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Enzymatic synthesis of lipids containing omega-3 fatty acids

Lyberg, Ann-Marie LU (2007)
Abstract (Swedish)
Popular Abstract in Swedish

Dokosahexaensyra (DHA) och eikosapentaensyra (EPA) är omega-3 fettsyror, som har hälsofrämjande effekter. De behöver skyddas, DHA i synnerhet, eftersom de är mycket känsliga för oxidation. Flera lipider som innehöll DHA utvärderades med avseende på lipidernas möjlighet att skydda DHA mot peroxidation genom att använda en HPLC metod som utvecklades som en del av detta arbete. Dessutom undersöktes hur fri DHA kunde skyddas i ett liposomsystem innehållande antioxidanter. Fosfolipiderna skyddade mot peroxidation, när DHA var inkorporerad i den ena positionen av antingen fosfatidylkolin eller fosfatidyletanolamin. Dessutom gav kombinationen alfa-tokoferol och askorbinsyra det bästa skyddet för fri DHA... (More)
Popular Abstract in Swedish

Dokosahexaensyra (DHA) och eikosapentaensyra (EPA) är omega-3 fettsyror, som har hälsofrämjande effekter. De behöver skyddas, DHA i synnerhet, eftersom de är mycket känsliga för oxidation. Flera lipider som innehöll DHA utvärderades med avseende på lipidernas möjlighet att skydda DHA mot peroxidation genom att använda en HPLC metod som utvecklades som en del av detta arbete. Dessutom undersöktes hur fri DHA kunde skyddas i ett liposomsystem innehållande antioxidanter. Fosfolipiderna skyddade mot peroxidation, när DHA var inkorporerad i den ena positionen av antingen fosfatidylkolin eller fosfatidyletanolamin. Dessutom gav kombinationen alfa-tokoferol och askorbinsyra det bästa skyddet för fri DHA i ett liposomsystem innehållande fosfatidylkolin.



Enzymatiska metoder utvecklades för att inkorporera antingen DHA eller EPA i position sn-1 av 2-acyl-lysofosfatidylkolin. Eftersom de använda enzymerna diskriminerade dessa fettsyror och DHA i synnerhet, utfördes ett förförsök för att finna ett lämpligt enzym. Bland de undersökta lipaserna gav de från Candida antarctica och Rhizopus arrhizus bäst prestanda med avseende på inkorporering av DHA respektive EPA. Inkorporeringen av dessa fettsyror kunde ökas genom att utföra reaktionerna vid vattenaktiviteten 0.22, vilket medförde att det förutom esterifikationen förekom viss hydrolys. Eftersom båda reaktionerna diskriminerade DHA och EPA, ökade innehållet av dessa fettsyror med reaktionstiden i den fosfatidylkolin som producerades.



Lipasernas förmåga att diskriminera DHA och EPA användes för att anrika dessa fettsyror i fiskolja och bläckfiskolja. Dessutom utvärderades ?konkurrensfaktorn? för både EPA och DHA i både marina oljor och en blandning av metylestrar. När hydrolys av alla substaten och etanolys av bläckfiskolja utfördes, reagerade framför allt andra fettsyror medan DHA och/eller EPA var kvar i substratet. Den uppnådda konkurrensfaktorn för DHA och EPA påverkades av flera egenskaper hos lipaset såsom fettsyraspecificitet, regiospecificitet, stereospecificitet och triglyceridspecificitet. Alltså påverkades konkurrensfaktorn av substratets struktur såsom metylester eller triglycerid. Placeringen av DHA i glyceridstrukturen påverkade också konkurrensfaktorn. Desutom påverkades konkurrensfaktorn av medsubstratet som i detta fallet var vatten eller etanol. När lipaser från Thermomyces lanuginosus, Pseudomonas cepacia och Pseudomonas fluorescens katalyserade etanolys, bildades inga DHA-etylestrar initialt. Inte heller EPA-etylestrar bildades i början av etanolysen, då reaktionen katalyserades av lipaserna från Pseudomonas. Detta kan förklaras med att dessa lipaser inte kunde etanolysera DHA som var bundet till en triglycerid och att lipaserna från Pseudomonas inte kunde etanolysera EPA bundet till en triglycerid. Etanolys gav högre konkurrensfaktorer än hydrolys för både EPA och DHA för fyra av de fem utvärderade lipaserna. Den högsta konkurrensfaktorn för DHA erhölls av lipaset från Thermomyces lanuginosus. Detta lipas betraktas som det bästa lipaset för DHA-anrikning på grund av dess höga DHA-utbyte tillsammans med hög DHA-anrikning. (Less)
Abstract
Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are omega-3 fatty acids, which have health promoting effects. Since they are highly prone to oxidation, they should be protected, in particular DHA. Several lipids containing DHA were evaluated with respect to the ability of the lipids to protect DHA against peroxidation using an HPLC method developed as part of this research. Furthermore, the protection of free DHA in a liposome system containing antioxidants was investigated. The phospholipids offered protection against peroxidation when DHA was incorporated at one position of either phosphatidylcholine or phosphatidylethanolamine. Furthermore, alfa-tocopherol combined with ascorbic acid offered the best protection to free DHA in... (More)
Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are omega-3 fatty acids, which have health promoting effects. Since they are highly prone to oxidation, they should be protected, in particular DHA. Several lipids containing DHA were evaluated with respect to the ability of the lipids to protect DHA against peroxidation using an HPLC method developed as part of this research. Furthermore, the protection of free DHA in a liposome system containing antioxidants was investigated. The phospholipids offered protection against peroxidation when DHA was incorporated at one position of either phosphatidylcholine or phosphatidylethanolamine. Furthermore, alfa-tocopherol combined with ascorbic acid offered the best protection to free DHA in a liposome system containing phosphatidylcholine.



Enzymatic methods were developed to incorporate either DHA or EPA at the sn-1 position of 2-acyl-lysophosphatidylcholine. Since the enzymes utilised discriminated against these fatty acids, in particular DHA, screening for the appropriate enzyme was performed. Among the investigated lipases, those from Candida antarctica and Rhizopus arrhizus showed the best performance in incorporating DHA and EPA, respectively. The incorporation of these fatty acids could be increased by performing the reaction at a water activity of 0.22, which allowed some hydrolysis to occur simultaneously with the esterification. Since DHA and EPA were discriminated against in both reactions, the content of these fatty acids in the synthesised phosphatidylcholine was increased with reaction time.



The ability of the lipases to discriminate against DHA and EPA was utilised to enrich these fatty acids in fish oil and squid oil. Furthermore, the competitive factors for both EPA and DHA were evaluated in both marine oils and a mixture of methyl esters. Hydrolysis of all substrates and ethanolysis of squid oil were performed, converting mainly other fatty acids and leaving DHA and/or EPA in the substrate. The competitive factors achieved for DHA and EPA were affected by several properties of the lipase, including the fatty acid specificity, regiospecificity, stereospecificity and triglyceride specificity. Hence, the competitive factors were influenced by the structure of the substrate, i.e. methyl esters or triglyceride. In the glyceride structure of the latter, the location of DHA also affected the competitive factors. Furthermore, the co-substrate, in this case water or ethanol, influenced the competitive factors. The lipases from Thermomyces lanuginosus, Pseudomonas cepacia and Pseudomonas fluorescens initially showed no formation of ethyl esters of DHA during ethanolysis. Neither did the Pseudomonas lipases provide any EPA ethyl esters during the early stages of ethanolysis. This could be explained by these lipases not being able to perform ethanolysis of DHA located in a triglyceride and the Pseudomonas lipases not being able to perform ethanolysis of EPA located in a triglyceride. Ethanolysis gave higher competitive factors than hydrolysis for both EPA and DHA for four of the five lipases evaluated. The lipase from Thermomyces lanuginosus gave the highest competitive factors for DHA and was considered to be the superior lipase for the enrichment of DHA due to its high DHA recovery together with high DHA enrichment. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Haraldsson, Gudmundur, Faculty of Science, University of Iceland, Reykjavik, Iceland
organization
publishing date
type
Thesis
publication status
published
subject
keywords
enzymologi, Proteiner, enzymology, Proteins, Enzymes, DHA, EPA, Biotechnology, Bioteknik
publisher
Department of Biotechnology, Lund University
defense location
Lecture Hall A, at the Center for Chemistry and Chemical Engineering, Sölvegatan 39, Lund
defense date
2007-06-13 13:15
external identifiers
  • Other:ISRN: LUTKDH/TKBT--07/1107--SE
ISBN
978-91-89627-54-3
language
English
LU publication?
yes
id
44435ff1-868d-4eca-95b5-d5cb93a3b45c (old id 548778)
date added to LUP
2007-10-09 12:42:35
date last changed
2016-09-19 08:45:09
@misc{44435ff1-868d-4eca-95b5-d5cb93a3b45c,
  abstract     = {Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are omega-3 fatty acids, which have health promoting effects. Since they are highly prone to oxidation, they should be protected, in particular DHA. Several lipids containing DHA were evaluated with respect to the ability of the lipids to protect DHA against peroxidation using an HPLC method developed as part of this research. Furthermore, the protection of free DHA in a liposome system containing antioxidants was investigated. The phospholipids offered protection against peroxidation when DHA was incorporated at one position of either phosphatidylcholine or phosphatidylethanolamine. Furthermore, alfa-tocopherol combined with ascorbic acid offered the best protection to free DHA in a liposome system containing phosphatidylcholine.<br/><br>
<br/><br>
Enzymatic methods were developed to incorporate either DHA or EPA at the sn-1 position of 2-acyl-lysophosphatidylcholine. Since the enzymes utilised discriminated against these fatty acids, in particular DHA, screening for the appropriate enzyme was performed. Among the investigated lipases, those from Candida antarctica and Rhizopus arrhizus showed the best performance in incorporating DHA and EPA, respectively. The incorporation of these fatty acids could be increased by performing the reaction at a water activity of 0.22, which allowed some hydrolysis to occur simultaneously with the esterification. Since DHA and EPA were discriminated against in both reactions, the content of these fatty acids in the synthesised phosphatidylcholine was increased with reaction time.<br/><br>
<br/><br>
The ability of the lipases to discriminate against DHA and EPA was utilised to enrich these fatty acids in fish oil and squid oil. Furthermore, the competitive factors for both EPA and DHA were evaluated in both marine oils and a mixture of methyl esters. Hydrolysis of all substrates and ethanolysis of squid oil were performed, converting mainly other fatty acids and leaving DHA and/or EPA in the substrate. The competitive factors achieved for DHA and EPA were affected by several properties of the lipase, including the fatty acid specificity, regiospecificity, stereospecificity and triglyceride specificity. Hence, the competitive factors were influenced by the structure of the substrate, i.e. methyl esters or triglyceride. In the glyceride structure of the latter, the location of DHA also affected the competitive factors. Furthermore, the co-substrate, in this case water or ethanol, influenced the competitive factors. The lipases from Thermomyces lanuginosus, Pseudomonas cepacia and Pseudomonas fluorescens initially showed no formation of ethyl esters of DHA during ethanolysis. Neither did the Pseudomonas lipases provide any EPA ethyl esters during the early stages of ethanolysis. This could be explained by these lipases not being able to perform ethanolysis of DHA located in a triglyceride and the Pseudomonas lipases not being able to perform ethanolysis of EPA located in a triglyceride. Ethanolysis gave higher competitive factors than hydrolysis for both EPA and DHA for four of the five lipases evaluated. The lipase from Thermomyces lanuginosus gave the highest competitive factors for DHA and was considered to be the superior lipase for the enrichment of DHA due to its high DHA recovery together with high DHA enrichment.},
  author       = {Lyberg, Ann-Marie},
  isbn         = {978-91-89627-54-3},
  keyword      = {enzymologi,Proteiner,enzymology,Proteins,Enzymes,DHA,EPA,Biotechnology,Bioteknik},
  language     = {eng},
  publisher    = {ARRAY(0xc71cd58)},
  title        = {Enzymatic synthesis of lipids containing omega-3 fatty acids},
  year         = {2007},
}