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Master thesis of EMQAL project

Evaluation of Extraction Methods for Recovery

of Fatty Acids from Marine Products

Liping Xiao 㙪Бᑇ

Supervisor: Svein Are Mjøs, Nofima Ingredients,

Bjørn Grung, University of Bergen

February 2010

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Abstract

The extraction efficiency of Soxhlet, acid hydrolysis and Bligh and Dyer were evaluated by using direct methylation on extracts and residues for calculating the mass balance of fatty acids for eight marine powders (fishmeals, krillmeals, cod filet, salmon filet and herring roe). The results show that Soxhlet gave lowest extracted fatty acid content, especially for the samples which contain a high amount of phospholipid. Acid hydrolysis and Bligh and Dyer extract gave comparable extracted fatty acid contents with direct methylation. The mass balance of fatty acids in extract and residue is close to 100% for the three extraction methods which indicate that fatty acid was not lost during the extraction procedures. The difference of extracted fatty acids is mainly due to the different extracting efficiency. The gravimetric lipid has limited correlation with total fatty acids, especially for

Soxhlet.

Analyses of the fatty acid profiles show ed that the Soxhlet extracts were different from the others. Extracts from the acid hydrolysis and Bligh and Dyer methods had similar fatty acid profiles as the direct methylation method. The precision of fatty acid analysis by direct methylation method for marine powders were also validated. The coefficient of variation was 5.11% for solid samples and 1.21% for liquid sample. Key words: direct methylation, one-step methylation, fatty acids, Soxhlet, acid hydrolysis, Bligh and Dyer

Table of Contents

List of Abbreviations.................................................................... 1

1 Introduction..............................................................................

2

1.1 Lipid nutrition in fish products.....................................................

2

1.2 Lipid soluble organic pollutants...................................................

2

1.3 Total lipid and fatty acid composition analysis................................

3

2 Theory ....................................................................................

5

2.1 Lipids...................................................................................

5

2.2 Fatty acids.............................................................................

5

2.3 Lipid classes: simple lipids and complex lipids...............................

7

2.4 Neutral and polar lipids............................................................

9

2.5 Lipid extraction principle...........................................................

10

2.6 Extraction methods and total lipid determination............................

11

2.6.1 Lipid extraction methods.......................................................

11

2.6.2 Commonly used methods.....................................................

12

2.7 Total fatty acids and fatty acid profile analysis...............................

15

2.7.1 Transmethylation /Methylation...............................................

15

2.7.2 Multistep methods vs. direct methylation methods.....................

16

2.7.3 GC analysis.......................................................................

17

2.8 Lipid class analysis.................................................................

17

3 Experimental Section.................................................................

19

3.1 Samples...............................................................................

19

3.2 Water content........................................................................

20

3.3 Extraction methods.................................................................

20

3.3.1 Soxhlet method..................................................................

20

3.3.2 Acid hydrolysis method.........................................................

22

3.3.3 Modified Bligh and Dyer method.............................................

23

3.4 T ransmethylation/ methylation method........................................

25

3.4.1 Preparation of methylation detergent and internal standard.........

25

3.4.2 Methylation procedure.........................................................

25

3.5 Fatty acid analysis by GC.........................................................

26

3.6 Lipid class analysis by LC.........................................................

27

3.7 Quality control........................................................................

28

3.8 Analysis of data......................................................................

29

3.9 Outline of the experiment..........................................................

29

4 Results and discussion..............................................................

31

4.1 Quality control result................................................................

32

4.1.1 Repeatability of fatty acid analysis by DM.................................

32

4.1.2 Intermediate precision for fish powder (A-I) and control oil

33

4.1.3 Comparison of the results of control oil................................

34

4.1.4 Comparison of the results of extraction methods........................

35

4.2 Samples...............................................................................

36

4.2.1 Fatty acid composition by GC................................................

36

4.2.2 Lipid classes by LC.............................................................

38

4.3 Mass balance of the extraction methods to direct methyaltion..........

39

4.3.1 Mass balance for the Soxhlet method......................................

40

4.3.2 Mass balance for the EU method............................................

41

4.3.3 Mass balance for the Bligh and Dyer method............................

42

4.3.4 Precision of the extraction methods........................................

44

4.4 Extracted total Fatty acids by three extraction methods...................

45

4.5 Gravimetric lipid content by extraction methods.............................

45

4.6 Influence of the extraction methods on fatty acid profiles.................

48

4.6.1 Introduction........................................................................

48

4.6.2 Fatty acid profile in extracts...................................................

49

4.6.3 Reconstructed fatty acid profile..............................................

51

4.6.4 Multivariate evaluation of the profiles.......................................

56

5 Conclusions............................................................................

57
59
60
66
Appendix A. Data for Direct methylation (Table A1- A10) 67
Appendix B. Data for Soxhlet method (Table B1-B12) 73
Appendix C. Data for acid hydrolysis method (Table C1-C12) 85
Appendix D. Data for Bligh and Dyer method (Table D1-D12) 97

Appendix E. Data for control oil

113

Appendix F. Soxhlet procedure for Nofima BioLab

115
Appendix G. Acid hydrolysis procedure for Nofima BioLab 117
Appendix H. Bligh and dyer method for Nofima BioLab 119
1

List of Abbreviations

AA Arachidonic acid (20:4 n-6)

AE acid hydrolysis extraction

ALA Alpha-linolenic acid (18:3 n-3)

AR acid hydrolysis reconstructed

B&D Bligh and Dyer

BE Bligh and Dyer extraction

BR Bligh and Dyer reconstructed

CADs charged aerosol detectors

C.O. control oil

CV coefficient of variance

DAG diacylglyero ls

DDT dichloro-diphenyl-trichloroethane

DHA docosahexaenoic ac id (22:6 n-3)

DM direct methylation

EPA eicosapentaenoic acid (20:5 n -3)

FA fatt y acid

FAME fatty ac id methyl ester

FFA free fa tty acid

FID flame ioniz ation detector

GC gas chromatography

IS Internal standard

LA linoleic ac id (18:3 n-3)

LC liquid chromatography

LPC lysophosphatidylcholine

LPE lyso-phosphatidyl ethanolamine

HPLC high-performance liquid chromatography

MAG monoacylglyerols

MUFA monounsaturated fatty acid

PC phosphatidylcholine

PCA principle Component analysis

PCB polychlorinated biphenyls

PE phosphatidylethanolamine

PI phoshatidyl inositol

PUFA polyunsaturated fatty acids

PL phospholipid

PS phosphatidylserine

SE Soxh let extraction

SR Soxhlet reconstructed

SFA saturated fatty acids

SOX Soxhlet

TAG triacy lglycerols

INTRODUCTION

2

1 Introduction

1.1 Lipid nutrition in fish products

Fish and fish products play an important role in human"s life. Fish lipids are excellent sources of the essential polyunsaturated fatty acids (PUFAs) in both the omega-3 and omega-6 families of fatty acids. Omega-6 PUFAs are also derived from vegetable oil, whereas long chain omega-3 PUFAs, such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) derive mainly from fish [1]. In recent years, the significance of polyunsaturated fatty acids analysis has gained much attention because of their various biological activities in health and disease, especially the n-3 and n-6 fatty acids. These fatty acids play an important role in the prevention and treatment of cardiovascular diseases, autoimmune diseases, eye sight and the improvement of learning ability [2]. The American Heart Association (AHA ) recommends that patients with cardiovascular disease eat a variety of fish (preferably oily) at least twice a week, or to consume about 1g of EPA+DHA per day, preferably from oily fish [3]. Fishmeal and fish oil are basically made from small, bony, and oily fish that otherwise are not suitable for human consumption and some is manufactured from by-products of seafood processing industries. Fishmeal and fish oil are among the major internationally traded food and feed commodities in the world. The trade in world fishmeal and fish oil totals about 4.0-4.5 million tonnes, of which fishmeal represents about 85%-90% [4]. They are globally important to livestock production, fish farming and human health. Although most of the oil usually gets extracted during processing of the fishmeal, the remaining lipid typically represents between 6% and 10% by weight but can range from 4% to 20%. The lipids in fishmeal not only impart an excellent source of essential fatty acids but also provide a high content of energy to the diet. The lipids in fishmeal are easily digested by all animals. The predominant omega-3 fatty acids in fishmeal and fish oil are linolenic acid, decosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA). Incorporation of DHA and EPA found in fish meal into the diets of fish and other farm animals is a convenient method to ensure a proper concentration of these important omega-3 fatty acids in the human diet [5].

1.2 Lipid soluble organic pollutants

The consumption of fish may also cause potential health risk because of the presence of lipophilic organic pollutants, such as DDT, dieldrin, heptachlor, PCBs and dioxines. These contaminants are present in low levels in lakes, rivers, seas and oceans, etc. However, the fish species can concentrate the

INTRODUCTION

4 environmental contaminants by bioaccumulation and biomagnifications. The fat soluble environmental contaminants concentrate in fatty tissue of fish. Thus, high levels of environmental contaminants may be stored in fatty tissue of fish and fish consumption is an import ant source of human exposure to the above-mentioned environmental contaminants [6]. Recently, more and more attention has been paid to the problem of optimizing the balance between the risk and benefit of fish intake [7, 8, 9]. To study the contaminants in fish products, usually the crude fat is extracted for further analysis and the lipid content is a key parameter to interpret data on organic contaminants [10, 11]. It is therefore necessary to have a good method to determine the lipid content and lipid composition in fish products for the following reasons:

To evaluate the nutrition of fish products;

To meet the requirements of international trade;

To manage the animal feeding;

To inspect chemical contaminants in fish products. This method should not only be accurate and reliable but also convenient, cost efficient and environmental sound.

1.3 Total lipid and fatty acid composition analysis

The lipid content is traditionally gravim etrically determined by solvent extractions. There are a large number of methods for lipid extraction. Soxhlet method [12], acid hydrolysis method [ 13], Bligh and Dyer [14] are most commonly used in fish industry. Different extraction methods vary in their lipid extraction efficiency. The total lipid by solvent extraction repres ents the content of crude fat, which may also contain non-fat material and often fails to accurately estimate nutritional values in biological materials. Total fatty acids are generally a better alternative for assessment of nutritional value than extractable lipids, especially for determination of digestible energy. Fatty acid component s need to be converted into fatty acid methyl ester (FAME) before analyzed by GC. FAME can either be prepared by multistep methods, consisting of lipid extraction followed by transmethylation, or by direct methylation m ethods. Direct methylation com bines extraction and transmethylation into one step. It overcomes several limit ations of the multistep methodology, giving rise to a simpler and faster analysis, consuming less organic solvent [15,16,17]. Another advantage by the direct methylation methods is that fatty acids are released from the matrix by br eaking the ester bonds. In general, direct methylation is therefore more efficient than extraction for recovering fatty acids in lipids t hat is tightly bound to the matrix, such as samples rich in phospholipids [15]. The main focus of the present study was to evaluate the efficiency of conventional lipid extraction methods in eight marine powders

INTRODUCTION

4

Lipid Extraction

(Soxhlet, Acid hydrolysis,

Bligh & Dyer)

GC analysis

Result 1

Trans esterification and extraction of FAME Trans esterification and extraction of FAME Trans esterification and extraction of FAME

GC analysis

Result 2

GC analysis

Result 3

(including lean fish, fat fish, fish meals and Krill meals) by using direct methylation on extracts and residues for calculating the mass balance of the fatty acids. Three classical extraction methods were studied: Soxhlet method, acid hydrolysis method and Bligh and Dyer. The analysis procedure is illustrated in Fig. 1 where the amount of fatty acids in Result 3 should be equalquotesdbs_dbs17.pdfusesText_23

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