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Amino acid composition and digestibility of Pacific oyster (Crassostrea gigas) proteins isolated from different parts
LWT - Food Science and Technology 116 (2019) 108591
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Amino acid composition and digestibility of Pacific oyster (Crassostrea gigas) proteins isolated from different parts
T
Suisui Jiang, Li Liu, Jinjin Xu, Mingyong Zeng∗, Yuanhui Zhao∗∗ College of Food Science and Engineering, Ocean University of China, Qingdao, 266003, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Essential amino acids Oyster proteins S–S group content Secondary structure Digestion
To understand the nutritional value and the relationship between structure and in vitro digestibility of Pacific oyster (Crassostrea gigas) proteins, the amino acid composition, physicochemical structure, and digestibility of different part proteins were investigated. All proteins contained significant amounts of essential amino acids, including lysine, leucine, and valine. The adductor protein showed the highest contents of SH (39.0 μmol/g protein) and S–S (11.7 μmol/g protein), whereas lower contents of SH (9.5 μmol/g protein) and S–S (5.1 μmol/g protein) in gill protein was observed. In addition, adductor protein contained more α-helix (64.7%) and β-sheet (11.2%) structures, whereas the proportions of α-helix (33.9%) and β-sheet (18.4%) structures in visceral mass protein were the lowest. The digestion results showed that gill protein undergoes rapid digestion in the stomach, and visceral mass protein shows the highest digestibility in the total digestive process. This work provides a greater understanding of the nutrition value and digestibility of oyster protein.
1. Introduction
Previous studies have demonstrated that protein digestion is a complex process that depends on the structure and physicochemical properties of proteins. The carbonyl groups present in proteins mainly come from the covalent bonding of proteins, carbohydrates and the oxidation products of carbohydrates or fatty acids, which are important indicators of the oxidation degree of proteins. Increasing carbonyl content can decrease the protein digestibility due to the formation of intra- or intermolecular cross-linking (Sante-Lhoutellier et al., 2007; Sun, Cui, Zhao, Zhao, & Yang, 2011a; Sun, Zhao, Yang, Zhao, & Cui, 2011b). A high sulfhydryl (SH) group content in protein usually implies a low degree of protein oxidation that is always accompanied by high digestibility (Xiong, Donkeun, & Tooru, 2009). During the heating process, many SH proteins can be oxidized to form S–S, which is the reason for the reduction in digestibility (Cui, Zhou, Zhao, & Yang, 2009; Sun et al., 2011a). Additionally, the protein aggregation state can affect the surface hydrophobicity, leading to varied digestibility (KaminBelsky, Brillon, Arav, & Shaklai, 1996; Liu & Xiong, 2000). Sun et al. reported that the increased surface hydrophobicity of a protein could cause a decrease in the α-helical content and an increase in the β-strand content, ascribed to the reduction of hydrogen bonds in the α-helix structure (Sun, Zhou, Zhao, Yang, & Cui, 2011c). Thus, the secondary structure composition of proteins must also be taken into account when determining protein digestion.
Dietary protein, particularly essential amino acids, is essential for humans to ensure body repair, cell regeneration, growth, and development (Bohrer, 2017). Dietary protein also plays a critical role in maintaining muscle mass, which is the basis of chronic disease prevention, functional capacity, and quality of life. However, after forty, skeletal muscle mass naturally starts to decline (Paddon-Jones & Rasmussen, 2009). Previous research has claimed that protein diets, especially high-quality protein, can attenuate natural muscle mass loss and promote muscle synthesis (Devries & Phillips, 2015). Therefore, high-quality protein is very important in our daily diet due to its’ high levels of essential amino acids (Mania, Wojciechowska-Mazurek, Starska, Rebeniak, & Postupolski, 2012; Willoughby, 2004). However, amino acid composition is not the only criterion of the nutritional quality of protein (Boye, Wijesinha-Bettoni, & Burlingame, 2012). To be absorbed, proteins must be hydrolyzed by proteases within the digestive tract to form amino acids and peptides. Vast accumulation of nonhydrolyzed or partially hydrolyzed proteins in the large intestine could increase colonic microbial fermentation to produce toxic metabolites, which can induce inflammation and further result in colon cancer (Dallas et al., 2017; Sante-Lhoutellier, Aubry, & Gatellier, 2007). Thus, digestibility also plays a significant role in protein quality.
∗
Corresponding author. Corresponding author. E-mail addresses: [email protected] (M. Zeng), [email protected] (Y. Zhao).
∗∗
https://doi.org/10.1016/j.lwt.2019.108591 Received 15 April 2019; Received in revised form 31 August 2019; Accepted 4 September 2019 Available online 05 September 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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et al. (2018) to determine the molecular weight (MW) distribution of the protein. Samples were electrophoresed on a 12% separating gel and a 5% stacking gel. Briefly, protein samples (80 μL, 10 mg/mL) were diluted using 5 × SDS-PAGE loading buffer (20 μL) at a final concentration of 2 mg/mL and then heated in boiled water for 10 min. After cooling to room temperature, 10 μL samples were added to each lane. A protein marker (5–245 kDa) was used for MW determination.
Oysters are one of the most valuable shellfishes, with the largest output in China, whose annual production in 2017 was approximately 4.89 million tons (Ministry of Agriculture and Rural Affairs of the People's Republic of China, 2018, p. 23). It is a high-protein food containing high amounts of essential amino acids and taurine (Je, Park, Jung, & Kim, 2005) and is an excellent source of high-quality protein. Furthermore, their delicate and delicious meat is one of the reasons why oysters are widely consumed by people. However, few studies have focused on determining the protein structure and digestibility of oyster proteins. The mantle, gill, visceral mass, and adductor are the main components of the oyster, and their structures and properties are substantially different. The purpose of this study was to explore the differences in the structures of different parts of oysters, including the mantle, gill, visceral mass, adductor and whole oyster. Moreover, the amino acid composition and the relationship between structure and in vitro digestion parameter of protein in different oyster parts were also evaluated. This work may help to better understand the nutritive value and digestion behavior of raw oysters.
2.5. Determination of the protein surface hydrophobicity The surface hydrophobicity of the protein was estimated using the method of Santé-Lhoutellier, Astruc, Marinova, Greve, and Gatellier (2008). The higher bound BPB value reflects a higher protein surface hydrophobicity. 2.6. Measurement of protein solubility Protein samples were dispersed in deionized water (1%, w/v), and the samples were adjusted to pH 2–12 using 1 N or 6 N HCl or NaOH and then stirred for 30 min at 25 °C. The mixture was centrifuged (8000g, 10 min), and the supernatant protein content was tested using the Biuret method. The sample was dissolved in 0.5 N NaOH, and its soluble protein content was expressed as the total protein content. Protein solubility was calculated by using Eq. (2):
2. Materials and methods 2.1. Materials Pacific oysters (Crassostrea gigas) were purchased from Qingdao local fish markets, kept on ice and immediately transported to the laboratory. Butylated hydroxytoluene (BHT), bromophenol blue (BPB), 2,4-dinitrophenylhydrazine (DNPH), 2,2′-dithiobis(5-nitropyridine) (DTNP), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), and a protein marker (5–245 kDa) were obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Pepsin (E.C. 3.4.23.1, > 250 U/ mg), trypsin (E.C. 3.21.4, 13,000–20,000 U/mg), and α-chymotrypsin (E.C. 3.4.21.1, > 40 U/mg) were obtained from Sigma-Aldrich Chemicals, St. Louis, MO, USA (China).
Solubility (%) =
(2)
2.7. Fourier-transform infrared spectroscopy (FTIR) measurements A Nicolet iS10 Fourier-transform spectrophotometer (Thermo Scientific Corp., Madison, WI, USA) was used to analyze the chemical structures of lyophilized protein (mantle, gill, visceral mass, adductor, and whole oyster) according to the method described by Jiang, Li, Chang, Xiong, and Sun (2017a). The spectra were acquired at wavenumbers of 4000–400 cm−1 in 64 scans with a resolution of 4 cm−1.
2.2. Protein isolation The whole oyster, mantle, gill, visceral mass, and adductor were collected and then homogenized and extracted in 50 mM phosphate buffer (pH 7.2) containing 0.1 M KCl for 1 h in an ice bath. The extraction was repeated twice, and the supernatants were collected and freeze-dried to obtain the protein power.
2.8. Measurement of SH and S–S protein content The SH group was measured using the method of Beveridge, Toma, & Nakai with slight modification (Beveridge, Toma, & Nakai, 1974). Protein samples (15 mg) were dispersed in 3 mL Tris-glycine buffer containing 8 M urea, 0.1% NaCl and 0.5% SDS and then incubated with 0.02 mL DTNB (4 mg/mL) at 25 °C for 30 min. After incubation, the mixture was centrifuged (8000 g, 5 min) at 4 °C. The absorbance of the supernatant at 412 nm (A412) was determined and the SH group level was estimated using Eq. (3):
2.3. Amino acid measurements The amino acid composition of the protein samples was determined using an amino acid analyzer (L-8500A, Hitachi Co., Tokyo, Japan) according to Cao et al. (Cao et al., 2009). Briefly, 0.1 g protein samples were suspended in 10 mL 6 M HCl in a test tube, and the mixture was heated at 110 °C for 24 h. The hydrolysates were cooled to room temperature and dried using a rotary evaporator (Laborota 4000, Heidolph Instruments GmbH & Co. KG, Schwabach, Germany) at 60 °C. The dried hydrolysate was dissolved in 5 mL 0.02 M HCl and filtered through a membrane with a pore size of 0.40 μm to remove impurities. An analytical C18 column was used to analyze the amino acid composition of the hydrolysate. The essential amino acid score (EAAS) was calculated as follows (Eq. (1)):
EAAS =
supernatant protein content × 100 total protein content
SH group level (μmol/g protein) = 73.53A412 × D/C
(3)
where D is the dilution coefficient (6.04) and C is the protein concentration in the tested sample (mg/mL). The S–S group content in oyster protein was determined using the method of Cui et al. (2009). The S–S group content was estimated by using Eq. (4): S–S group content (μmol/g protein) = 73.53A412 × D/C
(4)
where D is equal to 15.
mg of EAA in 1 g protein of test samples × 100 mg of EAA in 1g protein of FAO/WHO referencepattern
2.9. Circular dichroism (CD) spectroscopy
(1)
A JASCO J-715 CD spectrophotometer (JASCO Corp, Tokyo, Japan) was used to analyze the secondary structure of the oyster protein. The CD spectra of proteins (1 mg/mL) were obtained under constant nitrogen flushing at room temperature. The sample was recorded between 190 and 250 nm using a quartz cell of 0.1 cm path length. The
2.4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) SDS-PAGE was performed using the method of Semedo Tavares 2
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secondary structure fractions of the protein were calculated using the online circular dichroism website (http://dichroweb.cryst.bbk.ac.uk) (Jiang et al., 2017b). Each spectrum presented is the average of three consecutive measurements. 2.10. In vitro digestibility In vitro digestibility of the protein from different oyster parts was assessed using a digestion model as described by Sante-Lhoutellier (Sante-Lhoutellier et al., 2007). Protein samples were dispersed in 33 mM glycine buffer at pH 1.8 (1 mg/mL), then digested with pepsin (5 U/mg protein) at 37 °C for 120 min, followed by trypsin and αchymotrypsin (6.6 U and 0.33 U/mg protein, respectively) for 30 min. Digestibility and proteolysis rate of the protein samples were measured by adding trichloroacetic acid (15% final concentration, w/v) at different time points. The absorbance at 280 nm was recorded, which indicated the hydrolyzed peptide content during the digestion process. In addition, the optical density units per minute (ΔOD/min) showed the proteolysis rate of the protein. 2.11. Statistical analysis All values in the present study are expressed as the mean ± SD of triplicate measurements. The results were tested using one-way analysis of variance with the least significant difference test (SPSS 17.0 software, SPSS Inc., Chicago, IL, USA). The least significant differences (p < 0.05) among the treatments were accepted.
Fig. 1. Molecular mass distribution of proteins isolated from different parts of Pacific oyster (Crassostrea gigas). Under reducing condition, proteins (40 μg/ lane) were separated by 12% polyacrylamide gel and stained with coomassie brilliant blue. Lane 1 for molecular weight marker, lane 2 for whole oyster protein (A), lane 3 for visceral mass protein (B), lane 4 for gill protein (C), lane 5 for mantle protein (D), lane 6 for adductor protein (E). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
3. Results and discussion 3.1. Amino acid composition
3.2. Protein molecular weight distribution Essential amino acids cannot be synthesized in the human body and must be obtained from daily food. Thus, essential amino acid amounts usually determine the quality of protein. In this study, an essential amino acid score procedure that is a trustworthy and accurate method was applied to estimate the protein nutritional value (Cao et al., 2009; Synowiecki & Al-Khateeb, 2000). As shown in Table 1, all proteins contained significant amounts of essential amino acids including lysine, leucine, and valine. Almost all essential amino acid contents in the oyster protein approached the FAO/WHO pattern, except for the sulfurcontaining amino acids methionine and cysteine. More remarkably, the lysine content in the oyster protein was nearly twice as high as that in the FAO/WHO pattern. The EAAS of whole oyster protein and other organ proteins was much higher than that of shrimp head of Penaeus vannamei (EAAS was 340.29) (Cao et al., 2009), Penaeus vannamei (EAAS was 75) (Lin & Jiang, 2007, p. 28), Acetes chinensis (EAAS was 73) (Cao, Zhang, Chen, & Hong, 2001), closing to carp (EAAS was 413) and rainbow trout (EAAS was 433) protein (Chinkichi, 1980). This result demonstrated that oysters are composed of high-quality dietary proteins that contain high amounts of almost all essential amino acids.
The molecular weight distribution of proteins from different parts of the oyster was characterized using SDS-PAGE. All proteins showed at least 8 protein bands, ranging in molecular weight from 2 to 245 kDa (Fig. 1). The bands with molecular weights of 200, 100, 42, and 20 kDa were attributed to myosin heavy chains, paramyosin, actin, and myosin light chains in myofibril protein, respectively. The approximate molecular weight distribution of sarcoplasmic protein was 40, 30–33, and 11–12 kDa, corresponding to creatine kinase, phosphoglycerate mutase, and parvalbumin, respectively. Compared with other proteins, adductor protein did not show any bands with molecular weights of approximately 25–38 kDa. Only the visceral mass and whole oyster protein showed a band at 75 kDa, and the bands with a molecular weight greater than 75 kDa were not obvious. These results suggested that differences existed in the molecular weight distribution of mantle, gill, visceral mass, adductor, and whole oyster protein, suggesting there could be different protein species. Different resistant to digestive proteases might be induced by different species of proteins, further
Table 1 Essential amino acid content (mg/g protein) and score (EAAS) of proteins isolated from different parts of Pacific oyster (Crassostrea gigas). Amino acids
Thr Cys + Met Val Ile Leu Phe + Tyr Lys Total
FAO/WHO pattern
40 35 50 40 70 60 55 360
whole oyster
mantle
gill
adductor
visceral mass
content
EAAS
content
EAAS
content
EAAS
content
EAAS
content
EAAS
51.75 15.02 53.07 48.32 78.68 76.83 92.15 415.81
129.37 42.90 106.14 120.79 112.40 128.05 167.54 –
49.16 7.02 51.50 44.01 78.65 74.91 88.48 393.73
122.89 20.06 103.00 110.02 112.36 124.84 160.88 –
50.53 4.21 51.93 47.37 76.84 77.19 85.26 393.33
126.32 12.03 103.86 118.42 109.77 128.65 155.02 –
44.88 22.44 47.48 45.53 86.18 61.79 83.25 391.54
112.20 64.11 94.96 113.82 123.11 102.98 151.37 –
44.88 22.44 47.48 45.53 86.18 61.79 83.25 391.54
112.20 64.11 94.96 113.82 123.11 102.98 151.37 –
3
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Fig. 2. Surface hydrophobicity (a), solubility at different pH (2–12) (b), and FTIR spectra (c) of proteins isolated from different parts of Pacific oyster (Crassostrea gigas). The proteins with the higher bound BPB value were considered as to have a higher surface hydrophobicity; values not bearing common superscripts differ significantly (p < 0.05).
Kantachote, & Shahidi, 2007). Yu et al. also reported that protein solubility increased after alkali treatment (Yu, Yue, Wu, Xu, & Du, 2018). Adductor protein, with the highest hydrophobicity, showed a lower solubility than that of other proteins. Additionally, visceral mass, gill, and mantle protein had a higher solubility; it was presumed that there are more charged and polar groups on the surface of these proteins (Panyam & Kilara, 1996). These results proved that protein solubility was relevant to protein surface hydrophobicity.
affecting the digestibility of the protein (Sze-Tao & Sathe, 2000). 3.3. Surface hydrophobicity and solubility analysis Protein hydrophobicity negatively impacts the solubility (Hayakawa & Nakai, 1985). The surface hydrophobicity and solubility of proteins (mantle, gill, visceral mass, adductor, and whole oyster protein) are shown in Fig. 2a. Surface hydrophobicity, measured using BPB as a probe, is a suitable parameter to reflect the chemical and physical state of the protein. The proteins with the higher bound BPB value were considered to have a higher surface hydrophobicity. As shown in Fig. 2a, the bound BPB content of the mantle, gill, visceral mass, adductor, and whole oyster protein were 53.2, 59.9, 34.9, 86.7, and 42.9 μg, respectively. These results indicated that the hydrophobicity of the adductor protein was the highest, followed by that of the gill, mantle, whole oyster, and visceral mass protein. The high surface hydrophobicity of the proteins suggested that there were more nonpolar amino acids on the protein surface, resulting in lower solubility. Protein solubility is an important parameter that indicates amino acid position and intermolecular interactions in proteins, which can further affect other properties of proteins. The solubility of mantle, gill, visceral mass, adductor, and whole oyster protein in water at different pH values is shown in Fig. 2b. For all protein samples, a relatively lower solubility was shown between pH 2 and 5 due to the changes in the charges on the weakly acidic and basic side chain groups (Chobert, Bertrand-Harb, & Nicolas, 1988). This result was also confirmed by the research of Gbogori et al., who reported that the lowest solubility of salmon byproduct hydrolysates was at pH 4 (Gbogouri, Linder, Fanni, & Parmentier, 2004). With increasing pH, the protein surface appeared to have polar amino acid residues that could form hydrogen bonds with water, leading to an increase in solubility (Klompong, Benjakul,
3.4. FTIR analysis As depicted in Fig. 2c, all samples exhibited absorption bands at 1600-1700 cm−1 and 1500-1600 cm−1, which are attributed to the characteristics amide I and amide II bands of protein (Jiang et al., 2017a). For all protein samples, the absorption band in the range of 3110–3500 cm−1 corresponds to the O–H stretching vibration of the protein. Compared with other proteins, the mantle and adductor proteins exhibited sharper absorption bands, suggesting that increased hydrogen bonding was present. The formation of hydrogen bonds was ascribed to the interactions between carbonyl groups and amide groups in the protein secondary structure, which may enhance the structural stability of these proteins. 3.5. Analysis of SH and S–S groups The S–S and SH group contents are an important index of protein internal structure. The S–S and SH group levels in the mantle, gill, visceral mass, adductor, and whole oyster protein are presented in Fig. 3. The adductor protein showed the highest contents of SH (39.0 μmol/g protein) and S–S (11.7 μmol/g protein), whereas the lowest contents of SH (9.5 μmol/g protein) and S–S (5.1 μmol/g protein) were observed in gill protein. Furthermore, the SH and S–S group 4
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different. Adductor protein contained 42% α-helix, 34% β-sheet, 13% turn, and 11% unordered coil, whereas visceral mass protein was 33.9% α-helix, 18.4% β-sheet, 18.9% turn, and 28.8% unordered coil. Moreover, the secondary structures of the gill and visceral mass proteins are similar. The hydrogen bonds and carbonyl groups in the protein are important for maintaining the proportion of α-helical structures (Liu, Zhao, Xiong, Xie, & Qin, 2008). The S–S groups and hydrophobic interactions had a significant effect on the formation of the β-sheet, which had an adverse effect on digestibility (Carbonaro, Maselli, & Nucara, 2012). As a result, the adductor protein, possessing high contents of carbonyl groups and S–S and high hydrophobicity, was revealed to have a structure with high amounts of α-helices and β-sheets. In contrast, the relative contents of turns and unordered coils in the visceral mass protein were high. Furthermore, it has been reported that secondary structure elements play a critical role in affecting protein digestibility (Carbonaro et al., 2012). Hence, it could be concluded that different proteins from oysters with different secondary structures exhibited different digestibility behaviors.
Fig. 3. The content of total SH and S–S groups in proteins isolated from different parts of Pacific oyster (Crassostrea gigas). Results are expressed as means ± standard deviation from triplicate determinations. Values not bearing common superscripts differ significantly (p < 0.05).
3.7. In vitro digestibility contents of mantle and visceral mass proteins were similar and approximately half those of adductor protein. The different levels of SH and S–S were due to differences in the cysteine levels in the mantle, gill, visceral mass, adductor, and whole oyster protein. Sun et al. noted that the S–S group is formed by the oxidation of free thiol groups from cysteine residues (Sun et al., 2011a). The S–S group can stabilize the folded conformation of proteins and enhance the internal cross-linking of protein (Bulaj, 2005). Therefore, a higher S–S group content in protein may indicate that the internal structure of the protein is more compact due to aggregation of the protein, possibly contributing to low digestibility, which is discussed in later sections.
Although the in vitro digestion model cannot provide human digestive behavior with accuracy, it is still a common tool to estimate protein bioavailability. By measuring soluble amino acid and peptide contents, the efficiency of digestibility can be determined. The in vitro digestibility and digestion rate in proteins hydrolyzed by pepsin are shown in Fig. 5. For all proteins, the soluble amino acids and peptides increased rapidly during the first half hour and then plateaued (Fig. 5a), indicating that the proteins were mostly digested in the first half hour. In contrast, the digestion rates of all proteins decreased gradually over time possibly due to rapid protein consumption. With pepsin, the digestive product content and digestion rate of gill protein were higher than those of other proteins. Whole oyster protein exhibited the lowest digestive product content and digestion rate. There were no significant differences in digestive product content or digestion rate between mantle, adductor, or visceral mass protein. This result suggested that gill protein was rapidly digested by pepsin, while whole oyster protein was slowly digested. After digestion with trypsin and α-chymotrypsin, visceral mass protein exhibited the highest digestive product content and digestion rate (Fig. 5c and d). This suggests that visceral mass protein can be easily hydrolyzed by trypsin and α-chymotrypsin. Adductor protein had the lowest digestion rate and digestion product content. Moreover, the digestion rate in mantle and gill proteins did not gradually increase over time. The decreased digestion rate and increased digestive product content for all proteins over a prolonged period of time was also observed. The digestive process of gastric and pancreatic proteases occurs
3.6. Secondary structure of proteins The CD spectra and secondary structure content of proteins from different oyster parts were evaluated using far-UV CD. As seen in Fig. 4a, the spectra of all proteins were composed of two broad negative bands at 208–210 and 218–222 nm, which are due to π-π* and n-π* transfers for the peptide bond of an α-helix (Bode & Applequist, 2009). Thus, these proteins are α-helix-rich proteins. Compared with other proteins, the spectral intensity of adductor protein increased, indicating that the protein's secondary structure conformation was significantly different. The α-helix, β-sheet, turn, and unordered coil fractions are shown in Fig. 4b. The results showed that the secondary structure content of the mantle, gill, visceral mass, adductor, and whole oyster proteins were
Fig. 4. Far-UV CD spectra and secondary structure content of whole oyster, mantle, gill, visceral mass, and adductor protein. 5
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Fig. 5. Digestive product absorbance at 280 nm and proteolysis rate of different proteins treated by gastric pepsin (a, b) and pancreatic trypsin + α-chymotrypsin (c, d).
The amino acid composition and digestibility of oyster protein after different processing will be further investigated, which is also important for understanding the nutritional value of oyster protein.
in a sequential manner, and there are stacked effects on protein digestibility. In the total digestive process, visceral mass protein had the highest digestibility, followed by gill, mantle, whole oyster, and adductor protein. The reason for this was that the chemical groups, intramolecular force, and secondary structure of the proteins were different, leading to different digestibility values. On the one hand, protein recognition sites were buried due to protein aggregation by S–S group formation and hydrophobic interactions, leading to a decrease in the susceptibility to digestive enzymes (Carbonaro, Cappelloni, Nicoli, Lucarini, & Carnovale, 1997; Du et al., 2017). On the other hand, βsheets are a stable structure that had an adverse effect on the digestibility increment, and random coils were related to the increment in digestibility (Carbonaro et al., 2012). This result was also confirmed by the S–S, surface hydrophobicity, and secondary structure experimental results.
Funding This work was supported by the National Key R&D Program of China (2018YFD0901005), Shandong Provincial Key R&D Program (2018GHY115012) and Natural Science Foundation of Shandong Province (ZR2015CM011). References Beveridge, T., Toma, S. J., & Nakai, S. (1974). Determination of SH- and SS-groups in some food proteins using Ellman's reagent. Journal of Food Science, 39, 49–52. Bode, K. A., & Applequist, J. (2009). Improved theoretical π-π* absorption and circular dichroic spectra of helical polypeptides using new polarizabilities of atoms and NC'O chromophores. The Journal of Physical Chemistry A, 100(45), 17825–17834. Bohrer, B. M. (2017). Review: Nutrient density and nutritional value of meat products and non-meat foods high in protein. Trends in Food Science & Technology, 65, 103–112. Boye, J., Wijesinha-Bettoni, R., & Burlingame, B. (2012). Protein quality evaluation twenty years after the introduction of the protein digestibility corrected amino acid score method. British Journal of Nutrition, 108(S2), 183–211. Bulaj, G. (2005). Formation of disulfide bonds in proteins and peptides. Biotechnology Advances, 23(1), 87–92. Cao, W. H., Zhang, C. H., Chen, S. H., & Hong, P. Z. (2001). Analysis and nutritive evaluation of Acetes chinensis. Journal of Fujian Fisheries, 13(1), 8–15. Cao, W., Zhang, C., Hong, P., Ji, H., Hao, J., & Zhang, J. (2009). Autolysis of shrimp head by gradual temperature and nutritional quality of the resulting hydrolysate. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 42(1), 244–249. Carbonaro, M., Cappelloni, M., Nicoli, S., Lucarini, M., & Carnovale, E. (1997). Solubility−Digestibility relationship of legume proteins. Journal of Agricultural and Food Chemistry, 45(9), 3387–3394. Carbonaro, M., Maselli, P., & Nucara, A. (2012). Relationship between digestibility and
4. Conclusion The present investigation indicates that protein from different oyster parts contains high essential amino acid contents, which indicates high-quality protein. For the gastric digestion process, the digestion rate of gill protein was higher than that of the other proteins. However, for the total digestive process, visceral mass protein had the highest digestibility, followed by that of gill, mantle, whole oyster, and adductor protein. The S–S groups, carbonyl groups, intramolecular forces, and secondary structures in proteins may greatly affect protein digestibility. As a result, adductor protein, with relatively high hydrophobic interactions and S–S group, α-helix and β-sheet contents showed low digestibility. In contrast, the highest digestibility was in visceral mass protein due to its relatively low S–S group, α-helix and β-sheet contents. 6
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Amino acid composition and digestibility of Pacific oyster (Crassostrea gigas) proteins isolated from different parts
T
Suisui Jiang, Li Liu, Jinjin Xu, Mingyong Zeng∗, Yuanhui Zhao∗∗ College of Food Science and Engineering, Ocean University of China, Qingdao, 266003, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Essential amino acids Oyster proteins S–S group content Secondary structure Digestion
To understand the nutritional value and the relationship between structure and in vitro digestibility of Pacific oyster (Crassostrea gigas) proteins, the amino acid composition, physicochemical structure, and digestibility of different part proteins were investigated. All proteins contained significant amounts of essential amino acids, including lysine, leucine, and valine. The adductor protein showed the highest contents of SH (39.0 μmol/g protein) and S–S (11.7 μmol/g protein), whereas lower contents of SH (9.5 μmol/g protein) and S–S (5.1 μmol/g protein) in gill protein was observed. In addition, adductor protein contained more α-helix (64.7%) and β-sheet (11.2%) structures, whereas the proportions of α-helix (33.9%) and β-sheet (18.4%) structures in visceral mass protein were the lowest. The digestion results showed that gill protein undergoes rapid digestion in the stomach, and visceral mass protein shows the highest digestibility in the total digestive process. This work provides a greater understanding of the nutrition value and digestibility of oyster protein.
1. Introduction
Previous studies have demonstrated that protein digestion is a complex process that depends on the structure and physicochemical properties of proteins. The carbonyl groups present in proteins mainly come from the covalent bonding of proteins, carbohydrates and the oxidation products of carbohydrates or fatty acids, which are important indicators of the oxidation degree of proteins. Increasing carbonyl content can decrease the protein digestibility due to the formation of intra- or intermolecular cross-linking (Sante-Lhoutellier et al., 2007; Sun, Cui, Zhao, Zhao, & Yang, 2011a; Sun, Zhao, Yang, Zhao, & Cui, 2011b). A high sulfhydryl (SH) group content in protein usually implies a low degree of protein oxidation that is always accompanied by high digestibility (Xiong, Donkeun, & Tooru, 2009). During the heating process, many SH proteins can be oxidized to form S–S, which is the reason for the reduction in digestibility (Cui, Zhou, Zhao, & Yang, 2009; Sun et al., 2011a). Additionally, the protein aggregation state can affect the surface hydrophobicity, leading to varied digestibility (KaminBelsky, Brillon, Arav, & Shaklai, 1996; Liu & Xiong, 2000). Sun et al. reported that the increased surface hydrophobicity of a protein could cause a decrease in the α-helical content and an increase in the β-strand content, ascribed to the reduction of hydrogen bonds in the α-helix structure (Sun, Zhou, Zhao, Yang, & Cui, 2011c). Thus, the secondary structure composition of proteins must also be taken into account when determining protein digestion.
Dietary protein, particularly essential amino acids, is essential for humans to ensure body repair, cell regeneration, growth, and development (Bohrer, 2017). Dietary protein also plays a critical role in maintaining muscle mass, which is the basis of chronic disease prevention, functional capacity, and quality of life. However, after forty, skeletal muscle mass naturally starts to decline (Paddon-Jones & Rasmussen, 2009). Previous research has claimed that protein diets, especially high-quality protein, can attenuate natural muscle mass loss and promote muscle synthesis (Devries & Phillips, 2015). Therefore, high-quality protein is very important in our daily diet due to its’ high levels of essential amino acids (Mania, Wojciechowska-Mazurek, Starska, Rebeniak, & Postupolski, 2012; Willoughby, 2004). However, amino acid composition is not the only criterion of the nutritional quality of protein (Boye, Wijesinha-Bettoni, & Burlingame, 2012). To be absorbed, proteins must be hydrolyzed by proteases within the digestive tract to form amino acids and peptides. Vast accumulation of nonhydrolyzed or partially hydrolyzed proteins in the large intestine could increase colonic microbial fermentation to produce toxic metabolites, which can induce inflammation and further result in colon cancer (Dallas et al., 2017; Sante-Lhoutellier, Aubry, & Gatellier, 2007). Thus, digestibility also plays a significant role in protein quality.
∗
Corresponding author. Corresponding author. E-mail addresses: [email protected] (M. Zeng), [email protected] (Y. Zhao).
∗∗
https://doi.org/10.1016/j.lwt.2019.108591 Received 15 April 2019; Received in revised form 31 August 2019; Accepted 4 September 2019 Available online 05 September 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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et al. (2018) to determine the molecular weight (MW) distribution of the protein. Samples were electrophoresed on a 12% separating gel and a 5% stacking gel. Briefly, protein samples (80 μL, 10 mg/mL) were diluted using 5 × SDS-PAGE loading buffer (20 μL) at a final concentration of 2 mg/mL and then heated in boiled water for 10 min. After cooling to room temperature, 10 μL samples were added to each lane. A protein marker (5–245 kDa) was used for MW determination.
Oysters are one of the most valuable shellfishes, with the largest output in China, whose annual production in 2017 was approximately 4.89 million tons (Ministry of Agriculture and Rural Affairs of the People's Republic of China, 2018, p. 23). It is a high-protein food containing high amounts of essential amino acids and taurine (Je, Park, Jung, & Kim, 2005) and is an excellent source of high-quality protein. Furthermore, their delicate and delicious meat is one of the reasons why oysters are widely consumed by people. However, few studies have focused on determining the protein structure and digestibility of oyster proteins. The mantle, gill, visceral mass, and adductor are the main components of the oyster, and their structures and properties are substantially different. The purpose of this study was to explore the differences in the structures of different parts of oysters, including the mantle, gill, visceral mass, adductor and whole oyster. Moreover, the amino acid composition and the relationship between structure and in vitro digestion parameter of protein in different oyster parts were also evaluated. This work may help to better understand the nutritive value and digestion behavior of raw oysters.
2.5. Determination of the protein surface hydrophobicity The surface hydrophobicity of the protein was estimated using the method of Santé-Lhoutellier, Astruc, Marinova, Greve, and Gatellier (2008). The higher bound BPB value reflects a higher protein surface hydrophobicity. 2.6. Measurement of protein solubility Protein samples were dispersed in deionized water (1%, w/v), and the samples were adjusted to pH 2–12 using 1 N or 6 N HCl or NaOH and then stirred for 30 min at 25 °C. The mixture was centrifuged (8000g, 10 min), and the supernatant protein content was tested using the Biuret method. The sample was dissolved in 0.5 N NaOH, and its soluble protein content was expressed as the total protein content. Protein solubility was calculated by using Eq. (2):
2. Materials and methods 2.1. Materials Pacific oysters (Crassostrea gigas) were purchased from Qingdao local fish markets, kept on ice and immediately transported to the laboratory. Butylated hydroxytoluene (BHT), bromophenol blue (BPB), 2,4-dinitrophenylhydrazine (DNPH), 2,2′-dithiobis(5-nitropyridine) (DTNP), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), and a protein marker (5–245 kDa) were obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Pepsin (E.C. 3.4.23.1, > 250 U/ mg), trypsin (E.C. 3.21.4, 13,000–20,000 U/mg), and α-chymotrypsin (E.C. 3.4.21.1, > 40 U/mg) were obtained from Sigma-Aldrich Chemicals, St. Louis, MO, USA (China).
Solubility (%) =
(2)
2.7. Fourier-transform infrared spectroscopy (FTIR) measurements A Nicolet iS10 Fourier-transform spectrophotometer (Thermo Scientific Corp., Madison, WI, USA) was used to analyze the chemical structures of lyophilized protein (mantle, gill, visceral mass, adductor, and whole oyster) according to the method described by Jiang, Li, Chang, Xiong, and Sun (2017a). The spectra were acquired at wavenumbers of 4000–400 cm−1 in 64 scans with a resolution of 4 cm−1.
2.2. Protein isolation The whole oyster, mantle, gill, visceral mass, and adductor were collected and then homogenized and extracted in 50 mM phosphate buffer (pH 7.2) containing 0.1 M KCl for 1 h in an ice bath. The extraction was repeated twice, and the supernatants were collected and freeze-dried to obtain the protein power.
2.8. Measurement of SH and S–S protein content The SH group was measured using the method of Beveridge, Toma, & Nakai with slight modification (Beveridge, Toma, & Nakai, 1974). Protein samples (15 mg) were dispersed in 3 mL Tris-glycine buffer containing 8 M urea, 0.1% NaCl and 0.5% SDS and then incubated with 0.02 mL DTNB (4 mg/mL) at 25 °C for 30 min. After incubation, the mixture was centrifuged (8000 g, 5 min) at 4 °C. The absorbance of the supernatant at 412 nm (A412) was determined and the SH group level was estimated using Eq. (3):
2.3. Amino acid measurements The amino acid composition of the protein samples was determined using an amino acid analyzer (L-8500A, Hitachi Co., Tokyo, Japan) according to Cao et al. (Cao et al., 2009). Briefly, 0.1 g protein samples were suspended in 10 mL 6 M HCl in a test tube, and the mixture was heated at 110 °C for 24 h. The hydrolysates were cooled to room temperature and dried using a rotary evaporator (Laborota 4000, Heidolph Instruments GmbH & Co. KG, Schwabach, Germany) at 60 °C. The dried hydrolysate was dissolved in 5 mL 0.02 M HCl and filtered through a membrane with a pore size of 0.40 μm to remove impurities. An analytical C18 column was used to analyze the amino acid composition of the hydrolysate. The essential amino acid score (EAAS) was calculated as follows (Eq. (1)):
EAAS =
supernatant protein content × 100 total protein content
SH group level (μmol/g protein) = 73.53A412 × D/C
(3)
where D is the dilution coefficient (6.04) and C is the protein concentration in the tested sample (mg/mL). The S–S group content in oyster protein was determined using the method of Cui et al. (2009). The S–S group content was estimated by using Eq. (4): S–S group content (μmol/g protein) = 73.53A412 × D/C
(4)
where D is equal to 15.
mg of EAA in 1 g protein of test samples × 100 mg of EAA in 1g protein of FAO/WHO referencepattern
2.9. Circular dichroism (CD) spectroscopy
(1)
A JASCO J-715 CD spectrophotometer (JASCO Corp, Tokyo, Japan) was used to analyze the secondary structure of the oyster protein. The CD spectra of proteins (1 mg/mL) were obtained under constant nitrogen flushing at room temperature. The sample was recorded between 190 and 250 nm using a quartz cell of 0.1 cm path length. The
2.4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) SDS-PAGE was performed using the method of Semedo Tavares 2
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secondary structure fractions of the protein were calculated using the online circular dichroism website (http://dichroweb.cryst.bbk.ac.uk) (Jiang et al., 2017b). Each spectrum presented is the average of three consecutive measurements. 2.10. In vitro digestibility In vitro digestibility of the protein from different oyster parts was assessed using a digestion model as described by Sante-Lhoutellier (Sante-Lhoutellier et al., 2007). Protein samples were dispersed in 33 mM glycine buffer at pH 1.8 (1 mg/mL), then digested with pepsin (5 U/mg protein) at 37 °C for 120 min, followed by trypsin and αchymotrypsin (6.6 U and 0.33 U/mg protein, respectively) for 30 min. Digestibility and proteolysis rate of the protein samples were measured by adding trichloroacetic acid (15% final concentration, w/v) at different time points. The absorbance at 280 nm was recorded, which indicated the hydrolyzed peptide content during the digestion process. In addition, the optical density units per minute (ΔOD/min) showed the proteolysis rate of the protein. 2.11. Statistical analysis All values in the present study are expressed as the mean ± SD of triplicate measurements. The results were tested using one-way analysis of variance with the least significant difference test (SPSS 17.0 software, SPSS Inc., Chicago, IL, USA). The least significant differences (p < 0.05) among the treatments were accepted.
Fig. 1. Molecular mass distribution of proteins isolated from different parts of Pacific oyster (Crassostrea gigas). Under reducing condition, proteins (40 μg/ lane) were separated by 12% polyacrylamide gel and stained with coomassie brilliant blue. Lane 1 for molecular weight marker, lane 2 for whole oyster protein (A), lane 3 for visceral mass protein (B), lane 4 for gill protein (C), lane 5 for mantle protein (D), lane 6 for adductor protein (E). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
3. Results and discussion 3.1. Amino acid composition
3.2. Protein molecular weight distribution Essential amino acids cannot be synthesized in the human body and must be obtained from daily food. Thus, essential amino acid amounts usually determine the quality of protein. In this study, an essential amino acid score procedure that is a trustworthy and accurate method was applied to estimate the protein nutritional value (Cao et al., 2009; Synowiecki & Al-Khateeb, 2000). As shown in Table 1, all proteins contained significant amounts of essential amino acids including lysine, leucine, and valine. Almost all essential amino acid contents in the oyster protein approached the FAO/WHO pattern, except for the sulfurcontaining amino acids methionine and cysteine. More remarkably, the lysine content in the oyster protein was nearly twice as high as that in the FAO/WHO pattern. The EAAS of whole oyster protein and other organ proteins was much higher than that of shrimp head of Penaeus vannamei (EAAS was 340.29) (Cao et al., 2009), Penaeus vannamei (EAAS was 75) (Lin & Jiang, 2007, p. 28), Acetes chinensis (EAAS was 73) (Cao, Zhang, Chen, & Hong, 2001), closing to carp (EAAS was 413) and rainbow trout (EAAS was 433) protein (Chinkichi, 1980). This result demonstrated that oysters are composed of high-quality dietary proteins that contain high amounts of almost all essential amino acids.
The molecular weight distribution of proteins from different parts of the oyster was characterized using SDS-PAGE. All proteins showed at least 8 protein bands, ranging in molecular weight from 2 to 245 kDa (Fig. 1). The bands with molecular weights of 200, 100, 42, and 20 kDa were attributed to myosin heavy chains, paramyosin, actin, and myosin light chains in myofibril protein, respectively. The approximate molecular weight distribution of sarcoplasmic protein was 40, 30–33, and 11–12 kDa, corresponding to creatine kinase, phosphoglycerate mutase, and parvalbumin, respectively. Compared with other proteins, adductor protein did not show any bands with molecular weights of approximately 25–38 kDa. Only the visceral mass and whole oyster protein showed a band at 75 kDa, and the bands with a molecular weight greater than 75 kDa were not obvious. These results suggested that differences existed in the molecular weight distribution of mantle, gill, visceral mass, adductor, and whole oyster protein, suggesting there could be different protein species. Different resistant to digestive proteases might be induced by different species of proteins, further
Table 1 Essential amino acid content (mg/g protein) and score (EAAS) of proteins isolated from different parts of Pacific oyster (Crassostrea gigas). Amino acids
Thr Cys + Met Val Ile Leu Phe + Tyr Lys Total
FAO/WHO pattern
40 35 50 40 70 60 55 360
whole oyster
mantle
gill
adductor
visceral mass
content
EAAS
content
EAAS
content
EAAS
content
EAAS
content
EAAS
51.75 15.02 53.07 48.32 78.68 76.83 92.15 415.81
129.37 42.90 106.14 120.79 112.40 128.05 167.54 –
49.16 7.02 51.50 44.01 78.65 74.91 88.48 393.73
122.89 20.06 103.00 110.02 112.36 124.84 160.88 –
50.53 4.21 51.93 47.37 76.84 77.19 85.26 393.33
126.32 12.03 103.86 118.42 109.77 128.65 155.02 –
44.88 22.44 47.48 45.53 86.18 61.79 83.25 391.54
112.20 64.11 94.96 113.82 123.11 102.98 151.37 –
44.88 22.44 47.48 45.53 86.18 61.79 83.25 391.54
112.20 64.11 94.96 113.82 123.11 102.98 151.37 –
3
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Fig. 2. Surface hydrophobicity (a), solubility at different pH (2–12) (b), and FTIR spectra (c) of proteins isolated from different parts of Pacific oyster (Crassostrea gigas). The proteins with the higher bound BPB value were considered as to have a higher surface hydrophobicity; values not bearing common superscripts differ significantly (p < 0.05).
Kantachote, & Shahidi, 2007). Yu et al. also reported that protein solubility increased after alkali treatment (Yu, Yue, Wu, Xu, & Du, 2018). Adductor protein, with the highest hydrophobicity, showed a lower solubility than that of other proteins. Additionally, visceral mass, gill, and mantle protein had a higher solubility; it was presumed that there are more charged and polar groups on the surface of these proteins (Panyam & Kilara, 1996). These results proved that protein solubility was relevant to protein surface hydrophobicity.
affecting the digestibility of the protein (Sze-Tao & Sathe, 2000). 3.3. Surface hydrophobicity and solubility analysis Protein hydrophobicity negatively impacts the solubility (Hayakawa & Nakai, 1985). The surface hydrophobicity and solubility of proteins (mantle, gill, visceral mass, adductor, and whole oyster protein) are shown in Fig. 2a. Surface hydrophobicity, measured using BPB as a probe, is a suitable parameter to reflect the chemical and physical state of the protein. The proteins with the higher bound BPB value were considered to have a higher surface hydrophobicity. As shown in Fig. 2a, the bound BPB content of the mantle, gill, visceral mass, adductor, and whole oyster protein were 53.2, 59.9, 34.9, 86.7, and 42.9 μg, respectively. These results indicated that the hydrophobicity of the adductor protein was the highest, followed by that of the gill, mantle, whole oyster, and visceral mass protein. The high surface hydrophobicity of the proteins suggested that there were more nonpolar amino acids on the protein surface, resulting in lower solubility. Protein solubility is an important parameter that indicates amino acid position and intermolecular interactions in proteins, which can further affect other properties of proteins. The solubility of mantle, gill, visceral mass, adductor, and whole oyster protein in water at different pH values is shown in Fig. 2b. For all protein samples, a relatively lower solubility was shown between pH 2 and 5 due to the changes in the charges on the weakly acidic and basic side chain groups (Chobert, Bertrand-Harb, & Nicolas, 1988). This result was also confirmed by the research of Gbogori et al., who reported that the lowest solubility of salmon byproduct hydrolysates was at pH 4 (Gbogouri, Linder, Fanni, & Parmentier, 2004). With increasing pH, the protein surface appeared to have polar amino acid residues that could form hydrogen bonds with water, leading to an increase in solubility (Klompong, Benjakul,
3.4. FTIR analysis As depicted in Fig. 2c, all samples exhibited absorption bands at 1600-1700 cm−1 and 1500-1600 cm−1, which are attributed to the characteristics amide I and amide II bands of protein (Jiang et al., 2017a). For all protein samples, the absorption band in the range of 3110–3500 cm−1 corresponds to the O–H stretching vibration of the protein. Compared with other proteins, the mantle and adductor proteins exhibited sharper absorption bands, suggesting that increased hydrogen bonding was present. The formation of hydrogen bonds was ascribed to the interactions between carbonyl groups and amide groups in the protein secondary structure, which may enhance the structural stability of these proteins. 3.5. Analysis of SH and S–S groups The S–S and SH group contents are an important index of protein internal structure. The S–S and SH group levels in the mantle, gill, visceral mass, adductor, and whole oyster protein are presented in Fig. 3. The adductor protein showed the highest contents of SH (39.0 μmol/g protein) and S–S (11.7 μmol/g protein), whereas the lowest contents of SH (9.5 μmol/g protein) and S–S (5.1 μmol/g protein) were observed in gill protein. Furthermore, the SH and S–S group 4
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different. Adductor protein contained 42% α-helix, 34% β-sheet, 13% turn, and 11% unordered coil, whereas visceral mass protein was 33.9% α-helix, 18.4% β-sheet, 18.9% turn, and 28.8% unordered coil. Moreover, the secondary structures of the gill and visceral mass proteins are similar. The hydrogen bonds and carbonyl groups in the protein are important for maintaining the proportion of α-helical structures (Liu, Zhao, Xiong, Xie, & Qin, 2008). The S–S groups and hydrophobic interactions had a significant effect on the formation of the β-sheet, which had an adverse effect on digestibility (Carbonaro, Maselli, & Nucara, 2012). As a result, the adductor protein, possessing high contents of carbonyl groups and S–S and high hydrophobicity, was revealed to have a structure with high amounts of α-helices and β-sheets. In contrast, the relative contents of turns and unordered coils in the visceral mass protein were high. Furthermore, it has been reported that secondary structure elements play a critical role in affecting protein digestibility (Carbonaro et al., 2012). Hence, it could be concluded that different proteins from oysters with different secondary structures exhibited different digestibility behaviors.
Fig. 3. The content of total SH and S–S groups in proteins isolated from different parts of Pacific oyster (Crassostrea gigas). Results are expressed as means ± standard deviation from triplicate determinations. Values not bearing common superscripts differ significantly (p < 0.05).
3.7. In vitro digestibility contents of mantle and visceral mass proteins were similar and approximately half those of adductor protein. The different levels of SH and S–S were due to differences in the cysteine levels in the mantle, gill, visceral mass, adductor, and whole oyster protein. Sun et al. noted that the S–S group is formed by the oxidation of free thiol groups from cysteine residues (Sun et al., 2011a). The S–S group can stabilize the folded conformation of proteins and enhance the internal cross-linking of protein (Bulaj, 2005). Therefore, a higher S–S group content in protein may indicate that the internal structure of the protein is more compact due to aggregation of the protein, possibly contributing to low digestibility, which is discussed in later sections.
Although the in vitro digestion model cannot provide human digestive behavior with accuracy, it is still a common tool to estimate protein bioavailability. By measuring soluble amino acid and peptide contents, the efficiency of digestibility can be determined. The in vitro digestibility and digestion rate in proteins hydrolyzed by pepsin are shown in Fig. 5. For all proteins, the soluble amino acids and peptides increased rapidly during the first half hour and then plateaued (Fig. 5a), indicating that the proteins were mostly digested in the first half hour. In contrast, the digestion rates of all proteins decreased gradually over time possibly due to rapid protein consumption. With pepsin, the digestive product content and digestion rate of gill protein were higher than those of other proteins. Whole oyster protein exhibited the lowest digestive product content and digestion rate. There were no significant differences in digestive product content or digestion rate between mantle, adductor, or visceral mass protein. This result suggested that gill protein was rapidly digested by pepsin, while whole oyster protein was slowly digested. After digestion with trypsin and α-chymotrypsin, visceral mass protein exhibited the highest digestive product content and digestion rate (Fig. 5c and d). This suggests that visceral mass protein can be easily hydrolyzed by trypsin and α-chymotrypsin. Adductor protein had the lowest digestion rate and digestion product content. Moreover, the digestion rate in mantle and gill proteins did not gradually increase over time. The decreased digestion rate and increased digestive product content for all proteins over a prolonged period of time was also observed. The digestive process of gastric and pancreatic proteases occurs
3.6. Secondary structure of proteins The CD spectra and secondary structure content of proteins from different oyster parts were evaluated using far-UV CD. As seen in Fig. 4a, the spectra of all proteins were composed of two broad negative bands at 208–210 and 218–222 nm, which are due to π-π* and n-π* transfers for the peptide bond of an α-helix (Bode & Applequist, 2009). Thus, these proteins are α-helix-rich proteins. Compared with other proteins, the spectral intensity of adductor protein increased, indicating that the protein's secondary structure conformation was significantly different. The α-helix, β-sheet, turn, and unordered coil fractions are shown in Fig. 4b. The results showed that the secondary structure content of the mantle, gill, visceral mass, adductor, and whole oyster proteins were
Fig. 4. Far-UV CD spectra and secondary structure content of whole oyster, mantle, gill, visceral mass, and adductor protein. 5
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Fig. 5. Digestive product absorbance at 280 nm and proteolysis rate of different proteins treated by gastric pepsin (a, b) and pancreatic trypsin + α-chymotrypsin (c, d).
The amino acid composition and digestibility of oyster protein after different processing will be further investigated, which is also important for understanding the nutritional value of oyster protein.
in a sequential manner, and there are stacked effects on protein digestibility. In the total digestive process, visceral mass protein had the highest digestibility, followed by gill, mantle, whole oyster, and adductor protein. The reason for this was that the chemical groups, intramolecular force, and secondary structure of the proteins were different, leading to different digestibility values. On the one hand, protein recognition sites were buried due to protein aggregation by S–S group formation and hydrophobic interactions, leading to a decrease in the susceptibility to digestive enzymes (Carbonaro, Cappelloni, Nicoli, Lucarini, & Carnovale, 1997; Du et al., 2017). On the other hand, βsheets are a stable structure that had an adverse effect on the digestibility increment, and random coils were related to the increment in digestibility (Carbonaro et al., 2012). This result was also confirmed by the S–S, surface hydrophobicity, and secondary structure experimental results.
Funding This work was supported by the National Key R&D Program of China (2018YFD0901005), Shandong Provincial Key R&D Program (2018GHY115012) and Natural Science Foundation of Shandong Province (ZR2015CM011). References Beveridge, T., Toma, S. J., & Nakai, S. (1974). Determination of SH- and SS-groups in some food proteins using Ellman's reagent. Journal of Food Science, 39, 49–52. Bode, K. A., & Applequist, J. (2009). Improved theoretical π-π* absorption and circular dichroic spectra of helical polypeptides using new polarizabilities of atoms and NC'O chromophores. The Journal of Physical Chemistry A, 100(45), 17825–17834. Bohrer, B. M. (2017). Review: Nutrient density and nutritional value of meat products and non-meat foods high in protein. Trends in Food Science & Technology, 65, 103–112. Boye, J., Wijesinha-Bettoni, R., & Burlingame, B. (2012). Protein quality evaluation twenty years after the introduction of the protein digestibility corrected amino acid score method. British Journal of Nutrition, 108(S2), 183–211. Bulaj, G. (2005). Formation of disulfide bonds in proteins and peptides. Biotechnology Advances, 23(1), 87–92. Cao, W. H., Zhang, C. H., Chen, S. H., & Hong, P. Z. (2001). Analysis and nutritive evaluation of Acetes chinensis. Journal of Fujian Fisheries, 13(1), 8–15. Cao, W., Zhang, C., Hong, P., Ji, H., Hao, J., & Zhang, J. (2009). Autolysis of shrimp head by gradual temperature and nutritional quality of the resulting hydrolysate. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 42(1), 244–249. Carbonaro, M., Cappelloni, M., Nicoli, S., Lucarini, M., & Carnovale, E. (1997). Solubility−Digestibility relationship of legume proteins. Journal of Agricultural and Food Chemistry, 45(9), 3387–3394. Carbonaro, M., Maselli, P., & Nucara, A. (2012). Relationship between digestibility and
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