1. Folic acid, the synthetic form of folate vitamin has been shown to
reduce the incidence of neural tube defects in pregnancies. However, recent
studies indicated the excess intake of synthetic form of folate for the
population as a whole may not necessarily be beneficial. Conversely, the
natural forms of folate in physiological amounts are known to have no
adverse effects. Egg yolk has been reported as an excellent source of folate
(Strandler et al., 2011) with high bioavailability (house et al., 2003). with
folate principally restricted to the yolk fraction.
Scaling-up a process for the preparation of folate-enriched protein extracts from hen egg yolks
The objective of the present study was to develop and scale-up a
sequential separation method using water to fractionate native hen egg
yolk for the preparation of a folate-enriched extract. The composition of
fractions generated from laboratory-scale and pilot processes were
characterized for protein, lipid, cholesterol, folate and mass balances
were determined.
Introduction
Objective
Nassim Naderi1*, James D.House1,2 and Yves Pouliot1
1Institute of Nutraceuticals and Functional Foods (INAF), Department of Food Science and Nutrition, Université Laval, Québec, QC, Canada, G1V 0A6.
2Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, MB, Canada, R3T 2N2.
Methodology
Fractionation of egg yolk
Three dozen of fresh large eggs were purchased from local market
for each batch of experiments. Yolks were separated from albumen,
chalazae and vitelline membrane and diluted with Milli-Q water (1:1 v/v)
and gently homogenization by stirring. The method was based on
modification of the procedure described by Laca et al. (2010). The yolk
fractionation studies were performed by using a lab-scale centrifuge
(10,000×g; 4°C; 45 min) and a tubular bowl centrifuge (pilot-scale;
40,000×g).
The fractionation procedures at two scale is schematized in Figure
2.
Conclusion
• The granule fraction contains higher protein, lower lipid and cholesterol
proportion compared to the egg yolk.
• Granule fraction was mainly composed of high density lipoproteins
which are associated with higher protein (80%) and lower lipid (20%)
content (Guilmineau,et al., 2005).
• Folate analysis revealed that granule fraction was concentrated by a 3-
fold factor in terms of folate in comparison to native egg yolk.
• The increased ratio of protein/total soluble solids in the granule fraction
at pilot-scale separation compare to lab-scale separation was in
accordance with the ratio of folate/protein granule extracts. The folate is
likely protein bound however the nature of this protein remains to
be conclusively determined (Sherwood et al., 1993).
• Considering the recommended daily intake of 400 µg folate for adults,
the granule extracted from one egg (4 g) by our separation method can
provide 7-8 % of daily requirement.
References
1. Daneshfar, A., et al. (2009). J Chrom B Anal Tech Biomedical Life
Science, 877(4), 456–460.
2. Guilmineau, F., et al. 2005. J Agric Food Chem, 53, 9329-36.
3. House, J. D., et al. 2003. Journal of Agricultural and Food Chemistry, 51,
4461-4467.
4. Laca, A., et al. 2010. Food Hydrocolloids, 24, 434-443.
5. Lander, R., et al. 2005. BioProcess International, 32–40.
6. Naderi, N., et al. 2014, Journal of Food Engineering,
10.1016/j.jfoodeng.2014.05.011 (accepted)
7. Sherwood, T. A., et al. 1993. Archives of Biochemistry and Biophysics,
307, 66-72.
8. Strandler, H. S., et al. 2011. European Food Research & Technology,
233, 923-930.
Method 1- Centrifugation at Lab-scale:
(10,000g; 4◦C; 45min)
Results
Compositional analysis
• Protein content (Total N) using nitrogen gas analyzer system (LECO FP-
528).
• Lipids (Total) using the mojonnier method.
• Cholesterol extracted by Dispersive liquid-liquid microextraction
method (Daneshfar et al. 2009). The detection was done by HPLC system
(UV detection, 210 nm).
• Folate were extracted based on the method describe by House et al.
(2002) with minor modifications. Folate was detected by HPLC system
and the use of a Kinetex™ column (fluorescence detection, 290 nm
excitation and 360 nm emission; UV detection, 290 nm).
• Protein profile were studied by SDS-PAGE using commercially available
precast linear gradient polyacrylamide gels (4–20%; Bio-Rad, Canada).
Detection of phosphorylated proteins was achieved by adding aluminum
nitrate into the staining solution.
Table 1: Composition of egg yolk and fractions from lab- and pilot-scale
processes
Lab scale Pilot scale
Composition Sample Wet Dry matter Wet Dry matter
Dry matter (g/100g) Egg Yolk 50.88±0.27 - 50.54±0.52 -
Plasma 22.36±1.15 - 22.36±1.92 -
Granule 45.47±0.74a - 51.39±1.39b -
Protein (g/100g) Egg Yolk 16.15± 0.19 31.84±0.40 15.24± 0.37 30.15±0.37
Plasma 4.63± 0.19a 21.84±0.60 4.20 ± 0.36b 18.83±1.26
Granule 27.61± 0.35a 57.62±9.17 35.05± 1.57b 67.64±1.63
Fat (g/100g) Egg Yolk 28.65±1.41 56.50±2.85 31.52 ± 0.77 62.37±0.93
Plasma 14.05±0.96a 70.61±5.90 16.55 ± 0.61b 75.71±1.00
Granule 13.91± 0.57a 31.70±1.04 11.83 ± 1.17b 23.01±2.79
5-CH3-H4folate
(µg/100g)
Egg Yolk 203.84±12.55a 399.31±9.75 314 ± 143.5a 657.72±151.2
Plasma ND1 - ND -
Granule 670.05±34.82b 1474.41±41.78 1029.7 ± 286.1c 2078.8±307.4
Experiments were repeated on three separate occasions, and values are mean ±SD.
a-c Different letters in columns (lab-scale and pilot-scale) refer to significantly different values as calculated by ANOVA.
1ND: Not Detected
Figure 4: Demonstrates the cholesterol content (g/100g) of egg yolk,
plasma and granule fraction.
Table 2: Compositional characteristics of granule extracts compared to
egg yolk
Sample
Protein
(g/100g
solids)
[Folate]ug/
protein(g)
Ratio
[Folate]ug/
protein(g)
Folate
concentratio
n factor (CF)1
Egg Yolk 30 13 0.43 -
Granule Lab 52 24 0.46 3.29
Granule Pilot 72 29 0.40 3.28
1 CF= [Folate]G/[Folate]EY
Figure 2: Schematic presentation of egg yolk fractionation at lab- and
pilot-scale
Acknowledgement
Dilution (1:1 v/v) in
Granule
(Precipitate)
Plasma
(Supernatant)
Method 2- Centrifugation at pilot-scale:
(40,000g; 100mL/min)
Granule
(separated in cylinder)
Plasma
Diluted Yolk
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
Egg Yolk Plasma Granule
Cholesterol(g/100gdrymater)
Lab-scale Pilot-Scale
Figure 1: Represents the demand for a natural substitute for synthetic
folic acid.
Previous studies revealed that egg yolk can be fractionated into two
fractions of plasma and granules by dilution followed by centrifugation
(Laca et al., 2010) .
However those methods utilized strategies (e.g. pH adjustment, use
of salts with extreme water dilution) which all have compatibility for
ultimate use as a food or medicinal ingredient. The tubular bowl
centrifuges are well established efficient unit operations in the field of
biotechnology with highest centrifugal forces (Lander et al., 2005) and can
be used to scale-up an egg-yolk fractionation procedure.
Nassim Naderi is supported by a scholarship from the FAST
training program, an initiative of NSERC-CREATE Funding program.