Egg quality and isoflavone deposition due to dietary inclusion of isoflavone soy sauce by-product (ISSBP) in laying hens

The objective of the study was to evaluate the quality and deposition of isoflavones in the egg due to dietary inclusion of isoflavone soy sauce by-product (ISSBP) in laying hens. Experimental animals were 480 birds of 20-week old laying hens of Isa Brown strain, with initial body weight of 1,754 ± 42 g. The experiment was arranged in a completely randomized design (CRD), with four treatments and six replications (20 birds each). The treatments were dietary inclusion levels of isoflavone soy sauce by-product as follows: ISSBP0 = without isoflavones, ISSBP40 = 40 mg/100g, ISSBP80 = 80 mg/100g, and ISSBP120 = 120 mg/100 g feed. Dietary treatments were given for 10 weeks from week 20 until 30-week old. Parameters observed were egg cholesterol and isoflavone isomers in feed, blood, and eggs. Data of total cholesterol and isoflavone in the egg were statistically analysed and isoflavone isomer concentration in feed, blood, and egg were descriptively described. The results showed that feeding ISSBP significantly (P <0.05) decreased cholesterol and increased isoflavones in egg. Total cholesterol content in egg decreased up to 33.8%. However, isoflavones in the yolk were higher (28.9 mg/g) than those in blood (13.75 mg/g), and those deposited into the yolks indicated better quality because containing more aglicons isomers (87.5%), than those in feed (52%) and blood (68.4%). In conclusion, dietary inclusion of ISSBP to laying hens’s decreased cholesterol content and increased isoflavones deposition into the egg with better quality, so that the eggs can function as functional food.

and ISSBP120 = 120 mg/100 g feed. Dietary treatments were given for 10 weeks from week 20 until 30-

INTRODUCTION
Poultry is known to have excellent bioconvertion properties, such as the ability to convert feed components into a good food, egg or meat, and is beneficial for human growth and health. Therefore, egg could be designed to improve the nutritional compounds to become a functional food that is beneficial for humans. Isoflavones in addition to functioning as antioxidants (Akdemir and Sahin, 2009;Yang et al., 2011;Ni et al., 2012) and phytoestrogens (Ni et al., 2012;Shi et al., 2013;Elkomy and Elghalid, 2014), they could also be used to improve the quality of poultry meat (Jiang et al., 2014), and egg production (Cai et al. 2013;. Isoflavone could be transferred into the tissues of meat and eggs that is beneficial for human health (Markovic et al., 2015).
Research on feeding isoflavone from soy sauce by-product [ISSBP] to laying chicken in term of its deposition in egg and its quality has never been previously elucidated. This study is important for clarifying isoflavone soy sauce byproduct that has many advantages over other types of isoflavones as well as higher aglycone content. Soybean has glycoside content of 97.33%, while aglycon was only 2.67%, but after fermentation for 48 hours glycoside content decreased to 24.49% while aglycon increased to 75.51% (Silva et al., 2011). Increased isoplavone and active peptide aglycons have more health benefits (Hong et al., 2011).
The present study was conducted to evaluate feeding effect of ISSBP in laying chicken in relation to total cholesterol content and isoflavone deposition in yolk. The results of this study are expected to provide a new information concerning the development of poultry nutrition and feeding to produce the designed-egg rich in isoflavon antioxidant as functional food.

Experimental Animal and Diet
Experimental animals were 20-week old of 480 laying hens of Isa Brown strain with initial body weight of 1,754 ± 42 g, and were placed randomly in battery cages. Feed was composed of corn gluten meal, distillery dried grain with soluble, fish meal, meat bone meal, yellow corn, rice bran, vegetable oil, oyster shell, methionine and soy sauce by-product. The composition and nutrients content of experimental feed is presnted in Table 1.

Experimental Prosedures
The present research was conducted by experimental method, using completely randomized design, with 4 treatments and 6 replications with 20 birds each. Inclusion levels of soy sauce by-product were created as dietary treatment as follows: ISSBP0 : Control feed without isoflavon soy sauce by-product (ISSBP)

ISSBP40
: Feed contains ISSBP 40 mg/100 g; or equal to5.8% soy sauce byproduct ISSBP80 : Feed contains ISSBP 80 mg/100 g; or equal to 11.6% soy sauce by-product ISSBPP120 Feed contains ISSBP 120 mg/100 g; or equal to 17.4% soy sauce by-product Parameters observed were cholesterol and the distribution of isoflavone isomers in feed, blood and eggs.

Sampling Method and Analysis
After the chickens were provided dietary treatment for 10 weeks, 2.5 mL blood sample was taken on day 70 from one bird in each replication. Blood was taken through the veins of the wings, and collected in EDTA-containing test tube for isoflavones analysis. Isoflavones in egg was determined in a mixture sampels of egg white and yolk. Quantitative analysis of isoflavones was performed using high performance liquid chromatography (HPLC) according to the modified procedure of Harborne (1992), and total cholesterol analysis based on the method of Kleiner and Dotti (1962). One egg was taken from each replication unit.

Statistical Analysis
Data of total cholesterol, and isoflavone isomer in the egg were statistically analysed by analysis of variance and were continued to least significant different (LSD) test (Steel and Torrie, 1991). Isoflavone isomer concentration in feed, blood, and egg were descriptively analyzed.

Isoflavones Profiles in Feed, Blood and Eggs
Feeding isoflavones soy sauce by-product Isoflavone Soy Sauce by-product (ISSBP) for Laying Hens (A. Malik et al.) 189 *ISSBP0 = feed without isoflavones **Based on the laboratory analysis value of respective ingredients used for feed composition *** Calculated based on the formula of Carpenter and Clegg (1965) (ISSBP) indicated different effect on the isoflavones profiles in feed, blood, and eggs. Isoflavones analysis in feed, blood and eggs indicated varied content as presented in Table 2.
Results of the present study suggested that the transportation of isoflavones depending on the feed provided, and some interesting findings were found. First, in blood and egg samples of treatment without ISSBP inclusion (0 mg) did not find isoflavones at all either in whole form or isomers (Table 2, and Figure 1). Control feed (ISSBP0) did not composed of ingredients derived from the type of beans in general or soybean meal in particular, as the source of isoflavones. Second, in the contrary, isoflavones content in eggs yolk dramatically revealed the increasing values when the birds were given dietary inclusion of ISSBP (ISSBP40 until ISSBP120), even the treatments of feeding ISSBP at 80 mg/100g (ISSBP80) resulted significantly highest value both in whole form as well as in its isomers.

Egg Cholesterol
Total cholesterol in egg was significantly (P<0.05) decreased due to dietary inclusion of ISSBP. The highest cholesterol content of egg was found in group of laying hens provided control feed (Table 2 and Figure 2). When compared to control group (ISSBP0), feeding ISSBP40, ISSBP80, and ISSBP120 decreased egg cholesterol by 22.8, 33.8, and 29.9%, respectively. This condition indicated that ISSBPP was  Isoflavones derived from soybean in general serve as phytoestrogens source which are known to have weak estrogenic hormone activity due to their structure is similar to βestradiol. They can preferentially bind to estrogen receptor and mimicking the effects of estrogen in some tissues in one side, and blocking the effects of estrogen in other side. Due to its mode of action as eye scissors in which estrogenic effects in other tissues could help to maintain bone mineral density and improved egg production and shell quality (Markovic et al., 2015). On the other hand, because of their estrogenic blocking effects could improved blood lipid profiles and attributable to an impact on the decreased egg cholesterol found in the present study. Biochemical mechanism can be explained from the antagonistic activity of phytoestrogenes to depress endogenous estrogen produced by the developing follicle of the hens. This lead to the suppression of activity of HMG-CoA reductase enzyme which brought about the inhibition of cholesterol biosynthesis activity, consequently fewer endogenous cholesterol transported into the ovaries, but more were excreted through feces and urine (König et al., 2007;Vakili and Heravi, 2016).

Isoflavone Deposition in Eggs
Providing feed containing ISSBP significantly (P<0.05) increased yolk isoflavone content with the highest value was in ISSBP80 treatment, while hens fed control feed produced eggs without isoflavones. This phenomenon suggested that ISSBP in the feed can be transferred into the yolk depending on the level. This result was consistent with the report of Lin et al. (2004) who indicated that isoflavone genestein supplementation in quail feed increased the content of isoflavones in the yolk.The transportation mechanism of isoflavones into egg yolk have been clarified in connection with their changed into a conjugated form (Saitoh et al., 2004). Isoflavones were predicted to reduce by 30% during metabolism and approximately 70% were deposited into egg yolk in a conjugated form. Isoflavones changed to a conjugated form, a soluble isoflavones, causing it much easier to be transfered into egg yolk. Previous studies (Akdemir and Sahin, 2009;Lin et al., 2004) reported that isoflavone in egg yolk of quail increased due to dietary supplementation of soy isoflavones because the presence of genistein isomer form can be easier to be transferred into the yolk (Saitoh et al., 2004).This mechanism was likely seem to that of phytoestrogenes of ISSBP stimulated the liver to produce neutral fats and phospholipids to function as carrying substances. Therefore, conjugated form is absolutely possible to be constructed and carried by the blood into the ovaries for egg yolks formation.
The deposition rate of isoflavones in egg yolk which is mostly in the form of aglycone, was 87.6, 85.7, and 89.8% in ISSBP40, ISSBP80 and ISSBP120 treatments, respectively, and the remainder were isoflavones glycosides (Figure 3).

Figure 2. Cholesterol and Isoflavone in Yolk
A very high percentage of aglycone content in yolk when compared to isoflavones glycosides is assumed to be attributable to three factors. First, isoflavones in feed consisted of more aglycone around 54.21%, while glycoside only 12.21%, from total isoflavones content. Second, biotransformation process occured in the digestive tract brought about the change in glycoside into aglycone form. Third, isoflavones in the form of aglycone have higher absorptive rate than those in the form of glycosides. High aglycone concentration in egg yolk found in the present study is supported by the finding of Sanz and Luyten (2006) that the average absortive rate of aglycone (daidzein and genistein) was 83.5% while that of glycosides (daizine and genestin) was only 60%.
Isoflavones underwent a series of biotransformation process indicated by the difference in the number and composition of isoflavone isomers among feed, blood, and yolk ( Figure 1). The presence of isoflavones in feed were in four forms, namely glycosides, aglycons, malonilglycosides, and acetylglycosides, whereas in the blood and egg yolk only isoflavone glycosides (genistin and daidzin) and aglycons (genetein and daidzain). The form of isoflavones in the blood was 68.36% the type of aglycons   (genistein 31.27% and daidzein 37.09%) and that in egg yolk was equal to 87.45% (genistein 41.19% and daidzein 46.26%), these concentrations were higher than that in feed was 49.52% (genistein 25.08% and daidzein 18.25% (Figure 4). While isoflavones in the digestive tract, they still underwent biotransformation process (Turner et al., 2004) that could change their form of malonilglycosides and acetylglycosides into glycosides and aglycons. This transformation occurs due to the fermentation process under the influence of βglycosidase enzyme produced by intestinal microorganism. Similar phenomenon indicated that the glicitein isomer and its derivatives could be changed into other forms during the transformation process underwent in the chicken's body. Glicitin isomer and its derivatives were the most easily transformed group of isomers, during the processing of soybeans into soy sauce, and the change was also occured during transit time in the small intestine due to transformation.

CONCLUSION
Dietary inclusion of isoflavones soy sauce by-product (ISSBP) in laying hens, produce lower egg cholesterol content and rich in isoflavones dominated by aglycone, so that the eggs can function as functional food.