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Microalgae are procaryotic or eucaryotic photosynthetic micro-organism unified chiefly by their deficiency of roots, foliages, and stems that characterize higher workss. They can be found about anyplace with H2O and sunshine as cardinal demand including lakes, dirts, rivers, hotsprings, and the ocean. Marine algae are responsible for making the bulk of the O, where 40 % -50 % of the photosynthesis among them occurs on Earth each twelvemonth despite their photosynthetic biomass represents merely approximately 0.2 % of that on land ( Parker et al. , 2008 ) . Harmonizing to Mata EL Al. ( 2009 ) , microalgae with its unicellular or simple multicellular construction have high growing rate and are able to populate in rough status.

Microalgae contain the high value compounds like fatty acids ( I?- linolenic acid, arachidonic acid, eicosapentaenoic acid ( EPA ) , docosahexaenoic acids ( DHA ) , and etc ) , pigments ( chlorophyll and carotenoids ) , vitamin such as vitamin H, vitamin C and E, and others ( Converti et al. , 2009 ) . Owing to their high quality alimentary constituents, microalgae are able to heighten the nutritionary content of conventional nutrient readying, and are by and large applied in the field of human and carnal nutrition. Nowadays, it can besides be seen that microalgae for human nutrition are normally sold in the market in the signifier of tablets, capsules, and liquids as nutrient addendums and wellness nutrients. Indeed, the universe ‘s largest manufacturer Hainan Simai Enterprising has an one-year production of 200t of algal pulverization, which accounts for about 10 % of the universe end product ( Spolaore et al. , 2005 ) . For carnal nutrition, microalgae are primary nutrient beginning for rotifers, seawater runt, late larvae, juvenile fishes, and crustaceans in aquaculture ( Pratoomyot et al. , 2005 ) .

Microalgae rich in omega-3 polyunsaturated fatty acids ( n-3 PUFA ) chiefly EPA ( 20:5, n-3 ) and DHA ( 22:6, n-3 ) are ever the country of involvement in the human nutrition because they are believed to be indispensable for optimising human wellness. DHA is of import for encephalon and eyes development in pre-term and immature babies every bit good as support cardiovascular wellness in grownups. While, EPA is indispensable for the human metamorphosis, and is involved in the blood lipid equilibrium, prevent hypertriglyceridemia, anti-inflammatory, and so forth ( Kroes et al. , 2003 ; Ward & A ; Singh, 2005 ; Farjardo et al. , 2007 ) . Commercially, production of n-3 PUFA-rich microalgal oils has grown and microalgal oils are already used in 84 % of US infant expression over the last few old ages ( new wave Beelen et al. , 2009 ) .

Apart from that, microalgae contain a battalion of pigments peculiarly chlorophylls and carotenoids are besides of peculiar involvement. In add-on to chlorophylls as the primary photosynthetic pigment in microalgae, they are besides being used in the dyeing of groceries such as ice pick and cold drinks ( Macias-Sanchez et al. , 2005 ; Pulz and Gross, 2004 ) . On the other custodies, carotenoids which protect microalgae from inordinate solar radiation are besides found to be indispensable to human wellness and of import in commercial application ( Felti et al. , 2005 ) . For illustration, i??-carotene act as pro- vitamin A has been proven to forestall xeropthlamia ( Puah et al. , 2005 ) , astaxanthin act as natural colorant for musculus in marine animate beings like fish, xanthophyll, zeaxantin and canthaxantin for poulet tegument colour, or for pharmaceutical intents ( Pulz and Gross, 2004 ) , act as natural nutrient additive ( Del Campo et al. , 2000 & A ; Vilchez et al. , 1997 ) , and etc.

1.2. Statement of jobs

Chronic diseases including malignant neoplastic disease and cardiovascular diseases are the chief subscriber to decease in the universe ( Abd El-Baky et al. , 2007 ) . Due to the significant addition in the grounds base about the wellness benefits of n-3 PUFA peculiarly cardiovascular diseases, it is important to guarantee its sufficient consumption from diet ( Kris-Etherton et al. , 2009 ) . Virtually, animate beings and worlds lack the ability to synthesise the n-3 PUFA because efficiency of metabolic transition from I±- linolenic acid to EPA and DHA appear to be low ( Napier and Sayanova, 2005 ) . Therefore, bulk of these PUFAs must be obtained through diet. Based on National Health and Nutrition Examination Survey ( 1999-2000 ) , many of the US population ( including all ages and both genders ) are really non run intoing current recommendations for omega-3 fatty acid ( 500mg/day ) , with the average consumption of EPA and DHA is merely approximately 100mg/day ( Erwin et al. , 2004 ; Kris-Etherton et al. , 2009 ) . Besides, the n-3 PUFA consumption of the mean Malayan is presently low, estimated to be 0.3 % kcal ( recommended consumption is 0.3 – 1.2 % kcal ) , with 200 milligrams from EPA and DHA on fish ( Ng, 1995 ) .

Previously, fish and fish oil is the chief dietetic beginning of DHA and EPA. However, worsening beginnings of marine fish stocks and fish oil related to the serious environmental effects and uninterrupted development, which restricting the protective function of PUFA in human wellness has prompted the research into new beginnings of PUFA ( Burja et al. , 2007 ) . In add-on, certain disadvantages of fish oils such as unpleasant olfactory property, possible pollutants, and assorted fatty acid belongingss besides promoting the find of the alternate beginnings of PUFA ( Pulz and Gross, 2004 ) . As PUFA found in fish arising from microalgae consumed, it is sensible to see microalgae as possible beginning of PUFA.

On the other manus, carotenoids are besides having considerable public attending today. The nutritionary and curative relevancy of certain carotenoids is ever related to the possible pro-vitamin A activity, which can be converted into the vitamin A in the human organic structure ( Gouveia & A ; Empis, 2003 ) every bit good as other belongingss like antioxidants and anti-inflammatory. Harmonizing to Ishida & A ; Chapman ( 2009 ) , human must obtain carotenoids from dietetic beginnings as they can non synthesise it.

In most communities in developing states, carotenoids are the major beginning of vitamin A ( Tee, 1995 ) .Unfortunately, vitamin A lack ( VAD ) remains a important public wellness job globally with an estimated 190 million kids and 19 million pregnant adult females being affected ( WHO, 2009 ) . VAD is the taking cause of sightlessness in kids and is besides a major subscriber to morbidity and mortality from infections, particularly in kids and pregnant adult females from low and in-between income states. Prior to the 1950 ‘s, vitamin A lack in Malaysia has besides been reported to be a important upset impacting chiefly immature kids who taking imbalanced diet and from low socio-economic population ( National Coordinating Committee on Food and Nutrition, 2005 ) .

Many of the commercially available carotenoids, chiefly i??-carotene addendums ( more than 90 % ) are really produced by man-made mean ( Leon et al. , 2003 ) . Unlike natural carotenoids which is mixture of carotenoids compound, man-made signifier of carotenoids may non be readily metabolized quickly ( Puah et al. , 2005 ) . Besides, some surveies have besides shown that the natural carotenoids have antioxidant activity that the man-made signifier deficiencies ( Ben-Amotz & A ; Levy, 1996 ) . Consequently, it has driven the research and development on the production and usage of carotenoids from natural beginning of microalgae to run into the increasing demand for natural merchandises and act as replacement to the man-made signifier of the carotenoids ( Abd El-Baky, 2007 ; Del Campo et al. , 2000 ) .

Although microalgae contain of import biochemical composing ( peculiarly PUFA and carotenoids ) , the extraction methods and efficiencies for works stuff, particularly for algae, are less good established where there are really no standard extraction methods for finding of fatty acids contents and carotenoids in microalgae ( Wiltshire et al. , 2000 ) . Felti et Al. ( 2005 ) pointed out that absence of standard extraction method for carotenoids are really attributed to the broad spectrum of analysed stuffs ( groceries, works, animate being, and human samples ) and a broad scope of carotenoid nowadays. In fact, significant job associated with extraction methods may perplex the analysis of pigment and fatty acids composing of microalgae ( Wiltshire et al. , 1998 ) .

1.3. Significance of survey

Since n-3 PUFA and carotenoids play a major function in human wellness, it is necessary to happen out appropriate alternate nutrient beginnings to run into the demand of the public today. At present, microalgae offer great possibilities for the isolation of these substances has made it with a great trade of added value ( Macias-Sanchez et al. , 2009 ) . In add-on, there is besides a lifting tendency that people are traveling for natural merchandises. Therefore, functional ingredients such as n-3 PUFA, carotenoids, ( e.g. xanthophyll, lycopene, astaxanthin ) , and other pigments which are extracted from natural beginnings as like microalgae are preferred ( Herrero, Cifuentes & A ; Ibanez, 2005 ; Cohen & A ; Vonshak, 1991 ; Mahajan & A ; Kamat, 1995 ) . These functional ingredients have possible commercial applications as nutraceuticals, pharmaceuticals, and of import provender ingredient in marine civilization has besides greatly attracted attending from nutrient industries and agribusiness due to the economic and societal demands.

Due to high alimentary value of the biochemical composings of algae cells, chiefly fatty acids composings and carotenoids, research on quantitative finding of these biochemical composings from microalgae is therefore highly of import ( Deventer & A ; Heckman, 1996 ) . The informations can supply some penetrations to those research workers interested in this field of survey. Meanwhile, it can besides raise the consciousness of public on the good usage of microalgae. Besides, the information may besides beneficial to nutrient makers to heighten the nutritionary value of their nutrient merchandises. In Malaysia, they are merely limited research on finding of fatty acids composing and carotenoids in microalgae. Therefore, it is hoped that the informations are good in building nutrition database in the hereafter.

In order to find the sum of biochemical composing of involvement accurately, it is peculiarly of import to happen out efficient extraction methods, which can pull out the maximal output of biochemical composing from microalgae. Previously, assorted methods for the extraction of fatty acids from microbic biomass have been reported in many surveies ( Burja et al. , 2007 ; Lewis et al. , 2000 ; Grima et al. , 1994 ; Tran et al. , 2009 ; Lee et al. , 2009 ) . However, the different cell construction of microalgae may necessitate farther probe on usage of different organic dissolvers to pull out the fatty acids. In add-on, there is besides deficiency of surveies on the rating of extraction methods utilizing different organic dissolvers for fatty acids from marine microalgae. On the other manus, there is merely scarce surveies describing the extractability of carotenoids include an rating of extraction methods utilizing different organic dissolvers ( Armenta et al. , 2006 ) .

1.4. Aims

1.4.1General nonsubjective

To analyze the assorted extraction methods on fatty acids composing and carotenoids from Marine microalgae, Nannochloropsis oculata and Chaetoceros gracilis.

1.4.2 Specific aims

To find fatty acids composing in microalgae of different dissolvers extraction by utilizing gas chromatography ( GC ) .

To find carotenoids ( b-carotene, I±-carotene, and xanthophyll ) in microalgae of different dissolvers extraction by utilizing high public presentation liquid chromatography ( HPLC ) .

To compare the fatty acids composing between microalgae Nannochloropsis oculata and Chaetoceros gracilis utilizing different dissolvers extraction methods.

To compare the carotenoids between microalgae Nannochloropsis oculata and Chaetoceros gracilis utilizing different dissolvers extraction methods.

. Null hypothesis

H0: There is no important difference in the mean of fatty acids sum between microalgae Nannochloropsis oculata and Chaetoceros gracilis utilizing different dissolvers extraction methods.

2 ) H0: There is no important difference in the mean of carotenoids sum between microalgae Nannochloropsis oculata and Chaetoceros gracilis utilizing different dissolvers extraction methods.

Chapter 2


2.1. Microalgae

Alga is referred to as any being with chlorophyll a, and a thallus non differentiated into roots, stems, and leaves while the term microalgae refer to the microscopic algae, and the oxygenic photosynthetic bacteriums, such as the blue-green algae. Microalgae are present in all bing Earth ecosystems, non merely H2O but besides found on the surface of all type of dirts, stand foring a large assortment of species populating in a broad scope of environmental conditions ( Richmond, 2004 ) .

During the past decennaries, microalgae have begun attracted attending from research workers all over the universe where extended aggregations of microalgae have been created by research workers in different states. For illustration, University of Coimbra ( Portugal ) is considered as one of the universe ‘s largest research institute in the aggregation of fresh water microalgae, holding more than 4000 strains and 1000 species. This aggregation has revealed the benefit of the big assortment of microalgae in a wide diverseness of commercial applications, such as value added merchandises for pharmaceutical intents, nutrient harvests for human ingestion and as energy beginning ( Mata et al. , 2009 ) .

The alimentary quality of microalgae is dependent on several factors including biochemical composing, atom size and form, toxicity, and easiness of digestibleness ( related to cell wall construction and composing ) . The biochemical composing of microalgae is influenced by environmental conditions, including visible radiation, salt, temperature, foods and growing rate. Particularly, temperature has a major consequence on the biochemical composing of some microalgae. It has been reported that some microalgae respond to a lessening in temperature by increasing the ratio of unsaturated fatty acid to saturated fatty acids. Besides, high growing temperature has besides been related to important lessening in protein content, together with additions in lipoids and saccharides. However, several surveies found that the response of microalgae chemical composing to growing temperatures every bit good as other environment conditions stated supra is really varies from species to species ( Durmaz et al. , 2009 ; Renaud et al. , 1995 ; Renaud et al. , 2002 ) .

Harmonizing to Ariyadej et Al. ( 2004 ) , microalgae serve as the of import footing of the nutrient concatenation in the unfastened sea where they are primary nutrient beginning for larvae of many species of molluscs, crustaceans and fish. On the other manus, microalgae can besides assist to stabilise and better the quality of the civilization medium for Marine animate beings ( green-water technique ) attributed to its ability to better H2O quality by stabilisation of O and pH ( Chuntapa et al. , 2003 & A ; Muller-Feuga, 2000 ) . Recently, microalgae has besides been suggested as really good campaigners for fuel production attributed to their advantages of higher photosynthetic efficiency, higher biomass production, faster growing compared to other energy harvests, and much less land countries demand ( Widjaja et al. , 2009 ) .

Today, microalgae biomass and infusions from microalgae have gained a steadfast place on the market because there is an increasing demand for high-value merchandises from microalgae, such as polyunsaturated fatty acids ( PUFA ) , carotenoids, heat-induced proteins, or immunologically effectual compounds. In 2004, the microalgal biomass market produces about 5000t of dry matter/year and about US $ 1.25 x 109 /year of turnover is generated ( Pulz & A ; Gross, 2004 ) .

2.1.1 Nannochloropsis oculata

Nannochloropsis oculata is a marine unicellular algae belonging to the Eustigmatophyceae category. Eustigmatophytes are unicellular, with coccoid cells and polyose cell walls where the cells do non roll up amylum. Nannochloropsis are characterized by the absence of chlorophyll B and its carotenoids composing is comparative simple, incorporating i??-carotene, violaxanthin, and a vaucheraxanthin-thin pigments ( Rebolloso-Fuentes. , 2001 ) . Nannochloropsis are little and immotile domains with its diameter of about 2-4Aµm ( Figure 1 ) . All of its species do non show any other distinguishable morphological characteristics, and can non be distinguished by either visible radiation or negatron microscopy. Typically, they live in salt-water home grounds ( Hoek, Mann & A ; Jahns, 1995 ; Richmond, 2004 ) .

Figure 1: Microscopy position of Nannochloropsis sp. ( Adapted from Wikipedia, 2009 )

Presently, the eustigmatophyte Nannochloropsis is widely used in many aquaculture as the footing of an unreal nutrient concatenation. In Japan, Nannochloropsis oculata play an of import function as civilized provender for the rotifer Brachionus plicatilis, and its concentrated suspensions and frozen biomass are commercially available ( Chini Zittelli et al. , 1999 ) . The advantage of Nannochloropsis over other unicellular algae is chiefly its alone fatty acid composing, where it has been recognized as a good possible beginning of fatty acids such as eicosapentaenoic acid ( EPA ) and docosahexaenoic acid ( DHA ) , an of import polyunsaturated fatty acid in human diet ( Rodolfi et al. , 2003 ) . Previously, a survey assessed the fatty acerb composing of 15 species of Marine microalgae found that Nannochloropsis oculata exhibited comparative simple fatty acids profile, dominated by three components- 16:0, 16:1 ( n-7 ) , and 20:5 ( n-3 ) with a close entire absence of C18 PUFAs ( Zhukova & A ; Aizdaicher, 1995 ) .

In add-on, this microalga is besides good known as a beginning of different valuable pigment like carotenoids ( astaxanthin, zeaxanthin and canthaxanthin ) and chlorophyll a,

which is indispensable for commercial usage. ( Marcillaa et al. , 2009 ) . Indeed, the usage of Nannochloropsis has been recognized in human diets where several surveies have been conducted to look into its incorporation in nutrients such as noodles to better nutritionary profile ( Rebolloso- Fuentes, 2001 ) .

2.1.2 Chaetoceros gracilis

Chaetoceros sp. is likely the largest genus of Marine diatoms with about 400 species described. Diatoms are a major group of eucaryotic algae, and are one of the most common types of phytoplankton. Most diatoms are unicellular, although they can be as settlements in the form of fibrils or threads. Normally, diatoms are between 20-200 micrometers in diameter or length. However, some species of diatoms can make up to 2 millimeters in length. Besides, most diatoms are non-motile, although some may travel utilizing scourge. A alone characteristic of these diatom cells is that they are encased within a cell wall made of silicon oxide ( hydrous Si dioxide ) called frustule, which provide the diatom entree to the external environment for procedure such as waste remotion and mucilage secernment. Diatoms are belonging to group of heterokonts in which its yellowish-brown chloroplasts are the typical characteristic with four membranes and incorporating pigments such as the carotenoid ( fucoxanthin ) as shown in Figure 2 ( Wikepedia, 2009 ) .

They play an of import function in oceans, where they contributes up to 45 % of the entire pelagic primary production. Diatoms, in peculiar, are regarded as utile impersonal lipid beginnings. Indeed, some marine species of Chaetoceros are critical nutrient beginnings for Marine civilizations. For illustration, Chaetoceros gracilis has been shown to be an equal sole provender beginning for larvae and postlarvae of the runt Metapenaeus Ensis ( Chu, 1989 ) .

Besides, Tsitsa-Tzardis et Al. ( 1993 ) besides reported that Chaetoceros sp. is the most effectual phytoplankton in advancing juvenile oyster growing because its isolation contains cholesterin which is the major steroid alcohol in oysters. In add-on to high lipid content, diatoms are besides abundant of certain polyunsaturated fatty acids ( PUFAs ) such as big sums of eicosapentaenoic acids ( EPA ) . Therefore, the ability to pull strings the measure and quality of lipoid in Chaetoceros species is effectual for several applications such as renewable resource as fuel and nutrient ( McGinnis et al. , 1997 ) . A survey has besides shown that Chaetoceros gracilis contain chiefly C14, C16, and C20 fatty acids composing, specifically 14:0, 16:0, 16:1, and 20:5 under the different status of temperature, light strength and silicate ( Mortensen et al. , 1988 ) .

Figure 2: Microscopy position of Chaetoceros gracilis

( Adapted from )

2.2. Carotenoids

Carotenoids are natural pigments that are unambiguously synthesized in workss, algae, Fungis, and bacteriums ( Sandmann, 2001 ) . The pigments may change in xanthous, orange, and ruddy colour. The construction of the carotenoids is long, aliphatic, and conjugated dual bond systems ( Felti et al. , 2005 ) . They are isoprenoid polyenes formed by head-to-tail linkage of C5 isoprene unit, and merely in the centre of the molecule is a tail-to-tail lingkage that makes the molecules symmetrical ( Nollet, 2000 ) . Most of carotenoids contain 40 Cs atoms ( C40 carotenoids ) and can be divided into two groups, which are hydrocarbon ( made up of C and H atoms merely ) called as provitamin As and oxygenated derived functions of provitamin As ( incorporating at least one O map such as hydroxy, keto, epoxy, methoxy or carboxylic acid groups ) called as luteins ( Rodriguez-Amaya, 2001 ) . In nature, most carotenoids occur in the all-trans signifier. Cis isomers are often present in little sums ( Nollet, 2000 ) .

Some common constructions of carotenoids are shown as below:















( Adapted from hypertext transfer protocol: // )

2.2.1 Physiochemical belongingss of carotenoids

Carotenoids are lipotropic pigments, where they are indissoluble in H2O and soluble in organic dissolvers, such as propanone, intoxicant, ethyl quintessence, trichloromethane, and ethyl ethanoate. The solubility of the carotenoids depends on its specific structural. For illustration, provitamin As are readily soluble in crude oil quintessence, hexane, and methylbenzene ; luteins dissolve better in methyl alcohol and ethyl alcohol. Consequently, the proper choice of extraction method and pre-concentration agent are particularly required to obtain dependable consequence.

The presence of conjugated double-bond system which forms the light-absorbing chromophore gives carotenoids their intensely colour and provides the seeable soaking up spectrum. This characteristic is of import for their designation and quantification, where alterations or loss of colour during analysis may bespeak debasement or structural alteration of carotenoids. Indeed, the first diagnostic tool for the designation of carotenoids is the UV and seeable spectrum. The greater the figure of conjugated dual bonds, the higher the I»max values.

The extremely unsaturated carotenoids are susceptible to the isomerisation and oxidization peculiarly in the presence of heat, visible radiation, acids, hint of metal ions and handiness of O. Isomerization of usual constellation of carotenoids, which shift from trans-cis signifier, may hold consequence on the overall form of the carotenoids molecules and therefore its belongingss like loss of colour or provitamin A activity. On the other manus, extended losingss of carotenoids may originate from oxidization. Therefore, concern must be undertaken during carotenoids analysis. For illustration, carotenoids analysis carried out under vacuity and a N or Ar atmosphere can help to except O. Meanwhile, antioxidants ( e.g. butylated hydroxytoluene, BHT ) can besides be added to minimise oxidization particularly during drawn-out storage. Besides, carotenoids analysis must besides be done in hushed visible radiation. For illustration, vass incorporating carotenoids should be wrapped with aluminium foil to forestall from direct sunshine exposure. Due to thermolability of carotenoids, its infusions or solution should besides be concentrated in a rotary evaporator at decreased force per unit area and at temperature below 40oC and the dissolver used for extraction should be low boiling point. Yet, it is of import to forestall the infusions from complete waterlessness in rotary evaporator as it may do debasement of carotenoids particularly lycopene ( Felti et al. , 2005 ; Rodriguez-Amaya, 2001 ; van den Berg et al. , 2000 ) .

2.2.2 Health benefits of carotenoids

Human must obtain carotenoids from the dietetic beginnings since they can non synthesise it. Normally, workss serve as the primary beginnings of carotenoids to human ( Ishida & A ; Chapman, 2009 ) . These compounds are indispensable to the human wellness attributed to its physiological and biological maps. The indispensable function of carotenoids particularly i??-carotene as provitamin A has been best documented for many old ages ( Ishida & A ; Chapman, 2009 ; Holden et al. , 1999 ) . With its possible provitamin A activity in human diet, they are well-known to be capable of forestalling xeropthlamia, a dark sightlessness disease ( Takahashi et al. , 2006 ) . In add-on, epidemiological surveies have besides shown an consumption of carotenoids ( xanthophyll and zeaxanthin ) correlated with reduced hazard of cataract and age-related macular devolution ( AMD ) ( Basu et al. , 2001 ) .

Furthermore, carotenoids in peculiar lycopene and i??-carotene have besides been claimed as exposing anticancer activities. For illustration, overview of experimental epidemiologic surveies has shown that carotenoids are associated with decreased hazard of malignant neoplastic disease chiefly lung and tummy malignant neoplastic disease ( van Poppel & A ; Goldbohm, 1995 ) . In the instance of cardiovascular diseases ( CVD ) hazard, several ecologic, cross-sectional, and cohort surveies suggested a protective association between carotenoids ( i??-carotene ) and cardiovascular disease and its hazard index ( Kohlrneier & A ; Hastings, 1995 ) . Besides, some research has shown the efficiency of carotenoids ( lycopene and i??-carotene ) in tegument protection by supplying protection against ultraviolet radiation sunshine ( Sies & A ; Stahl, 2004 ) every bit good as holding immunomodulatory effects such as the decrease in UV-induced immunosuppression ( Fuller et. , 1992 ) .

In fact, protective effects of carotenoids against certain diseases stated above are associated to its antioxidant belongingss, which are capable of demobilizing reactive O species ( ROS ) , thereby assist hold or prevent oxidative harm ( O’Connell et al. , 2007 ) . In add-on, carotenoids besides act as free extremist scavenger ( Zanfini et al. , 2009 ) . Free extremist can do harm to the construction and map of cell membranes, DNA, and protein ( Ishida & A ; Chapman, 2009 ) .

Owing to functional chemical belongingss of carotenoids, they are progressively used in nutrients, pharmaceutical and cosmetics industries. Harmonizing to Lyn Patrick ( 2006 ) , more than 600 carotenoids have been isolated in nature. However, merely a little figure of carotenoids are used commercially, including i??-carotene, lycopene, astaxanthin, canthaxanthin, and xanthophyll ( Del Campo et al. , 2000 ) . They play critical function as natural nutrient colorants ( e.g orange juice ) , nutrient additives, and provender additives in agribusiness ( domestic fowl, fish ) ( Spolaore et al. , 2006 ) . For illustration, astaxanthin is best known for making pinkish ruddy colour in flesh of salmonids, runt, lobsters, and crayfish ( Jin & A ; Melis, 2003 ) . Annual worldwide gross revenues of this peculiar compound are estimated at US $ 200 million with its market value of US $ 2500/kg ( Hejazi & A ; Wijffels, 2004 ; Spolaore et al. , 2006 ) .

Until the early 1980s, commercial production of i??-carotene was all man-made signifier. During the 1970s, natural i??-carotene derived from microalgae ( i.e. Dunaliella ) has been discovered by research workers and this find is presently a significant and turning industry ( Richmond, 2004 ) .

2.3. Fatty acid

The construction of fatty acid comprised of hydrocarbon concatenation ( C and H atoms ) with a carboxyl group ( COOH ) at one terminal and a methyl group ( CH3 ) at the other terminal ( Figure 3 ) . Normally, fatty acid concatenation contains even figure of Cs and lengths vary from 2 to 80 with either presence or absence of dual bonds.

Figure 3: Structure of concentrated fatty acid

( Adapted from hypertext transfer protocol: // )

Fatty acids without dual bonds are known as concentrated fatty acid while fatty acids incorporating dual bonds are known as unsaturated fatty acids. If there is merely one dual bond nowadays in an unsaturated fatso acid, it is said to be a monounsaturated fatty acid ( MUFA ) while if there is two or more dual bonds present in an unsaturated fatso acid, it is called as polyunsaturated fatty acid ( PUFA ) . Unsaturated fatty acids are named by placing the figure of dual bonds and the place of the first dual bond counted from the methyl end point. For illustration, 18-carbon fatty acid with two dual bonds in the acyl concatenation and with the first dual bond on C figure six from the methyl end point is notated as 18:2, n-6. The common name of this fatty acid is linoleic acid ( Lunn & A ; Theobald, 2006 ) .

In general, fatty acids are critical for energy storage as triacylglycerol ( 3 fatty acids attached to a glycerin anchor ) , for cell membrane formation with a phospholipids bilayer ( 2 fatty acids attached to a phospholipid polar caput group ) , and for the formation of cholesteryl esters ( one fatso acid attached to liberate cholesterin ) . In human plasma, the most abundant fatty acids are the concentrated fatty acids: palmitic acid ( 16:0 ) and stearic acid ( 18:0 ) , the monounsaturated fatty acid: oleic acid ( 18:1n-9 ) , and the polyunsaturated fatty acid: linoleic acid ( 18:2n- 6 ) ( Johnson & A ; Schaefer, 2006 ) .

2.3.1 Physiochemical belongingss of fatty acids

Double bonds in unsaturated fatty acids can be arranged in one of two ways with either cis- or trans- coni¬?gurations. Double bonds with cis- constellation are largely found in nutrients, where both H atoms are found on the same side of the fatty acid. This fatty acid exists as liquid at room temperature since the presence of a cis- bond in a fatty acid lowers the runing point of the fatty acid. On the other manus, trans-fatty acids, where the H atoms are situated on opposite sides of the fatty acid, are less common in nature. Normally, they are produced during the hydrogenation ( indurating ) of unsaturated oils as was traditionally used in margarine industry ( Lunn & A ; Theobald, 2006 ) .

The susceptible of different fats to oxidative rancidity is besides varies, depending upon the grade of unsaturation of the constituent of fatty acids besides the handiness of antioxidants, and the presence of passage metal such as Fe and Cu. In other words, the greater the figure of dual bond in fatty acid, the more prone it is to peroxidation. Therefore, PUFA are more susceptible to oxidization as compared to MUFA ( Allen & A ; Hamilton, 1994 ) .

On the other manus, the physical belongingss of fatty acids such as runing point, H2O solubility, viscousness, and refractile index are besides depending on the length concatenation of fatty acids every bit good as its grade of unsaturation. Melting point of fatty acids will increase with C figure while the presence of dual bond in the fatty acids ( unsaturated fatty acids ) will hold lower runing point as compared to saturated fatty acids. Then, an addition in the concatenation length and grade of unsaturation will diminish the H2O solubility of fatty acids, yet increasing its refractile index. Unlike other physical belongingss, viscousnesss of fatty acid will increase with the concatenation length, but lessening with an increasing grade of unsaturation ( Allen & A ; Hamilton, 1994 ) .

2.3.2 Health benefits of PUFA

PUFA are ever claimed to be most good to human wellness among the assorted type of fatty acids. The prevailing PUFA are the omega-3 and omega-6 fatty acid. In human organic structure, there are two PUFA viz. omega-6 linoleic acid ( 18:2, n-6 ) and omega-3 I±-linolenic acid ( 18:3, n-3 ) can non be synthesized because they lack the desaturase enzymes necessary to synthesise these constituents. As a consequence, they must be obtained from works stuff in the diet and are known as indispensable fatty acids ( Tapiero et al. , 2002 ) . Once consumed, the organic structure can metabolise them by the debut of farther dual bonds ( desaturation ) and by lengthening the acyl concatenation ( elongation ) to organize other PUFAs such as arachidonic acid ( AA, 20:4, n-6 ) , eicosapentaenoic acid ( EPA ; 20:5, n-3 ) and docosahexaenoic acid ( DHA ; 22:6, n-3 ) that are more readily used in the organic structure ( Ng, 2006 ) . The tract of its transition is shown in Figure 4 ( Calder, 2004 ) .

Figure 4: Nerve pathway of the transition of linoleic and I±-linolenic acids into their longer concatenation derived functions

PUFA peculiarly n-3 fatty acids ( EPA and DHA ) , which supplied largely from fish or fish oil are believed to hold good consequence in bar and intervention of a broad scope of human diseases and upsets although their implicit in mechanism of action are still ill-defined ( Zittelli et al. , 1999 ; GISSI-Prevenzione Research workers, 1999 ) . They are considered as polar constituent in the human diet ( Napier & A ; Sayanova, 2005 ) .

Harmonizing to Ward & A ; Singh ( 2005 ) , wellness benei¬?ts of PUFAs has began pulling attending from research workers when it was noted that populations with high ingestion of fish had a much lower incidence of bosom disease. Indeed, there is significant grounds from epidemiological and instance control surveies shown the protective consequence of n-3 fatty acids in cut downing the hazard of cardiovascular mortality such as myocardial infarction ( Calder, 2004 ) . A survey besides suggested that ingestion of i¬?sh ( good beginning of n-3 fatty acids ) at least one time per hebdomad may cut down the hazard of sudden cardiac decease in work forces ( Albert et al. , 1998 ) . As information, high MUFA consumption has besides been shown associated with decreased cardiovascular disease hazard ( Kris-Etherton, 1999 ) .

Apart from that, the n -3 PUFAs such as EPA and DHA have besides been suggested to cut down hazard for metabolic syndrome, which is presently represent major public wellness job in industrialised societies by take downing plasma triglycerides, cut downing high blood pressure, better lipoprotein proi¬?le and insulin sensitiveness ( Graham et al. , 2004 ) . Metabolic syndrome is described as ‘clustering ‘ of several hazard factors for cardiovascular disease including fleshiness ( peculiarly abdominal fleshiness ) , dyslipidaemia, insulin opposition and high blood pressure. ( Nugent, 2004 ) .

There is besides increasing grounds from surveies shown that n -3 fatso acids play a cardinal function in encephalon and retina development in preterm babies and besides for the bar or detaining the procedure of dementedness and age-related macular devolution ( Dangour & A ; Uauy, 2008 ; Johnson & A ; Schaefer, 2006 ; Hoffman et al, 1993 ) . Furthermore, n-3 fatty acids are besides of import constituent of cell membrane phospholipids from some tissues like retina, encephalon, and myocardium ( Surette, 2008 ) . Membranes incorporating high proportions of the long concatenation PUFAs can guarantee efficient intracellular metamorphosis as they are most permeable to H2O ( Lunn & A ; Theobald, 2006 ) .

Recent surveies have revealed the importance of omega-6/omega-3 fatty acids ratio, whereby a really high omega-6/omega-3 ratio ( inordinate sums of omega-6 fatty acids ) as is found in today ‘s Western diets, promote the pathogenesis of many diseases, including cardiovascular disease, malignant neoplastic disease, and inflammatory and autoimmune diseases. On the contrary, lower omega-6/ omega-3 ratio ( increased degrees of omega-3 fatty acids ) exert suppressive effects. This implies the importance of little ratio of omega-6/omega-3 fatty acids in cut downing the hazard of many of the chronic diseases ( Simpoulos, 2001 ) .

Although there is increasing consciousness of general public sing health-benei¬?cial belongingss of dietetic ingestion of n-3 PUFA such as EPA and DHA, the natural resources that provide these oils are in danger of being exhausted and may inadequate for providing the spread outing market to run into its high demand ( Opsahl-Ferstad et Al. 2003 ; Sijtsma & A ; de Swaaf, 2004 ) . Recently, microalgae has been exploited and studied to be the alternate procedures for PUFA-production ( Sijtsma and de Swaaf, 2004 ) . However, DHA is the lone current algal PUFA commercially available ( Spolaore et al. , 2006 ) .

2.4. Mechanism of extraction

Extraction is the procedure by which analytes from sample matrix are selectively separated from other, frequently unsought compounds. Extraction of analytes from sample matrix is an of import measure prior the HPLC or GC analysis. One of the most common extraction methods is two-phase system for liquid-liquid extraction ( besides called biphasic aqueous/organic system ) , which depend on differential solvent solubility and solvent immiscibility. Virtually, the usage of two- stage aqueous organic systems has been proved to be an effectual manner to divide the ailing water-soluble compounds ( Leon et al. , 2003 ) .

A polar, aqueous solutions are frequently paired with non-polar organic dissolvers such as trichloromethane which is volatile, non reactive, non-miscible with and denser than H2O to organize a two-phase system for liquid-liquid extraction as shown in Figure 5. This allows the separation of analytes in either polar or non-polar dissolvers harmonizing to its comparative solubility, and for subsequent chromatographic separation and analysis ( Sana & A ; Fischer, 2007 ) .


Figure 5: Mechanism of two-phase system for liquid-liquid extraction

( Adapted from Sana & A ; Fischer, 2007 )

Basically, the sampled stuff matrix plays an of import function in extraction procedure as analyte may be strongly bound to the matrix, which affects the extraction efficiency ( Anklam & A ; von Holst, 2005 ) . In whole-cell systems, organic dissolver might be incorporated within membrane lipoids doing the break or change of membrane maps, inactivation or denaturation of membrane edge enzymes, prostration of conveyance mechanisms, and even cell lysis when a cell is exposed to biocompatible dissolver ( Cruz et al. , 2001 ) . In general, the cells will transport out the bioconversion when a biocompatible organic dissolver is in contact with the aqueous stage, where the biochemical compounds or analytes are continuously extracted into the organic stage due to a permeableness consequence of the dissolver on cell membrane ( Xu et al. , 2004 ) .

On the other manus, utilizing assorted dissolver has besides gained the involvement of research workers. Indeed, the usage of assorted dissolver compared to individual dissolver will increase the permeableness consequence of dissolvers and concentration of the dissolver accumulated in the cell membrane. Mojaat et Al. ( 2008 ) reported that the mixture dichloromethane-decane is able to pull out i??-carotene six times more expeditiously than the pure decane. Consequently, the choice of the dissolver is peculiar of import in order to guarantee extraction efficiency, where it must fulfill certain demands such as biocompatibility, maximal solubility and of import extraction ability.

2.4.1 Fatty acids extraction in microalgae

Fatty acids exist in a great assortment of molecules which differ in chemical and physical features poses a challenge with regard to the application of optimum extraction methods for quantitative extraction of the fatty acids from different matrices ( Jensen, 2008 ) . Indeed, different methods can really take to a treble difference in lipid contents ( Smedes, 1999 ) .

In malice of many extraction methods developed late including supercritical or subcritical i¬‚uid extraction, microwave and ultrasound assisted extraction, the engagement of organic dissolver in lipid extraction from microalgae including chloroform/methanol mixture, was still found superior in comparing to these techniques ( Mendes et al. , 2006 ) . The extraction outputs of lipids/fatty acids utilizing dissolvers dependent upon the nature of the microbic cell constructions, the sample-solvent ratio, temperature, clip of extraction and the extraction techniques used ( Chaiklahana et al. , 2008 ) .

In literature, Bligh & A ; Dyer method and Folch method are the one most common used to find the fatty acids in assorted nutrient beginnings including microalgae, where mixtures of trichloromethane and methyl alcohol were used to pull out lipids/fatty acids. ( Lewis, Nichols, & A ; McMeekin, 2000 ; Molina Grima et al. , 1994 ; Wiltshire et Al, 2000 ) . Other dissolvers may include hexane, ethyl alcohol, diethyl quintessence, methyl alcohol, propanone, crude oil quintessence, and so forth.

In fact, solvent extraction methods have been evaluated for the GC analysis of fatty acids/lipids from different nutrient merchandises ( Sahasrabudhe & A ; Smallbone, 1983 ; Somashekar et al. , 2001 ) , but it does non include marine microalgae. Many surveies sing the rating of extraction methods for fatty acids analysis from microalgae are focused on the different extraction techniques ( e.g. direct saponification, microwave-assistant extraction, supercritical fluid extraction, and others ) alternatively of rating of solvent extraction methods ( Burja et al. , 2007 ; Grima, E.M. , 1994 ) .

2.4.2 Carotenoids extraction

During carotenoids analysis of nutrients, important mistakes are typically originating from extraction processs and the mistake associated with chromatography is merely minor. However, it is hard to standardise the assortment of published methods available between research labs ( Rodriguez-Amaya et al. , 2006 ) .

In literature, the most widely accepted methods involve extraction of carotenoids with one or mixture organic dissolvers including hexanes, trichloromethane, methylene chloride, tetrahydrofuran, methyl alcohol, ethyl alcohol, ethyl ethanoate, n-butyl-alcohol, diethyl quintessence, 2-propanol, and crude oil ether. Many extraction processs require lyophilized stuff, involve saponification to take lipoids and chlorophylls and antioxidants use ( e.g. , butylated hydroxytoluene ( BHT ) or pyrogallic acid ) ( Howe & A ; Tanumihardjo, 2006 ) . Although legion extraction processs of carotenoids from nutrients involved assorted types of dissolver every bit good as solvent combinations have been proposed antecedently, the comparative efficaciousnesss of these bing methods have non yet been evaluated, particularly on microalgae ( Taungbodhitham et al. , 1998 ) . In any instance, proper choice of the dissolver for a peculiar carotenoid extraction is of chiefly importance ( Feltl et al. , 2005 ) .

2.5. High Performance Liquid Chromatography ( HPLC )

2.5.1 Principle

The implicit in rule of chromatography is that molecules non merely fade out in liquids but can besides absorb ( dissolve ) on to or interact with the surface of solids. If a molecule dissolved in a liquid ( nomadic stage ) is passed down a column of solid atoms ( stationary stage ) with which it interacts, it will take longer clip to go through down the column as it spends some clip fade out in the nomadic stage and some on the stationary stage. In contrast, to those molecules that do non interact with stationary stage, they will elute foremost from the column. The separation of molecules by chromatography varies, depending upon physical belongingss of the molecules in samples such as solubility in H2O, solubility in organic dissolvers, net positive or negative charge, and size. In amount, HPLC is a physical method of the separation of one type of molecule from others and therefore can be used to place and at the same time quantify many constituents of a complex mixture ( Bird, 1989 ) .

2.5.2 Application of reverse-phase HPLC ( RP-HPLC )

Among all different separation manners, RP-HPLC is the most widely used in modern column liquid chromatography ( more than 70 % ) which utilizes a non-polar stationary stage and a polar nomadic stage. In RP-HPLC, solutes are retained due to hydrophobic interactions with the non-polar stationary stage. Therefore, the higher the mutual opposition of the solutes, the more likely it is being eluted from column. Recently, RP-HPLC has become the method of pick for both qualitative and quantitative analysis of carotenoids besides finding of H2O and fat soluble vitamins, nutrient dyes, phenolic spirit compounds ( such as vanillin ) , and other pigments like chlorophyll ( Nielsen, 1998 ; Nollet, 2000 ) .

2.6. Gas chromatography ( GC )

2.6.1 Principle

GC is a powerful and widely used tool for the separation, designation and quantification of constituents in a mixture. In this technique, a sample is converted to the vapour province and a streamlined watercourse of inert bearer gas ( normally helium or H ) sweeps the sample into a thermally-controlled column. The column is normally packed with solid atoms that are coated with a non-volatile liquid, referred to as the stationary stage. As the sample mixture moves through the column, sample constituents that interact strongly with the stationary stage will pass more clip in the stationary stage than the traveling bearer gas. Consequently, more clip is required for sample constituents to travel through the column ( Truman State University CHEM 222 Lab Manual, 2008 ) .

2.6.2 Application of GC

GC is used loosely in assorted analyses, including finding of fatty acids, triglycerides, cholesterin, and other steroid alcohols, gases, solvent analysis, H2O, intoxicants, and simple sugars, every bit good as oligosaccharides, aminic acids, vitamins, nutrient additives, antioxidants, and so forth. However, GC is ideally suited merely to the analysis of thermally stable volatile substances. Harmonizing to Seppanen-Laakso et Al. ( 2002 ) , GC is a extremely applicable tool in micro-scale analytical work in a figure of research countries of fatty acids. Normally, GC requires an esterified sample of fatty acids such as the methyl ester ( FAME ) , and this addition the volatility lipids constituents, therefore supplying better separation ( Tran et al. , 2009 ) .

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