Marc Anton

Low density lipoproteins extracted from hen egg yolk by an easy method: cryoprotective effect on frozen–thawed bull semen

Hen egg yolk is widely used as a cryoprotective agent in semen freezing extenders in order to protect the spermatozoa against cold shock. The protective action of yolk is largely presumed to be due to low density lipoproteins (LDL). In recent years, arguments concerning the presence of cryoprotective antagonists in egg yolk, have reinforced interest in the use of only the LDL extracted from egg yolk in the extenders. However, current methods of LDL purification do not support the use of LDL in commercial extenders because they offer a poor recovery rate. Consequently, we have developed an easy method to extract LDL from egg yolk. Several concentrations of purified LDL (between 2.5 and 20%, w/v) were tested in freezing extenders for bull semen, and compared with commercial extenders. Our extraction method reached 97% purity and about 67% yield, and is easily reproducible on an industrial scale. Analysis of sperm motility showed that the motility and characteristics of spermatozoa movement were improved with LDL in the extender, as compared to a commercial extender containing egg yolk. The optimum LDL concentration in the extender was 8%. In conclusion, we propose that an extender containing LDL extracted from egg yolk could be used as cryoprotective media with a better efficiency than present commercial extenders.

Bull semen in vitro fertility after cryopreservation using egg yolk LDL: a comparison with Optidyl®, a commercial egg yolk extender

Low-density lipoproteins (LDL) have been previously isolated and identified as the cryoprotective fraction of yolk. The effect of LDL on sperm motility after freezing-thawing has been reported, but no study has been made to assess the effect of LDL on bull semen fertility. The aim of this study was to evaluate the fertility of bull semen cryopreserved in the presence of LDL. Motility of semen cryopreserved in LDL was analyzed and compared to semen cryopreserved with Optidyl, a commercial extender containing egg yolk. To evaluate the fertilizing ability of semen, we used in vitro fertilization test, whereas acrosome and plasma membrane integrity were also evaluated. The percentage of motile spermatozoa was two fold higher after freezing in LDL than in Optidyl 54.4% versus 30.2% (P < 0.05). The cleavage rate was significantly higher after fertilization with semen frozen in LDL than with Optidyl 63.0% versus 54.8% (P < 0.05). No significant difference was observed on the blastocyst rate after in vitro culture. Integrity of the acrosome and the plasma membrane were maintained in both extenders. In conclusion, LDL preserve bull semen quality and fertilizing ability, allowing also better semen motility, after the freeze-thaw process.

Chemical and structural characterisation of low-density lipoproteins purified from hen egg yolk

Abstract Low-density lipoproteins (LDL) are considered to be the main contributors to the exceptional emulsifying activity of hen egg yolk. However, the lack of understanding of the molecular basis for LDL functionality is a significant obstacle for good control of yolk emulsions. Consequently, we have attempted to link the structure and the characteristics of LDL with their emulsifying properties. After purification of LDL, we have determined their protein and lipid compositions, their ultrastructure, and then extracted their apoproteins for physicochemical characterisation. LDL are composed of about 12% of proteins and 87% of lipids and present a spherical shape with a mean diameter of about 35 nm. LDL solubility is high, whatever the medium conditions, because of their low density. LDL contain five major apoproteins out of which the apoprotein of 15 kDa is considered to be the most surface-active. After extraction, this apoprotein showed a high proportion of amphipathic α-helix chains, explaining the high capacity of this apoprotein to adsorb at the oil–water interface.

Characterisation of emulsion properties and of interface composition in O/W emulsions prepared with hen egg yolk, plasma and granules

Comprehension of hen egg yolk emulsifying properties remains incomplete because competition between its various emulsifiers (proteins and lipoproteins containing phospholipids) has not been clearly elucidated and colloidal interactions between yolk-stabilised oil droplets have not been documented. Recent studies emphasised the interest of the fractionation of yolk into plasma and granules to improve this comprehension. In the present study, we characterised, concurrently, emulsion properties (oil droplet size and stability against creaming) and interface attributes (interfacial concentrations of proteins and phospholipids, SDS-PAGE profiles of adsorbed proteins and zeta potential) in oil-in-water (O/W) emulsions prepared with yolk, plasma and granules. We observed these features at four physicochemical conditions (pH 3.0 or 7.0 and at 0.15 or 0.55 M NaCl). Emulsion properties in emulsions made with yolk or plasma varied similarly as a function of pH and NaCl concentration whereas granules emulsions exhibited distinct properties. Therefore the main contributors to yolk emulsifying properties are to be sought for among plasma constituents (proteinaceous or phospholipids). Since, in plasma emulsions, variations of emulsion stability against creaming correlated exclusively to variations of protein interfacial concentration, a driving contribution of the proteinaceous part of plasma, namely apo-LDL, was hypothesised. In the pH and ionic strength ranges studied, zeta potentials of the interfaces were low, excluding extended electrostatic repulsion between oil droplets. We deduced that steric repulsion is the main interaction opposing to droplet aggregation in food emulsions made with yolk.

Egg yolk: Structures, functionalities and processes

Hen egg yolk is an ideal example of natural supramolecular assemblies of lipids and proteins with different organization levels. These assemblies are mainly due to interactions between proteins and phospholipids, and these interactions are essential in understanding and controlling the production of food made with yolk, and particularly emulsions. Furthermore, these assemblies can be modulated by external constraints among which thermo-mechanical and high-pressure treatments. This review focuses on multi-scale structures present in egg yolk, and their modulation by processes, in relation with their emulsifying properties. Egg yolk is mainly composed of two fractions-plasma and granules-which are natural nano- and micro-assemblies. These two fractions possess different composition, structures and functionalities and exhibit specific behaviour under treatments such as high pressure and temperature. Plasma contains a large quantity of lipids structured as lipoproteins (low-density lipoproteins), whereas granules are mainly composed of proteins aggregated in micrometric assemblies. If plasma is responsible for the important emulsifying properties of yolk, granules bring interesting emulsifying properties when assemblies are in the form of micelles in presence of salts. High-pressure or thermal treatments, applied before or after emulsion fabrication, alter their functionalities and could be used to commercially exploit these fractions.

Filler effects of oil droplets on the rheology of heat-set emulsion gels prepared with egg yolk and egg yolk fractions

Hen egg yolk is a traditional ingredient used in a wide variety of food emulsions, especially fluid sauces. Industrial processing of these sauces generally involves heat treatments in order to pasteurise or sterilise them. These heat treatments may cause undesired gelation of the emulsion, because egg yolk proteins are particularly thermosensitive. Heat gelation of oil-in-water emulsions prepared with egg yolk may differ from that of egg yolk solutions, because of the influence of oil droplets on network formation. In this study, we investigated the influence of oil droplets on the gelation of oil-in-water emulsions made with yolk. We studied three pH values: 3.0, 5.0 and 7.0 with a constant NaCl concentration: 0.55 M. Oil droplet size was controlled after emulsification, gelation of solutions and emulsions was monitored in situ by coupling heating with recording viscoelastic properties, and transmission electron microscopy was conducted in heat-set emulsion gels. In an attempt to target the proteins that impose the kinetic of gelation of egg yolk, we repeated the experiment with plasma and granules, the main fractions of yolk. In situ rheology showed that, in our experimental conditions [especially oil volume fraction (0.3) and oil droplet size (d3.2=1 &mgr;m)], emulsions made with yolk and plasma have a similar gelation process with oil droplets acting as inactive fillers. Furthermore, transmission electron microscopy showed similar network characteristics between heated emulsions made with yolk and plasma. Moreover, we demonstrated that acidic conditions provided the fastest gelation of yolk solutions and emulsions. On the other hand, in emulsions prepared with granules, oil droplets behaved as active filler particles and reinforced the gel strength.

Adsorption at the oil–water interface and emulsifying properties of native granules from egg yolk: effect of aggregated state

Abstract Native and disrupted granules were used to prepare and stabilise oil-in-water emulsions containing washed sunflower oil. Native granules were in an insoluble aggregated form, whereas, after addition of sodium chloride, granules were disrupted and their proteins and lipoproteins were liberated and solubilised. Native granules were adsorbed at the oil–water interface as complex particles without dissociation of their individual proteins and lipoproteins. This aggregated state allowed a greater surface coverage than the molecular state following upon granules disruption, because of the convective mass-transport occurring during homogenisation. However, in emulsions made by means of these aggregates, the droplet size was larger, and stabilisation against creaming and coalescence were less efficient than that of emulsions made with proteins and lipoproteins liberated from disrupted granules. The electrophoretic study of emulsion droplets revealed that, whatever the native or disrupted form, all major granules proteins were adsorbed at the oil–water interface. Results suggest that it is possible to realise emulsions with native granules but that such emulsions are less fine, less homogenous and less stable than emulsions realised with disrupted granules.

The advantages of LDL (low density lipoproteins) in the cryopreservation of canine semen.

A medium containing LDL (Low Density Lipoproteins, the cryoprotective component of chicken egg yolk) was compared with egg yolk for the preservation canine spermatozoa during the freeze-thaw process. Twenty sperm samples taken from 10 dogs were frozen in liquid nitrogen at -196 degrees C in seven different media: one control medium containing 20% egg yolk, and six test media containing 4%, 5%, 6%, 7%, 8%, and 10% LDL, respectively. Following thawing, sperm motility was assessed using a Hamilton-Thorne Sperm Analyser equipped with the CEROS 12 software. The percentage of motile spermatozoa was 55.3% in the 6% LDL medium (optimal concentration) compared with 27.7% in the egg yolk based medium (p<0.05). In comparison with the egg-yolk medium, the LDL medium also resulted in an improved preservation of spermatozoa during the freezing process (p<0.05) in terms of acrosomal integrity (FITC-PSA test), flagellar plasma membrane integrity (HOS test), and DNA integrity (Acridine Orange test). In addition, six Beagle bitches were inseminated twice, via the intra-uterine route, at an interval of 24h; 200x10(6) spermatozoa that had been previously frozen in the 6% LDL medium were used per insemination. All of the bitches became pregnant (gestation rate of 100%). In conclusion, the 6% LDL medium provides improved protection of the spermatozoa during the freeze-thaw process and a marked improvement in the motility parameters of canine spermatozoa in comparison with the control medium containing egg yolk alone. Finally, the use of LDL as a cryoprotectant for canine semen does not interfere with fertility.

Surface properties of hen egg yolk low-density lipoproteins spread at the air–water interface

Abstract Hen egg yolk is largely used as food ingredient notably because of its exceptional emulsifying properties. Low-density lipoproteins (LDL) are the main egg yolk constituent. LDL and particularly apoLDL are thought to control largely emulsifying properties of egg yolk-based products. Nevertheless, few studies have concerned the interfacial behaviour of these lipoproteins at the oil–water interface and nothing has been published about the air–water interface. Controversies still remain about LDL adsorption mechanism at the oil–water interface even if a widely spread theory suggests their breaking at the interface, allowing then their constituents to spread. The Langmuir film balance and atomic force microscopy (AFM) were used in this study in the aim to characterise LDL surface behaviour in dynamic conditions at the air–water interface. The understanding of LDL adsorption mechanism and surface organisation at the air–water interface should provide useful information about LDL behaviour at the oil–water interface. LDL and lipids extracted from LDL—neutral lipids, phospholipids and total lipids (mixture of the two previous species)—were spread at the air–water interface to clarify the role of each constituent in the lipoprotein film. Results clearly show that LDL are disrupted at the interface to release notably neutral lipids from the lipoprotein core, enabling then their spreading. Each lipid class has been identified on the LDL film isotherm and seems to behave independently and individually at the interface within the lipoprotein film.

Bioactive egg components and their potential uses

The hens egg plays a crucial role in the embryonic development of the bird. It serves first as a source of energy and nutrients of high digestibility and, second, protects the embryonic bird against external aggressions. The unique structure of an egg with yolk containing the embryo surrounded by albumen and shell as physical barriers, is the first element of this protection. The second one consists of the specific composition of yolk, albumen and shell with many molecules possessing elevated biological properties. Thus, these molecules represent a major source of active principles usable by medical, pharmaceutical, cosmetic, nutraceutical and biotechnological industries. In this review, we will focus particularly on nutritional, health, and biotechnological activities of egg molecules and on the approaches, which are proposed by European research groups, to exploit this bioactive potential.