Effect of ultrasound assisted extraction on the physicochemical and functional properties of collagen from soft-shelled turtle calipash

Effect of ultrasound assisted extraction on the physicochemical and functional properties of collagen from soft-shelled turtle calipash

Accepted Manuscript Title: Effect of ultrasound assisted extraction on the physicochemical and functional properties of collagen from soft-shelled tur...

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Accepted Manuscript Title: Effect of ultrasound assisted extraction on the physicochemical and functional properties of collagen from soft-shelled turtle calipash Authors: Ye Zou, Li Wang, Panpan Cai, Pengpeng Li, Muhan Zhang, Zhilan Sun, Chong Sun, Weimin Xu, Daoying Wang PII: DOI: Reference:

S0141-8130(17)30784-5 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.03.011 BIOMAC 7174

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

12-10-2016 3-2-2017 2-3-2017

Please cite this article as: Ye Zou, Li Wang, Panpan Cai, Pengpeng Li, Muhan Zhang, Zhilan Sun, Chong Sun, Weimin Xu, Daoying Wang, Effect of ultrasound assisted extraction on the physicochemical and functional properties of collagen from soft-shelled turtle calipash, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2017.03.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Effect of ultrasound assisted extraction on the physicochemical and functional properties of collagen from soft-shelled turtle calipash

Ye Zoua, Li Wangb, Panpan Caic, Pengpeng Lia, Muhan Zhanga, Zhilan Suna, Chong Suna, Weimin Xua,*, Daoying Wanga,d,*

a

Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural

Sciences, Nanjing 210014, PR China b

College of Food Science and Engineering, Yangzhou University, Yangzhou 225127,

PR China c

d

Ginling College, Nanjing Normal University, Nanjing 210024, PR China Jiangsu Collaborative Innovation Center of Meat Production and Processing,

Quality and Safety Control, Nanjing Agricultural University, Nanjing 210095, PR China

*Corresponding author. Tel./fax: 86 25 84390065. E-mail address: [email protected] (D. Wang), [email protected] (W. Xu).

1

ABSTRACT The aim of the present study was to evaluate the physicochemical and functional properties of acid-soluble collagen by ultrasound assisted extraction (UASC) from calipash of soft-shelled turtle (Pelodiscus sinensis). The results showed the collagen content was increased by 16.3% in UASC over the collagen from the conventional extraction (ASC). Both ASC and UASC contained a moderate amount of imino acid (197 and 216 residues/1000 residues, respectively) and hydrophobic amino acid (353 and 391 residues/1000 residues, respectively) in amino acid composition. Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) analyses confirmed that the ultrasound treatment did not disrupt the triple-stranded helical structures in UASC. UASC had higher thermal stability compared with ASC by viscosity and differential scanning calorimetry (DSC) measurements, therefore, UASC might have the advantage to be used. In dynamic elastic behavior measurement, UASC showed a larger elasticity than ASC. With a mild modification by ultrasound, UASC had superior functional properties to ASC, including water/oil absorption capacity, water-holding capacity, emulsifying properties and foaming properties. These results suggested that UASC from the soft-shelled turtle calipash had a potential to be used widely in food, medicine, cosmetics and biomedical materials.

Keywords: Soft-shelled turtle calipash collagen; Ultrasound assisted extraction; Physicochemical properties 2

1. Introduction Collagen is the main component of the extracellular matrix and constitutes approximately 30% of the total protein in vertebrates. It is widely distributed in all kinds of connective tissues to support and protect the body and organs [1]. At least 29 types of collagens have been identified from diverse biological tissues, and each collagen type has a unique molecular structure and a different amino acid composition to play a specific role in tissues of vertebrates [2]. In addition, collagen has properties of holding water, gel strength, emulsification, low viscosity, biological compatibility, water absorption and moisturizing effect as natural biological resources, which can be widely used in food, medicine, cosmetics and biomedical materials [3]. Most of the collagen is derived from skins and bones of cow and pig [4]. The outbreaks of certain animal diseases such as bovine spongiform encephalopathy; and foot and mouth diseases have resulted in restrictions on the use of animal collagen [5]. In addition, Muslims and Jews do not accept any pig related food products while Hindus does not consume cow-based products [6]. In such circumstances, alternative sources, such as fish collagen of its high availability, no risk of disease transmission and no religious barriers, have received increasing attention for collagen extraction. The main differences of fish collagen from that of animal collagen are its high biological value, high essential amino acid content and low content of hydroxyproline (Hyp) [7, 8]. The soft-shelled turtle, Pelodiscus sinensis, is a commercially important and delicious aquatic species in Asian countries including Taiwan, China, Japan and Korea etc., due to its high nutritional and medicinal values, or sold as pet. Compared 3

to other coldwater fishes, the soft-shelled turtle is an ectothermic amniotic reptile, which is living in relatively higher ambient environment at around 30 °C [9]. The turtle collagens have higher denaturation temperature (Td) and may have the advantage of higher thermal stability in various applications. Moreover, the turtle calipash is rich in collagen. Some researchers have previously reported the extraction and characterization of collagen from turtle calipash [10] and a novel collagen gene fragment of 756 bp was cloned from a soft shelled turtle [11], whereas there is no information regarding the effect of ultrasonic extraction on characteristics and application of calipash from turtle is important. In recent years, ultrasound has been used as an alternative to improve the availability, acceptability and functional properties of kinds proteins, such as from eggs, milk, poultry and soy [12]. Ultrasound is defined as a wave of high frequency sound that exceeds the limit of human hearing (~20 kHz). Its functional mechanism is based on passing waves that create regions of high and low pressure; this variation in acoustic pressure is directly proportional to the amount of energy applied to the system. Ultrasound assisted extraction (UAE) is a new simple technique for the recovery of bioactive compounds from different sources [13, 14]. Its propagation in biological material also induces the compression and decompression of particles that changes the material’s physicochemical properties and improves the quality of protein in our previous study [15]. Therefore, the objectives of this study were to compare the collagen content of acid-soluble collagen from the soft-shelled turtle calipash between the conventional 4

and ultrasound assisted extraction using acetic acid (designated as ASC and UASC in this manuscript, respectively). It was also attempted to determine their amino acid composition and molecular structure of collagen, and further to investigate the physicochemical and functional properties of ASC and UASC. 2. Materials and methods 2.1. Materials and chemical reagents Fresh soft-shelled turtles were provided by Banquet King aquatic product Foods Ltd. in Suqian, Jiangsu, China (body weight of 500 ± 50 g). They were put in the fresh box with ice bag when transported to our laboratory by air express. The calipash tissues were dissected by using clean scalpel on crushed ice, cut into small pieces (1 × 0.5 cm), smashed in a blender until smooth and stored at -20 °C for further use. All experimental procedures were approved by the Animal Ethics Committee of Jiangsu Academy of Agricultural Sciences. L-hydroxyproline standard and dimethyl amine benzaldehyde was purchased from Sigma-Aldrich (Shanghai) Co., Ltd. All reagents used in this study were analytical grade. 2.2. Sample collection and calipash preparation The frozen calipash was first thawed at 4 °C and cut into small pieces, soaked in a solution of NaCl (20%, w/v) in Tris-HCl (0.05 M, pH 7.5) at a ratio of 1:20 (w/v) and stirred continuously for 12 h in magnetic stirring apparatus (78-1; Changzhou Guohua Electric Appliance Co., Ltd., Jiangsu, China) at 20 °C to remove non-collagenous substances. The mixture was centrifuged at 13,400 g for 20 min by a centrifuge (Vnicen MR, Herolab, Germany) and the precipitate was washed repeatedly with 5

distilled water to remove fats and bubbles, and the precipitate was stirred continuously for 24 h with a solution of Na2CO3 (0.5 M) at a ratio of 1:20 (w/v) to remove pigment. The Na2CO3 solution was renewed every 8 h. Calipash was de-mineralized using EDTA-2Na (0.3 M, pH 7.4) at a ratio of 1:20 (w/v) with stirring for 24 h using a magnetic stirrer to remove mineral substance. The EDTA-2Na solution was renewed every 8 h, too. The precipitate after centrifugation was soaked with isopropyl alcohol (10%, v/v) to remove fats and bubbles and washed repeatedly with distilled water at ratio of 1:20 (w/v) until samples reached a pH 7. At last, the pretreated calipash sample was kept at -20 °C until use. 2.3. Extraction and purification of collagen 2.3.1. Conventional extraction and purification of acid-soluble collagen (ASC) The ASC was extracted from soft-shelled turtle calipash according to the method described previously [16] with minor modification. The pretreated calipash particles were suspended in acetic acid (0.5 M, w/v) at liquid-to-solid ratios of 20:1 (mL:g) for 24 h. Subsequently, samples were centrifuged at 13,400 g for 15 min and collected the supernatant was collected. The residue from the above centrifugation was extracted again as the same process. The supernatants of calipash ACS were collected together and added with NaCl to salting-out for 12 h in a final concentration of 2.5 M and 1.0 M, respectively. The precipitate was collected after centrifugation at 13,400 g for 15 min. The precipitate from NaCl solution was dissolved in 0.5 M acetic acid (1:10, w/v) and then dialyzed in 0.1 M acetic acid (1:25, w/v), followed by distilled water for 48 h with changes in distilled water every 12 h. All processes were carried out at 4 °C. 6

ASC was lyophilized and then stored at -20 °C until further use. 2.3.2. Ultrasound assisted extraction and purification of acid-soluble collagen (UASC) In this section, the solution samples were extracted by an ultrasonic reactor (SCIENTZ-ⅡD, Ningbo Xinzhi ultrasonic technology co., LTD, Zhejiang, China) with a 1.5 cm flat tip probe operating in a pulsed on-time 2 s and off-time 3 s. The reaction worked with ultrasonic power of 200 W having a single frequency of 24 kHz in the ultrasonication experiments. A temperature controlled steel jacket passed through cold water was used in the ultrasonic processer. The cooling water temperature was set at 20 °C to avoid heating effects. Sonication was performed to samples for 24 min, and then it was centrifuged at 13400 g for 10 min. The next step was indicated as section 2.3.1. The ASC by ultrasonication (UASC) was lyophilized and then stored at -20 °C until further use. 2.4. Determination of hydroxyproline Hydroxyproline (Hyp) content was determined using the colorimetric method accordind to Messia etal. [17]. A calibration curve was built with absorbance value as the ordinate and Hyp concentration as the abscissa. Hyp content in the sample was calculated from the standard curve. 2.5. Amino acid composition A 5 mg sample was hydrolyzed with 6 mol/L HCl and placed in the oven at 110 °C for 24 h in a sealed tube using a dry bath incubator (DGG-9023, Senxin experimental instrument co., LTD). Amino acid compositions were determined by a Hitachi L-8800 automatic amino acid analyzer (Beckman, America). The amino acid composition was 7

reported as a proportion of the single amino acid to total amino acids. Tryptophan was not determined. 2.6. Fourier Transform Infrared (FTIR) Spectroscopy The lyophilized collagen powders were dessicated prior to FT-IR analysis. FT-IR spectra of the samples were recorded using Cary 600 Series FTIR spectrometer with attenuated total reflectance system (Agilent Technologies, U.S.) from 4000 to 650 cm-1. The measuring resolution was 4 cm-1 with automatic signal gain in 32 scans. 2.7. X-ray diffraction The crystal structures of lyophilized collagen samples were determined using a shimadzu X-ray diffraction instrument (Tokyo, Japan). The X-ray source was Cu Ka, tube voltage was 30 kV, tube current was 10 mA, scanning range was 5-35 ° (2θ), and the scanning speed was 0.06/s. 2.8. Determination of viscosity Determination of denaturation temperature (Td) was based on the method described by [18]. Briefly, the Ostwald's viscometer was filled with 0.1% (m/v) collagen solution in 0.1 M acetic acid. After immersing viscometer in the water bath at 15 °C, it was kept for 30 min to allow the collagen solution to equilibrate to the water bath temperature. The temperature was increased stepwise up to 50 °C and maintained at each temperature for 30 min. The viscosities of the collagen solution were measured at temperature intervals of about 5 °C from 10 °C up to 50 °C. Eight determinations were made at each temperature and the average was calculated. Then, the fractional viscosities were calculated for each temperature. The Td values of collagen were taken 8

as the mid-point of the linear portion of the sigmoidal curve obtained by plotting fractional viscosity against temperatures. 2.9. Differential Scanning Calorimetry (DSC) The melting temperature (Tm) was defined as the point at which the physical form of collagen was transformed from solid to liquid. The primary structure (sequence of amino acid) of collagen was destroyed when the temperature was higher than the Tm. The melting temperature of collagen was assessed with DSC. Samples were equilibrated at 4 °C for 16 h and heated from 20 °C to 150 °C at a rate of 10 °C min-1, respectively. An empty pan was used as reference. Tm was defined as the temperature of endothermic peak. The calorimetric results for ASC and UASC were determined on the average of at least three runs. 2.10. Rheological properties The rheological properties were performed in an oscillatory rheometer (MCR302, Anton Paar, Austria), using a parallel stainless steel plate with a diameter of 40 mm and a gap of 1 mm. The cross-linking behavior of collagen was assessed by dynamic frequency sweeps. Linear viscoelasticity range was determined at 1 Hz and 1% deformation was selected for frequency sweeps of all samples. In the rheological experiments, an appropriate volume (~2.5 mL) of the collagen samples was immediately transferred to the thermostatic inferior plate. The dynamic temperature sweeps were conducted within the linear range at a constant strain of 5% and a given frequency of 60 rad/s. Subsequently, the storage modulus (G’) and loss modulus (G’’) f the samples were recorded as functions of frequency ranging from 0.01 to 10 Hz. 9

2.11. Functional properties 2.11.1. Water absorption capacity Water absorption capacity (WAC) was determined by the method of Li [19] with some modifications. 1.0 g of the sample was spread onto a weighed dry culture dish (d = 10 cm). It was then placed into an incubator with a temperature of 30 °C and a relative humidity of 60%. The dish was weighed every 4 h until its weight changed little. Water absorption capacity was calculated with the following equation: WAC(%) =

(Wt −Ws ) W0

× 100

(1)

where W0 is the weight of the culture dish, Wt is the total weight of the culture dish and absorbed water by the sample, and Ws is the initial weight of the sample. 2.11.2. Water-holding capacity The collagen samples was weighed accurately and placed in a plastic tube containing aqueous PBS (pH 7.2-7.4, w/w=1:1) at 37 °C [19]. After shaken gently for 24 h, the water was absorbed into the samples and the total quality was measured. Water-holding capacity (WHC) was evaluated according to the following equation: WHC(%) =

(Wt −Ws ) Ws

× 100

(2)

where Ws is the weight of the initial weight of the sample and Wt is the total weight of the wet sample quality.

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2.11.3. Oil absorption capacity Oil absorption capacity (OAC) was determined according to the method of Al-Farga, [20] with some modifications. 0.3 g of the sample was taken into a scale centrifuge tube to which refined soybean oil (2.0 mL) was added and stirred vigorously. After being allowed to homogenize at 10,000 g for 1 min at 25 °C with a homogeniser (Universal Scientific Instrument Factory, Jintan, China), and stand at room temperature for 30 min, the mixture was centrifuged at 10,000 g for 15 min. The volume of free oil was recorded. Oil absorption was calculated using the following equation: OAC =

(V1 −V0 )

(3)

m0

where V0 and V1 is the volume of initial oil and free oil, respectively, and m0 is the weight of the sample. 2.11.4. Emulsifying properties The emulsifying activity index (EAI) and the emulsion stability index (ESI) were determined according to the method of Pearce [21] with slight modifications. Each fraction was measured under three pH conditions (4.0, 7.0 and 10.0). A volume of 2.0 mL of soybean oil and 6 mL of collagen solution (0.5%, w/v) were homogenized in a laboratory blender (Model T25 D S-25, IKA, Germany) at a speed of 10,000 g for 90 s. Emulsions were transferred out at 0 min and 10 min after homogenization and diluted 100 folds with SDS (0.1%, w/v) and mixed thoroughly for 10 s using a vortex mixer. The resulting dispersion was measured for absorbance at 500 nm using a spectrophotometer. The absorbance values measured immediately (A0) and 10 min 11

(A10) were used to calculate the EAI and ESI: 2

EAI ( m / g ) 

ESI (min) 

22.303  A0  dilution c (1 )10000

10  A0 A0  A10

(4)

(5)

where c represents the sample concentration before emulsification, and Φ is the oil volume fraction (v/v) of the emulsion (Φ= 0.25). 2.11.5. Foaming properties Foam expansion (FE) and foam stability (FS) were determined as described in by our previous study [22] with slight modification. Each fraction was measured under three pH conditions (4.0, 7.0, and 10.0). Collagen solution (2%, w/v) was transferred into a 100 mL cylinder and homogenized in a laboratory blender (Waring Commerical, 51BL30, CT, USA) at a speed of 10,000 g for 90 s at room temperature. The sample was allowed to stand for 60 min in a graduated cylinder. FE and FS were then calculated using the following equations: FE(%) =

𝑉𝑇

× 100

(6)

FS(%) = V t × 100

(7)

𝑉0 V

0

where VT is total volume after whipping, V0 is the original volume before whipping, and Vt is total volume after leaving at room temperature for 60 min.

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2.12. Statistical analysis All results were expressed as mean ± SD. The data were analyzed by one-way analysis of variance, using the software SPSS 16.0. The statistical differences between samples were determined using the LSD (least significant difference) test. The significance was established at P < 0.05. 3. Result and discussions 3.1. Determination of Collagen Content In this experiment, the collagen contents ASC and UASC obtained were 436.2 ± 3.3 and 507.5 ± 4.1g kg-1, respectively. Thus, the application of ultrasound treatment positively affected the collagen content. Under the ultraonication, the collagen content increased by 16.3% compared to that of ASC. Ultrasonication could lead to increases in the collagen content from soft-shelled turtle (Pelodiscus sinensis) calipash, which might be due to its cavitation effect and mechanical effect. Furthermore, the comparisons of physicochemical and functional properties between ASC and UASC need to be studied further to promote the commercial application of ultrasound in bioactive compounds. 3.2. Amino acid composition The amino acid composition of ASC and UASC from the turtle calipash was investigated and expressed as residues per 1,000 total residues. As shown in Table 1, Gly was the most abundant amino acid and accounted for above one fifth among the total amino acids in ASC (215 residues/1000 amino acid residues) and UASC (221 residues/1000 amino acid residues). According to previous studies, Gly was the most 13

abundant amino acid in all collagens [23, 24]. Both ASC and UASC samples had relatively high contents of Ala, followed by Pro, Glu, and Hyp. It was interesting to note that, for imino acid content, UASC had 127 residues/1000 amino acid residues of Pro and 84 residues/1000 amino acid residues of Hyp, representing one-fifth of the total amino acid had increased significantly by comparison with ASC (P < 0.05). In general, the molecular structure of collagen is maintained mainly by restrictions on changes in the secondary structure of the polypeptide chain, imposed by the pyrrolidine rings of Pro and Hyp, and also maintained partially by the hydrogen bonding ability of the hydroxyl group of Hyp [25]. Higher the imino acids content, greater is the stability of the helices and thermal stability of collagen [3, 26]. Moreover, the hydrophobic amino acids of UASC were significantly higher over those of ASC (P < 0.05). It has been reported that food-protein and their hydrolysates with high antioxidant activity are correlated to the hydrophobic amino acids, such as Leu or Val at the N-terminus and His, Tyr or Pro in the sequence. The reasons for these phenomena were the dual nature of fluctuation and energy by ultrasound, which could change the molecular structure of protein and destroy intra-molecular bond, causing solubility alteration of the sample [27]. The results indicated UASC can be used as an alternative source of antioxidant. Besieds, the contents of Tyr, Met, Cys, Trp were very low in ASC, and no content of Tyr and Trp was detected in UASC. The results suggested that both the conventional and ultrasound assisted extraction process could be easy to obtain collagen with high purity and quality. 3.3. Fourier Transform Infrared (FTIR) Spectroscopy 14

The major peaks of FTIR spectra of ASC and UASC are presented in Fig. 1. Both collagens from turtle calipash in our study by exhibited FTIR spectra were similar to that found in other skin collagens [28, 29] but the ASC and UASC showed discrepancy in their intensity. The amide A bands of ASC and UASC were found at a 3295.88 and 3292.38 cm-1, respectively, and associated with N-H (hydrogen bond) stretching vibrations and when the N-H group of a peptide was involved in hydrogen bond, the position was shifted to lower frequencies at around 3,300 cm-1. Therefore, the collagens obtained from our study are mainly stabilized by hydrogen bond as shown by a lower frequency than a free N-H stretching vibration (3400-3440 cm-1) [30]. On the other hand, it is known that amide B band is related to asymmetrical stretch of CH2 and absorption due to the −CH2 alkyl chain. The band was observed at ~2,938.02 cm−1 of ASC and UASC. FTIR spectra of ASC and UASC from the turtle calipash were similar, suggesting that they maintained their intact triple helical structure. The wave numbers of the amide I, II, and III bands are related to the collagen structure [31]. The amide I band with strong absorbance in the range of 1600-1700 cm-1 is primarily associated with C=O stretching vibrations along the polypeptide backbone, and a decrease of the molecular order causes a peak shift to a lower wave number [32]. The amide II band represents N-H bending vibrations coupled with C-N stretching vibrations. The amide I, II, and III bands of ASC and UASC were found at ~1,634, ~1,536, and ~1,235 cm-1, respectively. In addition, peak area ratios between amide III and the 1450-1452 cm-1 band ranged from 0.90 to 1.12 [33], confirming 15

ultrasound did not disrupt the triple-stranded helical structures in UASC. 3.4. X-ray diffraction The X-ray diffraction (XRD) diagrams of ASC and UASC are shown in Fig. 2. The XRD of ASC showed the diffraction peaks at 7.6° and 21.7°, and the XRD of UASC showed the diffraction peaks at 7.0° and 21.6°. The peak of around 7.0° presented the distance between the molecular chains of collagen fibers [34]. When the collagen was extracted with ultrasound, there seemed to be a little shift in the diffraction peak towards low degree, indicating a possible interaction with ultrasound. The distance between the molecular chains of ASC fibers was 1.16 nm, while the distance between the molecular chains of UASC fibers was 1.26 nm. The space between the molecular chains observed in UASC was greater than that of ASC, indicating ultrasound treatment could weaken the hydrogen bonding and van der Waals forces between the molecular chains. Therefore, ultrasound improved the property with drug delivery capacity of the collagen. The second diffraction peak was found to be around 20°. This reflected diffuse scatter caused by many structural layers of collagen fibers and indicated the characteristic inter-chain spacing of the collagen triple helix. This result was supported by the FTIR analysis, where the band at 1632 cm-1 indicated the triple helix structure of Type I in ASC and UASC [35]. The d value that corresponds to peak at around 20° had little difference between them.

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3.5. Viscosity and the denaturation temperature (Td) Denaturation temperature (Td) values are constructed from the specific viscosities, which refers to the temperature at the transition midpoint where the ratio of denatured to native state is 1/1, determined through viscosity measurements. The triple helix structure of collagen linked via hydrogen bonding could be transformed to an unordered coil configuration using thermal depolymerization, along with changes of physical characteristics, such as decreases in solubility, precipitation, and viscosity values. In Fig. 3, Td of ASC and UASC were judged to be 35.1 °C and 43.9 °C. The Td results of collagen from animal species were to be correlated with the imino acid content; Pro and Hyp [36]. ASC and UASC from the turtle calipash contained a higher amount of imino acids, compared with those from the skin of brown banded bamboo shark (204 and 207 residues/1000 residues, respectively), sailfish (213 and 221 residues/1000 residues, respectively) [37, 38]. Td value of 34.5 °C from the black drum was similar to ASC [39]. Td of ASC was about 2.0 °C lower than that of the collagen from the porcine skins. Generally, it was known that the Td of the collagens from land animals was higher than those from aquatic organisms and it was more correlated with the animal body temperature and environmental temperature of their habitat [40]. In contrast, a higher Td value of 43.9 °C was also observed for UASC. The higher Td value for collagen was attributed to higher imino acid content rather than Hyp and the change in its structure. The stability of collagen could have potential applications in biomedical and pharmaceutical products.

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3.6. Differential scanning calorimetry (DSC) Differential scanning calorimetry (DSC) is used to observe the changes for thermodynamic variables by heating samples. Two different endothermal peaks of ASC and UASC obtained by DSC pattern are showed in Fig. 4, which indicated that the Td was 43.98 °C and 35.19 °C and the Tm was 111.00 °C and 120.66 °C of ASC and UASC, respectively. The values of Td were almost in accordance with the above results. The Td and Tm of UASC was significantly higher than that of ASC (P < 0.05), which demonstrated that thermal behaviors of collagens correlated with their physicochemical transformations induced by ultrasound. It also proved that the levels of hydration and the numbers and nature of covalent cross-links were partially altered by ultrasound. Therefore, UASC may have more superiority of thermal stability and is advantageous in food processing and biomedical applications. 3.7. Rheological properties The comparison of elastic moduli (G’) and viscous modulus (G’’) of ASC and UASC is illustrated in Fig. 5. As can be shown in Fig. 11, the G’ moduli of UASC over the range from 0 to 4 Hz of measured frequencies was first lower and then larger than that of ASC in the next range (4.5-10.0 Hz) of frequencies. The results indicated that the elasticity of UASC decreased at first and then increased. Ultrasound could make the fibrils and bundles loose and the structure to be less compact and stiff, hence decreasing G’. On the other hand, a further increase in frequency caused the water molecules in collagen to compete for energy with the hydrogen bonds that maintained the triple-helical configuration causing the unwinding of the triple helix (denaturation) 18

and final formation of localized gel [41]. The G’’ modulus reflects the dissipated energy as a characteristic viscosity, which is consistent with the typical rheological properties of the gel structure [42]. The G’’ moduli of UASC were lower than those of ASC. This was due to the fibrils in the UASC gels were looser and more tenuous compared with ASC, due to the fibrils in the UASC gels were looser and more tenuous compared with ASC gels [43]. In addition, it was noticeable that the G’ was higher than the G’’ in two collagens, suggesting a greater contribution from the elasticity than the viscosity. In conclusion, the elastic behavior had been characterized in ASC and UASC. 3.8. Functional properties 3.8.1. Water absorption capacity Water absorption ability is one of major functional properties of collagen and their products. In general, it is expressed as the amount of water absorbed per gram collagen. The water absorption properties of ASC and UASC were examined and summarized in Table 2. UASC exhibited better water absorption ability with the value of 23% than ASC with 18%. The result implied UASC had higher ratio of hydrophilic groups. This was in accordance with that the collagens with higher water absorption ability were expected to have proportionally more polar residues, which could have the ability to form hydrogen bonds with water [44].

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3.8.2. Water-holding capacity Water-holding capacity of collagens can be influenced by many environmental factors including protein concentration and ion strength. From the result in Table 2, ultrasound treatment resulted in a significant increase in the water-holding capacity compared to the conventional acid extraction. The water-holding capacity of UASC improved by 75.3% compared with that of ASC from turtle calipash. A relevant difference could be detected between ASC and UASC group because of the different microstructure and physicochemical properties. It could also be inferred that the surface hydrophilicity and charged groups of collagen were increased after ultrasonication, which was beneficial for the cell biological response to the material [45]. Therefore, UASC showed good moisture absorption and retention property, and thus benefited for cell growth, defect repairing as a wound application materials and the cosmetic material. 3.8.3. Oil absorption capacity Oil absorption capacity of protein is an important attribute that not only influences the taste of the product but is also an important functional characteristic required especially for the meat and confectionary industry [46, 47]. Oil absorption of proteins or their hydrolysates is expressed as the amount of oil absorbed per gram protein, which is often determined by centrifugation method. Oil absorption capacity of UASC (6.28 mL/g) was about 144% higher than that of ASC in calipash (2.57 mL/g, P < 0.05, Table 2), which was possibly due to the differences in their peptide composition, non-polar side chains, a ratio of intermolecular cross-linking, such as hydroxylation of 20

Pro. It also could be affected by various factors such as protein type and its manufacture method, degree of hydrolysis [47], temperature and the oil used. 3.8.4. Emulsifying properties EAI and ESI of ASC and UASC at various pH values (4.0, 7.0 and 10.0) are shown in Fig. 6. The result presented that EAI and ESI were significantly affected by pH value. EAI and ESI of ASC at pH 7.0 was found to be significantly lower than those of pH 4.0 and 10.0 (P < 0.05). Compared with ASC, UASC demonstrated significantly higher EAI and ESI (P < 0.05), which might be ascribed to the changes in their peptide composition, molecular size and lipophilic-hydrophilic arrangement induced by ultrasound. The emulsification properties of them generally increased significantly at pH 10.0, followed by pH 4, as compared with those at pH 7.0 (P<0.05). This might be due to increased exposition of hydrophilic and hydrophobic residues of the collagen at higher pH values, which promoted a major interaction at the oil-in-water interface. Therefore, a monomolecular film was formed around the collagen particles. In general, EAI and ESI of UASC were superior to those of ASC, which implied that different physicochemical make-up of the collagen might play an important role in the emulsification properties. Owing to this emulsification property, UASC could be hopeful to be used in food processing as an emulsification agent.

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3.8.5. Foaming properties The foaming properties (foam ability (FA) and foam stability (FS)) of ASC and UASC samples as a function of ranging pH (4.0, 7.0 and 10.0) at 0.5% protein concentration are presented in Fig. 7. Both ASC and UASC had the highest FA and FS values at pH 10.0, followed by pH 4.0 and 7.0. Changes in the protein flexible and exposing the hydrophobic groups in an alkaline solution conditions could induce the formation and stability changes of foam. Moreover, FA and FS were improved when proteins were deviated from the isoelectric point. The reason for the lowest FA might due to its solubility, which was the lowest at isoelectric point. The reduced foam formation and stability may be due to aggregation of proteins which interfered with interactions between the protein and water needed for foam formation. Generally, transportation, penetration, and reorganization of protein molecules at the air-water interface controlled the formation of foam. For a good FA, a protein should be capable of migrating rapidly to the air-water interface, unfolding, and rearranging at the interface [48]. In addition, FA and FS of UASC were significantly higher than those of ASC at the same pH. The above results suggested a positive correlation between hydrophobicity of unfolded proteins and foaming characteristics induced by ultrasound.

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4. Conclusions In this study, ultrasound assisted extraction could facilitate the extraction of collagen by acetic acid and obtain moderate modification on its physicochemical properties compared with the conventional extraction. The collagen content of UASC was significantly higher than that of ASC, and functional properties, such as water/oil absorption capacity, emulsifying properties and foaming properties, of UASC were superior to those of ASC. Therefore, based on the functionalities and economic advantage, UASC from turtle calipash would be successfully utilized as a new resource for fish collagen and therefore shows great potentials to be applied in food, pharmaceutical, cosmetic and biomaterial industries. Acknowledgment This study was supported by National Natural Science Foundation of China (31401560), Science and Technology Project in Northern Jiangsu Province (BN2015148), China Postdoctoral Science Foundation (2016M600385), Policies to Guide Plan of Jiangsu Province (BY2015073-01), Fundamental Research Funds for Jiangsu Academy of Agricultural Sciences (ZX(16)2041) and Postdoctoral Foundation of Jiangsu Province (1601131C).

23

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Figure captions Fig. 1. Fourier transforms infrared spectra of ASC and UASC.

Fig. 2. X-ray diffraction (XRD) diagrams of the ASC and UASC.

Fig. 3. Thermal denaturation curves of ASC and UASC from turtle calipash.

Fig. 4. Differential scanning calorimetry (DSC) thermograms of ASC and UASC from calipash of turtle.

Fig. 5. Rheological spectra of ASC and UASC of turtle calipash. (A) storage modulus (G′); (B) loss modulus (G″).

Fig. 6. Effects of pH on the emulsification properties of ASC and UASC from turtle calipash. (A) EAI; (B) ESI. Different letters within the same pH indicate significant difference; P < 0.05.

Fig. 7. Effects of pH on the foaming properties of ASC and UASC from turtle calipash. (A) FA; (B) FS. Different letters within the same pH indicate significant difference; P < 0.05.

28

100

ASC UASC

Amide B Amide A

90

60

1633.89

50 40 4000

3500

3000

2500

2000

1500 -1

Wavenumbers (cm ) Fig. 1

29

1448.76 1335.95 1235.18 1080.91 852.86

70

AmideⅡ

1536.51

3295.68

%T

80

3078.56 2938.02

AmideⅠ

Amide Ⅲ

1000

500

Diffracted intensity (arbitrary unit)

1800

ASC UASC

1500

1200

900

600

300

0 10

20

30

Diffraction angle (degree) Fig. 2

30

40

1.0

ASC UASC

Fractional viscosity

0.8

0.6

0.4

0.2

0.0 10

20

30

40

Temperature (°C) Fig. 3

31

50

60

1

ASC UASC

0

Heat flow (mW/mg)

-1

43.98 -2

35.19 -3 -4

120.58

-5

110.00

-6 -7 0

20

40

60

80

100

Temperature (°C) Fig. 4

32

120

140

160

A

ASC UASC

12

Elastic Moduli G' (Pa)

10 8 6 4 2 0 -2 0

2

4

6

8

10

8

10

Frequency (Hz)

B

2.0

ASC UASC

Viscous Modulus G'' (Pa)

1.6

1.2

0.8

0.4

0.0 0

2

4

6

Frequency (Hz) Fig. 5

33

A

100

ASC UASC

b

a

60

a

2

EAI (m /g)

80

a 40

b a 20

0

7.0 pH

4.0

B

10.0

100

ASC UASC

b

80

ESI (min)

b

60

a

a b

40

a 20

4.0

7.0 pH

Fig. 6

34

10.0

A

120

b

b

ASC UASC

100

FA (%)

80

a

a

60

a a

40

20

0

4.0

B

10.0

7.0 pH

100

ASC UASC 80

b

b a

FS (%)

60

a

40

b a

20

0

4.0

7.0 pH

Fig. 7

35

10.0

Table 1 Amino acid composition of ASC and UASC from turtle calipash (residues/1,000 amino acid residues). Amino acid Asp Glu Ser His Gly Thr Arg Ala Pro Hyp Tyr Val Met Cys Trp Ile Leu Phe Lys Hydrophobic amino acids# Imino acid*

ASC

UASC b

50.1 ± 1.3a 91.4 ± 1.8a 39.6 ± 1.5a 8.7 ± 0.4a 221.7 ± 3.5a 31.6 ± 1.1b 62.5 ± 1.8a 140.6 ± 2.4b 127.1 ± 2.0b 87.8 ±1.2b -33.2 ±1.1b 0.7 ± 0.1b 0.1 ± 0a -13.9 ± 0.6a 34.6 ± 1.0b 10.2 ± 0.3b 41.0 ± 0.7b 391.9 ± 2.3b 215.9 ± 1.7b

59.4 ± 1.1 104.9 ± 2.1b 38.7 ± 1.0a 9.4 ± 0.3a 215.2 ± 3.3a 27.5 ± 0.5a 76.3 ± 1.5b 128.9 ± 2.2a 115.4 ± 2.3a 81.6 ± 1.7a 0.10 ± 0.0 27.1 ± 0.8a 1.1 ± 0.0 a 0.2 ± 0.0b 0.1 ± 0.0 15.4 ± 0.7a 30.2 ± 0.8a 7.8 ± 0.2a 34.9 ± 0.5a 353.4 ±2.1a 197.0 ±1.9a

#

Hydrophobic amino acids: Ala + Thr + Val + Pro + Ile + Leu + Met + Phe. Imino acid: Pro + Hyp. Different letters within the same line indicate significant difference; P < 0.05. *

36

Table 2 Water absorption capacity, water-holding capacity and oil absorption capacity of ASC and UASC from turtle calipash. ASC Water absorption capacity (%) Water-holding capacity (%) Oil absorption capacity (%)

UASC a

23 ± 1.2b 547 ± 64b 628 ± 52b

18 ± 0.7 312 ± 25a 257 ± 36a

Different letters within the same line indicate significant difference; P < 0.05.

37