CONTENT OF PROTEINS, LIPIDS AND SACCHARIDES IN EGG YOLK AND ALBUMEN OF DIFFERERNT HEN BREEDS

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1 CONTENT OF PROTEINS, LIPIDS AND SACCHARIDES IN EGG YOLK AND ALBUMEN OF DIFFERERNT HEN BREEDS PETRA VICAROVA 1, VOJTECH KUMBAR 2 1 Department of Chemistry and Biochemistry 2 Department of Technology and Automobile Transport Mendel University in Brno Zemedelska 1, Brno petra.vicarova@mendelu.cz Abstract: The first aim of this study was to determine the amount of proteins, lipids and saccharides in the hen egg yolk and albumen of different hen breeds. The second aim was to determine the correlation between amount of analysed components (proteins, saccharides and lipid) in hen albumen and amount of analysed components (proteins, saccharides and lipid) in hen yolk, during storage time. Hen eggs were stored at 6 C for 57 days and were sampled in two periods (29 June 2015, 23 February 2016). Two different breeds were used for this study Hybrid Isa Brown (36 rd week of lay) for first period and Hybrid Hisex Brown (13 rd week of lay) for second period. Total of 180 eggs were analysed. Biuret agent was used for determine of proteins in hen yolk and albumen. Concentrated sulfuric acid and 5% phenol were used for determination of saccharides in hen yolk and albumen. The amount of proteins and saccharides was determined by the UV-VIS absorption spectrometer (Helios Epsilon, Thermo Scientific). For determination of lipids saponification value was used. The differences in amounts of proteins, lipids and saccharides were not found between Hybrid Isa Brown and Hybrid Hisex Brown. Correlation was not found between amounts of proteins, lipids and saccharides in hen yolk and hen albumen for storage time. Key Words: proteins, lipids, saccharides, UV-VIS spectrometry, hen yolk, hen albumen INTRODUCTION Humans have utilized hen eggs as a nutritional food since ancient times (Yamamoto et al. 1997). Hen egg is the source of proteins known for their nutrition, biological and technological potential. (Machado et al. 2007) besides vitamins from the vitamin B complex (thiamine, riboflavin, niacin, pyridoxine and cyanocobalamin), they contain liposoluble vitamins (A, D, E and K). Further, egg contains minerals (iron, calcium, potassium, sodium, phosphorous and zinc). Hen egg is an important source of cholesterol and unsaturated fatty acids, mainly oleic acid (Kritchevsky 2004, Horbanczuk et al. 1999). In many publications the amount of proteins, saccharides and lipid in egg yolk and albumen was observed. (Aquino et al. 2010, Horbanczuk et al. 1999, Kritchevsky et al. 2004, Machado et al. 2007, Segura-Campos et al 2013) Analysis of proteins, lipids and saccharides are important for food industry and human nutrition. Content of proteins, lipids and saccharides in hen egg differs from breed hen age, storage, etc. (Janairo et al. 2011, Okutucu et al. 2007). Modern instrumental methods such as mass spectrometry, absorption spectroscopy, chromatography etc. are used for determination of proteins, lipids and saccharides, but these methods are expensive, difficult for manipulation and time-challenging. Traditional spectrophotometric and titration methods are cheap, fast, easy-working and the most common way to quantitate content of protein (Noble & Bailey 2009). The first goal of this study was to determine the content of proteins, lipids and saccharides in two different henbreeds Hybrid Isa Brown and Hybrid Hisex Brown. Second goal of this study was to analyse the influence of storage time of hen eggs to the amount of proteins, lipids and saccharides in hen yolk and hen albumen. 681 P age

2 MATERIAL AND METHODS Hen eggs were analysed in two periods - 29 June 2015, 23 February were stored at 6 C for 57 days. Two different breeds were used for this study Hybrid Isa Brown (36 rd week of lay) for first period and Hybrid Hisex Brown (13 rd week of lay) for second period. Nine sampling days were for both periods. A total of 180 hen eggs were analysed. Hens were kept in cages. Determination of proteins in hen yolk and hen albumen Biuret agent was used for determination of proteins in egg yolk and egg albumen. Biuret agent = copper sulfate pentahydrate CuSO 4.5 H 2O (c = 13.0 mmol/l), potassium sodium tartrate KNaC 4H 4O 6.4H 2O (c = 32.0 mmol/l), sodium hydroxide NaOH (c = 0.6 mol/l). Diluted sample was added into three tubes, further Biuret agent was added. The tubes were left 30 minutes at room temperature (~ 25 C). After 30 minutes the absorbance was measured by the UV-VIS spectrometer Helios Epsilon (Thermo Scientific) at 540 nm. Blank (mineralized water in place of sample) was used for determination of proteins in hen yolk and hen albumen. The protein assays were always performed in triplicates for result verification. Calculation of the amount of proteins was based on linear regression equation obtained by evaluation of standard curves of bovine serum albumen. Determination of saccharides in hen yolk and hen albumen Sulfuric acid (96%) and phenol (5%) were used for determination of saccharides in egg yolk and egg albumen. Diluted sample was added into three tubes, further sulfuric acid and phenol were added. The tubes were left 30 minutes at room temperature (~ 25 C). After 30 minutes the absorbance was measured by the UV-VIS spectrometer Helios Epsilon (Thermo Scientific) at 490 nm. Blank (mineralized water in place of sample) was used for determination of saccharides in hen yolk and hen albumen. Protein assays were always performed in triplicates for result verification. Calculation of the amounts of saccharides was based on linear regression equation obtained by evaluation of standard curves of standard solution of D-glucose. Determination of lipids in hen yolk and hen albumen Determination of saponification value was used for analysed amount of lipids in hen yolk and hen albumen. Sample was added into round bottom flask, further ethanolic solution of potassium hydroxide (0.5 mol/l) was added. This mixture was heated under reflux for 30 minutes. After 30 minutes phenolphthalein was added. Further this mixture was titrated (0.5 mol/l HCl) into the colorless. Blank (mineralized water in place of sample) was used for determination of saccharides in hen yolk and hen albumen. Protein assays were always performed in triplicates for result verification. Calculation of the amounts of lipids was based on saponification value (Eq. 1). SSSSSSSSSSSSSSSSSSSSSSSSSSSS vvvvvvvvvv = (aa bb). cc HHHHHH. MM KKKKKK mm a consumption of HCl for blank (ml) b consumption of HCl for sample (ml) c HCl concentration of HCl (mol/l) M KOH molar weight of KOH (g/mol) m weight of sample for determination of saponification value (g) Statistical analyses Statistical analyses of proteins content in albumen eggs and yolk eggs were made using one-way analysis of variance (ANOVA) and statistical significance was declared when p value was equal to or less than RESULTS AND DISCUSSION A total of 180 hen eggs were analysed (90 from each period). Determination of proteins in yolk and albumen for different hens breed Amount of proteins in hen yolk and hen albumen are shown in Figure 1. (1) 682 P age

3 Figure 1 Amount of proteins in hen yolk and hen albumen for different hens breed Legend: Data are presented as mean ±SE The amount of proteins in hen yolk (12.60% for Hybrid Isa Brown, 14.07% mg/ml for Hybrid Hisex Brown) were not statistically different (p<0.05) between both breeds. The amount of proteins in hen albumen for Hybrid Hisex Brown (9.40%) were statistically higher (p<0.05) than these those in albumen eggs for Hybrid Isa Brown (5.75%). Higher amount of proteins was in hen yolk than in hen albumen in both breeds. Studies (Salakova 2014, Yamamoto et al. 1997, Segura-Campos et al. 2013) demonstrated higher content of proteins in yolk egg than in albumen egg. Determination of saccharides in yolk and albumen for different hens breed Amount of saccharides in hen yolk and hen albumen have been shown in Figure 2. Figure 2 Amount of saccharides in hen yolk and hen albumen for different hens breed Legend: Data are presented as mean ±SE The amount of saccharides in hen yolk (1.88% for Hybrid Isa Brown, 1.87% for Hybrid Hisex Brown) and hen albumen (0.96% for Hybrid Isa Brown, 0.94% for Hybrid Hisex Brown) were not statistically different (p<0.05) between breeds. The same amount of saccharides may be caused by the storage time, because amount of saccharides was higher in yolk (1.89%) than in albumen (1.01%) for first day of experiment. On the other hand, amount of saccharides was higher in albumen (3.66%) than in yolk (1.50%) for last day of experiment. Salakova 2014 and Simenovova et al presented higher amount of saccharides in yolk than2 in albumen. Determination of lipids in yolk and albumen for different hen breeds Amount of lipids in hen yolk and hen albumen are shown in Figure 3. Figure 3 Amount of lipids in hen yolk and hen albumen for different hens breed 683 P age

4 Legend:Data are presented as mean ±SE The amount of lipids in hen yolk (40.35%for Hybrid Isa Brown, 40.91% for Hybrid Hisex Brown) and hen albumen (2.63% for Hybrid Isa Brown, 3.05% for Hybrid Hisex Brown) were not statistically different (p<0.05) between breeds. Higher amount of lipids was in hen yolk than in hen albumen. Aquino and Silva 2010 and Segura-Campos et al presented the same results showing the higher amount of lipids in egg yolk than in egg albumen. Amount of analysed f in hen yolk and hen albumen during storage time Correlation was used for evaluation results between the amount components (proteins, lipids, saccharides) of the selected egg. Correlations a between amounts of analysed components (proteins, saccharides and lipids) are shown in Figure 4 (A, B, C, D, E, F) Figure 4 Correlation between amount of proteins (A Hybrid Isa Brown, B Hybrid Hisex Brown), correlation between amount of saccharides (C Hybrid Isa Brown, D Hybrid Hisex Brown) and correlation between amount of lipids (E Hybrid Isa Brown, F Hybrid Hisex Brown) - n = 9 Legend: n = number of supply days Pearson correlation coefficient was used for comparison correlation between amount of analysed components (proteins, saccharides and lipid) in hen albumen and amount of analysed components (protein, saccharides and lipid) in hen yolk during storage time. Pearson correlation coefficient was 0.91 for amount of lipids in Hybrid Isa Brown eggs. For Hybrid Hisex Brown, Pearson correlation coefficient was Pearson correlation coefficient was low for amount of saccharides in hen eggs (Hybrid Isa Brown r = 0.74, Hybrid Hisex Brown r = 0.76). For amount of lipids, Pearson correlation coefficient was lower than for amount of saccharides (Hybrid Isa Brown r = 0.06, Hybrid Hisex Brown r = -0.33). We can say that amount of analysed components in hen yolk were not correlated with amount of analysed components in hen albumen. If amount of analysed folders was high in hen yolk, amount of analysed folders was low in hen albumen. Studies (Salakova 2014, Simeonovova et al. 1997, Aquino et al. 2010, Segura-Campos et al. 2013) presented that amount 684 P age

5 of analysed components (proteins, saccharides and lipids) in hen albumen decreases when amount of analysed components (proteins, saccharides and lipids) is increasing in hen yolk, and conversely. CONCLUSION Our results presented the differences in the amount of analysed components (proteins, saccharides and lipids) in hen yolk and hen albumen of different hen breeds (Hybrid Isa Brown and Hybrid Hisex Brown) and correlation between amount of analysed components (proteins, saccharides and lipid) in hen albumen and amount of analysed components (proteins, saccharides and lipid) in hen yolk during storage time. Differences in the amount of analysed components were not found between Hybrid Isa Brown and Hybrid Hisex Brown. Correlations were not found between amounts of analysed components in hen yolk and amount of analysed components in hen albumen. If amount of analysed components was higher in hen yolk, amount of analysed components was lower in hen albumen, and conversely. ACKNOWLEDGEMENT This research was supported by project TP 6/2015 Impact loading of agricultural products and foodstuffs financed by Internal Grand Agency FA MENDELU. REFERENCES Aquino, J., Silva, J.A Total lipids, cholesterol and fatty acids composition of ostrich eggs: a methodological approach. Revista do Instituto Adolfo Lutz [Online], 69(4): Available at: [ ]. Horbanczuk, J.O., Sales, J., Ziebra, G., Reklewski, T., Celeda, T., Kozaczynski, K Lipid cholesterol content and fatty acid composition of ostrich eggs as influenced by subspecies. Arch Geflügelkunde [Online], 63(5): Available at: [ ]. Janairo, G., Sy, M.L., Yap, L., Llanos-Lazaro, N., Robles, J Determination of the sensitivity range of biuret test for undergraduate biochemistry experiments. Journal of Science & Technology [Online], 5(6): Available at: [ ]. Kritchevsky, S.B A review of scientific research and recommendations regarding eggs. The Journal of the American College of Nutrition [Online], 23(6): Available at: [ ]. Machado, F.F., Coimbra, J.S.R., Rojas, E.E.G., Minim, L.A., Oliveira, F.C., Sousa, R.C.S Solubility and density of egg white proteins: Effect of ph and saline concentration. LWT-Food Science and Technology [Online], 40(7): Available at: [ ]. Noble, J.E., Bailey, M.J. A Champer 8 Quantitation of protein. Methods in Enzymology [Online], 463: Available at: [ ]. Okutucu, B., Dınçer, A., Habib, Ö., Zıhnıoglu, F Comparison of five methods for determination of total plasma protein concentration. Journal of Biochemical and Biophysical Methods [Online], 70(5): Available at: 22X [ ]. Salakova, A Hygiena a technologie drubeze, vajec a zveriny. Brno: VFU Brno. Segura-Campos, M., Perez-Hernandez, R., Chel-Guerrero, L., Castellanos-Ruelas, A., Gallegos- Tintore, S., Betancur-Ancona, D Physicochemical and functional properties of dehydrated Japanese quail (Coturnix japonica) egg white. Food and Nutrition Sciences [Online], 4(3): Available at: [ ]. 685 P age

6 Simeonovova, J. et al Technologie drubeze, vajec a minoritnich zivocisnych produktu. Brno: MZLU. Yamamoto, T., Juneja, L.R., Hatta, H Hen Eggs. USA: CRC Press LLC. 686 P age