Soutenance mémoire
SCALING-UP A PROCESS FOR THE PREPARATION OF FOLATEENRICHED PROTEIN EXTRACTS FROM HEN EGG YOLKS
FOLATE AND FOLIC ACID
Historical perspective
Folate, discovered in 1931, is a water-soluble B vitamin which is called vitamin B9. Folic acid received its name in 1941 when it was isolated from spinach and its structure was determined in the mid-1940s. The compound was consequently synthesized in pure crystalline form in 1943 and this finding proved that folic acid was composed of a pteridine ring, paraminobenzoic acid and glutamic acid. Afterward, it became evident that natural folates usually differed from pteroylglutamic acid. Today, folic acid refers to the fully oxidized chemical compound which does not exist in natural foods. The term ‘folate’ is designated to the large group of compounds having the same vitamin activity and includes natural folates and folic acid (Hoffbrand and Weir, 2001).
Structure and biochemistry
Folate is the generic term for folic acid (pteroylmonoglutamic acid) and related compounds exhibiting the biological activity of folic acid. The terms folacin, folic acids, and folates are used only as general terms for this group of heterocyclic compounds based on the N-[(6- pteridinyl)methyl]-p-aminobenzoic acid skeleton conjugated with one or more L-glutamic acid residues. Folates can consist of a mono- or polyglutamyl conjugate. The folates from most natural sources usually have a single carbon unit at N-5 and/or N-10; these forms participate in the metabolism of the single-carbon pool. The compound called folic acid is not present in living cells, being rather an artificial and manmade form of the vitamin. Folic acid (pteroylmonoglutamic acid) is an orange-yellow crystalline substance that is soluble in water but insoluble in ethanol or less polar organic solvents. It is unstable to light, acidic or alkaline conditions, reducing agents, and to heat (except in dry form). The UV absorption spectra of the folates are characterized by the independent contributions of the pterin and 4- aminobenzoyl moieties; most have absorption maxima in the region of 280–300 nm (Lavoisier, 2008).Naturally occurring folates, such as those found in foods and body tissues, exist in different forms. They contain a fully-reduced pteridine ring together with additional glutamic acid molecules . They are usually substituted by different one-carbon units at the N5 or N-10 positions, or have a single-carbon bridge spanning these positions. These onecarbon units can be at the oxidation level of methanol (5-methyltetrahydrofolate), or formaldehyde (5, 10-methylenetetrahydrofolate), or formate (5, 10- methenyltetrahydrofolate) (Scott and Weir, 1993).
The 5-methyltetrahydrofolate (5-MTHF) is the predominant natural form of folate in fruits and vegetables (Vahteristo et al., 1997). In animal products the 5-MTHF and tetrahydrofolate are predominate (Vahteristo et al., 1997) while cereal products contain 5- MTHF, 5-formyltrahydrofolate, and 10-formlytrahydro folate (Pfeiffer et al., 1997).
Importance of folate in human diet
The naturally occurring form of folate lacks stability during food storage and preparation and thereby the stable folic acid (Eitenmiller et al., 2007) was used for supplements and food fortification. Folate has a crucial role as a one-carbon source for DNA, RNA synthesis and protein methylation (Stover, 2009). A number of genetic polymorphisms affect critical components of folate pathways and metabolism, and have been associated with an increased risk for neural tube defects (NTD) (Molloy et al., 2009). However, the exact mechanism by which folic acid reduces the risk of NTDs is not known and remains an active area of research (Crider et al., 2011a). NTDs occur when the neural tube fails to close early in embryonic development, resulting in damage to the exposed underlying neural tissue. These birth defects can result in significant morbidity and mortality depending on the location and severity of the lesion (Crider et al., 2011a). The most severe lesions observed with spina bifida cause a range of morbidities, including urinary and fecal incontinence and paralysis of the lower limbs (Sutton et al., 2008).
The relationship between NTD occurrence and folate deficiency encouraged scientist to demonstrate the link between folic acid intake and the risk of birth defects. A randomized control trial study found that the risk of NTD occurrence can be reduced by 70% when women consume 400 µg of folic acid daily (MRC, 1991). In 1991, the Centers for Disease Control and Prevention recommended that women with a history of NTD affected pregnancy should consume 400 µg of folic acid daily before conception (CDC, 1991).
Subsequently, in 1992, the U.S. Public Health Service recommended that all women of childbearing age consume 400 µg of folic acid daily through fortification, supplementation, and diet to prevent NTDs (CDC, 1991). In 1998, the Institute of Medicine (IOM) recommended that women at the age of becoming pregnant should consume additional 400 µg of folic acid daily from fortified foods or supplements to that obtained through a normal diet (1998). In order to increase the folic acid level in the diet of all women of childbearing age, the regulations for mandatory fortification of wheat flour with folic acid has begun.
The mandatory folic acid fortification of enriched cereal grain products was authorized in 1996 in U.S. and fully implemented in 1998 (FDA, 1996). This program aimed to add 140 µg of folic acid/100 g of enriched cereal grain product in order to provide nearly 100–200 µg of folic acid/day for women of childbearing age (Quinlivan and Gregory, 2007).
FRACTIONATION METHODS FOR DEVELOPMENT OF FOOD-DERIVED BIOACTIVES
Separation, extraction and concentration processes, used in the food, nutraceutical, pharmaceutical and health ingredient industries, are essential to recover bioactive components from their natural matrices. The separations techniques usually intend to achieve removal of specific components (e.g. residue and/or the extracted components) in order to increase the added value of the products. Separation techniques, such as filtration, evaporation, dehydration, solid-liquid and gas-liquid extraction processes, are based on the nature (liquid, solid or gaseous) and different physicochemical properties (density, solubility, electrostatic charge, particle or molecular size and shape) of the sample and target components. However, most of the processes involved are of a physical nature (Grandison and Lewis, 1996).
The separation processes may be batch or continuous. A single separation process involves the contact of the solvent with the food. The continuous processes may be single or multiple stage processes (Grandison and Lewis, 1996).
Separation techniques for solid foods can be classified into two categories. The first category includes solid-solid separation processes that require the separation or segregation of particles. The other processes involve removing the discrete solid particles from liquid, gas or vapors. In solid-solid separation, the fractionation technique can be achieved on the basis of particle size from sorting of large food units down to the molecular level. The shape, electrostatic charge and degree of hydration are major factors that may affect the solid-solid separation (Brennan, 1990). The separation of liquid (oil, water, etc.) or air from solid matrix can achieve by processes such as pressing, extraction, dehydration, blanching.
FRACTIONATION CHALLENGE FOR PRODUCING FOLATE-ENRICHED PROTEIN EXTRACT FROM HEN EGG YOLK
Although the importance of hen egg as an economical and convenient source of metabolically active folate form has been well recognized (Seyoum and Selhub, 1998), less attention has subjected to separation of this compound. A number of different extraction methods have been employed to separate folate but many of them are based on the use of organic solvents. However, in most applications, these methods are not safe due to the possible solvent residuals in the final products (Laca et al., 2010) and furthermore the presence of food matrix may influence the bioavailability of folate (Gregory, 2001). It was shown that in the folate extract prepared from egg yolk, the presence of other egg yolk soluble components in extract has important influence on the extent of folate bioavailability in vivo. However, the degree of fat separation from egg yolk extract enhanced the concentration of folate in final egg yolk extract (McKillop et al., 2003). Therefore, there is a need to determine more efficient ways for folate separation and purification by using economical and easily scaled-up processes.
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Table des matières
CHAPTER 1: INTRODUCTION
CHAPTER 2: LITERATURE REVIEW
2.1. HEN EGG YOLK
2.1.1. Overall composition and structure
2.1.2. Nutritional and nutraceutical properties of egg yolk
2.1.3. Improving nutritional properties of egg yolk
2.2. FOLATE AND FOLIC ACID
2.2.1. Historical perspective
2.2.2. Structure and biochemistry
2.2.3. Importance of folate in human diet
2.3. FRACTIONATION METHODS FOR DEVELOPMENT OF FOOD-DERIVED BIOACTIVES
2.3.1. Overview of the separation methods in egg processing
2.4. FRACTIONATION CHALLENGE FOR PRODUCING FOLATE-ENRICHED PROTEIN EXTRACT FROM HEN EGG YOLK
CHAPTER 3: PROBLEMATIC, HYPOTHESISAND OBJECTIVES
3.1. PROBLEMATIC
3.2. HYPOTHESIS
3.3. MAIN AND SPECIFIC OBJETIVES
CHAPTER 4: SCALING-UP A PROCESS FOR THE PREPARATION OF FOLATEENRICHED PROTEIN EXTRACTS FROM HEN EGG YOLKS
4.1. CONTEXTUAL TRANSITION
4.2. RÉSUMÉ
4.3. ABSTRACT
4.4. INTRODUCTION
4.5. MATERIALS AND METHODS
4.5.1. Hen eggs
4.5.2. Chemicals
4.5.3. Fractionation of egg yolk
4.5.4. Pilot scale process
4.5.5. Chemical analyses
4.5.6. Extraction and analysis of folate content
4.5.7. Performance parameters of egg yolk fractionation
4.5.8. Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE)
4.5.9. Two dimensional electrophoresis
4.5.10. Statistical analysis
4.6. RESULTS AND DISCUSSION
4.6.1. Overall composition of the fractions
4.6.2. Folate enrichment
4.6.3. Protein profile of fractions obtained at lab- and pilot-scale
4.6.4. Mass balance and centrifugation performance
4.7. CONCLUSION
CHAPTER 5: EFFECT OF SELECTED PRETREATMENTS TO INCREASE THE FOLATE CONTENT OF GRANULE SUSPENSIONS PREPARED FROM HEN EGG YOLK
5.1. CONTEXTUAL TRANSITION
5.2. RÉSUMÉ
5.3. ABSTRACT
5.4. Introduction
5.5. MATERIALS AND METHODS
5.5.1. Materials and chemicals
5.5.2. Preparation of yolk and granules
5.5.3. Application of pre-treatments on granule suspensions
5.5.4. Chemical analyses
5.5.5. Determination of cholesterol
5.5.6. Fatty acid analysis
5.5.7. Determination of folate content
5.5.8. SDS–PAGE protein profiles
5.5.9. Statistical analysis
5.6. Results and discussion
5.6.1. Effect of ionic strenght
5.6.1. Effect of ultrasound treatment on granule and plasma
5.7. Conclusion
CHAPTER 6: UNDERSTANDING THE EFFECT OF IONIC STRENGTH AND MECHANICAL TREATMENTS ON THE COMPOSITION AND MICROSTRUCTURE OF GRANULE SEPARATED FROM HEN EGG YOLK
6.1. CONTEXTUAL TRANSITION
6.2. RÉSUMÉ
6.3. ABSTRACT
6.4. INTRODUCTION
6.5. MATERIALS AND METHODS
6.5.1. Materials and chemicals
6.5.2. Preparation of granules
6.5.3. Ionic strength modification
6.5.4. Mechanical treatments
6.5.5. Compositional analyses
6.5.6. Sodium Dodecyl Sulphate–Polyacrylamide Gel Electrophoresis
6.5.7. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)
6.5.8. Microstructure Determination by Confocal Laser Scanning Microscopy
6.5.9. Statistical analysis
6.6. RESULTS
6.6.1. Overall compositional changes induced in granules composition
6.6.2. Effect of treatments on protein distribution (2D-PAGE)
6.6.3. Microstructural changes following treatments
6.7. Discussion
6.8. Conclusion
CHAPTER 7: GENERAL CONCLUSION
7.1. Achievements and original contributions
7.2. Significance of the results
7.2.1. New knowledge on egg yolk granule structure
7.2.1. New options for processing egg yolk components
7.1. Research perspectives
References
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