapsulating agent for fish oil (FO) and the resultant FO: β-CD inclusion complex will be characterise using Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimeter (DSC) and Field Emission Scanning Electron Microscopy (FESEM). The stability of inclusion complex in comparison to control (original catfish oil) will also be investigated in this study.

Significance of study

This new technology to extract catfish oil using Pressurised Liquid Extraction (PLE) will have several advantages; more effective, rapid, selective in extraction and reliable compared to conventional methods. The possibility of changing several extractions variables such as temperature, pressure and volume of solvent is a promising characteristics of this analytical tool to get the optimise result in term of yield and also the marker compounds (EPA and DHA). This study also will be significant endeavor in microencapsulation of fish oil since the characteristics and stability of the resultant inclusion complex will be also determined. Water soluble powdered form of fish oil can be applied in various fields such as food, pharmaceutical and medical field. Besides, it can be used as a model study for future research by applying encapsulation technology to form inclusion complex of any fish oil that contain high in PUFAs especially EPA and DHA.

Objectives of study

The objectives of this study are to

develop Pressurised Liquid Extraction (PLE) method for catfish oil extraction in comparison to conventional extraction methods (rendering and hydrodistillation).

encapsulate and characterise β-cyclodextrin: catfish oil inclusion complexes.

determine the stability of the inclusion complex in comparison to control (catfish oil).

Scope and limitation

Application and optimisation of new extraction method (PLE) for catfish (Clarius batrachus) oil using water and solvent n-hexane. The resultant fish oil will be compared with conventional extraction techniques; rendering and hydrodistillation. The quality will be also compared with commercially available fish oil (Menhaden oil, Sigma). The extracted fish oil that have the highest quality in term of yield, EPA and DHA will be used for encapsulation study. Phase solubility study will be carried out to determine the molar ratio of encapsulation. The use of β-cyclodextrin as encapsulating agent for catfish oil will be also investigated. Characterisation of inclusion complex formed will be also carried out using instrumental analysis; FTIR, FESEM and DSC. The stability of the resultant inclusion complex and fish oil will be determined using Rancimat analysis. The analysis of EPA and DHA can be very challenging due to sensitivity of fish oil to oxidation. The unsaturation of fatty acids makes the fish oil more vulnerable for spoilage than the other oils. Sample for PLE extraction have to be carefully prepared in term of sample size and moisture content which can significantly effect the quality of resultant fish oil. The analysis of Response Surface Methodology (RSM) software can be difficult in order to get significant model and non significant lack of fit data.



2.1 Fish Oil (FO)

Fish oils are rich in polysaturated fatty acids (PUFAs) especially those of the omega-3 family, Docosahexaenoic Acid (DHA) and Eicosapentanaenoic Acid (EPA) which are the biological active or active agents in fish oil Chen et al. (2003). Due to its high content of PUFAs, including EPA and DHA, fish oils are highly susceptible to oxidative spoilage and it produces a rancid smell and flavor in the oil by reducing its nutritional quality, causing the formation of other undesirable substances (Frankell, 1996). However, attempts to incorporate fish oil into food formulations has had limited success because of "fishy" flavors in finished products-the main problem of food enrichment with omega-3 acids. One technology for overcoming these problems and also reducing oxidation of omega-3 fatty acids is encapsulation of fish oil.

2.2 Encapsulation

Encapsulation technology has been used in food industry for more than 60 years as a way to provide liquid and solid ingredients as an effective barrier for environmental and/ or chemical interactions until release is desired (Reineccius, 1994). The encapsulation process makes it possible to transform the oil into a powder in which small droplets of oil are surrounded by a "shell" of proteins or carbohydrate. This will improves the oxidative stability of fish oil and extends its shelf life to 12-24 months when stored in a dry cool environment. In addition, it has been proved that the use of fish oil microcapsules offers good results in the design of functional foods as cream to fill sandwich cookies (Borneo et al., 2007) and dairy products (yoghurt, fresh cheese, butter) (Kolanowski and Weiβbrodt, 2007).

Szente and Szejtli (2004) reviewed the applications of cyclodextrin (CD) as a food ingredient in association with encapsulation technology. In general, the cyclodextrin inclusion method is the better known technique for encapsulating active substances (He et al., 2007). CD can solubilise and stabilise active compounds on the molecular scale by applying the molecular inclusion method.

2.3 Cyclodextrin

Cyclodextrins are cyclic oligosaccharides consisting of six α-cyclodextrin, seven β-cyclodextrin, eight γ-cyclodextrin or more glucopyranose units linked by α-(1,4) bonds. The interior of the molecule is formed by hydrogen atoms and glycosidic oxygen bridge atoms, which give the cavity hydrophobic character and interact with various organic molecules. Guest molecules, with suitable dimensions to fit inside the interior, can be included into the cyclodextrin molecules to form agent-cyclodextrin inclusion complexes. This interaction is through the hydrophobic groups of the guest molecules with the walls of the cavity of the cyclodextrins. .

β-CD as a molecular encapsulant allows the flavour quality and quantity to be preserved to a greater extent and longer period compared to other encapsulants also it can provides longevity to the food item [21] S. Muñoz-Botella, B. del Castillo and M.A. MartyÌ‹n, Cyclodetrin properties and applications of inclusion complex formation, Ars. Pharm. 36 (1995), pp. 187-198. View Record in Scopus | Cited By in Scopus (13)(Munoz-Botella et al., 1995). [3] J. Szetjli, Introduction and general overview of cyclodextrin chemistry, Chem. Rev. 98 (1998), pp. 1743-1753.Klaypradit and Huang, 2007 reported that the encapsulation of fish oil significantly retards oxidation. It can mask the objectionable odours caused by volatile oxidation products and enhances the odours of fish oil-enriched products (Castro et al., 2004).

2.4 Inclusion complex techniques

2.4.1 Co-precipitation

Several techniques are used to form cyclodextrin complexes, co-precipitation, kneading, freeze drying, spray drying and suspension techniques. For co-precipitate method, this method is the most widely used method in the laboratory. Cyclodextrin is dissolved in water and the guest is added while stirring the cyclodextrin solution. The concentration of β-cyclodextrin can be as high as about 20% if the guest can tolerate higher temperatures. If a sufficiently high concentration is chosen, the solubility of the cyclodextrin-guest complex will be exceeded as the complexation reaction proceeds or as cooling is applied. In many cases, the solution of cyclodextrin and guest must be cooled while stirring before a precipitate is formed. The precipitate can be collected by decanting, centrifugation or filtration (Waleczek et al., 2003).

2.4.2 Kneading method

Kneading method is when the sample and cyclodextrin is mixing in mortar and kneads for 45 minutes before dried and sieved to form complex molecules (Zhang et al., 2005).

2.4.3 Physical mixture

Physical mixture is done by prepared dry-pestling in a mortar and kneaded for 5 minutes to obtain homogenous blend (Zhang et al., 2005).

2.5 Phase solubility study

Formation of inclusion complexes in solution can be detected by the phase solubility method described by Higuchi and Connors (1965). This method is carried out by adding increasing amounts of β-cyclodextrin to an aqueous suspension of an excess amount of guest (in this case is referred as fish oil). The suspensions are shaken at constant temperature until equilibrium is reached. The solid particles are removed and the solution is analyzed for the total concentration of guest dissolved. The solubility of the guest is now plotted against the β-cyclodextrin concentration.

2.6 Characterisation of inclusion complex

There are techniques used to characterize the inclusion complex and the interaction between them. In this study, Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimeter (DSC) and Field Emission Scanning Electron Microscopy (FESEM) will be used.

2.6.1 Fourier Transform Infrared Spectroscopy (FTIR)

A measurement technique for collecting infrared spectra. FTIR can detect very small alterations in bond lengths and angles in macromolecules and therefore has emerged as a powerful tool to investigate the structural changes in the molecules in detail (Garip et al., 2009). Zaibunnisa et al., 2009 in her study of inclusion complex between oleoresin and ɤ-cyclodextrin showed that cyclodextrin complex was formed with guest molecules processing carbonyl groups. The shift to higher wavelength numbers indicated that the conjugation in the oleoresin was reduced in the presence of cyclodextrin.

2.6.2 Differential Scanning Calorimeter (DSC)

This thermo analytical technique is used in studying phase transitions, such as melting, glass transitions or exorthemic decomposition. The disappearance of thermal events of guest molecules when they are examined as CD complexes is generally taken as proof of real inclusion (Pralhad and Rajendrakumar, 2004). Farcas et al., 2006 in his study of characterisation of furosemide complex in β-CD showed DSC plot of pure furosemide drug powder shows a sharp endorthermic peak near 210 oC, which attributed to ferosemide melting temperature while two endorthermic peaks were observed in the temperature range between 100-110 oC and 330 oC in the thermogram of β-CD. The DSC pattern of ferosemide: β-CD inclusion complex prepared by physical mixing show the presence of peaks of both pure compounds.

Figure 1 DSC thermograms of furosemide: β-CD inclusion complex by physical mixture.

Source: Farcas et al. (2006)

2.6.3 Field Emission Scanning Electron Microscopy (FESEM)

It is a type of electron microscope that contains information about the sample's surface topography, composition and electrical conductivity properties. It also gives information on the size and shape of samples. Farcas et al. (2006) in his study of characterisation of furosemide complex in β-CD by using FESEM measurement observed that pure furosemide (guest molecule) is characterised by the presence of crystalline particle of regular size while pure β-CD (host molecule) appears as crystalline particles without a definite shape. The physical mixture of furosemide: β-CD showed the crystalline structure of both furosemide and β-CD.

2.7 Quality and stability of fish oil

Fish oil is highly susceptibility to oxidative spoilage (Huss, 1988) because of its high content of polyunsaturated fatty acids. Oxidative stability is one of the most important quality indicators in fish oils. Peroxide value (PV) and thiobarbituric acid (TBA) analysis are commonly used to evaluate of fish oil stability and monitoring deterioration during storage.

Regulatory agencies have established the limits for quality and acceptability of oils for human consumption which 8 meq O2 /kg of oil is the limit of acceptability of PV and 7 -8 mg malonaldehyde/kg of oil (Huss, 1988) for TBA value. The original of fish oil in liquid form make it have limited shelf-life and difficulty in storage because the temperature will affect the formation of peroxide value. This was reported by Pak (2005) that during storage at 4oC, peroxide values of several fish oils reached the acceptable limit of 8meq/kg after 60 or 90 days and at -18 oC at least 150 days. This fact showed that the storage temperature had important effects on the storage stability of fish oil.

In fish oils, the induction period (IP) is the time before the highest increase of lipids oxidation. The new official method that allows the simultaneous and rapid determination of induction period or Oil Stability Indexes (OSI) to be performed is by using Metrohm's 743 Rancimat. The accelerated oxidative test Rancimat has been used extensively during the last years to determine oxidative stability of fats and oils under standardized conditions (Rossell, 1994). Concerning lipidic foods, application of Rancimat to intact foods would yield a more realistic representation of what may occur during storage than utilizing the extracted lipids, with the additional advantage of avoiding any previous, time-consuming handling of samples through the extraction step.

In the Rancimat, a stream of purified air is passed through a sample of oil or fat that is held in a heating block. The effluent air from the sample is then bubbled through a vessel containing deionised water. The conductivity of the water is continually monitored. Once the oil starts to oxidise, volatile organic acids, predominantly formic acid, are swept by the effluent stream, through the deionised water, causing an increase in the conductivity of the water. The OSI is defined as the point at which there is the maximum change in the rate of oxidation and is evaluated automatically in the Rancimat.



3.1 Sample preparation

Farmed Catfish is obtained from a local supplier in Seremban, Negeri Sembilan. The fish will be gutted, washed, minced in a commercial blender and stored at -20 OC until be analyzed.

3.2 Method of extraction

3.2.1 Rendering -Water cooking

Extraction followed the method by Sheu and Chen (2002). Flesh sample will be boiled with a sample/water ratio (1:2w/w) in glass beakers for 30, 40, or 50 min, separately. After removal of the flesh residues, the liquid will be cooled to room temperature and the fat layer will be removed using a separatory funnel.

3.2.2 Hydrodistillation

Extraction followed the method by Zaibunnisa et al., 2009. Flesh samples (300g) will be hydrodistilled for 8 hr using a Dean Stark- type apparatus. The distillates will be collected and liquid-liquid extractions will be carried out to separate the oil fraction from water.

3.2.3 Pressurised Liquid Extraction (PLE)

Sample will be ground and dried in oven at 40 oC for overnight for pre-treatment. Samples (5g) will be mixed together with diatomaceous earth (5g) with ratio before been placed into 22 ml cells with cellulose filter at the bottom end. Solvent is added. In this experiment, different solvents of n-hexane and water will be used to investigate the influence of solvent polarity on the yield of oil. The cell will be pressurized with 1000 psi, heated to desired temperature (70 oC) and sample is extracted statically for specific period time (5 min). Others condition will be standardized; flush volume, 100%; purge time, 60s and static cycle, 1.The extract will be removed from cell and cell will be flushed with fresh solvent. When extraction is complete, Nitrogen gas will be compressed to move all solvent from cell to vial for analysis. The extract will be evaporated to dryness (rotary evaporator) to calculate yield on dry weight basis. Optimization of PLE extraction can be getting by using Response Surface Methodology (RSM).

3.3 The physical and chemical analysis of fish oil

3.3.1 Determination of free fatty acid composition

The extracted lipid will undergo transesterification to produce fatty acid metyl ester (FAME) according to Kinsella et al. (1977). Sodium methoxide and hexane will be used for the preparation of the Fatty Acid Methyl Esters (FAME). The fatty acid methyl ester of samples will be analyzed by the gas chromatography (HEWLET PACKARD, HP 6890 Series, USA) which is equipped with Flame Ionization Detector (FID). A capillary column (SGE, 50 m length and 0.22 mm diameter) is used to separate the fatty acid components. The temperature of the injection port and detector will be set at 260°C. The oven temperature will be programmed to increase from 50 to 230°C at a rate of 4°C per min. One microliter of each sample will be injected manually in duplicates with the split 40. Fatty acid peaks in the samples will be identified by comparing the retention times with that of authentic reference standards. The results will be expressed as relative percentages.

3.3.2 Peroxide value analysis

The peroxide value will be determined using AOAC method (AOAC, 1995). Each fat sample (5g) will be mixed with 30 ml HOAc-CHCl3 and 0.5 ml saturated KI solution. After shaking for 1 min, 30 ml of distilled water will be added and mixed. A 0.01 N Na2S2O3 will be used for titration. Results will be expressed as mill equivalents/kg oil (meq/kg).

3.3.3 Thiobarbituric acid (TBA) value analysis

Thiobarbituric acid (TBA) value will be followed by distillation method (Tarladgis et al., 1960). Optical density (OD) will be measured against a reagent blank at 538nm. The TBA value will be obtained by multiplying the absorbance (OD) by a constant of 7.8.

3.3.4 Color analysis

Color of the samples will be measured using a minolta chroma meter (CR-200 Minolta, Japan). The color readings will be expressed by CIE (L* a* b*) system (Rafael et al., 2004). L*, a* and b* indicates whiteness/darkness, redness/greenness and blueness/yellowness, respectively. The maximum value for L* is 100, which would be a perfect reflecting diffuser. The minimum for L* would be zero, which would be black. The a* and b* axes have no specific numerical limits. Positive a* is red and negative a* is green. Positive b* is yellow and negative b* is blue.

3.4 Encapsulation and characterization catfish oil: β-cyclodextrin

Phase solubility studies will be carried out following method of Higuchi and Connors (1965) to determine the molar ratio of FO: β-CD. An excess amount of fish oil (20mg) will be added to screw-capped vials containing β-CD in 5.0 ml of ethanol: water (25:75 v/v) solution at various concentrations, ranging from 0 to 15 Mm for β-CD. The vials will be shaked at 30 OC for 48 hours in a water bath until reached equilibrium. The samples will be centrifuged at 3000rpm for 10 minutes. After attainment of equilibrium, the contents of the tube will be filtered through Whatman filter paper. The extracts will be pooled together and evaporated to 1 ml. Inclusion complex of catfish oil: β-CD will be prepared by using:

3.4.1 Co-precipitation method

Co-precipitation method will be carried out following method of Waleczek et al., 2003. FO will be added to screw-capped vials containing β-CD in ethanol: water (25:75 v/v) mixture of 5ml. The vials will be shaking at 30 OC until equilibrium reached in water bath for 48 hours. The samples will be centrifuged at 3000 rpm for 10 minutes. The supernatant will be decanted to form the complex as microcrystalline precipitate. The product obtained will be dried in oven at 40 OC for 48 hours. The dried mass will be sieved through 150µm mesh.

3.4.2 Kneading method

Kneading method will be carried out following method of Zhang et al., 2005. A molar ratio of β-CD and fish oil that will be determined by phase solubility study will be added in mortar and kneaded for 45minutes. While kneading, 40% of ethanol: water (25:75 v/v) mixture will be added to the mixture to maintain proper consistency. The products will be dried in oven at 40 OC for 48hours. The dried mass will be sieved through 150 µm mesh.

3.4.3 Physical method

Kneading method will be carried out following method of Zhang et al., 2005. For control, physical mixture of the same weight ratio of fish oil: β-CD will be prepared by dry-pestling in a mortar and kneaded for 5 minutes to obtain homogenous blend.

Characterisation of catfish oil: β-CD inclusion complex will be done by FTIR, FESEM and DSC.

3.5 Stability of fish oil

Oil Stability Index (OSI) will be determined at 100 OC and 20 ml air/hr using Rancimat apparatus following AOCS Method (AOCS, 1994). The Rancimat apparatus will be used with two evaluation modes, i) induction period (time corresponding to inflection point in the oxidation curve) and ii) time to delta k (time needed to achieve a specific difference in conductivity, here will be selected is in 25µS cm-1.

3.6 Statistical Analysis

Data analysis will be done by using analysis of variance (ANOVA). Comparison of means will be carried out by Duncan's multiple range tests (p <0.05). Statiscally analysis will be performed by using SPSS 10 for Windows.