Dietary Oregano (Origanum Onites) Maintained the Antioxidant Capacity and Haugh Unit, but Not Yolk Color of Stored Eggs up to 30 Days

Original Article Acta Veterinaria Eurasia 2021; 47(2): 76-81

Dietary Oregano (Origanum onites) Maintained the Antioxidant Capacity and Haugh Unit, but not Yolk Color of Stored Eggs up to 30 Days

Eyüp Eren GÜLTEPE1 , Aamir IQBAL1,2 , Cangir UYARLAR1 , Abdur RAHMAN3 , Ümit ÖZÇINAR1 , İbrahim Sadi ÇETİNGÜL1 , İsmail BAYRAM1 1Department of Animal Nutrition and Nutritional Diseases, Afyon Kocatepe University, Faculty of Veterinary Medicine, Afyonkarahisar, Turkey 2Facultyof Veterinary and Animal Science, University of Gomal, Dera Ismail Khan, Pakistan 3Department of Animal Sciences, College of Veterinary and Animal Sciences, University of Veterinary and Animal Sciences, Jhang, Pakistan Cite this article as: Gültepe, E. E., Iqbal, A., Uyarlar, C., Rahman, A., Özçınar, Ü., Çetingül, İ. S., & Bayram, İ. (2021) Dietary Oregano (Origanum onites) Maintained the Antioxidant Capacity and Haugh Unit, but not Yolk Color of Stored Eggs up to 30 Days. Acta Veterinaria Eurasia, 47(2), 76-81.

ORCID IDs of the authors: E.E.G. 0000-0002-2404-1232, A.I. 0000-0003-4473-2329, C.U. 0000-0002-7803-2454, I.S.C. 0000-0002-7608-6176,U.O. 0000-0002-1143-1215, I.B. 0000-0002-9993-7092,A.R. 0000-0002-3440-8106. Abstract

This study aimed to investigate the effects of dietary orega- fect eggshell breaking strength. On the 30th day, egg weight no leaves on the shelf-life of stored eggs for up to 30 days. A (p < .05) decreased in the 5% oregano group and Haugh unit total of 36 Babcock White laying hen hens were divided into increased (p < .05) in the 10% oregano group compared with 3 groups with 4 subgroups in each and fed diets containing the other groups. The yolk color in the treatment groups was 0%, 5%, and 10% oregano for 30 days. At the end of the study, lighter than the control (p < .05). Oregano did not affect the 8 eggs from each group were analyzed on the same day wit- TOS of stored eggs; however, the TAC was still high (p < .05) on th hout storage. The remaining eggs were stored at +4°C for 10, the 30 day. Therefore, dietary oregano leaves can be used to 20, and 30 days. Stored eggs were analyzed for egg traits, to- extend the storage time of eggs. tal antioxidant capacity (TAC), and oxidant status (TOS). The Keywords: Turkish oregano, thyme, total antioxidant status, results of this study showed that dietary oregano did not af- shelf-life

Introduction and Spain (Ozkan et al., 2010). It is a rich antioxidant source because of abundant thymol and carvacrol composition in addition The poultry industry plays a key role in the global economy, to some p-cymene and γ-terpinene content (Cimrin et al., 2019; and egg is a cheap source of available protein. The shelf-life of Ozkan et al., 2010). The potential antimicrobial and anticoccidi- the stored table egg is still a big challenge for global market- al effects of dietary thymol and carvacrol essential oils are well ing and public health (Yuceer & Caner, 2014). After laying the described and their antioxidant effects on poultry (Zhai et al., eggs, the external and internal quality of eggs decreases over 2018). Although the molecular structures of thymol and carvac- time, although it depends on storage conditions (Cetingul et rol are very similar to each other, different antimicrobial powers al., 2019; Cimrin et al., 2019; Gultepe et al., 2018; Rahman et al., are recorded for each of them (Michiels et al., 2009). Interestingly, 2017; Samli et al., 2005). The table egg includes not only abun- Bassole and Juliani (2012) have reported that their combination dant protein but also high phospholipid composition. There- had a higher synergistic antimicrobial effect than each other’s fore, lipid oxidation is one of the important challenges during individual effect. Furthermore, p-cymene has no antimicrobial storage owing to negative effects on egg nutritional quality, effect alone; however, it supports the mode of action of carvac- taste, flavor, and yolk color (Omri et al., 2019). rol via transporting it across the cell membrane (Oke et al., 2009). Therefore, we hypothesized that abundant active ingredients in Oregano (Origanum onites) belongs to the family Lamiaceae and dietary thyme leaves could show different synergetic metabolic grows in the Mediterranean area, including Turkey, Greece, Italy, effects rather than pure essential oil supplements.

Address for Correspondence: Eyüp Eren GÜLTEPE • E-mail: [email protected] This work is licensed under a Creative Received Date: June 29, 2020 • Accepted Date: January 29, 2021 • DOI: 10.5152/actavet.2021.20038 Commons Attribution-NonCommercial Available online at actavet.org 4.0 International License.

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Till date, there have been some efforts to improve the quality Eggshell breaking strength was measured by using Egg Force of stored eggs from laying hens supplemented with different Reader, (EF 0468-2011, Orka Food Tech. Ltd., Hong Kong, China), dietary herbals and their extracts owing to potential antioxi- and Haugh unit was calculated by measuring albumen height dant capacities (Canogullari Dogan et al., 2018; Cetingul et al., (digital micrometer). Egg yolk color was determined by using 2019; Cimrin et al., 2019; Gultepe et al., 2018; Omri et al., 2019; the Roch improved yolk color fan and comparing the color of Rahman et al., 2017). The results of the aforementioned studies yolks with 15 bands of the color fan. After the egg trait analysis, suggest that dietary management may affect the quality, fresh- the yolks of eggs were obtained into the Eppendorf-type mi- ness, and shelf-life of eggs during storage. crocentrifuge tube and were stored at −20°C until the laboratory assays. The TAC and TOS analysis were performed with colo- However, limited data are available on dietary oregano supple- rimetric enzyme-linked immunosorbent assay (ELISA) method mentation and their effect on egg quality traits in laying hens in yolk according to Chen et al. (2020). A commercial laboratory during different storage times. Considering the above points, (Derman Medical Diagnostic Lab, Konya, Turkey) performed the we hypothesized that dietary oregano leaves could help ex- analysis using an automated ELISA analyzer (Elisys Uno; Human tend the storage time of eggs with prolonged freshness and mbH, Wiesbaden, Germany) with commercial reagents (Rel As- quality owing to the proven antioxidant effects of the oregano say Diagnostics, Gaziantep, Turkey). plant and its byproducts. Method Statistical Analysis The model assumptions of normality and homogeneity of vari- Experimental Design and Management ance were examined by the Kolmogorov-Smirnov test, and A total of 36 Babcock White laying hens (30 weeks of age; initial log-transformation was used where needed. The statistical body weight 1,512.01 ± 127.19 g) were divided into 3 groups analysis was performed with MedCalc (MedCalc Software bvba, with 4 replication groups containing 3 hens in each subgroup. Oostend, Belgium). The independent variables were evaluated The birds were fed diets with 0%, 5%, and 10% Turkish orega- with one way analysis of variance (ANOVA), and dependent no (Oregano onites) for 30 days. Cages, management, and variables were evaluated with repeated measures ANOVA. Ei- environmental conditions were similar to our previous study ther Bonferroni or Tamhane’s T2 tests were performed as a post- (Gultepe et al., 2018). A total of 16 hours of light and 8 hours hoc analysis when there was a significant difference in the data. of dark were applied, and feed and water were supplied ad libi- The significance level was considered as p < .05, and all data - tum. In this study, all the treatment groups were fed a basal diet were expressed as X ± SEM. (~17% crude protein [CP], ~2,750 kcal/kg), which was prepared according to the requirement of the birds (NRC, 1994) (Table Results 1). There was no mortality record during the study. The orega- The results of the study showed that with the passage of time, no leaves were added to the relevant diet during the mixing eggshell breaking strength linearly decreased among the of feedstuffs. The leaves were provided by a commercial local groups (p < .05) at 5% and 10% oregano supplemented groups trader. The automatic nipple drinking system was used with an over control. However, the breaking strength was similar to the individual water tank for each group. control group at all time points of the treatment groups (Table Dry matter, ash, CP, and ether extract compositions of the 2). Egg weight significantly decreased p( < .05) in 5% oregano Oregano leaves were determined according to AOAC (1997) supplemented groups at 30 days of egg storage compared methods. The CP was determined by a nitrogen analyzer (Kjel- with other treatment and control groups (Table 2). Haugh unit Master K-375, Buchi Labortechnik AG, Flawil, Switzerland). Neu- significantly increased in 10% oregano supplemented groups tral detergent fiber (aNDFom with heat-stable α-amylase and at 30 days of egg storage compared with other treatment and sodium sulfite) and acid detergent fiber (ADFom) were deter- control groups. However, the Haugh unit decreased linearly (p mined according to Van Soest et al. (1991) using the Fibretherm < .05) among the control and the 5% oregano supplemented apparatus (Fibretherm®, C. Gerhardt GmbH & Co. KG, C., Konig- groups over time (Table 2). Egg yolk color decreases significant- swinter, Germany). Fiber values were expressed without residu- ly (p < .05) between and among the 5% oregano supplemented al ash (Mertens, 2002) (Table 1). group compared with 10% oregano supplemented and control groups during the 10th day, 20th day, and 30th day of storage (Ta- Data Collection and Analyses ble 2). At the end of the study, 8 eggs from each group were analyzed on the same day without storage. The remaining eggs were The TAC showed no difference between fresh (0 day) and 10 stored at +4°C. Then after 10th, 20th, and 30th days of storage, days stored eggs and was increased in both the 5% and 10% 8 eggs (totally 96 eggs) from each group were analyzed for in- inclusion groups on the 20th day of storage. On the 30th day of ternal egg quality parameters (egg weight, eggshell breaking storage, the TAC was still higher in the 10% thyme group (p < strength, yolk color index, and Haugh unit), total antioxidant .05) according to the 5% oregano and control groups (Table capacity (TAC), and total oxidant status (TOS). 3). The TOS results had no difference p( > .05) in all the orega-

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no supplemented groups compared with the control group Table 1 during 0 day, 10th day, 20th day, and 30th day of storage (Table 3). Ingredient Composition and Chemical Composition of Treatment Diets and Oregano (Origanum Onites L. ) Leaves (%, as Fed Basis) Discussion, and Conclusion and Recommendations Ingredients Control 5% Oregano 10% Oregano During the storage of table eggs, internal and external qualities Wheat 22.06 9.00 19.34 change over time because of the high lipid oxidation of yolk, Corn 30.49 35.13 19.29 loss of moisture from the pores, and decreased microbial qual- Vegetable oil 3.66 5.00 7.69 ity (Omri et al., 2019; Yuceer & Caner, 2014). Although Yuceer Sunflower meal (32% CP) 13.14 12.00 11.69 and Caner (2014) have observed some improvement in shell puncture strength through the coating of eggs, Rahman et al. Full fat soya 10.00 9.90 9.99 (2017) have observed that the dietary Mentha piperita did not Soybean meal (44% CP) 9.34 13.00 11.11 affect eggshell breaking strength during 0, 15, and 30 days of Oregano leaves 0 5.00 10.00 storage period of eggs of laying hens. Furthermore, Gultepe et Limestone 8.64 8.40 8.32 al. (2018) have observed no significant effect of dietary Pista- chia terebinthus seeds on eggshell breaking strength in laying Dicalcium phosphate 1.80 1.75 1.72 hens. In agreement with the aforementioned study, there was Common salt .40 .40 .40 no constant positive effect on eggshell breaking strength be- Vitamin-mineral premix1 .25 .25 .25 tween the groups as well as among the groups at 4°C tempera- th th th DL-Methionine .13 .14 .15 ture during the 0, 10 , 20 , and 30 days of storage.

L-Lysine HCl .10 .03 .04 Jin et al. (2011) reported a significant weight loss percent- Calculated values2 age in the eggs of laying hens until 10 days of storage even CP, % 17.7 17.7 17.7 at 5°C. However, dietary manipulations have shown no effect on egg weight loss during the storage periods in this study in ME, kcal/kg 2,750 2,750 2,742 agreement with previous study data, except on the 30th day of Ca 3.81 3.81 3.78 storage (Canogullari Dogan et al., 2018; Cetingul et al., 2019; Av.P .40 .40 .40 Gultepe et al., 2018; Rahman et al., 2017). Furthermore, Cim- Na,% .21 .21 .20 rin et al. (2019) have observed that, similar to our research, dietary thyme had no major impact on relative egg weight Met+Cys .75 .75 .75 loss during storage, neither at room temperature nor during Lysine, % .86 .86 .87 refrigeration. The aforementioned researchers suggested that Treonin, % .62 .64 .65 eggshell integrity may have a strong relationship with relative Triptophane, % .22 .24 .24 egg weight loss during storage. In this study, the lack of ef- Linoleic acid, % 3.51 5.35 4.20 fect of thyme supplementation on egg weight (or egg weight loss) could be supported by the fact that the supplement had Oregano leaves (% of DM) no beneficial effect on eggshell breaking strength and shell DM, % 92.52 integrity. CP 13.95 During storage, the Haugh unit progressively decreases over aNDFom 40.48 time owing to the loss of albumen height. Akter et al. (2014) ADFom 44.96 have suggested that the height loss of albumen is a result of Ether extract 1.18 degradation of the ovomucin and lysozyme complex in the Ash 1.11 egg. However, there are inconsistent results from the un- treated (control) eggs of previous egg storage studies. Some Note. 1Provided per kg of diet: vitamin A: 12,000,000 IU; vitamin D3: 3,000,000 IU; vitamin E: 35,000 IU; vitamin K3: 3,500 IU; vitamin B1: 2,750 IU; vitamin B2: 5,500 researchers concluded that the Haugh unit did not decrease IU; nicotinamide: 30,000 IU; Ca-D-pantothenate: 10,000 IU; vitamin B6: 4,000 IU; in control groups over time at refrigeration conditions (Cetin- vitamin B12: 15 IU; folic acid: 1,000 IU; d-biotin: 50 IU; choline chloride: 150,000 gul et al., 2019; Jin et al., 2011; Rahman et al., 2017), whereas IU; manganese: 80,000 mg; iron: 60,000 mg; zinc: 60,000 mg; copper: 5,000 mg; others reported a significant decrease in Haugh unit during iodine: 2,000 mg; cobalt: 500 mg; selenium: 150 mg; antioxidant: 15,000 mg 2NRC (1994) storage in the refrigerator without any treatment (Akter et al., CP = Crude protein; DM = Dry matter; ME = Metabolizable energy; Na = 2014; Canogullari Dogan et al., 2018; Gultepe et al., 2018; Yu- Sodium; Ca = Calcium; aNDFom = Ash-free neutral detergent fiber; ADFom ceer & Caner, 2014). In this study, the Haugh unit significant- = Ash-free acid detergent fiber; Av. P = Available Phosphorus; Met+Cys = ly decreased during storage in the control and 5% oregano Methionine+Cysteine. groups. However, the negative trend of Haugh unit reduction disappeared in the eggs of 10% dietary oregano supplemented

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Table 2 Effect of Different Levels of Oregano Leaves on Egg Quality Traits During Different Storage Time in Laying Hens (n = 8) Fresh 10th day 20th day 30th day - - - - Item X SEM X SEM X SEM X SEM p ESB, kg/cm2 Control 47.12 2.50 38.44 2.73 37.40 2.55 41.66 1.21 .10 5% Oregano 48.85a 1.72 40.60a,b 1.16 37.87b 1.48 35.63b 3.18 .003 10% Oregano 44.45a 2.37 40.00a,b 2.46 36.63a,b 1.74 34.09b 2.40 .02 p .31 .69 .89 .13 Egg weight, g Control 61.05 1.45 61.58 1.78 59.47 1.26 59.31A 1.35 .94 5% Oregano 60.77 1.07 56.87 1.33 57.33 1.56 54.37B 1.33 .07 10% Oregano 59.45 1.73 58.97 0.94 59.74 1.82 57.72A,B 1.48 .81 p .67 .07 .52 .049 Haugh unit Control 77.21a 1.94 65.19a,b 5.39 61.96a,b 1.19 57.46A,B,b 2.28 .03 5% Oregano 80.73a 1.62 66.26a,b 8.95 65.67a,b 2.72 59.80A,b 3.26 .01 10% Oregano 79.02 2.73 75.14 2.69 67.36 2.11 62.97B 3.30 .06 p .46 .06 .09 .02 Egg yolk color Control 8.27 .41 7.11A .48 8.00A .71 8.71A .52 .15 5% Oregano 8.09 .41 6.90B .60 6.33B .29 6.22B .15 .22 10% Oregano 8.64 .39 7.30A .62 7.50A .37 7.00B .42 .26 p .60 .001 .003 .001 Note. a,b,cValues with different superscripts in the same row differ significantlyp ( < .05) A,B,CValues with different superscripts in the same column differ significantlyp ( < .05) ESB = eggshell breaking strength.

Table 3 Effect of Different Levels of Oregano Leaves on TOS and TAC of Egg Yolks During Different Storage Time in Laying Hens (n = 8) Fresh 10th day 20th day 30th days - - - - Item X SEM X SEM X SEM X SEM p TAC mmol Trolox equiv./L Control .51 .14 .32 .07 .33A .08 .33A .01 .17 5% Oregano .38 .02 .39 .07 .60B .03 .45A,B .07 .19 10% Oregano .48 .12 .40 .04 .75B .02 .46B .02 .20 p .91 .52 .004 .046 TOS, pg/mL Control .34 .05 .39 .01 .39 .02 .40 .01 .58 5% Oregano .35 .01 .28 .07 .29 .08 .40 .02 .56 10% Oregano .33 .03 .42 .004 .40 .003 .43 .01 .10 p .81 .17 .31 .14 Note. a,b,cValues with different superscripts in the same row differ significantly (p < .05) A,B,CValues with different superscripts in the same column differ significantlyp ( < .05) TAC = Total antioxidant capacity; TOS = Total oxidant status.

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laying hens in this study. Although Rahman et al. (2017) and antioxidant activity than just fresh eggs over the control, al- Cetingul et al. (2019) did not observe any dietary/water sup- though there was no antioxidant improvement of carotenoids plement treatment effect on the Haugh unit of stored eggs, on stored eggs. However, lutein is the most abundant carot- Canogullari Dogan et al. (2018) and Gultepe et al. (2018) have enoid in oregano leaves (Young et al., 2003), but carotenoids reported significant improvements on Haugh unit during stor- are not the major active ingredients in the oregano. Carvacrol, age in agreement with the results of this study. Al-Harthi (2014) caffeic acid, and rosmarinic acid contribute 55% of the total observed an improvement in the Haugh unit of dietary vitamin phenolic compound of the oregano plant, and the phenolic E (antioxidant) supplemented laying hens. This evidence sug- compounds of oregano are highly responsible for its antioxi- gests that enriched antioxidant capacity (vide infra) with the dant activity (Pizzale et al., 2002). Our result showed that TAC dietary 10% oregano supplementation may help to prevent a remained unaffected in fresh and 10 days stored eggs; howev- significant decrease in the Haugh unit during storage of laid er, it increased in both the 5% and 10% inclusion groups on the eggs. The inconsistent results could also be explained by the 20th day of storage. On the 30th day of storage, the TAC was still different antioxidant capacities of the used herbal agents in the higher in the 10% oregano group than in the 5% group. This aforementioned studies. evidence suggests that dietary oregano may act as an antioxidant agent with phenolic compounds rather than carotenoids. In our study, the yolk color scores of stored eggs showed no The lighter yolk colors in the oregano supplemented groups th difference according to the time in storage until the 30 day support our hypothesis. similar to the study of Barbosa et al. (2011) who have reported that egg yolk pigmentation observed with egg yolk color fan In conclusion, dietary oregano leaves could be used in the di- was consistent with 2 weeks of storage in the refrigerator, al- ets of laying hens to extend the storage time of eggs as well as though it sharply decreased in room temperature after 7 days have well-known positive effects on animal health. The antiox- of storage. Furthermore, Christaki et al. (2011) observed no sig- idant effects of dietary oregano may help maintain the fresh- nificant difference in yellowness (b*) of the egg yolk of quails ness and quality of stored eggs at 4°C until 30 days. supplemented with oregano leaves compared with the control birds in agreement with our 0 day egg yolk color scores. How- ever, the yolk color scores of both the oregano groups were Ethics Committee Approval: Ethics committee approval was received significantly lighter than the control group at each time point for this study from the ethics committee of Afyon Kocatepe University in this study. Although Young et al. (2003) have observed that Faculty of Veterinary Medicine (case #49533702/142, date: November lutein, a powerful yolk color enhancer, is the main carotenoid in 08, 2016). Turkish oregano leaves, we believe the carotenoid content was very low with poor availability in our oregano leaves because of Peer-review: Externally peer-reviewed. very low ether extract content of the oregano leaves (1.18%) in Author Contributions: Concept - İ.B.; Design – İ.B., İ.S.Ç.; Supervision our study. Although Christaki et al. (2011) have reported high- – İ.B., İ.S.Ç., E.E.G.; Resources – C.U., E.E.G., A.R.; Materials – İ.B., E.E.G.; er ether extract content of the oregano leaves (6.4%) than our Data Collection and/or Processing – A.I., Ü.Ö., C.U., E.E.G.; Analysis study, they observed no significant effect on the egg yolk color. and/or Interpretation – E.E.G., İ.S.Ç., İ.B.; Literature Search – A.I., A.R., Nys (2000) suggested that the concentration of lutein was high E.E.G.; Writing Manuscript – E.E.G, A.I., İ.S.Ç., İ.B.; Critical Review – A.R., in forage plant leaves immediately after harvesting; however, C.U., İ.B. the concentration significantly decreases during the storage period as a dehydrated form. This evidence may explain why Acknowledgements: The authors thank Dr. Mustafa Kargioglu (Depart- the oregano leaves, assumed to have low carotenoid content, ment of Molecular Biology and Genetics, Afyon Kocatepe University, causes less yellow color during storage in this study. Turkey) for identifying the species of the plants (Origanum onites) and the staff of Afyon Kocatepe University Animal Teaching and Research During storage, TOS and TAC are important owing to the high Center (Afyonkarahisar, Turkey) for their assistance with this study. This phospholipid composition (Palacios & Wang, 2005) and high study was financially supported by the research projects commission of suspect lipid oxidation in the stored eggs, followed by altered Afyon Kocatepe University (grant number 17.VF.ALTY.02). nutritional quality, taste, flavor, and yolk color (Omri et al., 2019). Conflict of Interest: The authors have no conflicts of interest to de- There is limited data about the effects of dietary oregano on clare. TOS and TAC of stored eggs. Recently, Cimrin et al. (2019) have reported no significant effect of dietary thyme supplementa- Financial Disclosure: The authors declared that this study has received tion on thiobarbituric acid reactive substances (TBARS), a reli- no financial support. able lipid peroxidation marker in foods and in yolks. However, References Omri et al. (2019) have observed the effects of a carotenoid-rich dietary byproduct blend (linseeds, sweet red pepper, and to- Akter, Y., Kasim, A., Omar, H., & Sazili, A. Q. (2014). Effect of storage time mato) on TAC of eggs stored in a refrigerator for 30 days. They and temperature on the quality characteristics of chicken eggs. reported that the carotenoid-rich byproducts showed higher Journal of Food, Agriculture & Environment, 12(3&4), 87-92.

80 GÜLTEPE et al. Effects of Dietary Oregano on Stored Eggs Acta Veterinaria Eurasia 2021; 47(2): 76-81

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