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Subcritical fluid extraction treatment on egg yolk: Product characterization
Journal of Food Engineering 274 (2020) 109805
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Subcritical fluid extraction treatment on egg yolk: Product characterization Yujie Su a, 1, Mengyao Ji a, 1, Junhua Li a, Cuihua Chang a, Shijian Dong b, Yongdong Deng b, Yanjun Yang a, *, Luping Gu a, ** a
State Key Laboratory of Food Science and Technology, School of Food Science and Technology; Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, Jiangnan University, Wuxi, Jiangsu, China b Rongda Poultry Co.Ltd, Xuancheng, Anhui, 242000, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Subcritical fluid-propane extraction Egg yolk oil Low-fat egg yolk powder Functional properties
In this study, the method of subcritical fluid-propane extraction (SPE) used to coproduce egg yolk oil (EYO) and low-fat egg yolk powder from spray-dried egg yolk powder (EYP) was investigated. Results showed that the optimum parameters were: extraction temperature of 313.15 K, extraction time of 120 min, and solid-liquid ratio of 1:9 (g/mL). Under this condition, the residual oil rate of EYP was 21.04%, which was much lower than that of ethanol extraction (39.24%). Scanning electron microscopy reveled that serious disruptions of egg yolk sphere extracted by SPE were observed, facilitating the EYO extraction. In addition, oil extracted by SPE had lower acid value and peroxide value than that of ethanol extraction. Moreover, the emulsifying properties and solubility of low-fat EYP obtained by SPE were similar to the original egg yolk powder. This study provides a new technique for coproducing high-quality oil and protein.
1. Introduction Eggs, traditional nutrient-rich food, contain many important com ponents with superior nutritional value, such as proteins, essential fatty acids, phospholipids, vitamins and minerals (Paraskevopoulou et al., 1997). Due to their good functional properties, including emulsifying, gelling and foaming properties, eggs have extensive applications in food industry. However, there are some shortcomings about fresh eggs, for example, frangibility, transport difficulties, short storage period, etc. To address these issues, dried egg powder has been developed in the past few decades. Among these, egg yolk powder (EYP) is an important dried egg product, which almost fully retains functional properties of fresh egg yolk, especially in emulsifying property mainly. Moreover, EYP experi ences the pasteurization process before spray drying, and thus it is su perior to fresh egg yolk from the aspects of food safety. Although egg yolk is an important nutrition resource for human, many consumers avoid egg yolk in the diet, due to its high content of oil and cholesterol. Excessive fat intake from a long-term high-cholesterol diet can cause some diseases (e.g., hypertension and cardiovascular) (David Spence, 2016; Puertas and Vazquez, 2018). Therefore, it is meaningful to reduce oil and cholesterol in EYP, which could improve its nutritional value and
consumer acceptance. The most common techniques of oil extraction in food industry are press extraction and solvent extraction. And it was reported that tradi tional method used to extract oil from EYP was solvent extraction and organic solvents most common used were ethanol and hexane (Larsen and Froning, 1981). However, this method usually needs a long extraction period and further steps to separate the oil and solvent (Santos et al., 2015). In addition, there were some safety and quality problems on products, such as organic solvent residues and degenera tion of active components (Teixeira et al., 2018). Recently, supercritical carbon dioxide extraction (SCE) has been successfully applied in the removal of lipids and cholesterol from dried egg yolk. After SCE, the contents of oil and cholesterol in dried egg yolk were simultaneously reduced from 62.14%/3.18% – to 28.7%/2.54% under treating condition of 350 atm/333.15 K (Sun et al., 1995). Su percritical fluid extraction has overcome many deficiencies imposed by conventional organic solvent-oriented extraction methods and received much attention in food industry. The advantages of supercritical CO2 extraction of processed foods generally include freedom of residual solvent in extracted products, low to moderate temperature and minimal protein degradation. However, its pressure is indispensable to reach
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y. Yang), [email protected] (L. Gu). 1 The authors Yujie Su and Mengyao Ji are contributed equally to the manuscript. https://doi.org/10.1016/j.jfoodeng.2019.109805 Received 2 August 2019; Received in revised form 21 October 2019; Accepted 3 November 2019 Available online 18 December 2019 0260-8774/© 2019 Elsevier Ltd. All rights reserved.
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Journal of Food Engineering 274 (2020) 109805
about 350 atm, which is a great challenge for the manufacture of pres sure vessel. Fortunately, the pressure of SPE for oil extraction is at least an order of magnitude (tens of bar compared with hundreds of bar) lower than SCE (Lisiane et al., 2008). Meanwhile, it is promising for propane to be an alternative of CO2, due to its high solubility in lipids (Zanqui et al., 2014). Compared with supercritical CO2 extraction of oil from sapucaia (Lecythis pisonis) nuts, the extraction efficiency of subcritical propane (critical temperature of 370.15 K and critical pres sure of 4.19 MPa) was 93.38%, markedly higher than supercritical extraction efficiency (67.32%) under the same temperature of 333.13K and extraction time of 60 min (Teixeira et al., 2018). Subcritical propane has been successfully used to extract oils from a wide range of vegetable sources such as seed (Coelho et al., 2016), palm (Jesus et al., 2013) and so on. However, no information could be found in the literatures regarding the egg yolk oil extraction using subcritical propane. Hence, the object of this study was to extract oil from EYP through SPE and to prepare low-fat EYP. The effects of extraction parameters including temperature, extraction time and solid-liquid ratio on extraction efficiencies of oil and cholesterol were studied. The physi cochemical properties of egg yolk oil (EYO) extracted and low-fat EYP were also investigated.
2.2.2. Solvent extraction (SE) EYO extracted by a solvent extraction method was used as a control. The experiment was performed according to the method described by Su et al. (2015) with slight modifications. Briefly, EYP and ethanol (10 mL/g yolk powder) were added to a jacket type reactor, and then the mixture was stirred at 342.15 K for 10 h. After that, solvent was removed in a vacuum rotary evaporator at 323.15K (IKA RV10 Basic, Germany). EYO was kept in an amber glass vessel and stored at 277.15 K until further analysis. The residual low-fat EYP extracted using ethanol (LFY-E) was stored in a dryer at room temperature for analysis. 2.3. The properties of egg yolk oil 2.3.1. Acid value and peroxide value measurement Acid value (AV) and peroxide value (POV) of the lipids were ob tained according to the methodology of Sun et al. (2018). The samples evaluated were oil from ethanol extraction and oil obtained from subcritical propane extraction. 2.3.2. Analysis of fatty acid methyl esters The fatty acid composition of extracted oil was analyzed by gas chromatography (GC) system (Agilent GC 7820 A, Agilent Technologies, Santa Clara, CA, USA) equipped with a flame ionization detector (FID) and a TR-FAME fused silica capillary column (60 mⅹ0.32 mmⅹ2.5 μm, Thermo Fisher Scientific, Franklin, MA, USA). The fatty acid methyl esters (FAMEs) were prepared by transesterification of the extracted oil, according to the method described by Stoffel et al. (1959). The column temperature program for GC analysis was set at initial temperature of 333.15 K for 3 min, followed by a linear increase of temperature of 278.15 K/min to 448.15 K, and a linear increase of temperature to 493.15 K at the rate of 275.15 K/min, with the final temperature of 493.15 K lasting for 10 min. The injector and detector temperature were held at 493.15 K and 523.15 K, respectively. Nitrogen (99.99% purity) was used as carrier gas at a constant velocity of 1.0 mL/min. The in jection volume was 1 μL. And the relative concentrations of the fatty acids were calculated from peak areas.
2. Materials and methods 2.1. Materials Spray-dried EYP was provided by Anhui Rongda Poultry Co., Ltd. (Xuancheng, China). Sunflower seed oil (COFCO) was purchased from a local supermarket (Wuxi, China). All chemical reagents of analytical grade were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). 2.2. Sample preparation 2.2.1. Subcritical fluid extraction (SPE) SPE was applied to extract EYO from spray-dried EYP. Apparatus for SPE was shown in Fig. 1. The SPE procedure was performed using pro pane as a solvent. 70.0 g of EYP was filled into the extractor tank, and then. EYO was extracted according to the following parameters: extraction temperature (303.15 K–323.15 K), extraction time (30 min–150 min) and ratio of powder to liquefied propane (1:3 g/mL - 1:11 g/mL). Then the oil was collected with a glass tube under the evapora tion tank. Finally, EYO was kept in an amber glass vessel and stored at 277.15 K until further analysis. The residual low-fat EYP extracted by propane (LFY-P) was stored in a dryer at room temperature for analysis.
2.4. The properties of residual low-fat egg yolk powder 2.4.1. Composition analysis Moisture of the sample was determined by using an oven drying procedure (16.002,), and protein was measured by Kjeldahl method (N ⅹ 6.25). Cholesterol was measured by the method of R. Sun et al. (1995). And lipid content of residual was measured by the method of Floch et al. (Folch et al., 1957).
Fig. 1. Schematic diagram of SPE unit used in this work. (1) an extraction tank; (2) an evaporation tank; (3) a buffer tank; (4) a condenser; (5) three solvent tanks; (6) a hot water tank; (7) a hot water pump; (8) a compressor; (9) a vacuum pump; (10) a flame arrester and (11) a control panel. 2
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2.4.2. Particle size and color analysis The particle size distribution of samples was measured by a laser particle size analyzer (S3500, Microtrac lnc, Florida, USA). Color values (L , a , b , representing lightness, redness and yellowness, respectively) were measured using an UltraScan Pro1166 spectrometer (HunterLab, Reston, USA).
microscope slide for observation. The images were recorded and for further analysis. 2.5. Data analysis Each sample was analyzed in triplicate at least. The results were expressed as mean � standard deviation and analyzed by SPSS 17.0 (IBM Corporation, New York, USA). One-way analysis of variance (ANOVA) and Duncan’s multiple range tests were carried out for dif ference analysis (P < 0.05).
2.4.3. Dissolution properties 2.4.3.1. Solubility. The solubility of sample was determined according to the method of Fabien et al. (2005). Firstly, samples were dispersed in Milli-Q water (20 g kg 1) at 302.15 K with moderate magnetic stirring for 1 h. The solution was then centrifuged at 5000 rpm for 20 min. After that the supernatant was decanted and filtered by filter paper. Protein content was then determined by the Kjeldahl method (N � 6.25). Sol ubility (S) was calculated using the following equation:
3. Results and discussion 3.1. Extraction efficiency of EYO Organic solvents extraction has been applied to EYO isolation from egg yolk. When using these solvents, only small amount of unavoidable processing agent presenting no danger to human is allowed to be left in residual yolk powder. This risk disappeared when a subcritical extrac tion method was used to extract EYO. In this study, propane was chosen for its high solvation properties and non-toxicity (Piva et al., 2018). The effects of extraction time, extraction temperature and solid-liquid ratio on extraction efficiency of EYO were studied. One point should be noted that temperature is the determinant factor in the subcritical region instead of pressure in the supercritical region (Teixeira et al., 2018). Therefore, pressure was not taken into account in the study of SPE. Initially, total oil content in EYP was determined and it occupied for 58.26%. In order to evaluate extraction efficiency of EYO, the oil content in residual EYP treated by subcritical extraction was studied. The lower oil content in residual EYP, the higher of the extraction efficiency. There was a significant difference in the oil content in residual EYP at different temperatures with a constant extraction time and solid-liquid ratio. Fig. 2A clearly showed that the lowest residual oil rate (21.04%) of EYP occurred at 313.15K. Specifically, with an increase in extraction temperature from 303.15 K to 313.15 K, the oil content in residual EYP decreased from 26.8% to 21.8%, indicating improved extraction effi ciency. This may be ascribed to the fact that as the temperature increased, the motion rate of propane molecules and the mass transfer coefficient of propane increased, resulting in an increase in yolk oil dissolution (Piva et al., 2018). However, a further increase in temper ature caused a decrease in oil extraction efficiency, which may due to decreases in the density and intermolecular force of propane, leading to a decrease in oil dissolving capacity of propane. Meanwhile, under the condition of 250 atm/317.15 K in SCE process, the total lipids in residual EYP were 33.45%, higher than SPE (Sun et al.). The results proved that propane has a higher efficiency for the oil extraction. Subcritical fluid technology introduced the new solvent (propane) with each extraction, which helped to break the equilibrium in the degreasing process. As can be seen from Fig. 2B, no significant difference was observed with extraction time longer than 120 min, and thus the optimal extraction time was chosen as 120 min. This fact was related to the concept that the oil located inside the particle took more time to cross the solid-fluid interface, when compared to the oil on the surface of the particle (Da Silva et al., 2017). Compared with the other materials using SPE to extract oils, such as sapucaia (Lecythis pisonis) nuts and Elaeis spp, extraction equilibrium time was about 60 min (Da Silva et al., 2017; Teixeira et al., 2018). This phenomenon was due to the solid properties of the raw material, propane can’t penetrate the interior resulting in a short equilibrium time. In order to characterize the equilibrium dissolution property of EYO between the two phases, effect of solid-liquid ratio on extraction effi ciency of EYO was studied. As shown in Fig. 2C, with an increase in solid-liquid ratio, the residual oil rate gradually decreased. And when the solid-liquid ratio was higher than 1:9 g/mL, a steady trend was observed. This may be because the volume of liquefied propane had basically covered the raw material (EYP), and more solvents will only
(1)
Sð%Þ ¼ ðm1 = m2 Þ � 100
where m1 was the protein content of the supernatant and m2 was the total protein content of the sample. 2.4.3.2. Stability coefficient. Stability coefficient was measured by the method of Tang et al. (2019). The yolk powder solution was prepared at 1% (w/v) concentration with deionized water and stirred magnetically at 305.15 K for 1 h. The sample was then centrifuged at 3000 rpm for 20 min. The supernatant was diluted for 100 times and scanned in the range of 250–350 nm to calculate the maximum absorbance. The stability coefficient (R) was calculated using the following equation: (2)
Rð%Þ ¼ ðA1 = A2 Þ � 100
where A1 is the absorbance of the supernatant at the maximum ab sorption wavelength in the range 250–350 nm and A2 is the absorbance of the sample prior to centrifugation at the maximum absorption wavelength. 2.4.4. Emulsifying properties The emulsifying activity index (EAI) and emulsion stability index (ESI) of samples were determined according to turbidimetric Method (Jing et al., 2011).The EYP (2%,w/v) was dispersed in water and stirred magnetically at room temperature for 1 h. 10 mL sunflower oil and 30 mL of sample solution was mixed by an Ultra-Turrax blender (IKA T25 Basic, Staufen, Germany) at 10,000 rpm for 1 min at 302.15K. 100 μL coarse emulsions were pipetted from the bottom of the tube into 10 mL of SDS solutions (0.1%, w/v) at 0 min and 10 min after homogenization. The turbidity of the diluted solutions was determined at 500 nm. The EAI was the absorbance taken immediately after emulsification. The ESI values were calculated using the following equations: ESIðminÞ ¼ A0 ⅹ10 = ðA0
A10 Þ
(3)
where A0 and A10 are the absorbances of diluted emulsions at 0 and 10 min, respectively. 2.4.5. Determination of egg yolk powder microstructure Microstructures of EYP were observed by a scanning electron mi croscope (SEM) (su1510, HITACHI, Japan). The powder was sprinkled on aluminum stubs using a double-sided adhesive tape. The samples were then sputter-coated with gold to produce the conductive surface and observed. Representative micrographs were taken at 500ⅹ and 2000ⅹmagnification. In order to further observe the changes of oil and protein in EYP, a laser scanning confocal microscope (CLSM) (LSM710, zeiss, Germany) was used. Firstly, samples (1 mL) were prepared by dispersing EYP in deionized water (1:20, w/v) and stained with 10 μL of FITC and Nile Red (0.1%, v/v). After 20 min, dyed sample solution was smeared on the 3
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Journal of Food Engineering 274 (2020) 109805
Fig. 2. Extraction of EYO by SPE: (A) extraction temperature (303.15 K–323.15 K), extraction time (120 min) and solid-liquid ratio (1:10 g/mL); (B) extraction temperature (313.15 K), extraction time (30–150 min) and solid-liquid ratio (1:10 g/mL); (C) extraction temperature (313.15 K), extraction time (120 min) and solidliquid ratio (1:3–1:11 g/mL). Different lower-case letters represent the significant difference (p < 0.05).
increase the cost. Hence, 1:9 g/mL was the appropriate solid-liquid ratio for subcritical propane extraction of EYO. According to the results above, the optimum parameters for SPE on EYO were: 313.15K, solid-liquid ratio of 1:9 g/mL and extraction time of 120 min. Under this condition, the oil content in residual EYP was reduced from 58.26% to 21.04%. The oil extraction yield reached 63.88%, even higher than the best oil extraction yield (62.58%) in the SCE process (Sun et al., 1995).
SE. Oleic and linoleic acids were the main unsaturated fatty acids, while palmitic and stearic acids were the main saturated fatty acids, whose contents were in the following order: oleic acid > palmitic acid > linoleic acid > stearic acid. In addition, EYO has 15.6% polyunsaturated fatty acids, including linoleic acid (18:2), linolenic acid (18:3) and arachidonic acid (20:4), and linoleic acid was the main component (14.6%). Linoleic acid was a recognized essential fatty acid, which was highly valued for lowering blood cholesterol and preventing athero sclerosis. Consequently, EYO is a good novel food source that could be used for developing functional food.
3.2. Properties of egg yolk oil 3.2.1. AV and POV Initially, the appearance of egg yolk oil extracted by SPE and SE was shown in Fig. 3. It was observed that EYO extracted by SPE was less red and more transparent, indicating a better quality of oil. In order to confirm this, two common indexes (AV and POV) were determined and the lower of AV and POV, the better quality of oil. As shown in Table 1, oil extracted by SPE had lower AV and POV than that of SE, indicating weaker oxidation of EYO occurred during SPE process. Ethanol extrac tion required a solvent removal process, and the temperature of this process may result in an increase of the peroxidation and acidity. The results were in accordance with the report that AV and POV of Antarctic krill lipid extracted by subcritical propane were lower than those of lipid extracted by ethanol (Sun et al., 2018).
3.3. Properties of residual egg yolk powder 3.3.1. General composition After oil was extracted from EYP, there was a significant change on the composition of residual EYP. Table 3 showed that the composition of original egg yolk powder (OYP) was 31.55% protein and 58.26% lipids. For residual EYP extracted by SE (LYP-E), protein and lipid content were changed into 44.74% and 39.24%, respectively. However, after SPE, the total lipid content was reduced about 63.88%, which was consistent with the conclusion that propane had better lipid solubility. Addition ally, the protein content increased by about twice accordingly. The reason for this increased protein content was that most of the total lipids were extracted from samples. Moreover, it was noticeable that the cholesterol content was reduced from 20.35 mg/g to 7.61 mg/g, which was highly beneficial to broaden the application of EYP extracted by SPE (LYP-P). Consequently, compared with LYP-E, LYP-P could be a better nutritional ingredient for those who pursued high quality protein with low fat and cholesterol. Especially, LYP-P could be used to develop food
3.2.2. Fatty acid composition The fatty acids composition of EYO was further analyzed to evaluate the oil quality. As shown in Table 2, no significant difference was observed between fatty acid composition of EYO extracted by SPE and 4
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Table 3 Composition analysis of egg yolk powder. Sample
Protein (%)
Total Lipid (%)
Moisture (%)
Cholesterol (mg/ g)
OYP LYP-P LYP-E
31.55 � 0.65c 61.43 � 0.53a 44.74 � 0.23b
58.26 � 0.74a 21.04 � 0.31c 39.24 � 0.42b
3.83 � 0.10c 6.62 � 0.13b 10.00 � 0.16a
20.35 � 0.52a 7.61 � 0.08c 13.21 � 0.03b
Different lower-case letters in the same list represent the significant difference (p < 0.05).
for the infant and elderly. 3.3.2. Color difference analysis It could be seen in Table 4 that the yellow value (b) and red value (a) of LYP-P both decreased obviously. The original egg yolk powder (OYP) had the higher yellow value and red value because OYP contains the lipid-soluble pigments such as carotene and lutein. With the oil extrac ted, the content of carotene and lutein also declined in the residual EYP. Although SE can also extract EYO to some extent, but the b and a of LYPE were higher than that of LYP-P. The results were related with the re sidual oil rate of EYP. On the contrary, the a of LYP-E was even higher than OYP. Such behavior could be attributed to the higher temperature and longer extraction time during SE process, leading to oxidation of pigment (Long et al., 2018). In addition, the morphology of powder particles shown in Fig. 4 seemed to be related to the lipid content. The OYP was more viscous due to its higher lipid content compared to low-fat yolk powder. Due to the denaturation and oxidation of proteins, LYP-E was more agglomerated. However, LYP-P was more evenly distributed. The results indicated that SPE was indeed a better technique to coproduce low-fat yolk powder. Fig. 3. Picture of the EYO extracted by different methods (SPE and SE).
3.3.3. Particle size distribution As can be seen from Fig. 5, OYP showed bimodal particle size dis tributions. Compared to OYP, the particle size distribution of LYP-P was more uniform. This measurement result was consistent with the visual observation of Fig. 4. As expected, the peak particle size of LYP-E moved towards a larger value due to its aggregation. Table 5 showed the surface area weighted average diameter (D [3,2]) and volume weighted average diameter (D [4,3]) of the samples. The average size of larger particle was typically characterized by the volumeweighted mean diameter (D [4,3]) (Borges et al., 2015). Consistent with Fig. 5, the volume-weighted mean diameter (D [4,3]) followed the order: LYP-E > OYP > LYP-P. The results showed that the particle size of LYP-P was significantly smaller and more uniform, while LYP-E was more agglomerated.
Table 1 Physicochemical properties of egg yolk oil obtained by different methods. Parameters
oil(subcritical-propane)
oil(ethanol)
(AV)/(mg KOH/g) (POV)/(meq/kg)
25.67 � 0.93b 3.88 � 0.02b
30.84 � 1.02a 4.76 � 0.03a
Different lower-case letters in the same row represent the significant difference (p < 0.05). Table 2 Fatty acid composition of EYO obtained by different methods (%). Fat acids
Subcritical propane extraction
Ethanol extraction
C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:1 C20:4 Others ΣMUFAa ΣPUFAb Σ PUFA þΣ MUFA ΣSFAc
0.37 � 0.02b 24.36 � 0.04b 4.23 � 0.01a 6.16 � 0.02a 47.67 � 0.05a 14.62 � 0.03b 0.46 � 0.02a 0.24 � 0.01b 0.50 � 0.02a 1.39 � 0.02a 52.14 � 0.04a 15.08 � 0.02b 67.22 � 0.06a 30.89 � 2.05b
0.50 � 0.03a 26.64 � 0.01a 3.30 � 0.01b 5.54 � 0.01b 47.25 � 0.06b 14.67 � 0.02a 0.46 � 0.03a 0.30 � 0.02a 0.52 � 0.03a 0.82 � 0.03b 50.85 � 0.05b 15.13 � 0.03a 65.98 � 0.04b 32.68 � 1.95a
3.3.4. Solubility and stability coefficient Proteins and lipids are the two major components of egg yolk. Sol ubility is a functional property used as a key prerequisite for the po tential application of protein components in beverages, liquid foods and emulsions under different processing conditions (Sousa et al., 2007). Such property can influence other functional protein properties. In general, soluble proteins are expected to possess excellent emulsion, foam, gelation and other characteristics. As shown in Fig. 6, the solu bility of OYP was 18.06%, and the solubility of LYP-P was 12.31%, Table 4 Color difference analysis of egg yolk powder.
Different lower-case letters in the same row represent the significant difference (p < 0.05). a Monounsaturated fatty acids. b Polyunsaturated fatty acids. c Saturated fatty acids.
Sample
L
a
b
OYP LYP-P LYP-E
83.58 � 0.37b 88.73 � 0.29a 67.66 � 0.32c
8.98 � 0.28b 3.91 � 0.34c 12.51 � 0.21a
46.74 � 0.41a 26.26 � 0.25c 36.98 � 0.35b
Different lower-case letters in the same list represent the significant difference (p < 0.05). 5
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Fig. 4. Macroscopic pictures of egg yolk powder. (A) OYP: original egg yolk powder; (B) LYP-P: the low-fat egg yolk powder obtained by SPE; (C) LYP-E: the low-fat egg yolk powder obtained by SE.
Fig. 6. Dissolution properties of samples. Different letters represent the sig nificant difference (p < 0.05).
which may be due to its insolubility as previously mentioned in Fig. 6. And then the emulsifying activity and emulsion stability of OYP and LYP-P were analyzed. Fig. 7 denoted that compared with OYP, the emulsifying activity of LYP-P decreased slightly, and its emulsifying stability was significantly improved. The increased stability of emulsion may be related with the decreased particle size of LYP-P which can form more uniform and smaller emulsion droplets.
Fig. 5. Particle size distribution of samples. Table 5 Surface area-weighted mean diameter (D diameter (D [4,3]) of egg yolk powder. Sample
D
OYP LYP-P LYP-E
59.61 � 2.34c 65.20 � 1.68b 83.72 � 1.94a
[3,2]
(μm)
[3,2])
and volume-weighted mean D
[4,3]
(μm)
95.05 � 2.05b 92.08 � 2.18c 100.5 � 2.35a
3.3.6. Microstructural analysis Microstructural property of EYP after treatment of oil extraction was investigated by SEM (Fig. 8). It can clearly be seen that OYP consisted of granules with various sizes and rough surfaces, which may be induced by spray drying on egg yolk. Other work also reported that after spray drying, the surface of egg yolk granules was rough and some tiny granules were observed, which results from the collapse or wrinkling of spherical particles (Hammes et al., 2015). After oil extraction by SPE, the granules were severely damaged and the internal structure was completely exposed, which showed that one granule contains many smaller granules. The destruction and exposure could promote the release of oil, which was beneficial to oil extraction. However, for LYP-E, the damage degree of the particle was weaker and just some cracks were observed on the surface, leading to less contact area between solvent and yolk powder. And these results were in accordance with the higher extraction efficiency of EYO by SPE. And then CLSM was further operated to observe distribution of proteins and lipids in EYP. As illustrated in Fig. 9, the majority of pro teins in egg yolks were organized into micellar and granular structures, together via gathering of polar and non-polar lipid molecules, which was also reported by literature (Kiosseoglou, 2003). And consistent with SEM images, many holes can be clearly observed on the surface of LYP-P
Different lower-case letters in the same list represent the significant difference (p < 0.05).
slightly lower than OYP. However, LYP-E was almost insoluble, possibly due to the severe denaturation of proteins at higher temperatures during SE process, which was similar to the results of defatted walnut powder obtained by Mao and Hua (Xiao-Ying and Yu-Fei, 2014). The stability of different samples is also shown in Fig. 6. The stability coefficient of LYP-P (16.22%) was significantly (P < 0.05) higher than that of OYP (25.42%) and LYP-E (11.47%), suggesting that a more stable dispersions of low-fat yolk powder was obtained after SPE process. Overall, LYP-E had lost its solubility function, whose application would be very limited. In contrast, LPY-P exhibited good performance of sol ubility and dispersity and had a promising prospect in food industry. 3.3.5. Emulsifying properties Emulsifying property is a significant functional property of egg yolk, which gives the product the desired taste and texture properties. As shown in the inset of Fig. 7, both OYP and LYP-P had good stability of emulsion. However, LYP-E completely lost its emulsifying properties, 6
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Fig. 7. Emulsifying properties of samples.
Fig. 9. CLSM micrographs of samples. A: FITC labeled proteins; B: Nile Red labeled lipids; C: Diagram of proteins and lipids synthesis. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
4. Conclusions Overall, the SPE showed some interesting advantages than tradi tional solvent method (such as ethanol). AV, POV and fatty acid composition analysis indicated that EYO extracted by SPE had better quality than that by SE. Except for the obvious decrease of oil and cholesterol, LYP-P also exhibited better solubility and emulsification characteristics. Consequently, subcritical technique shows unique ad vantages in the preparation of egg yolk oil and low-fat EYP.
Fig. 8. Scanning electron microscope images of samples. The magnification A:500ⅹf; B: 2000ⅹf.
and the size of particles reduced. Compared with those of OYP, some free lipids were also observed in LYP-P and LYP-E, which may be due to the destruction of lipoprotein structure. In addition, extraction operations could lead to the migration of oil from the interior of the lipoprotein to the surface of the yolk particles. And due to higher oil extraction effi ciency by SPE, the oil on the surface of LYP-P was obviously less than that of LYP-E.
Declaration of competing interest No conflict of interest exits in the submission of this manuscript, and 7
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manuscript is approved by all authors for publication. I would like to declare, on behalf of my co-authors, that the work described herein is original and has not been published previously. This work is not under consideration for publication elsewhere, in whole or in part.
Lisiane, D.S.F., Jos� e Vladimir, D.O., Cl� audio, D., Rosangela Assis, J., Elina Bastos, C.O., 2008. Extraction of grape seed oil using compressed carbon dioxide and propane: extraction yields and characterization of free glycerol compounds. J. Agric. Food Chem. 56 (8), 2558–2564. Long, J., Wang, F., Jiao, A., Xu, X., Jin, Z., 2018. Preparation, characterization and physicochemical properties of novel low-phosphorus egg yolk protein. J. Sci. Food Agric. 99, 1740–1747. Paraskevopoulou, A., Panayiotou, K., Kiosseoglou, V., 1997. Functional properties of egg yolk concentrate with a reduced cholesterol content prepared by treatment with supercritical CO2. Food Hydrocolloids 11 (4), 385–391. Piva, G.S., Weschenfelder, T.A., Franceschi, E., Cansian, R.L., Paroul, N., Steffens, C., 2018. Extraction and modeling of flaxseed (Linnum usitatissimum ) oil using subcritical propane. J. Food Eng. 228, 50–56. Puertas, G., Vazquez, M., 2018. Advances in techniques for reducing cholesterol in egg yolk: a review. Crit. Rev. Food Sci. Nutr. 1–11. Santos, K.A., Bariccatti, R.A., Cardozo-Filho, L., Schneider, R., Palú, F., Silva, C.D., Silva, E.A.D., 2015. Extraction of crambe seed oil using subcritical propane: kinetics, characterization and modeling. J. Supercrit. Fluids 104, 54–61. Sousa, R.D.C.S., Coimbra, J.S.R., Rojas, E.E.G., Minim, L.A., Oliveira, F.C., Minim, V.P.R., 2007. Effect of pH and salt concentration on the solubility and density of egg yolk and plasma egg yolk. LWT Food Sci. Technol. 40 (7), 1253–1258. Stoffel, W., Chu, F., Ahrens Jr., E.H., 1959. Analysis of long-chain fatty acids by gasliquid chromatography. Micromethod for preparation of methyl esters. Anal. Chem. 31 (2), 307–308. Su, Y., Tian, Y., Yan, R., Wang, C., Niu, F., Yang, Y., 2015. Study on a novel process for the separation of phospholipids, triacylglycerol and cholesterol from egg yolk. J. Food Sci. Technol. 52 (7), 4586–4592. Sun, R., Sivik, B., Larsson, K., 1995. The fractional extraction of lipids and cholesterol from dried egg-yolk using supercritical carbon-dioxide. Fett Wissenschaft Technologie-Fat Sci. Technol. 97 (6), 214–219. Sun, D.W., Cao, C., Li, B., Chen, H.J., Li, J.W., Cao, P.R., Liu, Y.F., 2018. Antarctic krill lipid extracted by subcritical n-butane and comparison with supercritical CO2 and conventional solvent extraction. LWT Food Sci. Technol. 94, 1–7. Tang, S., Zhou, X., Gouda, M., Cai, Z., Jin, Y., 2019. Effect of enzymatic hydrolysis on the solubility of egg yolk powder from the changes in structure and functional properties. LWT Food Sci. Technol. 110, 214–222. Teixeira, G.L., Ghazani, S.M., Corazza, M.L., Marangoni, A.G., Ribani, R.H., 2018. Assessment of subcritical propane, supercritical CO2 and Soxhlet extraction of oil from sapucaia (Lecythis pisonis) nuts. J. Supercrit. Fluids 133, 122–132. Xiao-Ying, M., Yu-Fei, H., 2014. Chemical composition, molecular weight distribution, secondary structure and effect of NaCl on functional properties of walnut (Juglans regia L) protein isolates and concentrates. J. Food Sci. Technol. 51 (8), 1473–1482. Zanqui, A.B., Morais, D.R.d., Silva, C.M.d., Santos, J.M., Chiavelli, L.U.R., Bittencourt, P. R.S., Eberlin, M.N., Visentainer, J.V., Cardozo-Filho, L., Matsushita, M., 2014. Subcritical extraction of salvia hispanicaL. oil with n-propane: composition, purity and oxidation stability as compared to the oils obtained by conventional solvent extraction methods. J. Braz. Chem. Soc. 26 (2), 282–289.
Acknowledgments The authors would like to thank for the financial supporting from the National Key Research and Development Program of China (2018YED0400303) and the National Natural Science Foundation of China (No. 31801483, 31671809). References Borges, G.R., Farias, G.B., Braz, T.M., Santos, L.M., Amaral, M.J., Fortuny, M., Franceschi, E., Dariva, C., Santos, A.F., 2015. Use of near infrared for evaluation of droplet size distribution and water content in water-in-crude oil emulsions in pressurized pipeline. Fuel 147, 43–52. Coelho, R., Kanda, L.R.S., Hamerski, F., Masson, M.L., Corazza, M.L., 2016. Extraction of kiwifruit seed (actinidia deliciosa) oil using compressed propane. J. Food Process. Eng. 39 (4), 335–344. Da Silva, C.M., Zanqui, A.B., Da Silva, E.A., Gomes, S.T.M., Filho, L.C., Matsushita, M., 2017. Extraction of oil from elaeis spp. using subcritical propane and cosolvent: experimental and modeling. J. Supercrit. Fluids 133, 401–410. David Spence, J., 2016. Dietary cholesterol and egg yolk should be avoided by patients at risk of vascular disease. J. Trans. Intern. Med. 4 (1), 20–24. Fabien, G., Ingolf, K., Ulrich, K., 2005. Efficient analysis of egg yolk proteins and their thermal sensitivity using sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing and nonreducing conditions. J. Agric. Food Chem. 53 (24), 9329. Folch, J., Lees, M., Stanley, G.H.S., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 495–509. Hammes, M.V., Englert, A.H., Nore~ na, C.P.Z., Cardozo, N.S.M., 2015. Study of the influence of soy lecithin addition on the wettability of buffalo milk powder obtained by spray drying. Powder Technol. 277, 237–243. Jesus, A.A., Almeida, L.C., Silva, E.A., Filho, L.C., Egues, S.M.S., Franceschi, E., Fortuny, M., Santos, A.F., Araujo, J., Sousa, E.M.B.D., 2013. Extraction of palm oil using propane, ethanol and its mixtures as compressed solvent. J. Supercrit. Fluids 81 (8), 245–253. Jing, H., Yap, M., Wong, P.Y.Y., Kitts, D.D., 2011. Comparison of physicochemical and antioxidant properties of egg-white proteins and fructose and inulin maillard reaction products. Food Bioprocess Technol. 4 (8), 1489–1496. Kiosseoglou, V., 2003. Egg yolk protein gels and emulsions. Curr. Opin. Colloid Interface Sci. 8 (4), 365–370. Larsen, J.E., Froning, G.W., 1981. Extraction and processing of various components from egg yolk. Poult. Sci. 60 (1), 160–167.
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Journal of Food Engineering journal homepage: http://www.elsevier.com/locate/jfoodeng
Subcritical fluid extraction treatment on egg yolk: Product characterization Yujie Su a, 1, Mengyao Ji a, 1, Junhua Li a, Cuihua Chang a, Shijian Dong b, Yongdong Deng b, Yanjun Yang a, *, Luping Gu a, ** a
State Key Laboratory of Food Science and Technology, School of Food Science and Technology; Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, Jiangnan University, Wuxi, Jiangsu, China b Rongda Poultry Co.Ltd, Xuancheng, Anhui, 242000, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Subcritical fluid-propane extraction Egg yolk oil Low-fat egg yolk powder Functional properties
In this study, the method of subcritical fluid-propane extraction (SPE) used to coproduce egg yolk oil (EYO) and low-fat egg yolk powder from spray-dried egg yolk powder (EYP) was investigated. Results showed that the optimum parameters were: extraction temperature of 313.15 K, extraction time of 120 min, and solid-liquid ratio of 1:9 (g/mL). Under this condition, the residual oil rate of EYP was 21.04%, which was much lower than that of ethanol extraction (39.24%). Scanning electron microscopy reveled that serious disruptions of egg yolk sphere extracted by SPE were observed, facilitating the EYO extraction. In addition, oil extracted by SPE had lower acid value and peroxide value than that of ethanol extraction. Moreover, the emulsifying properties and solubility of low-fat EYP obtained by SPE were similar to the original egg yolk powder. This study provides a new technique for coproducing high-quality oil and protein.
1. Introduction Eggs, traditional nutrient-rich food, contain many important com ponents with superior nutritional value, such as proteins, essential fatty acids, phospholipids, vitamins and minerals (Paraskevopoulou et al., 1997). Due to their good functional properties, including emulsifying, gelling and foaming properties, eggs have extensive applications in food industry. However, there are some shortcomings about fresh eggs, for example, frangibility, transport difficulties, short storage period, etc. To address these issues, dried egg powder has been developed in the past few decades. Among these, egg yolk powder (EYP) is an important dried egg product, which almost fully retains functional properties of fresh egg yolk, especially in emulsifying property mainly. Moreover, EYP experi ences the pasteurization process before spray drying, and thus it is su perior to fresh egg yolk from the aspects of food safety. Although egg yolk is an important nutrition resource for human, many consumers avoid egg yolk in the diet, due to its high content of oil and cholesterol. Excessive fat intake from a long-term high-cholesterol diet can cause some diseases (e.g., hypertension and cardiovascular) (David Spence, 2016; Puertas and Vazquez, 2018). Therefore, it is meaningful to reduce oil and cholesterol in EYP, which could improve its nutritional value and
consumer acceptance. The most common techniques of oil extraction in food industry are press extraction and solvent extraction. And it was reported that tradi tional method used to extract oil from EYP was solvent extraction and organic solvents most common used were ethanol and hexane (Larsen and Froning, 1981). However, this method usually needs a long extraction period and further steps to separate the oil and solvent (Santos et al., 2015). In addition, there were some safety and quality problems on products, such as organic solvent residues and degenera tion of active components (Teixeira et al., 2018). Recently, supercritical carbon dioxide extraction (SCE) has been successfully applied in the removal of lipids and cholesterol from dried egg yolk. After SCE, the contents of oil and cholesterol in dried egg yolk were simultaneously reduced from 62.14%/3.18% – to 28.7%/2.54% under treating condition of 350 atm/333.15 K (Sun et al., 1995). Su percritical fluid extraction has overcome many deficiencies imposed by conventional organic solvent-oriented extraction methods and received much attention in food industry. The advantages of supercritical CO2 extraction of processed foods generally include freedom of residual solvent in extracted products, low to moderate temperature and minimal protein degradation. However, its pressure is indispensable to reach
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y. Yang), [email protected] (L. Gu). 1 The authors Yujie Su and Mengyao Ji are contributed equally to the manuscript. https://doi.org/10.1016/j.jfoodeng.2019.109805 Received 2 August 2019; Received in revised form 21 October 2019; Accepted 3 November 2019 Available online 18 December 2019 0260-8774/© 2019 Elsevier Ltd. All rights reserved.
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about 350 atm, which is a great challenge for the manufacture of pres sure vessel. Fortunately, the pressure of SPE for oil extraction is at least an order of magnitude (tens of bar compared with hundreds of bar) lower than SCE (Lisiane et al., 2008). Meanwhile, it is promising for propane to be an alternative of CO2, due to its high solubility in lipids (Zanqui et al., 2014). Compared with supercritical CO2 extraction of oil from sapucaia (Lecythis pisonis) nuts, the extraction efficiency of subcritical propane (critical temperature of 370.15 K and critical pres sure of 4.19 MPa) was 93.38%, markedly higher than supercritical extraction efficiency (67.32%) under the same temperature of 333.13K and extraction time of 60 min (Teixeira et al., 2018). Subcritical propane has been successfully used to extract oils from a wide range of vegetable sources such as seed (Coelho et al., 2016), palm (Jesus et al., 2013) and so on. However, no information could be found in the literatures regarding the egg yolk oil extraction using subcritical propane. Hence, the object of this study was to extract oil from EYP through SPE and to prepare low-fat EYP. The effects of extraction parameters including temperature, extraction time and solid-liquid ratio on extraction efficiencies of oil and cholesterol were studied. The physi cochemical properties of egg yolk oil (EYO) extracted and low-fat EYP were also investigated.
2.2.2. Solvent extraction (SE) EYO extracted by a solvent extraction method was used as a control. The experiment was performed according to the method described by Su et al. (2015) with slight modifications. Briefly, EYP and ethanol (10 mL/g yolk powder) were added to a jacket type reactor, and then the mixture was stirred at 342.15 K for 10 h. After that, solvent was removed in a vacuum rotary evaporator at 323.15K (IKA RV10 Basic, Germany). EYO was kept in an amber glass vessel and stored at 277.15 K until further analysis. The residual low-fat EYP extracted using ethanol (LFY-E) was stored in a dryer at room temperature for analysis. 2.3. The properties of egg yolk oil 2.3.1. Acid value and peroxide value measurement Acid value (AV) and peroxide value (POV) of the lipids were ob tained according to the methodology of Sun et al. (2018). The samples evaluated were oil from ethanol extraction and oil obtained from subcritical propane extraction. 2.3.2. Analysis of fatty acid methyl esters The fatty acid composition of extracted oil was analyzed by gas chromatography (GC) system (Agilent GC 7820 A, Agilent Technologies, Santa Clara, CA, USA) equipped with a flame ionization detector (FID) and a TR-FAME fused silica capillary column (60 mⅹ0.32 mmⅹ2.5 μm, Thermo Fisher Scientific, Franklin, MA, USA). The fatty acid methyl esters (FAMEs) were prepared by transesterification of the extracted oil, according to the method described by Stoffel et al. (1959). The column temperature program for GC analysis was set at initial temperature of 333.15 K for 3 min, followed by a linear increase of temperature of 278.15 K/min to 448.15 K, and a linear increase of temperature to 493.15 K at the rate of 275.15 K/min, with the final temperature of 493.15 K lasting for 10 min. The injector and detector temperature were held at 493.15 K and 523.15 K, respectively. Nitrogen (99.99% purity) was used as carrier gas at a constant velocity of 1.0 mL/min. The in jection volume was 1 μL. And the relative concentrations of the fatty acids were calculated from peak areas.
2. Materials and methods 2.1. Materials Spray-dried EYP was provided by Anhui Rongda Poultry Co., Ltd. (Xuancheng, China). Sunflower seed oil (COFCO) was purchased from a local supermarket (Wuxi, China). All chemical reagents of analytical grade were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). 2.2. Sample preparation 2.2.1. Subcritical fluid extraction (SPE) SPE was applied to extract EYO from spray-dried EYP. Apparatus for SPE was shown in Fig. 1. The SPE procedure was performed using pro pane as a solvent. 70.0 g of EYP was filled into the extractor tank, and then. EYO was extracted according to the following parameters: extraction temperature (303.15 K–323.15 K), extraction time (30 min–150 min) and ratio of powder to liquefied propane (1:3 g/mL - 1:11 g/mL). Then the oil was collected with a glass tube under the evapora tion tank. Finally, EYO was kept in an amber glass vessel and stored at 277.15 K until further analysis. The residual low-fat EYP extracted by propane (LFY-P) was stored in a dryer at room temperature for analysis.
2.4. The properties of residual low-fat egg yolk powder 2.4.1. Composition analysis Moisture of the sample was determined by using an oven drying procedure (16.002,), and protein was measured by Kjeldahl method (N ⅹ 6.25). Cholesterol was measured by the method of R. Sun et al. (1995). And lipid content of residual was measured by the method of Floch et al. (Folch et al., 1957).
Fig. 1. Schematic diagram of SPE unit used in this work. (1) an extraction tank; (2) an evaporation tank; (3) a buffer tank; (4) a condenser; (5) three solvent tanks; (6) a hot water tank; (7) a hot water pump; (8) a compressor; (9) a vacuum pump; (10) a flame arrester and (11) a control panel. 2
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2.4.2. Particle size and color analysis The particle size distribution of samples was measured by a laser particle size analyzer (S3500, Microtrac lnc, Florida, USA). Color values (L , a , b , representing lightness, redness and yellowness, respectively) were measured using an UltraScan Pro1166 spectrometer (HunterLab, Reston, USA).
microscope slide for observation. The images were recorded and for further analysis. 2.5. Data analysis Each sample was analyzed in triplicate at least. The results were expressed as mean � standard deviation and analyzed by SPSS 17.0 (IBM Corporation, New York, USA). One-way analysis of variance (ANOVA) and Duncan’s multiple range tests were carried out for dif ference analysis (P < 0.05).
2.4.3. Dissolution properties 2.4.3.1. Solubility. The solubility of sample was determined according to the method of Fabien et al. (2005). Firstly, samples were dispersed in Milli-Q water (20 g kg 1) at 302.15 K with moderate magnetic stirring for 1 h. The solution was then centrifuged at 5000 rpm for 20 min. After that the supernatant was decanted and filtered by filter paper. Protein content was then determined by the Kjeldahl method (N � 6.25). Sol ubility (S) was calculated using the following equation:
3. Results and discussion 3.1. Extraction efficiency of EYO Organic solvents extraction has been applied to EYO isolation from egg yolk. When using these solvents, only small amount of unavoidable processing agent presenting no danger to human is allowed to be left in residual yolk powder. This risk disappeared when a subcritical extrac tion method was used to extract EYO. In this study, propane was chosen for its high solvation properties and non-toxicity (Piva et al., 2018). The effects of extraction time, extraction temperature and solid-liquid ratio on extraction efficiency of EYO were studied. One point should be noted that temperature is the determinant factor in the subcritical region instead of pressure in the supercritical region (Teixeira et al., 2018). Therefore, pressure was not taken into account in the study of SPE. Initially, total oil content in EYP was determined and it occupied for 58.26%. In order to evaluate extraction efficiency of EYO, the oil content in residual EYP treated by subcritical extraction was studied. The lower oil content in residual EYP, the higher of the extraction efficiency. There was a significant difference in the oil content in residual EYP at different temperatures with a constant extraction time and solid-liquid ratio. Fig. 2A clearly showed that the lowest residual oil rate (21.04%) of EYP occurred at 313.15K. Specifically, with an increase in extraction temperature from 303.15 K to 313.15 K, the oil content in residual EYP decreased from 26.8% to 21.8%, indicating improved extraction effi ciency. This may be ascribed to the fact that as the temperature increased, the motion rate of propane molecules and the mass transfer coefficient of propane increased, resulting in an increase in yolk oil dissolution (Piva et al., 2018). However, a further increase in temper ature caused a decrease in oil extraction efficiency, which may due to decreases in the density and intermolecular force of propane, leading to a decrease in oil dissolving capacity of propane. Meanwhile, under the condition of 250 atm/317.15 K in SCE process, the total lipids in residual EYP were 33.45%, higher than SPE (Sun et al.). The results proved that propane has a higher efficiency for the oil extraction. Subcritical fluid technology introduced the new solvent (propane) with each extraction, which helped to break the equilibrium in the degreasing process. As can be seen from Fig. 2B, no significant difference was observed with extraction time longer than 120 min, and thus the optimal extraction time was chosen as 120 min. This fact was related to the concept that the oil located inside the particle took more time to cross the solid-fluid interface, when compared to the oil on the surface of the particle (Da Silva et al., 2017). Compared with the other materials using SPE to extract oils, such as sapucaia (Lecythis pisonis) nuts and Elaeis spp, extraction equilibrium time was about 60 min (Da Silva et al., 2017; Teixeira et al., 2018). This phenomenon was due to the solid properties of the raw material, propane can’t penetrate the interior resulting in a short equilibrium time. In order to characterize the equilibrium dissolution property of EYO between the two phases, effect of solid-liquid ratio on extraction effi ciency of EYO was studied. As shown in Fig. 2C, with an increase in solid-liquid ratio, the residual oil rate gradually decreased. And when the solid-liquid ratio was higher than 1:9 g/mL, a steady trend was observed. This may be because the volume of liquefied propane had basically covered the raw material (EYP), and more solvents will only
(1)
Sð%Þ ¼ ðm1 = m2 Þ � 100
where m1 was the protein content of the supernatant and m2 was the total protein content of the sample. 2.4.3.2. Stability coefficient. Stability coefficient was measured by the method of Tang et al. (2019). The yolk powder solution was prepared at 1% (w/v) concentration with deionized water and stirred magnetically at 305.15 K for 1 h. The sample was then centrifuged at 3000 rpm for 20 min. The supernatant was diluted for 100 times and scanned in the range of 250–350 nm to calculate the maximum absorbance. The stability coefficient (R) was calculated using the following equation: (2)
Rð%Þ ¼ ðA1 = A2 Þ � 100
where A1 is the absorbance of the supernatant at the maximum ab sorption wavelength in the range 250–350 nm and A2 is the absorbance of the sample prior to centrifugation at the maximum absorption wavelength. 2.4.4. Emulsifying properties The emulsifying activity index (EAI) and emulsion stability index (ESI) of samples were determined according to turbidimetric Method (Jing et al., 2011).The EYP (2%,w/v) was dispersed in water and stirred magnetically at room temperature for 1 h. 10 mL sunflower oil and 30 mL of sample solution was mixed by an Ultra-Turrax blender (IKA T25 Basic, Staufen, Germany) at 10,000 rpm for 1 min at 302.15K. 100 μL coarse emulsions were pipetted from the bottom of the tube into 10 mL of SDS solutions (0.1%, w/v) at 0 min and 10 min after homogenization. The turbidity of the diluted solutions was determined at 500 nm. The EAI was the absorbance taken immediately after emulsification. The ESI values were calculated using the following equations: ESIðminÞ ¼ A0 ⅹ10 = ðA0
A10 Þ
(3)
where A0 and A10 are the absorbances of diluted emulsions at 0 and 10 min, respectively. 2.4.5. Determination of egg yolk powder microstructure Microstructures of EYP were observed by a scanning electron mi croscope (SEM) (su1510, HITACHI, Japan). The powder was sprinkled on aluminum stubs using a double-sided adhesive tape. The samples were then sputter-coated with gold to produce the conductive surface and observed. Representative micrographs were taken at 500ⅹ and 2000ⅹmagnification. In order to further observe the changes of oil and protein in EYP, a laser scanning confocal microscope (CLSM) (LSM710, zeiss, Germany) was used. Firstly, samples (1 mL) were prepared by dispersing EYP in deionized water (1:20, w/v) and stained with 10 μL of FITC and Nile Red (0.1%, v/v). After 20 min, dyed sample solution was smeared on the 3
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Fig. 2. Extraction of EYO by SPE: (A) extraction temperature (303.15 K–323.15 K), extraction time (120 min) and solid-liquid ratio (1:10 g/mL); (B) extraction temperature (313.15 K), extraction time (30–150 min) and solid-liquid ratio (1:10 g/mL); (C) extraction temperature (313.15 K), extraction time (120 min) and solidliquid ratio (1:3–1:11 g/mL). Different lower-case letters represent the significant difference (p < 0.05).
increase the cost. Hence, 1:9 g/mL was the appropriate solid-liquid ratio for subcritical propane extraction of EYO. According to the results above, the optimum parameters for SPE on EYO were: 313.15K, solid-liquid ratio of 1:9 g/mL and extraction time of 120 min. Under this condition, the oil content in residual EYP was reduced from 58.26% to 21.04%. The oil extraction yield reached 63.88%, even higher than the best oil extraction yield (62.58%) in the SCE process (Sun et al., 1995).
SE. Oleic and linoleic acids were the main unsaturated fatty acids, while palmitic and stearic acids were the main saturated fatty acids, whose contents were in the following order: oleic acid > palmitic acid > linoleic acid > stearic acid. In addition, EYO has 15.6% polyunsaturated fatty acids, including linoleic acid (18:2), linolenic acid (18:3) and arachidonic acid (20:4), and linoleic acid was the main component (14.6%). Linoleic acid was a recognized essential fatty acid, which was highly valued for lowering blood cholesterol and preventing athero sclerosis. Consequently, EYO is a good novel food source that could be used for developing functional food.
3.2. Properties of egg yolk oil 3.2.1. AV and POV Initially, the appearance of egg yolk oil extracted by SPE and SE was shown in Fig. 3. It was observed that EYO extracted by SPE was less red and more transparent, indicating a better quality of oil. In order to confirm this, two common indexes (AV and POV) were determined and the lower of AV and POV, the better quality of oil. As shown in Table 1, oil extracted by SPE had lower AV and POV than that of SE, indicating weaker oxidation of EYO occurred during SPE process. Ethanol extrac tion required a solvent removal process, and the temperature of this process may result in an increase of the peroxidation and acidity. The results were in accordance with the report that AV and POV of Antarctic krill lipid extracted by subcritical propane were lower than those of lipid extracted by ethanol (Sun et al., 2018).
3.3. Properties of residual egg yolk powder 3.3.1. General composition After oil was extracted from EYP, there was a significant change on the composition of residual EYP. Table 3 showed that the composition of original egg yolk powder (OYP) was 31.55% protein and 58.26% lipids. For residual EYP extracted by SE (LYP-E), protein and lipid content were changed into 44.74% and 39.24%, respectively. However, after SPE, the total lipid content was reduced about 63.88%, which was consistent with the conclusion that propane had better lipid solubility. Addition ally, the protein content increased by about twice accordingly. The reason for this increased protein content was that most of the total lipids were extracted from samples. Moreover, it was noticeable that the cholesterol content was reduced from 20.35 mg/g to 7.61 mg/g, which was highly beneficial to broaden the application of EYP extracted by SPE (LYP-P). Consequently, compared with LYP-E, LYP-P could be a better nutritional ingredient for those who pursued high quality protein with low fat and cholesterol. Especially, LYP-P could be used to develop food
3.2.2. Fatty acid composition The fatty acids composition of EYO was further analyzed to evaluate the oil quality. As shown in Table 2, no significant difference was observed between fatty acid composition of EYO extracted by SPE and 4
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Table 3 Composition analysis of egg yolk powder. Sample
Protein (%)
Total Lipid (%)
Moisture (%)
Cholesterol (mg/ g)
OYP LYP-P LYP-E
31.55 � 0.65c 61.43 � 0.53a 44.74 � 0.23b
58.26 � 0.74a 21.04 � 0.31c 39.24 � 0.42b
3.83 � 0.10c 6.62 � 0.13b 10.00 � 0.16a
20.35 � 0.52a 7.61 � 0.08c 13.21 � 0.03b
Different lower-case letters in the same list represent the significant difference (p < 0.05).
for the infant and elderly. 3.3.2. Color difference analysis It could be seen in Table 4 that the yellow value (b) and red value (a) of LYP-P both decreased obviously. The original egg yolk powder (OYP) had the higher yellow value and red value because OYP contains the lipid-soluble pigments such as carotene and lutein. With the oil extrac ted, the content of carotene and lutein also declined in the residual EYP. Although SE can also extract EYO to some extent, but the b and a of LYPE were higher than that of LYP-P. The results were related with the re sidual oil rate of EYP. On the contrary, the a of LYP-E was even higher than OYP. Such behavior could be attributed to the higher temperature and longer extraction time during SE process, leading to oxidation of pigment (Long et al., 2018). In addition, the morphology of powder particles shown in Fig. 4 seemed to be related to the lipid content. The OYP was more viscous due to its higher lipid content compared to low-fat yolk powder. Due to the denaturation and oxidation of proteins, LYP-E was more agglomerated. However, LYP-P was more evenly distributed. The results indicated that SPE was indeed a better technique to coproduce low-fat yolk powder. Fig. 3. Picture of the EYO extracted by different methods (SPE and SE).
3.3.3. Particle size distribution As can be seen from Fig. 5, OYP showed bimodal particle size dis tributions. Compared to OYP, the particle size distribution of LYP-P was more uniform. This measurement result was consistent with the visual observation of Fig. 4. As expected, the peak particle size of LYP-E moved towards a larger value due to its aggregation. Table 5 showed the surface area weighted average diameter (D [3,2]) and volume weighted average diameter (D [4,3]) of the samples. The average size of larger particle was typically characterized by the volumeweighted mean diameter (D [4,3]) (Borges et al., 2015). Consistent with Fig. 5, the volume-weighted mean diameter (D [4,3]) followed the order: LYP-E > OYP > LYP-P. The results showed that the particle size of LYP-P was significantly smaller and more uniform, while LYP-E was more agglomerated.
Table 1 Physicochemical properties of egg yolk oil obtained by different methods. Parameters
oil(subcritical-propane)
oil(ethanol)
(AV)/(mg KOH/g) (POV)/(meq/kg)
25.67 � 0.93b 3.88 � 0.02b
30.84 � 1.02a 4.76 � 0.03a
Different lower-case letters in the same row represent the significant difference (p < 0.05). Table 2 Fatty acid composition of EYO obtained by different methods (%). Fat acids
Subcritical propane extraction
Ethanol extraction
C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:1 C20:4 Others ΣMUFAa ΣPUFAb Σ PUFA þΣ MUFA ΣSFAc
0.37 � 0.02b 24.36 � 0.04b 4.23 � 0.01a 6.16 � 0.02a 47.67 � 0.05a 14.62 � 0.03b 0.46 � 0.02a 0.24 � 0.01b 0.50 � 0.02a 1.39 � 0.02a 52.14 � 0.04a 15.08 � 0.02b 67.22 � 0.06a 30.89 � 2.05b
0.50 � 0.03a 26.64 � 0.01a 3.30 � 0.01b 5.54 � 0.01b 47.25 � 0.06b 14.67 � 0.02a 0.46 � 0.03a 0.30 � 0.02a 0.52 � 0.03a 0.82 � 0.03b 50.85 � 0.05b 15.13 � 0.03a 65.98 � 0.04b 32.68 � 1.95a
3.3.4. Solubility and stability coefficient Proteins and lipids are the two major components of egg yolk. Sol ubility is a functional property used as a key prerequisite for the po tential application of protein components in beverages, liquid foods and emulsions under different processing conditions (Sousa et al., 2007). Such property can influence other functional protein properties. In general, soluble proteins are expected to possess excellent emulsion, foam, gelation and other characteristics. As shown in Fig. 6, the solu bility of OYP was 18.06%, and the solubility of LYP-P was 12.31%, Table 4 Color difference analysis of egg yolk powder.
Different lower-case letters in the same row represent the significant difference (p < 0.05). a Monounsaturated fatty acids. b Polyunsaturated fatty acids. c Saturated fatty acids.
Sample
L
a
b
OYP LYP-P LYP-E
83.58 � 0.37b 88.73 � 0.29a 67.66 � 0.32c
8.98 � 0.28b 3.91 � 0.34c 12.51 � 0.21a
46.74 � 0.41a 26.26 � 0.25c 36.98 � 0.35b
Different lower-case letters in the same list represent the significant difference (p < 0.05). 5
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Journal of Food Engineering 274 (2020) 109805
Fig. 4. Macroscopic pictures of egg yolk powder. (A) OYP: original egg yolk powder; (B) LYP-P: the low-fat egg yolk powder obtained by SPE; (C) LYP-E: the low-fat egg yolk powder obtained by SE.
Fig. 6. Dissolution properties of samples. Different letters represent the sig nificant difference (p < 0.05).
which may be due to its insolubility as previously mentioned in Fig. 6. And then the emulsifying activity and emulsion stability of OYP and LYP-P were analyzed. Fig. 7 denoted that compared with OYP, the emulsifying activity of LYP-P decreased slightly, and its emulsifying stability was significantly improved. The increased stability of emulsion may be related with the decreased particle size of LYP-P which can form more uniform and smaller emulsion droplets.
Fig. 5. Particle size distribution of samples. Table 5 Surface area-weighted mean diameter (D diameter (D [4,3]) of egg yolk powder. Sample
D
OYP LYP-P LYP-E
59.61 � 2.34c 65.20 � 1.68b 83.72 � 1.94a
[3,2]
(μm)
[3,2])
and volume-weighted mean D
[4,3]
(μm)
95.05 � 2.05b 92.08 � 2.18c 100.5 � 2.35a
3.3.6. Microstructural analysis Microstructural property of EYP after treatment of oil extraction was investigated by SEM (Fig. 8). It can clearly be seen that OYP consisted of granules with various sizes and rough surfaces, which may be induced by spray drying on egg yolk. Other work also reported that after spray drying, the surface of egg yolk granules was rough and some tiny granules were observed, which results from the collapse or wrinkling of spherical particles (Hammes et al., 2015). After oil extraction by SPE, the granules were severely damaged and the internal structure was completely exposed, which showed that one granule contains many smaller granules. The destruction and exposure could promote the release of oil, which was beneficial to oil extraction. However, for LYP-E, the damage degree of the particle was weaker and just some cracks were observed on the surface, leading to less contact area between solvent and yolk powder. And these results were in accordance with the higher extraction efficiency of EYO by SPE. And then CLSM was further operated to observe distribution of proteins and lipids in EYP. As illustrated in Fig. 9, the majority of pro teins in egg yolks were organized into micellar and granular structures, together via gathering of polar and non-polar lipid molecules, which was also reported by literature (Kiosseoglou, 2003). And consistent with SEM images, many holes can be clearly observed on the surface of LYP-P
Different lower-case letters in the same list represent the significant difference (p < 0.05).
slightly lower than OYP. However, LYP-E was almost insoluble, possibly due to the severe denaturation of proteins at higher temperatures during SE process, which was similar to the results of defatted walnut powder obtained by Mao and Hua (Xiao-Ying and Yu-Fei, 2014). The stability of different samples is also shown in Fig. 6. The stability coefficient of LYP-P (16.22%) was significantly (P < 0.05) higher than that of OYP (25.42%) and LYP-E (11.47%), suggesting that a more stable dispersions of low-fat yolk powder was obtained after SPE process. Overall, LYP-E had lost its solubility function, whose application would be very limited. In contrast, LPY-P exhibited good performance of sol ubility and dispersity and had a promising prospect in food industry. 3.3.5. Emulsifying properties Emulsifying property is a significant functional property of egg yolk, which gives the product the desired taste and texture properties. As shown in the inset of Fig. 7, both OYP and LYP-P had good stability of emulsion. However, LYP-E completely lost its emulsifying properties, 6
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Journal of Food Engineering 274 (2020) 109805
Fig. 7. Emulsifying properties of samples.
Fig. 9. CLSM micrographs of samples. A: FITC labeled proteins; B: Nile Red labeled lipids; C: Diagram of proteins and lipids synthesis. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
4. Conclusions Overall, the SPE showed some interesting advantages than tradi tional solvent method (such as ethanol). AV, POV and fatty acid composition analysis indicated that EYO extracted by SPE had better quality than that by SE. Except for the obvious decrease of oil and cholesterol, LYP-P also exhibited better solubility and emulsification characteristics. Consequently, subcritical technique shows unique ad vantages in the preparation of egg yolk oil and low-fat EYP.
Fig. 8. Scanning electron microscope images of samples. The magnification A:500ⅹf; B: 2000ⅹf.
and the size of particles reduced. Compared with those of OYP, some free lipids were also observed in LYP-P and LYP-E, which may be due to the destruction of lipoprotein structure. In addition, extraction operations could lead to the migration of oil from the interior of the lipoprotein to the surface of the yolk particles. And due to higher oil extraction effi ciency by SPE, the oil on the surface of LYP-P was obviously less than that of LYP-E.
Declaration of competing interest No conflict of interest exits in the submission of this manuscript, and 7
Y. Su et al.
Journal of Food Engineering 274 (2020) 109805
manuscript is approved by all authors for publication. I would like to declare, on behalf of my co-authors, that the work described herein is original and has not been published previously. This work is not under consideration for publication elsewhere, in whole or in part.
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