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The investigation of the changes in physicochemical, texture and rheological characteristics of salted duck egg yolk during salting
Accepted Manuscript The investigation of the changes in physicochemical, texture and rheological characteristics of salted duck egg yolk during salting Min-Min Ai, Shan-Guang Guo, Quan Zhou, Wei-Liang Wu, Ai-Min Jiang PII:
S0023-6438(17)30751-X
DOI:
10.1016/j.lwt.2017.10.013
Reference:
YFSTL 6576
To appear in:
LWT - Food Science and Technology
Received Date: 4 July 2017 Revised Date:
5 October 2017
Accepted Date: 6 October 2017
Please cite this article as: Ai, M.-M., Guo, S.-G., Zhou, Q., Wu, W.-L., Jiang, A.-M., The investigation of the changes in physicochemical, texture and rheological characteristics of salted duck egg yolk during salting, LWT - Food Science and Technology (2017), doi: 10.1016/j.lwt.2017.10.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The investigation of the changes in physicochemical, texture and rheological
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characteristics of salted duck egg yolk during salting
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4 Min-Min Ai a, Shan-Guang Guo a, Quan Zhou a, Wei-Liang Wu b, Ai-Min Jiang a, *
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College of Food Science, South China Agricultural University, Guangzhou, 510642,
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P. R. China
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b
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Control and Prevention, Guangzhou, 511430, Guangdong, P. R. China
Institute of Nutrition and Food Safety, Guangdong Provincial Center for Disease
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*
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Ai-Min Jiang, Professor
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College of Food Science, South China Agricultural University, Guangzhou, 510642,
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P. R. China.
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Tel: +86-20-85280268
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E-mail: [email protected]
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Corresponding author:
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Abstract The relationships between the maturation of salted duck egg yolk and the changes
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of physicochemical, texture and rheological properties were investigated by
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instrumental analyses. For the physicochemical properties, the apparent oil yield rate
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and oil extraction rate of salted duck egg yolk respectively increased during salting.
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The surface hydrophobicity also had an increasing trend and achieved a maximum
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value of 96.961S0, however, the particle size and zeta potential had significant
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decrement because of the protein accumulation. For texture, the parameters of
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hardness (858.91 to 2396.46 N), adhesiveness (2.64 to 5.11 Ns), springiness (0.28 to
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0.43 mm), gumminess (352.34 to 918.29 N), and chewiness (139.39 to 417.45 N) had
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prominent increases during salting, while cohesiveness had a reverse trend. For the
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rheological characteristics, the gelatinization of yolk caused by salt resulted in the
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increments of elastic and viscous modulus. These significant alterations on yolk
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indicated that the micro-structure of yolk protein had changes that induced by NaCl in
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the process of curing. The equilibriums of three properties achieved during 20–25
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days salting implied the maturation of salted duck egg yolk. Meanwhile, the
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composition and polypeptide chains of yolk did not degrade or aggregate during
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salting according to the gel electrophoresis.
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Key words: Salted duck egg; Yolk; Texture profile; Rheological property; Sodium
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chloride
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Chemical compounds studied in this article
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Sodium chloride (Pub Chem CID: 5234 ); silver nitrate (Pub Chem CID: 24470);
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8-aniline-1-naphthalenesulfonic acid (Pub Chem CID: 1369).
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1 Introduction The salted duck egg, one of the traditional foods in China, is typically processed by
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using salt. Because of its richness in taste and high nutrition components, the salted
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duck egg yolk is widely accepted and commonly applied in moon cakes, pasta and
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other kinds of food for the purpose of improving their quality and flavor (Yang &
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Chen, 2001; Chiang & Chuang, 1986; Chi & Tseng, 1998). In the process of salting,
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salt solution gradually permeates and diffuses into the inside of eggs through the
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eggshell and the eggshell membrane, which causes the moisture to migrate from the
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egg yolk to the egg white, and as a result, the egg yolk solidifies from the exterior to
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the central part accompanied by different biochemical reactions in the interior egg
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yolk (Chi & Tseng, 1998).
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The characteristics of the salted duck egg yolk include the solidification of egg yolk,
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oily texture, and fragrance. Chi and Tseng(1998), as well as Thammarat, Soottawat,
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and Wonnop(2009) reported that the microstructure of the interior region of the salted
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duck egg yolk observed by scanning electron microscopy congealed tightly due to the
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dehydration in the process of salting, and that the more the water was removed, the
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tighter the spheres congealed. However, the exterior and central regions of the yolk
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sphere congealed loosely so that it could offer space for the increasing flowing oil to
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fill. In terms of the rheological behavior, viscosity of the salted duck egg yolk would
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increase in the process of salting (Ibarz, 2010). According to these results, it indicated
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that the reactions in the egg in the process of salting would influence the changes of
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physicochemical and texture properties of the egg yolk. Cooked salted duck egg yolk
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with a granular texture was generally considered to be desirable for customers
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(Chiang & Tseng, 1986; Wang 1992), but the researches about the characteristics of
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the salted duck egg yolk primarily focused on the changes of physics, chemistry and
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microstructure (Thammarat, Soottawat, & Wonnop, 2009; Lai, Chuang, Jao, & Hsu,
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2010; Chi & Tseng, 1998). Many studies have been done to make contributions to investigate the changes that
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occurred inside the egg yolk in the process of salting. Furthermore, one of the aims in
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this paper is to further to characterize the mechanism of the salted duck egg yolk by
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making use of the physicochemical and texture characteristics and to explore the
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relationship between the maturation of the salted duck egg yolk and the changes of
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these characteristics in the process of salting including granule particle size, zeta
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potential, rheological properties, texture, and electrophoretic patterns of proteins.
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Moreover, the influence mechanism of salt on the micro-structures of the egg yolk in
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the process of curing is also revealed through the detection of the indicators above.
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2 Materials and methods
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2.1 Chemicals
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Sodium chloride (NaCl) was obtained from a local supermarket. Anhydrous ethanol,
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coomassie brilliant blue G-250, coomassie brilliant blue R-250 and ammonium ferric
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sulfate (NH4Fe(SO4)2·12H2O) were purchased from Tianjin Fuyu Fine Chemical Co.,
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Ltd (Tianjin, China). Potassium thiocyanate (KSCN), silver nitrate (AgNO3) and
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nitrate (HNO3) were obtained from Sinopharm Chemical Reagent Co., Litd (Shanghai,
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China). Sodium dodecyl sulfate (SDS) and 8-aniline-1-naphthalenesulfonic acid
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(ANS) were supplied by Sigma (St. Louis, MO, USA). Unless indicated otherwise,
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the chemicals were analytical grade and were used as received.
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2.2 Duck egg collections and sample preparation
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Fresh duck eggs, ranging from 55 to 65g, were purchased from a local market.
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These eggs were cleaned with flowing tap water and checked for any crack on egg
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shell prior to being salted. The sodium chloride aqueous solution used for salting was
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then the fresh duck eggs were soaked and immersed into this solution with a sealed
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container (Wei & Tong, 2011). During processing, ten eggs were taken out at 5, 10, 15,
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20, 25 days for determination and analysis, respectively. Salted duck eggs were placed
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into boiling water for 10 min poaching. After that, egg white of each sample was
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carefully separated from egg yolk for texture profile determination immediately. The
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rest of separated samples wrapped by preservative film were kept at 4 ºC refrigerator
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until instrumental identification
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2.3 Determination of moisture and NaCl contents of salted duck egg yolk
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Moisture and NaCl contents of salted duck egg yolk samples were determined
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according to the methods of AOAC (2000). The procedure for NaCl determination as
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follow: twenty millimeters of 0.1 mol/L AgNO3 and 10 mL of 30 mL/100mL HNO3
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were added into 5g egg yolk samples. The mixtures were boiled gently on a hot plate
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until all solids were dissolved except AgCl2. After that, the mixture was cooled down
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at ambient temperature (24~26 ), and 5 mL of 5 g/100g FeNH4(SO4)2·12H2O were
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added. The mixtures were then titrated with 0.1 mol/L KSCN standard solution until
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the solution became permanently light brown. The percentage of NaCl of samples was
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calculated by the following equation:
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Salt(g/100 g) = 5.8 × [(V1 × N 1 ) − (V2 × N 2 )] / W
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(1)
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where V1 is the volume of AgNO3 solution (mL), N1 is the molar concentration of
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AgNO3 solution (mol/L), V2 is the consumed volume of KSCN solution (mL), N2 is
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the molar concentration of KSCN solution (mol/L), W is the weight of the sample.
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2.4 Determination of apparent oil yield rate
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Whole salted eggs including eggshell were completely cooked and weighted as M1,
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and then egg shell was removed and weighed as M2. After that, masses of egg whites
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were weighted as M3 after separating egg whites and egg yolk completely. Several
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oil-absorbing papers, of which the mass noted as M4, were tiled under egg yolk for oil
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absorption, and then the weight of oil-absorbing papers was measured as M5. The
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parameter was calculated by Eq (2) as follows:
apparent oil yield rate(g/100g) = ( M 5 − M 4 ) /( M 1 − M 2 − M 3 ) ×100
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2.5 Extraction of oil from salted duck egg yolk
Extraction of oil from salted duck egg yolk was conducted according to the method
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described by Liu et al. (2013). Five grams salted duck egg yolk was added into a flask
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and then placed the flask in a water bath at 82
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ethanol to extract oil. After that, the mixture was centrifuged at a speed of 2683×g for
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15min. The supernatant was transferred into a round-bottom flask and concentrated at
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50
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removed in a drying cabinet (DHG-9073BS-III, Shanghai, China) at 65
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weight became constant. The parameter was calculated by Eq (3) as follows:
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for 52 min with 12-fold absolute
Oil extraction rate(g/100g) = m1 / m2 ×100
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(3)
2.6 Determination of surface hydrophobicity
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Protein surface hydrophobicity was measured according to the method described by
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Benjakul, Visessanguan, Ishizaki, and Tanaka (2001) using ANS as a probe. Four
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millimeters of salted duck egg yolk solution, concentrations ranging from 0 to 0.30
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mg/mL protein in 0.1mol/L sodium phosphate buffer (pH=6.8), and 20 µL of 8
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mmol/L ANS in 0.1mol/L sodium phosphate buffer (pH=6.8) were mixed sufficiently
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in flask by a vortex. Fluorescence intensity (FI0) was measured using a fluorescent
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spectrophotometer (RF-5301PC, Shimadzu, Tokyo, Japan) with an excitation at 390
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nm and emission at 470 nm after the mixture stands still for 15 min in the dark place.
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In order to eliminate the effect of turbidity of the protein solution to the fluorescence
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without addition of the fluorescent probe was also measured as FI1. The slope of the
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initial phase was the surface hydrophobicity (S0 ANS) of the protein molecule, with
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the protein concentration as the abscissa and the corrected FI (the difference between
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FI0 and FI1) as the ordinate.
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2.7 Determination of granule particle size and zeta potential
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Different weights of salted duck egg yolk were dissolved respectively into
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demonized water to prepare two levels of solution including 1 g/L and 0.1 g/L with
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stirring. After standing still for 1h, the supernatants of these two solutions were
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determined by nano-meter analyzer (B1-90PLus, Brookhaven, NewYork, US) to
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obtain the data of granule particle size and zeta potential. The measurements of
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characteristics of zeta potential were as follows: the cuvette was a 1cm polystyrene
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cell with a pair of 0.45 cm2 platinum electrodes at spacing 0.4 cm. The temperature
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was 25
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2.8 Determination of texture profile analysis (TPA) of salted duck egg yolk
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and the temperature equilibrium time was 4 min.
TPA was performed with a TA-XT Plus texture analyzer (Stable MicroSystems,
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Surrey, England) as described by Bourne (1978). Salted duck egg yolk was rolled on a
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filter paper (Whatman No.1) to remove the egg white. Prepared samples were
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compressed twice to half of their original height with a compression cylindrical
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aluminum probe (50 mm diameters). Re and post-test speeds were 1 and 5 mm/s,
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respectively. The time between two compression cycles was 5 s. Force-distance
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deformation curves were recorded at a cross head speed of 5 mm/s. Parameters
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including hardness, adhesiveness, springiness, cohesiveness, resilience, gumminess
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and chewiness were obtained by using the MicroStable software (Stable Micro
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Systems, Surrey, England).
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2.9 Determination of rheological property of salted duck egg yolk The rheological properties of the samples were determined by MCR101 (Anton
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Parr, Austria). A 50 mm stainless steel parallel plate geometry with a 1 mm gap was
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used for determination. To scan the frequency at 25 , oscillation mode and linear
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viscous area under the conditions of frequency scanning range of 10-700rad/s, control
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strain of 0.5%. The elastic modulus (G') and viscous modulus (G") of salted duck egg
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yolk were measured under the vibration frequency changes.
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2.10 SDS-polyacrylamide gel electrophoresis (SDS–PAGE)
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Protein patterns of salted duck egg yolk were determined using 5 g/mL stacking gel
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and 12 g/mL separating gel according to the method described by Laemmli (1970).
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Three grams of salted duck egg yolk was added to 27 mL of 5 g/100g SDS and
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homogenized using a homogenizer for rapid dispersion at 16992×g for 1 min
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(Polytron, PT 2100, Kinematica AG, Luzern, Switzerland). The homogenate was
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incubated at 85
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temperature using a centrifuge (Sorvall, Model RC-B Plus, Newtown, CT, USA). The
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supernatant of protein concentration was determined by the Biuret method (Robinson
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& Hodgen, 1940) using bovine serum albumin (BSA) as a standard. The protein
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concentration was adjusted to 1 mg/mL with the sample treatment solution, and 20 µL
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of sample and 10 µL of standard protein marker (the weight of standard proteins was
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ranged from 20.1-97.2kDa) was loaded onto the gel. Electrophoresis was performed
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using a vertical gel electrophoresis unit (Mini-protein II; Bio-Rad Laboratories,
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Richmond, CA, USA) at the constant voltage of 200 V in the resolving gel. The gels
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were stained with Coomassie Brilliant Blue R-250 0.125 g/100g, 25 mL/100mL
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methanol and 10 mL/100mL acetic acid. Destaining was performed using 40
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mL/100mL methanol and 10 mL/100mL acetic acid.
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for 1 h followed by centrifugation at 7500×g for 10 min at room
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2.11 Statistical analysis All data were expressed as the mean ± standard deviation of triplicate
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determinations. One-way analysis of variance (ANOVA) was carried out and mean
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comparisons were performed by Duncan’s multiple range tests (Steel et al. 1980).
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Statistical analyses were measured using the statistical program (SPSS16.0 for
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windows, SPSS Inc., Chicago, IL, USA).
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3 Results and discussion
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3.1 The changes of moisture and NaCl contents of salted duck egg yolk
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Moisture and NaCl contents decreased or increased respectively (p<0.05), which
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were presented in Fig.1 as the salting time was prolonged. These findings were
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consistent with the results of Lai, Chi, and Ko (1999). When fresh duck eggs were
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soaked into NaCl solution, the water of the egg yolk was gradually transferred to egg
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white. After 10 days’ salting, the exterior egg yolk became harder, while the inside
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region was still in a liquid state, as a result of the dehydration of egg yolk and the
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penetration of salt (Chi & Tseng, 1998). Salt penetrated through the pores in eggshell
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and permeated slowly into egg white to improve the level of osmotic pressure via
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eggshell membrane, and thus, the rise of osmotic pressure in egg white promoted the
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salt to continue penetrating to egg yolk. Furthermore, the stable emulsification system
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collapsed. At the same time, chemical bonds were gradually strengthened because of
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the demulsification of egg yolk under the high concentration of salt. The interspace of
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egg yolk granules became narrower, causing the solidification of exterior egg yolk
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and the enhancement of osmotic pressure in the interior of the egg yolk. The
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solidification part of the egg yolk gradually extended to the interior part until the
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osmotic pressure of interior yolk realized equilibrium with exterior yolk.
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Consequently, the content of moisture and salt reached a steady state after salting up
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to 20 days.
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3.2 Changes in apparent oil yield rate and oil extraction rate Egg yolk grains in the salted duck egg yolk were in the form of lipoprotein-yolk
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high phosphoprotein bonded by calcium-phosphorus bridge complex. Ca2+ in the
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calcium-phosphorus bridge replaced by Na+ resulted in the destroyed structure of egg
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yolk particles in the gradual penetration of the salt (Causeret, Matringe, & Lorient,
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2010). The increase of salt content caused the occurrence of salt-solubilization of
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protein in the emulsified system (Zheng, Peng, Lin, & Xue-Juan, 2013). And weak gel
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was formed by yolk protein on account of hydrophobic interactions and
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hydrogen-bond
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(Paraskevopoulou, Kiosseoglou, Alevisopoulos, & Kasapis, 2000). Feeney, Weaver,
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Jones, and Rhodes (1956) reported that the increasing water migration from albumin
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to yolk diluted the free lipid-protein from the surface of the egg yolk, which could be
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attributed to the weakened yolk membrane in the process of salting, thus leading to oil
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exudation. Therefore, the results of apparent oil yield rate and extraction oil rate all
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showed an increasing trend (p<0.05), which were presented in Table 1. Meanwhile,
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after salting for 20 days, there was no significant change in oil extraction (p<0.05),
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which indicated that the oil exudation of salted duck egg yolk had reached the
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maximum in the process of salting for up to 20-25 d and it was at the stage of best
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oily texture. The reverse trend occurred between the variation of oil extraction and
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moisture content, indicating the relative oil content of egg yolk increased together
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with the water migration.
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3.3 The changes of surface hydrophobicity
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the
lipid
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protein
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separated
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(Li-Chan & Nakai, 1991). Surface hydrophobicity depends on the interaction
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between the water molecules repelled by the nonpolar groups which are caused by
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the gathering of proteins. The level of hydrophobic interaction protein can be
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expressed by the value of the surface hydrophobicity to be combined with
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8-anilino-1-naphthalenesulfonate (ANS) as a probe, which further interacts with
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anionic proteins (Smith, Galazka, Wellner, & Sumner, 2000). The results of the
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measurement are reflected in Fig.2, showing that the surface hydrophobicity of
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salted duck egg yolk remarkably increased (p<0.01) before 15d, and in contrast,
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decreased slowly during the later time (p>0.05).
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It is found that the pH of salted duck egg yolk decreased from 6.42 to 6.03 in the
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process of salting, and therefore, the surface of egg yolk particles was covered by a
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layer of hydrophobic groups with negative charges because the protein isoelectric
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point of egg yolk is about 5.0 (Rong, Zhang, & Han, 2012). At the same time, the
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calcium-phosphorus
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lipovitelenin was replaced by Na+, leading to the fact that part of the bridge might be
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broken, combined with the separation of lipid and protein from lipoprotein. As a
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result, the content of anionic proteins increased, indicating that surface
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hydrophobicity rose as well. When the concentration of ions in the egg yolk reached
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a certain level, it indicated the protein accumulating in the egg yolk reached a state
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of equilibrium. The enhancement of ion interaction also drove the Na+ to replace the
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hydrophobic groups in the protein surface because of the further diffusion of salt and
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Martinez, Riscardo, & Jose, 2007). Therefore, the surface hydrophobicity of the
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protein declined slightly. On the other hand, when the increasing Na+ in the interior
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region of the egg yolk interacted with the hydrophobic groups with negative charges,
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the structure of lipoprotein and the balance between oil and moisture were broken.
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In this way, the lipid was free from the interior region of hydrophobic structure and
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hence gathered to form big drops of lipid because of the similarity-intermiscibility.
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This is the reason why the salted duck egg yolk forms its unique features of oil
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permeation and sandy texture in the later stage of salting.
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3.4 Particle size analysis and zeta potential
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The results of particle size and zeta potential of salted duck egg yolk are marked
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in Fig.3. The size of the granule particles decreased significantly (p<0.05) in the
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process of salting for up to 15 days, while this change was not significant any longer
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(p>0.05) after 15 days. Furthermore, it was indicated that sandy texture was formed
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after salting for 15 days. Our observation was consistent with the results reported by
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Thammarat, Soottawat , and Wonnop (2009). The lipid in the egg yolk granules and
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a certainty of free lipid from low-density lipoprotein permeated as several bonds
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between protein and lipid were unfolded and the emulsion system and the structure
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of egg yolk granules were broken (Bee & Cotterill, 2006). Meanwhile, the colloid
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system of egg yolk was broken, showing the significant decrease of zeta potential
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(p<0.01), which was a measurement of the intensity of the repulsion or attraction
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between the particles. The results of surface hydrophobicity illustrated that the
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became steady even though some slight float occurred during the later salting time
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(15-25d). However, the accumulation of counter ions near the surface shielded the
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surface charge, thus reducing the zeta potential with salt concentration increasing
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even at the later salting time.
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Zeta potential can be used to reflect the stability of colloidal dispersion. The
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smaller the molecular or dispersed particles were, the higher the zeta potential was
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and the more stable the system was. That is, the dissolution or dispersion of
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molecular or dispersed particles could resist aggregation when higher zeta potential
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appeared. Because of its small size, it thus caused more stable emulsified system. On
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the contrary, the lower zeta potential caused more tend to concentration or
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agglomeration, indicating the attraction exceeded the repulsive force and that the
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dispersion system was destroyed, thus leading to coagulation or condensation. The
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egg yolk emulsion system was destroyed when salt content increased, and the zeta
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potential declined from 13.5mV down to 1.9mV, indicating the accumulation of
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protein in egg yolk, which was the reason why the sandy texture was formed. And
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the sandy taste is the major factor impacting consumers’ acceptance of salted duck
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egg (Chi & Tseng, 1998).
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3.5 The changes of textural properties
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The changes of TPA of salted duck egg yolk in the process of salting were
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summarized in Table 2. The data indicated that hardness significantly increased
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(p<0.01) from 1341.77N to 2396.36N during the later stage of salting, demonstrating
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that the changes occurred in the structure of the salted duck egg yolk including the - 13 -
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dehydration. The increase of salt concentration in the process of salting contributed to
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physical and chemical changes of the egg yolk including the decrease of mobility and
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the decline of sulfydryl groups as a result of the interaction between the disulfide
319
bond and sulfydryl groups as well as the oxidation of sulfydryl groups (Huang et al.,
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2012). These changes are the major factors causing the increasing hardness of egg
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yolk, which are in accordance with the reasons reported by Thammarat, Soottawat,
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and Wonnop (2009). The parameters of springiness, cohesiveness and chewiness of
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egg yolk significantly increased (p<0.05), in contrast, significant decline (p<0.05) of
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cohesiveness was observed in the process of salting. With regard to the resilience,
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there was no significant (p>0.05) change in this parameter in the process of salting.
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Our findings were consistent with the results published by Thammarat, Soottawat, and
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Wonnop (2009). However, there was no remarkable change in adhesiveness,
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springiness and gumminess of parameters (p>0.05) after 20 days’ salting, which
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suggested that the best texture of salted duck egg yolk was formed during the latter
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salting time.
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Springiness is a factor on how much the gel structure is broken down by the initial
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compression. When the gel structure was broken down into a few large pieces during
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the first TPA compression, the results of springiness showed high significantly, but
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became lower because the gel structure was broke down into many small pieces (Lau,
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Tang, & Paulson, 2000). The parameters of springiness showed slightly changes and
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the values were low after salting up to 10d in spite of the increasing springiness of egg
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yolk, demonstrating egg yolk was broken down into many small pieces in the process
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of TPA compression. This further showed the dehydration and the changes in the
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interior structure made the exterior egg yolk present a state of semi-solidness or
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intact network structure. Gradually smaller and slight changing cohesiveness
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indicated that the unequal osmotic pressure inside and outside of the egg yolk reduced
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the ability to maintain the internal network structure of the egg yolk, which might lead
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to the gradual accumulation of proteins in the egg yolk. The increase of hardness and
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the decrease of chewiness and particle size demonstrated that the tight combination of
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egg yolk granules during the later salting time made sure of the integrity of the egg
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yolk contributing to the sandy texture after cooking.
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3.6 The changes of rheological properties
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The changes of rheological properties of the salted duck egg yolk during different
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salting times were shown in Fig.4. G' of the egg yolk increased significantly (p<0.05)
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with the change of salting time. In the process of salting, the effects of NaCl promoted
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the fractures of the egg yolk spheres to release the egg yolk granules for yolk
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gelatinization, which resulted in the occurrence of semi-solidness or solidness of the
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egg yolk with the increase of G' (Peng, Lin, Xiao, Huang, & Zhang, 2011). Variation
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of changes in G" was similar to G', which had a significant increase (p<0.05), thus
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indicating that the internal viscosity of egg yolk increased with the increase of the
357
salting time. Several types of particles including spheres, granules and low-density
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lipoproteins in egg yolk, which were suspended in a protein solution or plasma, had
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the ability to form gel (Woodward & Cotterill, 2006). Therefore, the G' and G" varied
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when egg yolk became an elastic gel after salting. Our results were consistent with the
361
results discovered by Kaewmanee, Benjakul, Visessanguan, & Gamonpilas (2013),
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indicating that the addition of NaCl could regulate the visco-elastic behavior of the
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duck egg yolk by delaying the gel network formation. The presence of native yolk
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lipids and emulsified oil droplets also influenced the rheological properties of salted
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salting time, indicating that the elasticity was a major rheological property in the
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system of the salted duck egg yolk (Wang & Weil, 2011). The changes of G' and G"
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were consistent with the changes of springiness and adhesiveness in the texture
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analysis, revealing that the elasticity and viscosity of the egg yolk have partially
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changed because of the texture changes of the salted duck egg yolk.
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3.7 SDS - polyacrylamide gel electrophoresis (SDS-PAGE)
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The changes of the SDS-PAGE patterns of proteins in the salted duck egg yolk at
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different salting stages were analysed by the protein relative rate by using standard
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protein as the control. The SDS-PAGE results showed that the molecular weights of
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the standard proteins were 20.1–97.2 kDa, while the molecular weights of proteins in
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the salted duck egg yolk from different salting times ranged from 26.2 to 80.9 kDa
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(Fig.5). It indicated that the molecular weights of proteins in the salted duck egg yolk
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did not change after salting. However, these results were quite different from the
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results reported by Thammarat, Soottawat, and Wonnop (2009). They pointed out that
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the molecular weights of egg yolk proteins were between 30 and 220 kDa. The
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lipoproteins in the egg yolk could be classified into low-density lipoprotein (LDL)
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and high-density lipoprotein (HDL) (Martin, Augustyniak, & Cook, 1964; Burley &
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Cook, 1961). The molecular weight of LDL apoprotein was about 80.9 kDa (Sikorski,
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2001), while the molecular weight of HDL apoprotein was about 52.2 kDa (Raikos,
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Hansen, Campbell, & Euston, 2006). The results of electrophoresis in each sample
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showed that the changes of molecular weights of lipoproteins were not observed in
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the process of salting. There was no difference among the electrophoretograms of
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proteins in the salted duck egg yolk from different salting times. Our results indicated
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that dehydration and high concentration of salt (20 g/100g) in the salting process did
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structure of the protein, even if the salt content of the salted duck egg yolk was high
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after salting. In comparison with the electrophoretograms of peptides between the
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salted duck egg yolk and the fresh egg yolk, similar changes were observed, which
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indicated that the peptide chain did not aggregate or degrade to form a new protein
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fragment in the process of salting.
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4 Conclusion
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The investigation on the relationship between the maturation of the salted duck
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egg yolk and the changes of physicochemical, texture and rheological properties was
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carried out to find out the best curing period for the sandy and oily texture of the
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salted egg yolk. The results revealed that the physicochemical, texture, and
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rheological characteristics of the yolk went through changes caused by the
402
dehydration and the high salt content, which had an influence on the micro-structure.
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After salting 20–25d, the sandy and oily salted duck egg yolks were obtained when
404
the characteristics achieved equilibrium. Furthermore, the electrophoretograms
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showed that the proteins and peptides in the salted duck egg yolk did not degrade or
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aggregate at different salting times. This study further illustrated the mechanism
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changes of the duck egg yolk in salt solution, but it’s deeper and complex changes
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need to be further explored.
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Acknowledgements
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This study was sponsored for the foundations of poultry products processing
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engineering research and development center construction project, and poultry - 17 -
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products precision machining and secure national local joint engineering research
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center construction project. The authors would like to express sincere thanks to these
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foundations for their financial support.
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provided the methods and some feasible idea to investigate salted duck egg yolk
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with scientific ways. And most importantly, we could make comparison with the
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results of these key references when we conducted experiment.
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Fig. 1 The changes of moisture and NaCl contents of raw and cooked salted egg yolks
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during salting. (●) raw egg yolk; (■) cooked egg yolk.
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Fig. 2 The change of surface hydrophobicity of egg yolk during salting.
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Fig. 3 The change of granule particle size and zeta potential of salted egg yolk during salting. (●) curing for 0 day;(∆) curing for 5 day; (○) curing for 10 day; (□) curing for 15 day; (▲) curing for 20 day; (■) curing for 25 day.
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Fig.4 The changes of storage modulus (G′) and loss modulus(G″) of salted egg yolks in different salting period. (■) curing for 0 day; (●) curing for 5 day; (▲) curing
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for 10 day; (▼) curing for 15 day; (♦) curing for 20 day; (◄) curing for 25 day.
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Fig.5 The changes in SDS-PAGE patterns of proteins in salted duck egg yolk for different salting times. 0EY: fresh egg yolk; 5EY: egg yolk after 5 days salination; 10EY: egg yolk after 10 days salination; 15EY: egg yolk after 15 days salination; 20EY: egg yolk after 20 days salination; 25EY: egg yolk after 25 days salination.
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Table 1 The changes of apparent oil yield rate and oil extraction rate of egg yolk during salting. Apparent oil yield rate(g/100g)
Oil extraction rate(g/100g)
0
0.68±0.05d
29.71±1.09e
5
0.86±0.04d
30.04±1.29d
10
1.19±0.04c
15
1.24±0.07c
20
2.02±0.04b
25
2.95±0.07a
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Salting time(d)
A
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different salting periods
39.81±1.46b 40.86±1.15a
41.27±0.72a
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33.07±1.59c
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Table 2 The changes of textural properties during salting A 5d
10d
15d
858.91±17.41f
1341.77±22.33e
1462.04±38.52d
Adhesiveness (Ns)
2.64±0.07d
2.86±0.24d
3.25±0.04c
Springiness (mm)
0.28±0.02b
0.34±0.01b
0.39±0.01a
Cohesiveness
0.52±0.03a
0.49±0.01b
Gumminess (N)
352.34±72.49d
Chewiness (N) Resilience
3
A
1870.42±36.15c
2056.05±104.86b
2396.46±46.69a
4.04±0.33b
5.08±0.23a
5.11±0.18a
0.41±0.00a
0.42±0.01a
0.43±0.02a
0.45±0.01c
0.44±0.00c
0.41±0.01c
0.39±0.02c
463.56±4.13c
824.85±25.33b
838.35±12.89b
914.43±29.33a
918.29±17.87a
139.39±11.03e
259.42±10.54d
334.04±17.09c
345.05±10.95c
386.29±26.30b
417.45±29.66a
0.18±0.01a
0.17±0.01a
0.14±0.01a
0.14±0.00a
0.14±0.00a
0.16±0.01a
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Different lower letters in the same column illustrated the significant differences (p<0.05) in textural properties during different salting times
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20d
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Highlights
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3.Patterns of the proteins electrophoresis were not significantly different.
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S0023-6438(17)30751-X
DOI:
10.1016/j.lwt.2017.10.013
Reference:
YFSTL 6576
To appear in:
LWT - Food Science and Technology
Received Date: 4 July 2017 Revised Date:
5 October 2017
Accepted Date: 6 October 2017
Please cite this article as: Ai, M.-M., Guo, S.-G., Zhou, Q., Wu, W.-L., Jiang, A.-M., The investigation of the changes in physicochemical, texture and rheological characteristics of salted duck egg yolk during salting, LWT - Food Science and Technology (2017), doi: 10.1016/j.lwt.2017.10.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Manuscrip
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The investigation of the changes in physicochemical, texture and rheological
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characteristics of salted duck egg yolk during salting
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4 Min-Min Ai a, Shan-Guang Guo a, Quan Zhou a, Wei-Liang Wu b, Ai-Min Jiang a, *
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College of Food Science, South China Agricultural University, Guangzhou, 510642,
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P. R. China
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b
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Control and Prevention, Guangzhou, 511430, Guangdong, P. R. China
Institute of Nutrition and Food Safety, Guangdong Provincial Center for Disease
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*
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Ai-Min Jiang, Professor
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College of Food Science, South China Agricultural University, Guangzhou, 510642,
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P. R. China.
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Tel: +86-20-85280268
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E-mail: [email protected]
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Corresponding author:
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Abstract The relationships between the maturation of salted duck egg yolk and the changes
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of physicochemical, texture and rheological properties were investigated by
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instrumental analyses. For the physicochemical properties, the apparent oil yield rate
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and oil extraction rate of salted duck egg yolk respectively increased during salting.
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The surface hydrophobicity also had an increasing trend and achieved a maximum
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value of 96.961S0, however, the particle size and zeta potential had significant
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decrement because of the protein accumulation. For texture, the parameters of
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hardness (858.91 to 2396.46 N), adhesiveness (2.64 to 5.11 Ns), springiness (0.28 to
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0.43 mm), gumminess (352.34 to 918.29 N), and chewiness (139.39 to 417.45 N) had
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prominent increases during salting, while cohesiveness had a reverse trend. For the
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rheological characteristics, the gelatinization of yolk caused by salt resulted in the
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increments of elastic and viscous modulus. These significant alterations on yolk
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indicated that the micro-structure of yolk protein had changes that induced by NaCl in
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the process of curing. The equilibriums of three properties achieved during 20–25
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days salting implied the maturation of salted duck egg yolk. Meanwhile, the
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composition and polypeptide chains of yolk did not degrade or aggregate during
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salting according to the gel electrophoresis.
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Key words: Salted duck egg; Yolk; Texture profile; Rheological property; Sodium
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chloride
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Chemical compounds studied in this article
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Sodium chloride (Pub Chem CID: 5234 ); silver nitrate (Pub Chem CID: 24470);
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8-aniline-1-naphthalenesulfonic acid (Pub Chem CID: 1369).
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1 Introduction The salted duck egg, one of the traditional foods in China, is typically processed by
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using salt. Because of its richness in taste and high nutrition components, the salted
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duck egg yolk is widely accepted and commonly applied in moon cakes, pasta and
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other kinds of food for the purpose of improving their quality and flavor (Yang &
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Chen, 2001; Chiang & Chuang, 1986; Chi & Tseng, 1998). In the process of salting,
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salt solution gradually permeates and diffuses into the inside of eggs through the
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eggshell and the eggshell membrane, which causes the moisture to migrate from the
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egg yolk to the egg white, and as a result, the egg yolk solidifies from the exterior to
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the central part accompanied by different biochemical reactions in the interior egg
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yolk (Chi & Tseng, 1998).
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The characteristics of the salted duck egg yolk include the solidification of egg yolk,
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oily texture, and fragrance. Chi and Tseng(1998), as well as Thammarat, Soottawat,
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and Wonnop(2009) reported that the microstructure of the interior region of the salted
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duck egg yolk observed by scanning electron microscopy congealed tightly due to the
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dehydration in the process of salting, and that the more the water was removed, the
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tighter the spheres congealed. However, the exterior and central regions of the yolk
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sphere congealed loosely so that it could offer space for the increasing flowing oil to
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fill. In terms of the rheological behavior, viscosity of the salted duck egg yolk would
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increase in the process of salting (Ibarz, 2010). According to these results, it indicated
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that the reactions in the egg in the process of salting would influence the changes of
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physicochemical and texture properties of the egg yolk. Cooked salted duck egg yolk
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with a granular texture was generally considered to be desirable for customers
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(Chiang & Tseng, 1986; Wang 1992), but the researches about the characteristics of
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the salted duck egg yolk primarily focused on the changes of physics, chemistry and
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microstructure (Thammarat, Soottawat, & Wonnop, 2009; Lai, Chuang, Jao, & Hsu,
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2010; Chi & Tseng, 1998). Many studies have been done to make contributions to investigate the changes that
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occurred inside the egg yolk in the process of salting. Furthermore, one of the aims in
78
this paper is to further to characterize the mechanism of the salted duck egg yolk by
79
making use of the physicochemical and texture characteristics and to explore the
80
relationship between the maturation of the salted duck egg yolk and the changes of
81
these characteristics in the process of salting including granule particle size, zeta
82
potential, rheological properties, texture, and electrophoretic patterns of proteins.
83
Moreover, the influence mechanism of salt on the micro-structures of the egg yolk in
84
the process of curing is also revealed through the detection of the indicators above.
85
2 Materials and methods
86
2.1 Chemicals
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Sodium chloride (NaCl) was obtained from a local supermarket. Anhydrous ethanol,
88
coomassie brilliant blue G-250, coomassie brilliant blue R-250 and ammonium ferric
89
sulfate (NH4Fe(SO4)2·12H2O) were purchased from Tianjin Fuyu Fine Chemical Co.,
90
Ltd (Tianjin, China). Potassium thiocyanate (KSCN), silver nitrate (AgNO3) and
91
nitrate (HNO3) were obtained from Sinopharm Chemical Reagent Co., Litd (Shanghai,
92
China). Sodium dodecyl sulfate (SDS) and 8-aniline-1-naphthalenesulfonic acid
93
(ANS) were supplied by Sigma (St. Louis, MO, USA). Unless indicated otherwise,
94
the chemicals were analytical grade and were used as received.
95
2.2 Duck egg collections and sample preparation
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Fresh duck eggs, ranging from 55 to 65g, were purchased from a local market.
97
These eggs were cleaned with flowing tap water and checked for any crack on egg
98
shell prior to being salted. The sodium chloride aqueous solution used for salting was
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100
then the fresh duck eggs were soaked and immersed into this solution with a sealed
101
container (Wei & Tong, 2011). During processing, ten eggs were taken out at 5, 10, 15,
102
20, 25 days for determination and analysis, respectively. Salted duck eggs were placed
103
into boiling water for 10 min poaching. After that, egg white of each sample was
104
carefully separated from egg yolk for texture profile determination immediately. The
105
rest of separated samples wrapped by preservative film were kept at 4 ºC refrigerator
106
until instrumental identification
107
2.3 Determination of moisture and NaCl contents of salted duck egg yolk
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Moisture and NaCl contents of salted duck egg yolk samples were determined
109
according to the methods of AOAC (2000). The procedure for NaCl determination as
110
follow: twenty millimeters of 0.1 mol/L AgNO3 and 10 mL of 30 mL/100mL HNO3
111
were added into 5g egg yolk samples. The mixtures were boiled gently on a hot plate
112
until all solids were dissolved except AgCl2. After that, the mixture was cooled down
113
at ambient temperature (24~26 ), and 5 mL of 5 g/100g FeNH4(SO4)2·12H2O were
114
added. The mixtures were then titrated with 0.1 mol/L KSCN standard solution until
115
the solution became permanently light brown. The percentage of NaCl of samples was
116
calculated by the following equation:
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(1)
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where V1 is the volume of AgNO3 solution (mL), N1 is the molar concentration of
119
AgNO3 solution (mol/L), V2 is the consumed volume of KSCN solution (mL), N2 is
120
the molar concentration of KSCN solution (mol/L), W is the weight of the sample.
121
2.4 Determination of apparent oil yield rate
122
Whole salted eggs including eggshell were completely cooked and weighted as M1,
123
and then egg shell was removed and weighed as M2. After that, masses of egg whites
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were weighted as M3 after separating egg whites and egg yolk completely. Several
125
oil-absorbing papers, of which the mass noted as M4, were tiled under egg yolk for oil
126
absorption, and then the weight of oil-absorbing papers was measured as M5. The
127
parameter was calculated by Eq (2) as follows:
apparent oil yield rate(g/100g) = ( M 5 − M 4 ) /( M 1 − M 2 − M 3 ) ×100
129
(2)
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2.5 Extraction of oil from salted duck egg yolk
Extraction of oil from salted duck egg yolk was conducted according to the method
131
described by Liu et al. (2013). Five grams salted duck egg yolk was added into a flask
132
and then placed the flask in a water bath at 82
133
ethanol to extract oil. After that, the mixture was centrifuged at a speed of 2683×g for
134
15min. The supernatant was transferred into a round-bottom flask and concentrated at
135
50
136
removed in a drying cabinet (DHG-9073BS-III, Shanghai, China) at 65
137
weight became constant. The parameter was calculated by Eq (3) as follows:
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for 52 min with 12-fold absolute
Oil extraction rate(g/100g) = m1 / m2 ×100
until the
(3)
2.6 Determination of surface hydrophobicity
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Protein surface hydrophobicity was measured according to the method described by
141
Benjakul, Visessanguan, Ishizaki, and Tanaka (2001) using ANS as a probe. Four
142
millimeters of salted duck egg yolk solution, concentrations ranging from 0 to 0.30
143
mg/mL protein in 0.1mol/L sodium phosphate buffer (pH=6.8), and 20 µL of 8
144
mmol/L ANS in 0.1mol/L sodium phosphate buffer (pH=6.8) were mixed sufficiently
145
in flask by a vortex. Fluorescence intensity (FI0) was measured using a fluorescent
146
spectrophotometer (RF-5301PC, Shimadzu, Tokyo, Japan) with an excitation at 390
147
nm and emission at 470 nm after the mixture stands still for 15 min in the dark place.
148
In order to eliminate the effect of turbidity of the protein solution to the fluorescence
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150
without addition of the fluorescent probe was also measured as FI1. The slope of the
151
initial phase was the surface hydrophobicity (S0 ANS) of the protein molecule, with
152
the protein concentration as the abscissa and the corrected FI (the difference between
153
FI0 and FI1) as the ordinate.
154
2.7 Determination of granule particle size and zeta potential
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Different weights of salted duck egg yolk were dissolved respectively into
156
demonized water to prepare two levels of solution including 1 g/L and 0.1 g/L with
157
stirring. After standing still for 1h, the supernatants of these two solutions were
158
determined by nano-meter analyzer (B1-90PLus, Brookhaven, NewYork, US) to
159
obtain the data of granule particle size and zeta potential. The measurements of
160
characteristics of zeta potential were as follows: the cuvette was a 1cm polystyrene
161
cell with a pair of 0.45 cm2 platinum electrodes at spacing 0.4 cm. The temperature
162
was 25
163
2.8 Determination of texture profile analysis (TPA) of salted duck egg yolk
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and the temperature equilibrium time was 4 min.
TPA was performed with a TA-XT Plus texture analyzer (Stable MicroSystems,
165
Surrey, England) as described by Bourne (1978). Salted duck egg yolk was rolled on a
166
filter paper (Whatman No.1) to remove the egg white. Prepared samples were
167
compressed twice to half of their original height with a compression cylindrical
168
aluminum probe (50 mm diameters). Re and post-test speeds were 1 and 5 mm/s,
169
respectively. The time between two compression cycles was 5 s. Force-distance
170
deformation curves were recorded at a cross head speed of 5 mm/s. Parameters
171
including hardness, adhesiveness, springiness, cohesiveness, resilience, gumminess
172
and chewiness were obtained by using the MicroStable software (Stable Micro
173
Systems, Surrey, England).
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2.9 Determination of rheological property of salted duck egg yolk The rheological properties of the samples were determined by MCR101 (Anton
176
Parr, Austria). A 50 mm stainless steel parallel plate geometry with a 1 mm gap was
177
used for determination. To scan the frequency at 25 , oscillation mode and linear
178
viscous area under the conditions of frequency scanning range of 10-700rad/s, control
179
strain of 0.5%. The elastic modulus (G') and viscous modulus (G") of salted duck egg
180
yolk were measured under the vibration frequency changes.
181
2.10 SDS-polyacrylamide gel electrophoresis (SDS–PAGE)
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Protein patterns of salted duck egg yolk were determined using 5 g/mL stacking gel
183
and 12 g/mL separating gel according to the method described by Laemmli (1970).
184
Three grams of salted duck egg yolk was added to 27 mL of 5 g/100g SDS and
185
homogenized using a homogenizer for rapid dispersion at 16992×g for 1 min
186
(Polytron, PT 2100, Kinematica AG, Luzern, Switzerland). The homogenate was
187
incubated at 85
188
temperature using a centrifuge (Sorvall, Model RC-B Plus, Newtown, CT, USA). The
189
supernatant of protein concentration was determined by the Biuret method (Robinson
190
& Hodgen, 1940) using bovine serum albumin (BSA) as a standard. The protein
191
concentration was adjusted to 1 mg/mL with the sample treatment solution, and 20 µL
192
of sample and 10 µL of standard protein marker (the weight of standard proteins was
193
ranged from 20.1-97.2kDa) was loaded onto the gel. Electrophoresis was performed
194
using a vertical gel electrophoresis unit (Mini-protein II; Bio-Rad Laboratories,
195
Richmond, CA, USA) at the constant voltage of 200 V in the resolving gel. The gels
196
were stained with Coomassie Brilliant Blue R-250 0.125 g/100g, 25 mL/100mL
197
methanol and 10 mL/100mL acetic acid. Destaining was performed using 40
198
mL/100mL methanol and 10 mL/100mL acetic acid.
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2.11 Statistical analysis All data were expressed as the mean ± standard deviation of triplicate
201
determinations. One-way analysis of variance (ANOVA) was carried out and mean
202
comparisons were performed by Duncan’s multiple range tests (Steel et al. 1980).
203
Statistical analyses were measured using the statistical program (SPSS16.0 for
204
windows, SPSS Inc., Chicago, IL, USA).
205
3 Results and discussion
206
3.1 The changes of moisture and NaCl contents of salted duck egg yolk
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Moisture and NaCl contents decreased or increased respectively (p<0.05), which
208
were presented in Fig.1 as the salting time was prolonged. These findings were
209
consistent with the results of Lai, Chi, and Ko (1999). When fresh duck eggs were
210
soaked into NaCl solution, the water of the egg yolk was gradually transferred to egg
211
white. After 10 days’ salting, the exterior egg yolk became harder, while the inside
212
region was still in a liquid state, as a result of the dehydration of egg yolk and the
213
penetration of salt (Chi & Tseng, 1998). Salt penetrated through the pores in eggshell
214
and permeated slowly into egg white to improve the level of osmotic pressure via
215
eggshell membrane, and thus, the rise of osmotic pressure in egg white promoted the
216
salt to continue penetrating to egg yolk. Furthermore, the stable emulsification system
217
collapsed. At the same time, chemical bonds were gradually strengthened because of
218
the demulsification of egg yolk under the high concentration of salt. The interspace of
219
egg yolk granules became narrower, causing the solidification of exterior egg yolk
220
and the enhancement of osmotic pressure in the interior of the egg yolk. The
221
solidification part of the egg yolk gradually extended to the interior part until the
222
osmotic pressure of interior yolk realized equilibrium with exterior yolk.
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Consequently, the content of moisture and salt reached a steady state after salting up
224
to 20 days.
225
3.2 Changes in apparent oil yield rate and oil extraction rate Egg yolk grains in the salted duck egg yolk were in the form of lipoprotein-yolk
227
high phosphoprotein bonded by calcium-phosphorus bridge complex. Ca2+ in the
228
calcium-phosphorus bridge replaced by Na+ resulted in the destroyed structure of egg
229
yolk particles in the gradual penetration of the salt (Causeret, Matringe, & Lorient,
230
2010). The increase of salt content caused the occurrence of salt-solubilization of
231
protein in the emulsified system (Zheng, Peng, Lin, & Xue-Juan, 2013). And weak gel
232
was formed by yolk protein on account of hydrophobic interactions and
233
hydrogen-bond
234
(Paraskevopoulou, Kiosseoglou, Alevisopoulos, & Kasapis, 2000). Feeney, Weaver,
235
Jones, and Rhodes (1956) reported that the increasing water migration from albumin
236
to yolk diluted the free lipid-protein from the surface of the egg yolk, which could be
237
attributed to the weakened yolk membrane in the process of salting, thus leading to oil
238
exudation. Therefore, the results of apparent oil yield rate and extraction oil rate all
239
showed an increasing trend (p<0.05), which were presented in Table 1. Meanwhile,
240
after salting for 20 days, there was no significant change in oil extraction (p<0.05),
241
which indicated that the oil exudation of salted duck egg yolk had reached the
242
maximum in the process of salting for up to 20-25 d and it was at the stage of best
243
oily texture. The reverse trend occurred between the variation of oil extraction and
244
moisture content, indicating the relative oil content of egg yolk increased together
245
with the water migration.
246
3.3 The changes of surface hydrophobicity
247
the
lipid
and
protein
were
effectively
separated
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It is reported that the hydrophobic interaction of proteins is prominently important - 10 -
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249
(Li-Chan & Nakai, 1991). Surface hydrophobicity depends on the interaction
250
between the water molecules repelled by the nonpolar groups which are caused by
251
the gathering of proteins. The level of hydrophobic interaction protein can be
252
expressed by the value of the surface hydrophobicity to be combined with
253
8-anilino-1-naphthalenesulfonate (ANS) as a probe, which further interacts with
254
anionic proteins (Smith, Galazka, Wellner, & Sumner, 2000). The results of the
255
measurement are reflected in Fig.2, showing that the surface hydrophobicity of
256
salted duck egg yolk remarkably increased (p<0.01) before 15d, and in contrast,
257
decreased slowly during the later time (p>0.05).
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It is found that the pH of salted duck egg yolk decreased from 6.42 to 6.03 in the
259
process of salting, and therefore, the surface of egg yolk particles was covered by a
260
layer of hydrophobic groups with negative charges because the protein isoelectric
261
point of egg yolk is about 5.0 (Rong, Zhang, & Han, 2012). At the same time, the
262
calcium-phosphorus
263
lipovitelenin was replaced by Na+, leading to the fact that part of the bridge might be
264
broken, combined with the separation of lipid and protein from lipoprotein. As a
265
result, the content of anionic proteins increased, indicating that surface
266
hydrophobicity rose as well. When the concentration of ions in the egg yolk reached
267
a certain level, it indicated the protein accumulating in the egg yolk reached a state
268
of equilibrium. The enhancement of ion interaction also drove the Na+ to replace the
269
hydrophobic groups in the protein surface because of the further diffusion of salt and
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between
high-density
lipoprotein
(HDL)
and
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ACCEPTED MANUSCRIPT dehydration of egg yolk (Anton & Gandemer, 1997; Anton & Martinet, 2003;
271
Martinez, Riscardo, & Jose, 2007). Therefore, the surface hydrophobicity of the
272
protein declined slightly. On the other hand, when the increasing Na+ in the interior
273
region of the egg yolk interacted with the hydrophobic groups with negative charges,
274
the structure of lipoprotein and the balance between oil and moisture were broken.
275
In this way, the lipid was free from the interior region of hydrophobic structure and
276
hence gathered to form big drops of lipid because of the similarity-intermiscibility.
277
This is the reason why the salted duck egg yolk forms its unique features of oil
278
permeation and sandy texture in the later stage of salting.
279
3.4 Particle size analysis and zeta potential
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The results of particle size and zeta potential of salted duck egg yolk are marked
281
in Fig.3. The size of the granule particles decreased significantly (p<0.05) in the
282
process of salting for up to 15 days, while this change was not significant any longer
283
(p>0.05) after 15 days. Furthermore, it was indicated that sandy texture was formed
284
after salting for 15 days. Our observation was consistent with the results reported by
285
Thammarat, Soottawat , and Wonnop (2009). The lipid in the egg yolk granules and
286
a certainty of free lipid from low-density lipoprotein permeated as several bonds
287
between protein and lipid were unfolded and the emulsion system and the structure
288
of egg yolk granules were broken (Bee & Cotterill, 2006). Meanwhile, the colloid
289
system of egg yolk was broken, showing the significant decrease of zeta potential
290
(p<0.01), which was a measurement of the intensity of the repulsion or attraction
291
between the particles. The results of surface hydrophobicity illustrated that the
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ACCEPTED MANUSCRIPT gathering of yolk proteins had reached a state of equilibrium, so the size of granules
293
became steady even though some slight float occurred during the later salting time
294
(15-25d). However, the accumulation of counter ions near the surface shielded the
295
surface charge, thus reducing the zeta potential with salt concentration increasing
296
even at the later salting time.
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Zeta potential can be used to reflect the stability of colloidal dispersion. The
298
smaller the molecular or dispersed particles were, the higher the zeta potential was
299
and the more stable the system was. That is, the dissolution or dispersion of
300
molecular or dispersed particles could resist aggregation when higher zeta potential
301
appeared. Because of its small size, it thus caused more stable emulsified system. On
302
the contrary, the lower zeta potential caused more tend to concentration or
303
agglomeration, indicating the attraction exceeded the repulsive force and that the
304
dispersion system was destroyed, thus leading to coagulation or condensation. The
305
egg yolk emulsion system was destroyed when salt content increased, and the zeta
306
potential declined from 13.5mV down to 1.9mV, indicating the accumulation of
307
protein in egg yolk, which was the reason why the sandy texture was formed. And
308
the sandy taste is the major factor impacting consumers’ acceptance of salted duck
309
egg (Chi & Tseng, 1998).
310
3.5 The changes of textural properties
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The changes of TPA of salted duck egg yolk in the process of salting were
312
summarized in Table 2. The data indicated that hardness significantly increased
313
(p<0.01) from 1341.77N to 2396.36N during the later stage of salting, demonstrating
314
that the changes occurred in the structure of the salted duck egg yolk including the - 13 -
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ACCEPTED MANUSCRIPT solidification of egg yolk, which was caused by the increase of salt content and
316
dehydration. The increase of salt concentration in the process of salting contributed to
317
physical and chemical changes of the egg yolk including the decrease of mobility and
318
the decline of sulfydryl groups as a result of the interaction between the disulfide
319
bond and sulfydryl groups as well as the oxidation of sulfydryl groups (Huang et al.,
320
2012). These changes are the major factors causing the increasing hardness of egg
321
yolk, which are in accordance with the reasons reported by Thammarat, Soottawat,
322
and Wonnop (2009). The parameters of springiness, cohesiveness and chewiness of
323
egg yolk significantly increased (p<0.05), in contrast, significant decline (p<0.05) of
324
cohesiveness was observed in the process of salting. With regard to the resilience,
325
there was no significant (p>0.05) change in this parameter in the process of salting.
326
Our findings were consistent with the results published by Thammarat, Soottawat, and
327
Wonnop (2009). However, there was no remarkable change in adhesiveness,
328
springiness and gumminess of parameters (p>0.05) after 20 days’ salting, which
329
suggested that the best texture of salted duck egg yolk was formed during the latter
330
salting time.
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Springiness is a factor on how much the gel structure is broken down by the initial
332
compression. When the gel structure was broken down into a few large pieces during
333
the first TPA compression, the results of springiness showed high significantly, but
334
became lower because the gel structure was broke down into many small pieces (Lau,
335
Tang, & Paulson, 2000). The parameters of springiness showed slightly changes and
336
the values were low after salting up to 10d in spite of the increasing springiness of egg
337
yolk, demonstrating egg yolk was broken down into many small pieces in the process
338
of TPA compression. This further showed the dehydration and the changes in the
339
interior structure made the exterior egg yolk present a state of semi-solidness or
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341
intact network structure. Gradually smaller and slight changing cohesiveness
342
indicated that the unequal osmotic pressure inside and outside of the egg yolk reduced
343
the ability to maintain the internal network structure of the egg yolk, which might lead
344
to the gradual accumulation of proteins in the egg yolk. The increase of hardness and
345
the decrease of chewiness and particle size demonstrated that the tight combination of
346
egg yolk granules during the later salting time made sure of the integrity of the egg
347
yolk contributing to the sandy texture after cooking.
348
3.6 The changes of rheological properties
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The changes of rheological properties of the salted duck egg yolk during different
350
salting times were shown in Fig.4. G' of the egg yolk increased significantly (p<0.05)
351
with the change of salting time. In the process of salting, the effects of NaCl promoted
352
the fractures of the egg yolk spheres to release the egg yolk granules for yolk
353
gelatinization, which resulted in the occurrence of semi-solidness or solidness of the
354
egg yolk with the increase of G' (Peng, Lin, Xiao, Huang, & Zhang, 2011). Variation
355
of changes in G" was similar to G', which had a significant increase (p<0.05), thus
356
indicating that the internal viscosity of egg yolk increased with the increase of the
357
salting time. Several types of particles including spheres, granules and low-density
358
lipoproteins in egg yolk, which were suspended in a protein solution or plasma, had
359
the ability to form gel (Woodward & Cotterill, 2006). Therefore, the G' and G" varied
360
when egg yolk became an elastic gel after salting. Our results were consistent with the
361
results discovered by Kaewmanee, Benjakul, Visessanguan, & Gamonpilas (2013),
362
indicating that the addition of NaCl could regulate the visco-elastic behavior of the
363
duck egg yolk by delaying the gel network formation. The presence of native yolk
364
lipids and emulsified oil droplets also influenced the rheological properties of salted
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366
salting time, indicating that the elasticity was a major rheological property in the
367
system of the salted duck egg yolk (Wang & Weil, 2011). The changes of G' and G"
368
were consistent with the changes of springiness and adhesiveness in the texture
369
analysis, revealing that the elasticity and viscosity of the egg yolk have partially
370
changed because of the texture changes of the salted duck egg yolk.
371
3.7 SDS - polyacrylamide gel electrophoresis (SDS-PAGE)
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The changes of the SDS-PAGE patterns of proteins in the salted duck egg yolk at
373
different salting stages were analysed by the protein relative rate by using standard
374
protein as the control. The SDS-PAGE results showed that the molecular weights of
375
the standard proteins were 20.1–97.2 kDa, while the molecular weights of proteins in
376
the salted duck egg yolk from different salting times ranged from 26.2 to 80.9 kDa
377
(Fig.5). It indicated that the molecular weights of proteins in the salted duck egg yolk
378
did not change after salting. However, these results were quite different from the
379
results reported by Thammarat, Soottawat, and Wonnop (2009). They pointed out that
380
the molecular weights of egg yolk proteins were between 30 and 220 kDa. The
381
lipoproteins in the egg yolk could be classified into low-density lipoprotein (LDL)
382
and high-density lipoprotein (HDL) (Martin, Augustyniak, & Cook, 1964; Burley &
383
Cook, 1961). The molecular weight of LDL apoprotein was about 80.9 kDa (Sikorski,
384
2001), while the molecular weight of HDL apoprotein was about 52.2 kDa (Raikos,
385
Hansen, Campbell, & Euston, 2006). The results of electrophoresis in each sample
386
showed that the changes of molecular weights of lipoproteins were not observed in
387
the process of salting. There was no difference among the electrophoretograms of
388
proteins in the salted duck egg yolk from different salting times. Our results indicated
389
that dehydration and high concentration of salt (20 g/100g) in the salting process did
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ACCEPTED MANUSCRIPT not influence the structure of the protein peptide chain, but did affect the spatial
391
structure of the protein, even if the salt content of the salted duck egg yolk was high
392
after salting. In comparison with the electrophoretograms of peptides between the
393
salted duck egg yolk and the fresh egg yolk, similar changes were observed, which
394
indicated that the peptide chain did not aggregate or degrade to form a new protein
395
fragment in the process of salting.
396
4 Conclusion
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The investigation on the relationship between the maturation of the salted duck
398
egg yolk and the changes of physicochemical, texture and rheological properties was
399
carried out to find out the best curing period for the sandy and oily texture of the
400
salted egg yolk. The results revealed that the physicochemical, texture, and
401
rheological characteristics of the yolk went through changes caused by the
402
dehydration and the high salt content, which had an influence on the micro-structure.
403
After salting 20–25d, the sandy and oily salted duck egg yolks were obtained when
404
the characteristics achieved equilibrium. Furthermore, the electrophoretograms
405
showed that the proteins and peptides in the salted duck egg yolk did not degrade or
406
aggregate at different salting times. This study further illustrated the mechanism
407
changes of the duck egg yolk in salt solution, but it’s deeper and complex changes
408
need to be further explored.
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409 410
Acknowledgements
411
This study was sponsored for the foundations of poultry products processing
412
engineering research and development center construction project, and poultry - 17 -
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ACCEPTED MANUSCRIPT 413
products precision machining and secure national local joint engineering research
414
center construction project. The authors would like to express sincere thanks to these
415
foundations for their financial support.
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416 References
418
AOAC (2000) Official methods of analysis, 17th ed. Association of Official
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ACCEPTED MANUSCRIPT The reason why we choose these five key references: The key references have
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provided the methods and some feasible idea to investigate salted duck egg yolk
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with scientific ways. And most importantly, we could make comparison with the
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results of these key references when we conducted experiment.
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Fig. 1 The changes of moisture and NaCl contents of raw and cooked salted egg yolks
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during salting. (●) raw egg yolk; (■) cooked egg yolk.
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Fig. 2 The change of surface hydrophobicity of egg yolk during salting.
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Fig. 3 The change of granule particle size and zeta potential of salted egg yolk during salting. (●) curing for 0 day;(∆) curing for 5 day; (○) curing for 10 day; (□) curing for 15 day; (▲) curing for 20 day; (■) curing for 25 day.
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Fig.4 The changes of storage modulus (G′) and loss modulus(G″) of salted egg yolks in different salting period. (■) curing for 0 day; (●) curing for 5 day; (▲) curing
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for 10 day; (▼) curing for 15 day; (♦) curing for 20 day; (◄) curing for 25 day.
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Fig.5 The changes in SDS-PAGE patterns of proteins in salted duck egg yolk for different salting times. 0EY: fresh egg yolk; 5EY: egg yolk after 5 days salination; 10EY: egg yolk after 10 days salination; 15EY: egg yolk after 15 days salination; 20EY: egg yolk after 20 days salination; 25EY: egg yolk after 25 days salination.
Table
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Table 1 The changes of apparent oil yield rate and oil extraction rate of egg yolk during salting. Apparent oil yield rate(g/100g)
Oil extraction rate(g/100g)
0
0.68±0.05d
29.71±1.09e
5
0.86±0.04d
30.04±1.29d
10
1.19±0.04c
15
1.24±0.07c
20
2.02±0.04b
25
2.95±0.07a
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Salting time(d)
A
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different salting periods
39.81±1.46b 40.86±1.15a
41.27±0.72a
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33.07±1.59c
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Table 2 The changes of textural properties during salting A 5d
10d
15d
858.91±17.41f
1341.77±22.33e
1462.04±38.52d
Adhesiveness (Ns)
2.64±0.07d
2.86±0.24d
3.25±0.04c
Springiness (mm)
0.28±0.02b
0.34±0.01b
0.39±0.01a
Cohesiveness
0.52±0.03a
0.49±0.01b
Gumminess (N)
352.34±72.49d
Chewiness (N) Resilience
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A
1870.42±36.15c
2056.05±104.86b
2396.46±46.69a
4.04±0.33b
5.08±0.23a
5.11±0.18a
0.41±0.00a
0.42±0.01a
0.43±0.02a
0.45±0.01c
0.44±0.00c
0.41±0.01c
0.39±0.02c
463.56±4.13c
824.85±25.33b
838.35±12.89b
914.43±29.33a
918.29±17.87a
139.39±11.03e
259.42±10.54d
334.04±17.09c
345.05±10.95c
386.29±26.30b
417.45±29.66a
0.18±0.01a
0.17±0.01a
0.14±0.01a
0.14±0.00a
0.14±0.00a
0.16±0.01a
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25d
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Hardness (N)
20d
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Highlights
ACCEPTED MANUSCRIPT Highlights 1.The best salting and texture periods was 20-25d. 2.Physicochemical and texture properties were investigated under 20wt% NaCl.
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3.Patterns of the proteins electrophoresis were not significantly different.
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