**3. Analysis of enriched fatty acid in marine algae**

The reagents commonly used for acid-catalyzed transesterification are methanolic, hydrochloric and sulfuric acid, and boron trifluoride in methanol. All of them are suitable for lipid transesterification and also free-fatty-acid methylation. However, at ambient temperature neither acid-catalyzed nor boron-fluoridecatalyzed reactions proceed; in both cases the reaction requires heating. Among the mentioned reagents, boron trifluoride-methanol reagent (12–14% w/v) is the

most often used for transesterification of all types of lipids and it is being the best and very useful reagent for lipid esterification. Under the conditions recommended (heating at 100°C), transesterification is complete within 2 min for free fatty acids, within 10 min for phosphoglycerides, within 30 min for triglycerides and within 90 min for sphingomyelin. For biological samples boron fluoride-methanol reagent is used for transesterification of lipids following the procedure given below: into a screw-capped tube (Teflon cap liner) a small aliquot of the lipid extract (dissolved in chloroform) is added; to it add 0.5–1 ml boron fluoride-methanol reagent (140 g/l, containing an required amount of BHT as antioxidant); the tube is then closed and heated at 90°C for 2 h. This methodology proved to be administered complete transesterification of all lipids; the addition of BHT can prevent rancidity of PUFA.

After the transesterification reaction completed, FAMEs are extracted by adding n-hexane and water twice to the proportion of sample. This is the most simple and effective procedure. Even though, this method is most popular, boron tri-fluoridemethanol has few disadvantages. Unless refrigerated, the reagent has only a limited shelf life [17]. The use of recent or too focused solutions might lead to the assembly of artifacts or loss of PUFAs.

If the sample contains plasmalogens, aldehydes area unit liberated by the chemical agent and area unit regenerate into dimethyl acetals (DMAs), that area unit nearly not possible to break free the major carboxylic acid methyl esters including methyl palmitate. There is wide usage of anhydrous methanolic hydrochloric acid and methanolic sulfuric acid for lipid esterification varying under different conditions, in particular, different reaction temperatures, different acid concentrations, and different reaction times. The methanolic acid boron fluoride-methanol works as methylating free fatty acids very rapidly so can be used to transesterify all the lipids which are typically present in the biological samples. With a concentration of 5% methanolic hydrochloric acid complete transesterification can be carried out by heating the sample in the reagent for about 2 h under refluxion. This reaction can also be carried out at 50°C overnight. In the same way a solution of 1–2% (v/v) concentrated sulfuric acid in methanol can be used for the transesterification of lipid samples. This method using methanolic, hydrochloric and sulfuric acids also have the disadvantage like boron trifluoride-methanol that DMAs are formed during transesterification from plasmalogens. Acetyl chloride and aluminum chloride are the other reagents used for transesterification. Both these reagents shown complete transesterification in the samples without prior extraction of the lipid, whereas, aluminum chloride has the disadvantage that it does not esterify free fatty acids.

#### **4. PUFA as phospholipids fractions**

Phospholipids are major constituents of cell membranes and play essential roles in biochemistry and physiology of the cell functions. Phospholipids in fish and marine species are highly enriched with the long-chain n-3 type polyunsaturated fatty acids. About 40–50% content of EPA and DHA is not uncommon in some phospholipid classes in fish. The role of n-3 polyunsaturated fatty acids in phospholipid moiety is in adjusting the membrane integrity and functions presumably at lower temperature, and also to the membrane fluidity and mobility as a result of their higher unsaturation. In the case of fish among the phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are by far the most abundant in the flesh, especially PC make up to 50–60% of the total phospholipid content [18]. The composition of individual phospholipid classes is remarkably similar among fish species as is the characteristic fatty acid composition of each

**151**

*Bioconcentration of Marine Algae Using Lipase Enzyme DOI: http://dx.doi.org/10.5772/intechopen.87026*

fraction, 58 and 69%, respectively.

**5. Application of PUFA**

class. Lecithins present in plant and vegetable origin are popularly using as health supplements. The vegetable oil is highly enriched with n-6 fatty acids, which so as in the case of the n-3 fatty acid fish oils. On the other hand, purified fish lecithins, which are highly enriched with n-3 polyunsaturated fatty acids phospholipids, are not available on the market at all. This is because tedious extraction procedure is required for obtaining lecithin from fish oil unlike plant or vegetable lecithins. Here, certain attempts for the preparation of such phospholipids, highly enriched with EPA and DHA, from the more readily available plant or animal lecithins is explained. Pure phosphatidylcholine can be obtained from egg yolk after purification by preparative HPLC and was treated under the acidolysis reaction using the *Mucor miehei* lipase. There is observed reaction as anticipated in which the rate of the reaction involving the phospholipids that possess the zwitterionic head group. This is much lower as compared to the natural triacylglycerol substrates. Therefore, large quantities of lipase were required, which will resulted in high extent of hydrolysis side reaction. The optimal reaction conditions is offered in a mixture of phospholipids of approximately 40% desired for phosphatidylcholine and lysophosphatidylcholine (LPC) whereas, 20% of glycerol phosphatidylcholine (GPC). In LPC only one of the acyl moiety will be hydrolyzed and in glycerol phosphatidylcholine both acyl groups will get hydrolyzed. When pure EPA was used, both the PC and the LPC fractions were highly enriched with EPA, particularly the LPC

PUFA had many helpful effects for human health so it considered as unit important elements in human nutrition. The intake of PUFA in diet, together with n-6 fatty acids, is understood to modulate the inflammatory processes among different cell functions. Although many of the species exhibited high amounts of SFA, some *Phaeophyta* and *Rhodophyta* species show higher concentrations of PUFA, and PUFA/SFA ratios higher than 1 (*H. scoparia*, 1.46; *T. atomaria*, 1.33; *C. spongiosus*, 1.77; *Peyssonnelia* sp., 1.33). Whereas, the lowest ratios were discovered in algae from the phylum Chlorophyta (0.27–0.68) [19]. It seems that this phylum incorporates a lower potential, examination to the opposite two phyla studied, as a nutritional source of PUFA for human consumption. However, not all PUFA are associated with the promotion of health benefits. For example, in the inflammation process, eicosanoids derived from n-6 PUFA are generally considered as pro-inflammatory or as promoters of other cell harmful effects, whereas n-3 PUFA derivatives are considered less inflammatory or even anti-inflammatory [20].

Since the synthesis pathway of those fatty acids depends on identical enzymes

The World Health Organization (WHO) recommends a ∑n-6/∑n-3 magnitude relation not up to 10. Almost all algae can be considered as a good source of dietary PUFA, since they showed ratios ranging between 0.29 and 6.73 [21]. The exception was *Chaetomorpha* sp., during which the ∑n-6/∑n-3 magnitude relation was the best from all the studied species (31.25) and in *D. spiralis* during which no n-3 fatty acids were detected. Besides associate degree applicable nutritionary profile, these

Many of the PUFA thought-about powerful molecules against many diseases and area unit already employed in totally different medical specialty applications. For example, several reports suggest that n-3 fatty acids, mainly EPA and DHA, may have a significant potential in the treatment of autoimmune and inflammatory

for n-3 and n-6 PUFA, the health promoting effects area unit keen about the

n-6/n-3 magnitude relation of PUFA obtained through diet.

macroalgae can also be exploited for pharmaceutical purposes.

*Bioconcentration of Marine Algae Using Lipase Enzyme DOI: http://dx.doi.org/10.5772/intechopen.87026*

*Microalgae - From Physiology to Application*

of PUFA.

of artifacts or loss of PUFAs.

**4. PUFA as phospholipids fractions**

most often used for transesterification of all types of lipids and it is being the best and very useful reagent for lipid esterification. Under the conditions recommended (heating at 100°C), transesterification is complete within 2 min for free fatty acids, within 10 min for phosphoglycerides, within 30 min for triglycerides and within 90 min for sphingomyelin. For biological samples boron fluoride-methanol reagent is used for transesterification of lipids following the procedure given below: into a screw-capped tube (Teflon cap liner) a small aliquot of the lipid extract (dissolved in chloroform) is added; to it add 0.5–1 ml boron fluoride-methanol reagent (140 g/l, containing an required amount of BHT as antioxidant); the tube is then closed and heated at 90°C for 2 h. This methodology proved to be administered complete transesterification of all lipids; the addition of BHT can prevent rancidity

After the transesterification reaction completed, FAMEs are extracted by adding n-hexane and water twice to the proportion of sample. This is the most simple and effective procedure. Even though, this method is most popular, boron tri-fluoridemethanol has few disadvantages. Unless refrigerated, the reagent has only a limited shelf life [17]. The use of recent or too focused solutions might lead to the assembly

If the sample contains plasmalogens, aldehydes area unit liberated by the chemi-

cal agent and area unit regenerate into dimethyl acetals (DMAs), that area unit nearly not possible to break free the major carboxylic acid methyl esters including methyl palmitate. There is wide usage of anhydrous methanolic hydrochloric acid and methanolic sulfuric acid for lipid esterification varying under different conditions, in particular, different reaction temperatures, different acid concentrations, and different reaction times. The methanolic acid boron fluoride-methanol works as methylating free fatty acids very rapidly so can be used to transesterify all the lipids which are typically present in the biological samples. With a concentration of 5% methanolic hydrochloric acid complete transesterification can be carried out by heating the sample in the reagent for about 2 h under refluxion. This reaction can also be carried out at 50°C overnight. In the same way a solution of 1–2% (v/v) concentrated sulfuric acid in methanol can be used for the transesterification of lipid samples. This method using methanolic, hydrochloric and sulfuric acids also have the disadvantage like boron trifluoride-methanol that DMAs are formed during transesterification from plasmalogens. Acetyl chloride and aluminum chloride are the other reagents used for transesterification. Both these reagents shown complete transesterification in the samples without prior extraction of the lipid, whereas, aluminum chloride has the disadvantage that it does not esterify free fatty acids.

Phospholipids are major constituents of cell membranes and play essential roles in biochemistry and physiology of the cell functions. Phospholipids in fish and marine species are highly enriched with the long-chain n-3 type polyunsaturated fatty acids. About 40–50% content of EPA and DHA is not uncommon in some phospholipid classes in fish. The role of n-3 polyunsaturated fatty acids in phospholipid moiety is in adjusting the membrane integrity and functions presumably at lower temperature, and also to the membrane fluidity and mobility as a result of their higher unsaturation. In the case of fish among the phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are by far the most abundant in the flesh, especially PC make up to 50–60% of the total phospholipid content [18]. The composition of individual phospholipid classes is remarkably similar among fish species as is the characteristic fatty acid composition of each

**150**

class. Lecithins present in plant and vegetable origin are popularly using as health supplements. The vegetable oil is highly enriched with n-6 fatty acids, which so as in the case of the n-3 fatty acid fish oils. On the other hand, purified fish lecithins, which are highly enriched with n-3 polyunsaturated fatty acids phospholipids, are not available on the market at all. This is because tedious extraction procedure is required for obtaining lecithin from fish oil unlike plant or vegetable lecithins. Here, certain attempts for the preparation of such phospholipids, highly enriched with EPA and DHA, from the more readily available plant or animal lecithins is explained. Pure phosphatidylcholine can be obtained from egg yolk after purification by preparative HPLC and was treated under the acidolysis reaction using the *Mucor miehei* lipase. There is observed reaction as anticipated in which the rate of the reaction involving the phospholipids that possess the zwitterionic head group. This is much lower as compared to the natural triacylglycerol substrates. Therefore, large quantities of lipase were required, which will resulted in high extent of hydrolysis side reaction. The optimal reaction conditions is offered in a mixture of phospholipids of approximately 40% desired for phosphatidylcholine and lysophosphatidylcholine (LPC) whereas, 20% of glycerol phosphatidylcholine (GPC). In LPC only one of the acyl moiety will be hydrolyzed and in glycerol phosphatidylcholine both acyl groups will get hydrolyzed. When pure EPA was used, both the PC and the LPC fractions were highly enriched with EPA, particularly the LPC fraction, 58 and 69%, respectively.
