Supercritical Fluid Fractionation Sff Of Marine Fish Oil Biology Essay


The triumphs of treating nutritional deficiencies to improve health and prevent and treat disease throughout medical history have and continue to abound. Potential dietary manipulation that is beginning to emerge is the potential impact of correcting low levels of omega-3 fatty acids to prevent a variety of medical conditions from occurring or progressing such as: arthritis, some cancers, cardiovascular disease, depression, maternal/fetal well-being, and neurological disease. Omega-3 fatty acids are a group of PUFA, the essential fatty acids that the human body cannot produce. Both n−3 and n−6 PUFA are entirely derived from the diet and necessary for human health. An n−6:n−3 fatty acid ratio of 5:1 or less is desired, as suggested by nutrition experts. However, nowadays food habits in western society are characterized by a high consumption of meat, seed oils, fast food (such as pizzas and hamburger) and baked and snack food ( such as cakes, biscuits and bread), that contain a large amount of saturated fatty acids and a low proportion of PUFA. However, since changing the nutritional habits of a whole society is very difficult, in the last years, many products enriched with n−3, like nutritional supplements or functional foods, have been developed to supplement the diet and reach a good n−6:n−3 ratio in blood without changing the diet too much.

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Omega-3, are essential in human nutrition since they play an important role in the organism and prevent several diseases. Fish has been the traditional source to obtain omega-3 enriched oil and concentrates, although alternative sources as marine microorganisms or transgenic plants have been proposed recently. Supercritical fluid extraction has been demonstrated as good methods in the production of omega-3 oil and omega-3 concentrates, mainly in their ethyl esters derivative form, avoiding the use of high temperatures and organic solvents. The use of omega-3 fatty acids, especially EPA and DHA, as active compounds in pharmacology or functional ingredients in industrial food requires a previous concentration step into a chemical form easily metabolised by human organism and with a good stability against oxidation. In the last years many processes have been developed to isolate, fractionate or concentrate n−3 PUFA from fish oil, although the majority of them are referred to omega-3 ethyl esters formed by the esterification or saponification of triglycerides with ethanol. Nowadays, SFF is generally considered a useful technology to replace traditional concentration processes. Therefore, SFF is a promise technology to be scaled up to indusial scale in order to produce good quality n−3 concentrates.

Functional food products enriched with n−3 fatty acids have been the type of functional foods whose production in Europe and USA has increased the most in the last years. There is nowadays a wide variety of commercial food products enriched with omega-3, as bakery products, milk and derivatives, spreadable fats, eggs, juices and soft drinks, meat and poultry products, etc. In most cases, fish oil is the natural source of n−3 fatty acids to be incorporated into conventional food products, following different strategies to avoid important modifications in the sensorial quality of the products, and prevent the oxidation to which n−3 fatty acids are prone to.

The fortified food must be acceptable to and consumed by the target population, must provide iron in a stable and highly bioavailable form and the chosen fortificant must not adversely affect the organoleptic qualities and shelf-life of the food vehicle. The vehicle used in fortification programs is determined by the locally available foods and the dietary patterns of the target populations. To this end, a number of vehicles have been investigated including rice, milk, salt, sugar, soy sauce, fish sauce and fruit juices with wheat and maize flours being the most used in iron-fortification programs. The relatively low cost and widespread consumption of wheat flour bread makes it a particularly appealing vehicle for ω-3 fatty acids -fortification programs.

Objective of the proposed research

The major objectives of the proposed research are:

Supercritical Fluid Fractionation (SFF) of marine fish oil for the production of ω-3 fatty acid concentrates.

Effects of ω-3 fatty acid fortification on quality and storage attributes of wheat bread.

Experimental approach and methods

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Objective 1: Supercritical Fluid Fractionation (SFF) of marine fish oil for the production of ω-3 fatty acid concentrates

Supercritical Fluid Fractionation (SFF) of marine fish oil

Optimization of process parameters

ω-3 fatty acids profile of concentrate and studies on oxidative stability.

SFF is a relatively new fractionation process that may circumvent some of the problems associated with the use of conventional separation techniques. Application of SFE technique will be evaluated for the extraction of ω-3 fatty acids from fish oil with special reference to sardine oil. The major process parameters will be optimized using Response Surface Methodology to enhance the yield. The parameters include experimental pressure and temperature ranges. The amount of ω-3 fatty acids will be quantified using HPLC method.

Objective 2: Effects of ω-3 fatty acid enrichment on quality attributes of wheat bread.

Effects of ω-3 fatty acid on the bread dough quality

Physicochemical analysis of bread containing ω-3 fatty acid

Sensory analysis of ω-3 fatty acid fortified bread

Storage studies of ω-3 fatty acid fortified bread


Farinograph tests

Dough fortified with ω-3 fatty acid at various levels will be tested according to the ICC-standard method. The ω-3 fatty acid concentrate will be first mixed well with the wheat flour into the mixing bowl (300 g) of the Farinograph, and water absorption, dough development time, and dough stability will be determined.

Extensograph tests

The control (unfortified) and the ω-3 fatty acid enriched doughs will be prepared in the 300 g mixing bowl of the Farinograph. The wheat flour will be first mixed well with the ω-3 fatty acid at different concentration levels, before salt and water addition, to produce the dough samples. Water will be added to produce dough with a required consistency of 500 BU (Brabender Units), followed by 5 min of mixing. A test piece (150 g) will be rounded into a ball, shaped into a cylinder and clamped into the holder. After 45, 90, and 135 min resting times in the fermenting cabinet at 30-32 _C, each dough piece will be stretched in the Extensograph by a hook until rupture, as described in the ICC-Standard 114/1 method (1992). The stretching force will thus be recorded as a function of time, and the resistance to constant deformation after 50 mm stretching (R50) and the extensibility (E) will be obtained.

Alveograph tests (Rheological properties)

Bread making performance of flour has been shown to be reliably predicted from an alveograph test. Rheological properties of the dough samples (with or without ω-3 fatty acid) will be determined using an alveograph according to the approved methods. Alveogram parameters include dough tenacity (P), extensibility (L), stability (P/L), swelling index (G), and work input (W). The colour of dough will be measured using a Hunter Lab system.

Color measurements:

The dough colour will be measured using a Hunter Lab system and results will be expressed as L*, a* and b* values, where L* indicates whiteness (value 100) or blackness (value 0), a* indicates red (positive value) or green (negative value), and b* indicates yellow (positive value) or blue (negative value). Mean values of four observations will be recorded from each dough and the means of at least 4 readings will be taken for comparison purposes.

Microbiological analysis of bread dough

Bread dough with or without ω-3 fatty acids will be used for plating to determine lactic acid bacteria, total plate count and yeast and molds. The microbial load will be measured by suspending the respective dough in 0.5% sterile saline and plating it out at appropriate dilutions. de Man, Rogos and Sharpe (MRS) media, potato dextrose agar (PDA) and nutrient agar (NA) media will be used for enumerating the number of lactic acid bacteria, yeast and molds, and mesophilic aerobes respectively employing spread plate and pour plate method. Colonies will be counted after 24 h incubation at 30 °C (PDA) and 37 °C (NA and MRS) and the results will be expressed as cfu/g of dough.


Loaf volume and weight will be determined 1 h after baking at ambient temperature according to reported procedures, and expressed as the specific volume. A container will be filled with a known volume of rapeseed. The amount of seed displaced when the loaf is introduced will be measured in a graduated cylinder, and measured as the loaf volume.

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Bread crust and crumb color: The bread image will be acquired with a digital camera and will used to evaluate the crust color of each baked sample. This will allow measurement of a larger bread surface area than attained with conventional colorimeters. The digital image will be analyzed using an appropriate software and will be converted to a 256 gray level scale. Sample lightness varied from 0 (black) to 255 (white). The array of pixel values will average to determine the average crust lightness. The standard deviation of pixel values will be used to measure the color variation. Parallally, the crumb colour will be measured using a Hunter Lab system and results will be expressed as L*, a* and b* values, where L* indicates whiteness (value 100) or blackness (value 0), a* indicates red (positive value) or green (negative value), and b* indicates yellow (positive value) or blue (negative value). The colour will be measured 1 h after baking. Mean values of four observations will be recorded from each loaf and the means of at least 4 breads will be taken for comparison purposes.

Moisture will be determined by oven-drying at 101 C for 17 h as described by Chen, Long, Raun, and Labuza (1997); incineration at 550 1C for ash (AOAC, 1998; method 923.03); and Water activity (aw) of the center of the loaf (crumb) will also be measured.

Microstructure of Bread:

For scanning electron microscopy (SEM) analysis, bread crumb samples containing various levels of ω-3 fatty acid will be prior dried in a forced convection oven (at 70 _C for 12 h) and powdered. Dried bread samples will be mounted on specimen stub using conducting silver paint and sputter coated using gold-palladium target prior to the examination. The specimen stub will be then mounted on a specimen holder and put in the machine. The microstructures of the samples will viewed on an appropriate scanning electron microscope and will be observed at a magnification level of 800x and 400x.

Storage test

Samples of the highest fortification level, showing no significant change in overall sensory quality, will be selected for a storage test. The test breads will be packaged in polyethylene bags for storage at 22±2 degree C. Breads will be stored whole, and analysis of bread samples will be performed on 0 (fresh bread), 2, 4, 6, and 8 days.

Sensory evaluation

An appropriate number of well trained panellists (10 or more) will participate in the sensory evaluation based on scoring various bread samples according to five attributes: sweetness, porosity, astringency, stickiness, crumb attributes (elasticity, crumb grain structure), crust colour, shape regularity (height/diameter ratio), colour, flavour and overall acceptability. In terms of corresponding intensity, each attribute will be ranked as low, medium and high grades gaining 1, 2, 3 score, respectively. The rating scale for elasticity ranges from 1 (unacceptable) to 5 (excellent), for crumb grain structure; it ranges from 1 (distinctively coarse) to 5 (spongy), for crumb grain uniformity; it ranges from 1 (uneven pore distribution) to 5 (even pore distribution), and crust colour from 1 (pale or burned) to 5 (deep golden). Flavour will be determined using a 5-point hedonic rating scale ranging from 1 (dislike extremely) to 5 (like extremely). Sensory evaluation will be conducted on three batches of bread. The mean scores and corresponding standard deviation values for each attribute will be calculated for comparison.