Cholesterol Content and Fatty Acids Profile in Conventional and Omega-3 Enriched Eggs

This study aimed to produce table eggs enriched with n-3 polyunsaturated fatty acids by diet modification and at the same time, determine the fatty acid profile and cholesterol content in egg yolk, and compare them with that of conventionally


INTRODUCTION
Eggs are a source of protein, fat and micro-nutrients having a significant role in the basic human diet.Ceylan et al. (2011) reported that eggs contain 195-230 mg of cholesterol in average, a compound that is linked to an atherosclerosis development.That is why some people do not want to consume eggs.However, eggs are a valuable source of essential amino acids and other biological ingredients that, apart from nutrition, also have a biological function or effect physiological processes (Mazalli et al., 2004;Laca et al., 2010;Liu et al., 2010;Kralik et al., 2020).Eggs from conventional farming are poor in α-linolenic acid (ALA, 18:3n3), and contain neither eicosapentaenoic (EPA,20:5n3) nor docosahexaenoic (DHA, C20: 6n3) fatty acids (Souza et al., 2008).In the last decade, beneficial changes have been achieved in egg yolks fatty acid profile by adding fish and flaxseed oil or seeds to laying hens (Cachaldora et al., 2006).In detail, n-6/n-3 PUFA ratio in designed eggs (canola and fish oil) is reduced to the desired range of 4 to 10, in comparison with 35 in regular eggs (sunflower oil) (Mazalli et al., 2004).Cholesterol in eggs is often noted as a debatable ingredient.There are several researches that have been successful in efforts to reduce egg yolks cholesterol by using modified diets in the laying hens feeding (Sari et al., 2002;Sujatha & Narahari, 2011;Promila et al., 2017).There are data in the literature that state that the use of linseed eRBCA-2020-1412 eRBCA-2020-1412 oil and seeds, as well as fish oil in diets, increases the levels of n-3 polyunsaturated fatty acids in egg yolks and reduces cholesterol content at the same time (Basmacioglu et al., 2003;Loh et al., 2009;Mattioli et al., 2016).The aforementioned authors assume that the egg yolks cholesterol content is related to the n-3 polyunsaturated fatty acids (n-3 PUFA) concentration.Namely, the aim of this research was to design a laying hens diet supplemented with fish, rapeseed and flaxseed oil, leading to the production of eggs enriched with omega 3 fatty acids.Also, the aim was to determine the effect of this diet on fatty acid profiles and cholesterol content of the enriched eggs.

Animals and diets
All procedures involving work with animals used in the research were carried out according to protocols approved by the Bioethics Committee for Animal Research, Faculty of Agrobiotechnical Sciences Osijek, Josip Juraj Strosmayer University of Osijek.The study was conducted on a total of 80 laying hens of Tetra SL hybrids being 31 weeks old at the beginning of the study.The hens were randomly allocated into 2 experimental groups (conventional-control (C) and n-3 PUFA -experimental (E)), and each group was further divided into 5 replicants of 8 hens in enriched cages.The laying hens were kept in the same microclimatic conditions while feed and water were provided ad libitum.The diets for laying hens differed in the oil content.The C group of laying hens consumed conventional diet with 5% soybean oil whereas the E group of hens fed diet with 1.5% fish oil, 1.5% rapeseed oil and 2% linseed oil.Table 1 shows the diets composition, and Table 2 the fatty acids profile in diets, which were analyzed in triplicate.The eggs were sampled for the fatty acids and cholesterol analysis after 5 weeks of feeding the laying hens.For the purposes of these analyses, 40 eggs were collected, i.e. 20 eggs per group of laying hens (10 eggs for fatty acid analysis, 10 eggs for cholesterol analysis).

Fatty Acids Analyses
The fatty acid profile in egg yolk and diets was analyzed as follows: the fat of the homogenized samples was extracted using the method of Folch et al. (1957) All solvents used were of ultrapure grade from Sigma-Aldrich (Schnelldorf, Germany).Butylated hydroxytoluene (100 mg/L) was added to the extraction mixture (chloroform: methanol,

Cholesterol Analysis
Cholesterol content was determined by the modified method of Albuquerque et al. (2016) as follows: 5 mL of 0.4 M KOH in ethanol were added to 0.5 g of egg yolk and the solution was shaked well in a vortex.The samples were incubated in a 50 °C water bath for 30 minutes.After cooling at room temperature, the cholesterol extraction was done twice with 10 mL of n-hexane.The extracts were combined and an aliquot of 3 mL was dried and replenished with 3 mL of mobile phase.Shimazdu HPLC system equipped with UV-VIS detector SPD-10AV VP and SIL-10AD VP auto-injector, Shimadzu Shim-pack GIST (250 x 4.6 mm I.D., 5 µm particle size) column were used for separation and quantification of cholesterol.The mobile phase was the solution of isopropanol: acetonitrile (50:50 v/v).Prior using, the mobile phase was filtered through a 0.20 µm membrane filter and degassed in an ultrasonic bath.The column temperature was 37 o C, the flow rate 1.2 mL/min and the injected volume 10 µL.Cholesterol was detected using a UV-VIS detector set at a wavelength of 210 nm.Total analysis time was 10 minutes.

Statistical Analyses
The research results were processed using TIBC Statistica™ version 13.4.0.14.(Soft Inc., © 1984-2018).Descriptive statistics and analysis of variance were implemented (ANOVA).If the P value for the analysis of variance was statistically significant, differences between the groups were tested by the Fisher LSD test.

RESULTS
The fatty acid profile of C and E diets is shown in Table 2.The main differences were found in the proportion of MUFA, n-6 PUFA and n-3 PUFA (p<0.05).The n-6/n-3 PUFA ratio was 3.14 to 10.41 i.e. three times lower (p<0.05).Table 3 shows the effect of feeding treatment on the fatty acid profile in the yolks of both C and E eggs.Designing the hens diet by using a mixture of oils (fish, flaxseed and rapeseed oil) increased the content of ALA, EPA and DHA in hen diet which resulted in a beneficial fatty acid profile in the yolks of E group eggs compared to conventionally produced eggs.Conventional eggs contained more palmitic acid (C16:0) and total saturated fatty acids (ΣSFA, p<0.001).Enriched eggs with n-3 PUFAs contained more heptadecenoic acid (C17:1, p<0.05) and less palmitoleic (C 16:1, p<0.001).Differences in MUFA between the egg groups were not statistically significant (p>0.05).The arachidonic content (C20:4n6) was higher in the C than in the E eggs (p<0.01)whereas the difference between the groups in Σn-6 PUFA was not statistically significant either (p>0.05).

Cholesterol Content and Fatty Acids Profile in Conventional and Omega-3 Enriched Eggs
A significant difference was found between E and C group of eggs in the content of ALA, EPA and DHA (p<0.001).The content of Σn-3 PUFA in E group was 5.7 times higher than in the C group (p<0.01).The n-6/n-3 PUFA ratio was significantly more favorable in the E (5.11) compared to C (23.73).
Figure 1 shows the content of cholesterol in the E and C group of eggs (mg/100 g of egg yolk).Eggs supplemented with n-3 PUFA contained on average 1179 mg and control eggs 1287 mg cholesterol per 100 g of yolk.According to our results the eggs enriched with n-3 PUFAs contained less cholesterol than the control eggs by 108.21 mg/100 g of egg yolk i.e. 8.4%, although this difference was not significant (p>0.05).

DISCUSSION
Omega-3 fatty acids are desirable in the functional products food sector.This has prompted egg producers to increase the content of omega-3 fatty acids in table eggs, a very important component of the food chain, by using innovative technologies.The content of some fatty acids in diets, especially of the long chain was affected by the type and amount of added oils.Although the diet for conventional egg production did not contain EPA and DHA (Table 2), these fatty acids were found in the eggs lipids of this group (Table 3).The reason for this is that hens can synthesize EPA and DHA in limited amounts if there is enough ALA in the feed.This has been affirmed by our former researches (Kralik et al., 2008;Kralik et al., 2017).By adding the fish oil in the diet, containing EPA and DHA as well as rapeseed and linseed oils, being the important ALA sources, are reabsorbed from the feed by the laying hens.On the other hand they formed long chain n-3 PUFAs via their metabolism through the elongation and desaturation processes depositing them in the egg yolks, a fact that is in line with Yang et al. (2000) findings.Increasing of blood LD cholesterol content by consuming food rich in saturated fatty acids is the risk factor for cardiovascular diseases occurance in humans.During the fatty acid profile analysis in the egg yolks of our investigated groups it was noticed that palmitic, stearic and oleic saturated fatty acids were mostly present.Palmitic fatty acid is generally believed to raise blood cholesterol more than stearic fatty acid (Fattore et al. 2014).Thus, the combination of oils used in laying hens diets of the omega-3 PUFA group in our study is justified since the eggs yolk of this group significantly (p<0.001)reduces the palmitic fatty acid proportion compared to conventionally produced eggs.In our study, the arachidonic fatty acid content in the E group was significantly reduced (p<0.05)compared to eggs from C group (E = 1.30 % i.e.C = 1.70 %).This was also stated by Baucells et al. (2000) and Cachaldora et al. (2006).The phenomenon is attributed to the greater use of Δ-6 saturase in the synthesis of n-3 PUFA compared to n-6 PUFA.This enzyme acts in both directions (Ayerza and Coates, 2000) and there is competition between n-3 PUFAs and n-6 PUFAs in their biosynthesis (Mazalli et al. 2004).High levels of ALA limit the synthesis of arachidonic fatty acid from linoleic since ALA competes with linoleic for the same enzyme Δ-6 desaturase as reported by Ceylan et al. (2011).These authors consider that the fish oil addition into the laying hens diets reduces the synthesis of arachidonic from linoleic fatty acid.They also state that EPA and DHA can reduce the arachidonic fatty acid production.The observations of these authors are also noticed in our study, because in eggs from E group, where laying hens consumed a diet with the addition of fish, flaxseed and rapeseed oil combination, the content of arachidonic fatty acid decreased with increasing ALA.The results of the fatty acid profiles analysis (Table 3) showed a statistically significant lower ∑SFA content in yolk lipids of eggs from E group compared to the C group (30.55 : 35.80 %, p<0.001).However, differences in the content of ∑MUFA and ∑n-6 PUFAs between the egg groups were not statistically significant (p>0.05).
The trend of cholesterol reduction in eggs enriched with n-3 PUFA was also shown by the results of Loh et al. (2009) and Promila et al. (2017).The results of our study also show lower cholesterol content in n-3 PUFA eggs compared to the eggs from the conventional production, although differences were not significant (p>0.05).Sari et al. (2002) found that the addition of flaxseed to laying hens increased the n-3 PUFA content linearly (p<0.001), the n-6/n-3 PUFA ratio was decreased, as did the cholesterol level (p<0.05).Similar results were obtained by Promila et al. (2017) using flaxseed oil in diets for laying hens.They found a reduction in cholesterol of 13.57-12.05mg/g yolk (p<0.05), an increase in n-3 PUFA and a decrease in n-6/n-3 PUFA ratio from 8.46 to 1.79.Since the designed diet in our study had a combination of three oils, one of which was flaxseed, we can assume that contributed to ALA increase and thus reduced the n-6/n-3 PUFA ratio in n-3 PUFA eggs.
Ansari et al. ( 2010) found that linseed oil enriched with copper resulted in the cholesterol reduction (p<0.05).At the same time, the SFA content was reduced and n-3 PUFA content increased, especially ALA.Basmacioglu et al. (2003) used fish oil in combination with linseed in laying hens feeding.They found lower levels of yolk cholesterol in enriched eggs compared to the control ones (12.56 and 12.97: 13.71 mg/g).Our results are consistent with the results of the aforementioned authors for the content of SFA and cholesterol in the yolks of eggs from E group whereas the content of total n-3 PUFA in these eggs increased.
Poultry are capable of producing 10 times more cholesterol per kg of liver than humans.Due to the intensive production of cholesterol, treatments related to the reduction of cholesterol in eggs through feeding are not always effective (Faitarone et al., 2013).According to Shafey & Cham (1994) cholesterol in eggs is a change resistant, and essential for embryo development.Milinks et al. (2003) and Mattioli et al. (2016) stated that the cholesterol modulation in the yolk depends on feeding, i.e. concentration of the structural ingredients in eggs.However, laying hens are capable of changing the profile of polyunsaturated fatty acids relative to the source of fat in diet.Unlike mammals poultry can absorb fat and transport it through the portal bloodstream to the liver where lipogenesis takes place (Van Elswyk et al., 1994).Unlike the abovementioned authors, the addition of vegetable oils to laying hens diets cannot reduce eggs cholesterol content (Faitarone et al., 2013).Hargis (1988) pointed out that the cholesterol content of eggs can be altered up to 25% by the diet.Addition of copper at 150-250 mg/kg in the diets of laying hens resulted in the insignificant cholesterol content decrease in eggs (Balevi & Coskun, 2004).Eliat-Adar et al. (2013) considered that egg cholesterol has a very limited effect on blood cholesterol content.Keum et al. (2018) did not find that a fat source (lard, flaxseed oil or conjugated linolic acid -CLA) affected the egg yolks cholesterol content.However, CLA lowers cholesterol in eggs if added in laying hens diet (Yin et al., 2008).From the aforementioned studies, it can be concluded that the researchers' opinions in terms of the effect of feeding the designed diets on the fatty acid profile are consistent (omega-3 fatty acids increase and the n-6/n3 PUFA ratio decreases).However, they differ in cholesterol content.Today, the lifestyle of people often involves the so-called fast food consumption being adverse from a nutritional point of view as it is rich in saturated fatty acids and has an unfavorable n-6/n-3 PUFA ratio.All the above mentioned affects the occurrence of various diseases of the cardiovascular system (Simopoulos, 2002).Simopoulos (2009) pointed out that the n-6/n-3 PUFA ratio is significant for the cardiovascular diseases prevention recommending 4:1 ratio.An acceptable 3:1 n-6/n-3 PUFA ratio by up to 5:1 was stated by Griffin (2008).The American Heart Association (AHA) recommended that n-6 PUFAs participate with 5-10% of total meal energy consumed (Harris et al., 2009).Some authors recommend reducing n-6 PUFA consumption while others recommend increasing n-3 PUFA consumption, especially EPA and DHA (Candela et al., 2011).
In our research, eggs from E group contained 4.73% and eggs from C group 0.82% of the n-3 PUFA in total fatty acids (p<0.001).The n-6/n-3 PUFA ratio in E eggs was 5.11 and C eggs 23.73 i.e. 4.6 times higher (p<0.001).At the same time the E eggs contained cholesterol of 1179 mg/100 g of yolk and C eggs of 1287 mg/100 g of yolk (p>0.05),indicating a numerical decrease by 8.4%.The analysis of all results justifies the use of this diet aiming to produce eggs enriched with n-3 PUFA that have a more favourable fatty acid profile.
/v) as an antioxidant.Subsequently, fatty acidcontaining lipids were transmethylated by the basecatalyzed sodium methoxide.Gas chromatography was conducted on a Bruker 430-GC apparatus (Billerica, MA, USA), equipped with a FAMEWAX (RESTEK, Bellefonte, PA, USA) type capillary column (30 m x 0.32 mm internal diameter, 0.25 μm film) and flame ionization detector.The characteristic operating conditions were as follows: temperature injector, 220 °C; temperature detector, 230 °C; helium flow, 25 ml/ min.The oven temperature was graded from 50 to 225 °C at 6 °C/min and held for 21 min at 225 °C.A fatty acid standard mixture (Supelco 37 Component FAME Mix, SUPELCO® Analytical, Bellefonte, PA, USA) was used to identify the individual fatty acids in the chromatogram.Portions of individual and total fatty acids were shown as the percentage of total fatty acids in the diets, and in eggs in mg/100 g edible part.

Table 2 -
Fatty acids composition in diets (% FA in total fatty acids, x ± s).

Table 1 -
Composition and chemical analysis of basic diet.

Table 3 -
Effect of feeding treatment on fatty acid profile in yolks of both C and E eggs (% FA in total fatty acids, x ± s).