Gel properties of protein hydrolysates from trypsin-treated male gonad of scallop (Patinopecten yessoensis)

Accepted Manuscript Gel properties of protein hydrolysates from trypsin-treated male gonad of scallop (Patinopecten yessoensis) Jia-Nan Yan, Meng Zhang, Jun Zhao, Yue Tang, Jia-Run Han, Yi-Nan Du, Hui Jiang, Wen Gang Jin, Hai-Tao Wu, Bei-Wei Zhu PII:

S0268-005X(17)31354-1

DOI:

https://doi.org/10.1016/j.foodhyd.2018.12.050

Reference:

FOOHYD 4854

To appear in:

Food Hydrocolloids

Received Date: 4 August 2017 Revised Date:

28 November 2018

Accepted Date: 28 December 2018

Please cite this article as: Yan, J.-N., Zhang, M., Zhao, J., Tang, Y., Han, J.-R., Du, Y.-N., Jiang, H., Jin, W.G., Wu, H.-T., Zhu, B.-W., Gel properties of protein hydrolysates from trypsin-treated male gonad of scallop (Patinopecten yessoensis), Food Hydrocolloids (2019), doi: https://doi.org/10.1016/ j.foodhyd.2018.12.050. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Scallop male gonads

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trypsin

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lyophilized gel formation 50mg/ml

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Gel

properties

of

Protein

Hydrolysates

from

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Trypsin-treated Male Gonad of Scallop (Patinopecten

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yessoensis)

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Jia-Nan Yana,*, Meng Zhanga,*, Jun Zhaob, Yue Tanga, b, Jia-Run Hana, Yi-Nan Dua,

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Hui Jianga, Wen Gang Jinc, Hai-Tao Wua, b, †, Bei-Wei Zhua, b, †

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a

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China

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National Engineering Research Center of Seafood, Dalian 116034, PR China

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c

School of Bioscience and Engineering, Shaanxi University of Technology, Hanzhong 723000, PR

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China

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School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, PR

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*These authors contributed equally to this work. † To whom correspondence should be addressed. Tel:

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86-411-86318731, Fax: 86-411-86318655. E-mail address: [email protected]

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Abbreviations: SMGHs, scallop male gonad hydrolysates; SMGs, scallop male gonads; SDS-PAGE,

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sodium dodecyl sulfate-polyacrylamide gel electrophoresis; LF-NMR, Low-field NMR; DH, degree

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of hydrolysis; Mw, molecular weight; LVR, linear viscoelastic region; T2, Transverse spin-spin

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Abstract In order to provide a foundation for comprehensive utilization of scallop (Patinopecten

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yessoensis), the gel properties of hydrolysates from scallop male gonad (SMGHs) were studied. The

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hydrolysates were obtained from scallop male gonads (SMGs) by using trypsin. The changes in

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molecular mass of proteins were determined by SDS-PAGE and HPLC with a peptide separation

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column. The gel properties of SMGHs were investigated by using rheometer and texture instrument in

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comparison with that of ι-carrageenan and gelatin. The results showed that the proteins of SMGs were

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significantly degraded after hydrolysis with trypsin at a dosage of 3000 U/g protein. The SMGHs

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showed an appearance of gelation after hydrolysis for 30 to 180 min and cooling down. Under various

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sweep models, the values of storage modulus (G'), loss modulus (G''), viscosity (ƞ) of SMGHs were

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obvious higher than those of SMGs. The textural parameters containing firmness, cohesiveness and

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adhesive force of SMGHs at dosages of 50 and 75 mg/mL were similar to those of ι-carrageenan and

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gelatin at 25 mg/mL, respectively. For the mixtures, the gel property of SMGHs/ι-carrageenan

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SMGHs/gelatin increased significantly. The Low-field NMR (LF-NMR) also reflected the T23 of

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SMGHs/ι-carrageenan, T21 of SMGHs/ι-carrageenan and SMGHs/gelatin mixture decreased

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significantly in comparison with SMGHs alone. These results suggest that SMGHs could be applied as

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a potential gelling and thickening agent in food formulations.

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Keywords: Scallop; Male gonad; Hydrolysates; Gel properties; ι-Carrageenan

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1. Introduction Scallop (Patinopecten yessoensis) is a kind of bivalve molluscs widely cultured in East Asia. In

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China, the production of scallop has been up to 1.8 million tons by 2015 (FAO, 2015). With the

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continuous expansion of aquaculture scale of scallop P. yessoensis, the demand of scallop processing is

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increasing. Gonad is the edible part of scallop P. yessoensis. Male gonad from scallop P. yessoensis

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displayed gelation characteristics by treating with appropriate enzymes. Our previous studies have

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reported that neutrase-treated hydrolysates from scallop P. yessoensis male gonad (SMGHs) exhibited

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specific gelation property with a cellular three-dimensional network (Jin, Wu, Zhu, & Ran, 2012; Jin et

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al., 2014). Moreover, SMGHs contain abundant essential amino acids, accounting about 42% of the

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total amino acids, can be used as protein supplement (Jin, Wu, Zhu, & Ran, 2012). However, the

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knowledge about gel properties of SMGHs is still limited. It is necessary to study the gel properties,

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especially rheological characters, of this potential marine resource in detail.

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Enzymatic hydrolysis is considered as an effective way to obtain novel food ingredients with

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modified functionalities. Several studies have demonstrated enzymatic hydrolysis promoted the gel

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properties of protein products. Most of reports have been focused on the plant proteins, including

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sunflower globulin (Sanchez & Burgos, 1997), soybean protein (Zhong, Wang, Xu & Shoemaker, 2007;

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Lamsal, Jung & Johnson, 2007; Lv, Guo & Yang, 2009), canola protein (Pinterits & Arntfield, 2007),

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pea protein(Tamm, Herbst, Brodkorb & Drusch, 2016), coconut protein (Thaiphanit, Schleining &

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Anprung, 2016). Animal proteins have also been reported such as casein (Aberg, Chen, Olumide,

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Raghavan & Payne,

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al., 2009; Leeb, Götz, Letzel, Cheison & Kulozik, 2015; Tarhan, Spotti, Schaffter, Corvalan &

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Campanella, 2016), egg yolk protein (Orcajo, Marcet, Paredes & Díaz, 2013), bovine sodium caseinate

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2004), whey proteins (Spellman, Kenny, O'Cuinn & Fitzgerald, 2005; Pouliot et

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ACCEPTED MANUSCRIPT (Hidalgo et al, 2015), shark protein (Diniz & Martin, 1997), catfish skin protein (Yin et al., 2010) and

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crayfish protein (Felix, Romero, Rustad & Guerrero 2017). Especially for marine materials, even

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barely be restricted to fish-based protein as the knowledge we can acquire. Thus, it is essential to

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examine the contribution of enzymatic hydrolysis to the gel properties of SMGHs. Marine protein and

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isolated peptides have attracted much attention due to their excellent physicochemical and functional

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characteristics (Vijaykrishnaraj & Prabhasankar, 2015). Although most studies have focus on the gel

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properties of hydrolysates from both plant and animal resources, knowledge about marine protein

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hydrolysates are still limited.

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Recently, it is notable that protein/polysaccharide and protein/protein complexes always exhibit

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various functional properties and show texturization and stabilization in food system (Lam &

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Nickerson, 2014 ab; Abbasi & Dickinson, 2004; Devi, Buckow, Hemar & Kasapis, 2014). Traditionally,

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ι-carrageenan and gelatin, as a kind of polysaccharide and protein, respectively, are two important types

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of food macromolecules and widely used as effective co-gelator for gelation. Lam & Nickerson (2014 a)

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reported that whey protein/ι-carrageenan mixture show a certain enhancement in rheological

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characterization in comparison to whey protein alone, where electrostatic attraction drove the cross

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linking among them. Wang, Tong, Luo, Xu & Ren (2016) proposed that the addition of 0.3 g/kg

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ι-carrageenan improved the textural and rheological properties of low-fat cheese. Devi et al. (2014)

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revealed that under proper high pressure processing, the whey protein/gelatin mixture exhibited

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improved gel property with increasing concentration of whey protein, the effect was might contributed

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to the hydrophobic interactions. In addition, mixing fish gelatin and κ-carrageenan is also a favorable

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way to identify new and modified gelling systems (Yasin, Babji & Ismail, 2016). However, knowledge

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about the interaction between protein hydrolysates and ι-carrageenan or gelatin is still limited.

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Therefore, SMGHs can be taken as a valuable precursor to study the gel formation of marine protein

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hydrolysates in the presence of ι-carrageenan or gelatin. In this study, the prospective gel-likeness of SMGHs was obtained by treatment with trypsin. The

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molecular weight distribution of SMGHs was estimated by SDS-PAGE and HPLC. The effects of

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hydrolysis time and hydrolysate concentration on the rheological behavior of SMGHs were determined

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by rheometer. The gel strength of SMGHs was evaluated in comparison with commercial hydrocolloids,

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including ι-carrageenan and gelatin by texture analyzer. The synergistic effect of ι-carrageenan and

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gelatin on rheological properties of SMGHs was as well studied. Furthermore, the water migration in

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the mixture of SMGHs combined with ι-carrageenan and gelatin was reported as well.

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2. Materials and methods

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2.1. Materials and chemicals

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Scallops P. yessoens harvested from Ocean Island, China, were purchased from Changxing

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Aquatic Products Market (Dalian, China) at developmental stage of 30th, Mar. in 2016. The male

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gonads were collected, boiled for 10 min to inactive the endogenous enzymes and then freeze-dried.

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The scallop male gonads (SMGs) were stored at -80℃ before use.

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Trypsin was obtained from Sigma-Aldrich Co., Ltd (St. Louis, MO, USA). Bovine serum albumin

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was purchased from Sangon Biotech Co., Ltd (Shanghai, China). The standard molecular weight

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markers were supplied by Takara Bio Co., Ltd (Dalian, China). Iota-carrageenan was obtained from

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Aladdin Co., Ltd (Shanghai, China) and gelatin was obtained from Sangon Biotech Co., Ltd

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(Shanghai, China). All other chemical reagents used were of analytical grade.

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2.2. Preparation of scallop male gonad hydrolysates Lyophilized powder of SMGs was mixed with deionized water at protein concentration of 4%

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ACCEPTED MANUSCRIPT (w/v). The suspension was adjusted to pH 8.0 by using 0.1 M NaOH and heated with continuously

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stirring at 37℃. Trypsin (3000 U/g protein) was added to initiate the reaction. The mixture was

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maintained at pH 8.0 and hydrolyzed for 30-180 min. After enzymatic hydrolysis, the mixture was

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boiled for 10 min to inactivate the enzyme.

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After inactivation of the enzyme, partial SMGHs (30-180 min) were collected directly for

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SDS-PAGE analysis and induction of gel formation. Other SMGHs (30-180 min) were freeze-dried,

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and the powders were stored at -80 ℃ for further analysis.

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2.3. Gel formation

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Samples of SMGHs at different hydrolysis time (30-180 min) were directly collected. After

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inactivation of the enzyme, SMGHs were kept at 4℃ to promote the gel formation. In order to ensure

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the complete hydration during gel formation, the suitable incubation time was determined as 16 h in the

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preliminary experiments.

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For studying the gel properties of SMGHs at different concentrations, the powder of SMGHs

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(hydrolyzed for 180 min) was suspended with deionized water at the concentrations of 25, 50, 75 and

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100 mg/mL, respectively. The mixture was boiled for 10 min. The samples were centrifuged at 5000×g

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for 10 min to remove the bubbles after cooling to room temperature. Then the obtained suspensions

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were kept at 4℃ for 16 h to promote the gel formation.

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For the gel mixture, ι-carrageenan or gelatin were first dissolve in deionized water (50mg/mL) for

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swelling with continuously stirring at 60℃ overnight. The powder of SMGHs (hydrolyzed for 180 min)

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was mixed with commercial hydrocolloids (ι-carrageenan or gelatin) solution as obtained above at a

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concentration of 50 mg/mL and 25 mg/mL, respectively. The mixture was boiled for 10 min. The

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samples were centrifuged at 5000×g for 10 min to remove the bubbles after cooling to room

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temperature. Then the obtained suspensions were kept at 4℃ for 16 h to promote the gel formation.

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2.4. Determination of hydrolysis degree

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The degree of hydrolysis (DH) was measured by pH-stat method as described previously (Adler-Nissen et al., 1986) and expressed as below: DH（%）=

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B × Nb 1 1 × × ×100 Mp α htot

where Mp is the protein mass (g); B is the NaOH amount (mL) consumed in enzymatic reaction to keep

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pH constant; Nb is the NaOH normality; htot is the total number of peptide bonds in the substrate, which

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is 7.5 meq/g as previously described (Jin et al. 2012); α is the average dissociation degree of the

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released α-NH2 groups during hydrolysis as shown below:

α= 142

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10 pH -pK 1 + 10 pH - pK

where pH is the value at which the hydrolysis was conducted, pK is 7.5 in the present study.

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2.5. Determination of TCA-soluble oligopeptide content

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Aliquots (2 mL) of suspension obtained after hydrolysis for 0, 30, 60, 90, 120, 150 and 180 min,

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respectively, were fully mixed with same volumes of 20% (w/v) TCA solution. The mixtures were

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placed at room temperature for 20 min, and centrifuged at 5000×g for 20 min. The TCA-soluble

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oligopeptide content of supernatant was evaluated by the Lowry’s method (Lowry, Rosenbrough, Farr,

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& Randae, 1951) using bovine serum albumin as a standard protein. The content of TCA-soluble

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oligopeptide was calculated as:

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TCA-soluble oligopeptide content (%) = N1 × 100% / N0

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where N0 is the total protein in the suspension (g), N1 is the TCA-soluble oligopeptide in the

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supernatant (g).

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2.6. Protein degradation of SMGs during hydrolysis

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electrophoresis apparatus (AE-6450, ATTO Co., Ltd, Japan). The SMGHs were mixed with sample

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buffer [250 mM Tris-HCl buffer, pH 7.5, 8 M urea, 5% SDS (w/v), 5% mercaptoethanol (w/v)] at a

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ratio of 1:1 (v/v). The mixture was boiled for 10 min then was continuously shaken over night at room

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temperature. After centrifugation at 12,000×g for 10min, the supernatant was analyzed by

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electrophoresis with 15% running gel and 5% stacking gel. The gel was stained with 0.05% coomassie

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brilliant blue R-250 (w/v). After destaining, the gel was imaged using Gel Capture software (DNR

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Bio-Imaging Systems, Jerusalem, Israel).

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2.7. Determination of molecular weight (Mw) distribution

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The powder of SMGHs at different trypsin-hydrolyzed time was dissolved in deionized water at 4

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mg/mL. An Elite P230 HPLC system (Elite Analytical Instruments Co., Ltd., Dalian, China) equipped

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with a Superdex Peptide 10/300 GL column (GE Healthcare Co., Little Chalfont, Buchinghamshire,

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UK) was used. After filteration, 10 µL SMGHs solution was injected onto the column and monitored at

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220 nm. The isocratic elution was carried out at a flow rate of 0.4 mL/min containing deionized water,

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acetonitrile, trifluoroacetic acid at a ratio of 700:300:1. Cytochrome c (12,500 Da), aprotinin (6512 Da),

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vitamin B12 (1355 Da), glutathion (307 Da) and glycine (75 Da) were used as standards. The standards

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had a linear log Mw (y) versus retention time (x) regression curve: y= -0.0713x + 5.6943 (R2 =0.9906).

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The Mw distribution of SMGHs was obtained through the peak area of the standard curve. The relative

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proportion of different Mw fractions was estimated by the percentage to the total peak area detected.

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2.8. Rheological measurement

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All rheological properties of samples were determined by a rheometer (Discovery HR-1, TA

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Instruments Menu Co., Ltd, USA) equipped with parallel plate geometry (d=40 mm). Oscillatory stress

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viscoelastic region (LVR). For steady tests, samples were sheared continuously at a rate from 0.5-50 s-1

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to measure the apparent viscosity (η). It has been reported that rheological behavior of many protein

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gels was measured at 5-20°C (Devi et al., 2014; Pang, Deeth & Bansal, 2015; Ramasubramanian,

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D’Arcy, Deeth & Oh, 2014). To avoid the production of condensed water during the measurement,

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15°C was selected as a desired temperature to conduct rheological measurement. The gap of all tests

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was 1 mm.

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2.9. Textural measurement

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The textural properties of samples were evaluated with a Texture Analyzer (TA-XT2i, Stable

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Micro Systems, Co., Ltd, UK). Briefly, 20 mL semisolid sample was compressed using a type A/BE

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acrylic cylinder probe. The equipment was set as followed: pre-test speed of 1.0 mm/s; test speed of 1.0

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mm/s; post-test speed of 10 mm/s; compression degree of 10%; trigger force of 0.5 g. Parameters like

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firmness (g), cohesiveness (g·s) and adhesive force (g) were immediately obtained through the

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instrument software after the test was finished.

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2.10. NMR relaxometry

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NMR relaxometry experiments were performed as previously described (Kang, Li, Ma & Chen,

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2016) with minor modifications. The sample (2 g) was added in a 2 mL glass vial, then inserted into the

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sample bed of NMR scanner. Transverse spin-spin relaxation (T2) measurement was performed on the

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MesoMR23-060V-1 Analyzer equipped with a 0.5 T permanent magnet (Niumag Co., Ltd., Shanghai,

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China). The proton resonance frequency was 23.2 MHz at 32 ℃. The spin-spin relaxation time T2 was

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measured by using Carr-Purcell-Meiboom-Gill sequence. The parameters were as followed:

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τ-value=100 (time between 90° and 180°pulses), the number of echoes NECH=5000, and TW (the

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and every sample was performed at least triplicate. The NMR T2 data distributed exponential fitting of

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Carr-Purcell-Meiboom-Gill decay curves were obtained by MultiExp Inv analysis software (Niumag

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Electric Corporation, Shanghai, China).

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2.11. Statistical analysis

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All data were expressed as mean ± standard deviation. The significant difference among means was analyzed by Student's t-test. A level of P<0.05 was considered as significant.

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3. Results

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3.1. Gel formation of SMGHs during hydrolysis with trypsin

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The lyophilized powder of SMGs containing 83.7% of protein were hydrolyzed by trypsin at 37℃

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and pH 8.0. The hydrolysisi profile of SMGs was an incessant process up to 180 min as shown in Fig.

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1A. A rapid increase in DH (%) was observed in the initial stage (0-30 min), and then a slower increase

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in DH (%) occurred during 30-90 min. After 90 min, the DH (%) began to increase at an extremely low

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rate and reached a plateau with a value of 12.3% at 180 min.

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The data obtained from TCA-soluble oligopeptide content of SMGHs corresponded well to

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hydrolysis profile, showing increase tendency with extending hydrolysis time (Fig. 2B). After trypsin

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treatment 30 min, the TCA-soluble oligopeptide content of SMGHs significantly increased from 0.14%

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to 8.54 % (P<0.05), about 61-folds increment. Until 90 min, TCA-soluble oligopeptide content of

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SMGHs reached to 9.79%, significantly higher than that of 30 and 60 min (P<0.05). However, the

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values of TCA-soluble oligopeptide content from 90-180 min showed no difference (P>0.05).

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After hydrolysis, the SMGHs treated by trypsin for different hydrolysis time (30-180 min) were

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storage at 4℃ for 16 h for gel formation. As shown in Fig. 1C, it was obvious that the lyophilized

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homogeneous and slimy gradually with the prolonging of hydrolysis time. At 30 min, the SMGHs

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showed a little delamination appearance, while the insoluble substance was vanished gradually at 60

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and 90 min. Furthermore, the SMGHs showed uniform gel likeness performance at 120, 150 and 180

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min, respectively. These results suggest that SMGHs exhibit a specific gel performance by treatment

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with trypsin.

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3.2. Molecular weight changes of SMGHs during hydrolysis with trypsin

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In order to confirm the proteolysis of SMGs during hydrolysis with trypsin, the protein profile of

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SMGs during hydrolysis was analyzed by SDS-PAGE. As shown in Fig. 2A, SMGs contained the most

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abundant proteins, with the Mw of 51.5, 43.1, 16.2, 15.3, 12.8 kDa, respectively. With the prolonging

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of hydrolysis time, the SDS-PAGE pattern of SMGHs exhibited an obvious smearing. At 30 and 60

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min, there was still a small amount of proteins with Mw of 16.2, 15.3 and 12.8 kDa. After hydrolysis

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for 90 min, SMGHs at a Mw region above 14.3 kDa significantly disappeared. Moreover, the

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consequent generation degraded products in SMGHs with Mw less than 14.3 kDa were also detected

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after hydrolysis for 30-180 min.

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To further understand the migration trend of the consequent peptides, the Mw distribution of

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SMGHs was investigated by using HPLC equipped with a gel filtration column. On the basis of the

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retention time, the elution curve was separated into seven fractions, including above 10 kDa, 5-10 kDa,

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3-5 kDa, 1-3 kDa, 0.5-1 kDa, 0.2-0.5 kDa, and below 0.2 kDa. As shown in Fig. 2B, changes in the

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percentage contents of Mw distribution of SMGHs at different hydrolysis time were similar. Through

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horizontal comparison, a clear Mw transition of SMGHs from high to low was observed with extending

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of hydrolysis time. In this work, the Mw of SMGHs mainly distributed between 0.5 kDa and 10 kDa,

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Moreover, at the beginning of hydrolysis, only 0.14% of TCA-soluble oligopeptide was detected in

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SMGs protein, while the trypsin treatment effectively increased the release of TCA-soluble

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oligopeptide to 8.54% at 30 min (about 61 folds as comparison with that at 0 min). Thus, we expected

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that the response signal of SMGHs at 0 min through HPLC system might be extremely low so that the

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oligopeptide distribution at before trypsin treatment was finally not added in Fig.2B. Moreover, the

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content of the fractions above 3 kDa decreased from 29.97 to 21.10% after hydrolysis from 30-180 min.

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Meanwhile, the fractions below 3 kDa increased from 70.03% to 78.90%. Combining the results in Fig.

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1C, it is reasonable to suggest that the gel formation of SMGHs is contributed to the generation of

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peptides.

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3.3. The rheological behavior of SMGHs at different hydrolysis time

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In order to investigate the gel properties of SMGHs, the rheological behavior of SMGHs was

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investigated. Oscillation strain sweep is a typical model to ascertain the LVR and n-LVR of samples.

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The elastic modulus (G′) and viscous modulus (G″) in oscillation strain sweep were used to reflect the

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rheological properties. As showed in Fig. 3A, the sweep ension of SMGs (0 min) showed no

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rheological behavior. The SMGHs at 30 min also had extremely low response for rheological behavior,

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similar with SMGs (0 min), (Data not shown). While, SMGHs presented a similar pattern in G' and G''

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corresponding to oscillatory strain at different hydrolysis time (60-180 min). The profile showed a LVR

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region where G' and G'' showed constant values at oscillation strain less than 2%, followed by a n-LVR

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region, showing a downward trend of G'. In terms of G'', it increased significantly at the initial stage of

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n-LVR region and decreased rapidly after the crossover occurring. Before the crossover, all examined

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SMGHs showed G' values much higher than G" at the same oscillation strain, indicating an elastic

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occurred. Moreover, the values of G', G" as well as the modulus value of crossover for each SMGHs

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were also found to be dependent on the trypsin-hydrolyzed time. With the time extending, the values

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mentioned above increased notably. Taken the modulus value of crossover as example, it shifted from

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2.49 Pa to 15.49 Pa at 60 min, 180 min, respectively.

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To determine the flow behavior of SMGHs, the apparent viscosity (η) at different shear rates for

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each SMGHs was shown in Fig. 3B. The viscosity of the SMGHs (60-180 min) decreased sharply with

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the increasing of shear rate between 0.5-50 s-1, then slowed down after 13 s-1. As in parallel with Fig.

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3A, SMGs (0 min) showed no viscosity characteristics. Moreover, at a fixed shear rate value of 0.5 s-1,

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the viscosity value increased with the increasing hydrolysis time and reached 23.0 Pa·s at 180 min.

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These results suggest that SMGHs possess a non-Newtonian shear thinning behavior which was

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promoted by the prolonging of trypsin-hydrolyzed time.

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3.4. Rheological behavior of SMGHs at different concentration

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Although the gel property of SMGHs is expected to rise even above 180 min, we selected 180 min

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as a desired hydrolysis time in consideration of the enzymolysis efficiency and the prevention of

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microbial growth. In order to investigate the gel properties of trypsin-hydrolyzed SMGHs in detail,

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SMGHs were prepared after hydrolysis with trypsin time for 180 min and freezed-dried to obtain the

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powder. The SMGHs powder was suspended in water at concentrations of 50, 75, and 100 mg/mL,

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respectively, for gel formation. As shown in Fig. 4, it was demonstrated that the rheological behavior of

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SMGHs was in a dose-dependent manner. In Fig. 4A, both G' and G'' of SMGHs increased with the

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increasing concentration. At the oscillation strain of 1%, the values of G' and G'' for SMGHs at 100

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mg/mL reached to 224 Pa and 17 Pa, respectively, obviously higher than those at 50mg/L and 75mg/L.

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The SMGHs at 100 mg/mL showed an elastic characteristic in the appearance (Fig. 4A). In agreement with Fig. 4A, the viscosity of SMGHs also exhibited a dose-dependent manner. At

289

the fixed shear rate of 0.5 s-1, the highest value of SMGHs at concentration of 100 mg/mL reached to

290

53 Pa. These results suggest that the SMGHs form gel in a dose-dependent manner.

291

3.5. Gel strength of SMGHs in comparison with commercial hydrocolloids at different

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concentration

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In order to explore the potential application of SMGHs, the gel strength of SMGHs was

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determined in comparison with ι-carrageenan and gelatin by using a texture analyzer. The indices of

295

firmness (g), cohesiveness (g·sec) and adhesive force (g) were used to evaluate the gel strength. As

296

shown in Fig. 5, all the samples showed a dose-dependent manner in terms of firmness, cohesiveness

297

and adhesive force at 25-75 mg/mL. At the same concentration, gelatin showed the best gel strength

298

following by ι-carrageenan and SMGHs. The gel strength of SMGHs showed firmness of 352 g and

299

adhesive force of 152 g at 50 mg/mL, which were close to those of ι-carrageenan at 25 mg/mL

300

(P>0.05). Moreover, SMGHs at 75mg/mL with firmness of 833 g, cohesiveness of 5397 g·s and

301

adhesive force of 310 g were similar to gelatin at 25 mg/mL (P>0.05). The above results suggest that

302

the gel strength of SMGHs can comparable with that of ι-carrageenan and gelatin, and have a potential

303

application in food industry.

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3.6. The synergistic effect of commercial hydrocolloids on gel formation of SMGHs

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To investigate the commercial hydrocolloids effect on gel formation of SMGHs, the gel mixture

306

was prepared for rheology analysis. The mechanical spectra of G' and G'' versus oscillation strain of the

307

SMGHs combined with or without ι-carrageenan and gelatin were shown in Fig. 6. Both G' and G'' of

308

gelatin at 25 mg/mL were higher than those of ι-carrageenan at 25 mg/mL and SMGHs at 50 mg/mL.

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ACCEPTED MANUSCRIPT The values of G' and G'' of ι-carrageenan (25 mg/mL) and SMGHs (50 mg/mL) were similar. These

310

results above are in accordance with those in Fig. 5. At the fixed oscillation strain value of 1%, G' of

311

SMGHs, ι-carrageenan and gelatin was 47, 51, and 603 Pa, respectively. However, ι-carrageenan and

312

gelatin significantly enhanced the G' of SMGHs to 870 and 795 Pa, respectively. It was notable that the

313

synergistic effect of ι-carrageenan on gel formation of SMGHs was much higher than that of gelatin.

314

Moreover, the synergistic effect of SMGHs on gel formation of ι-carrageenan was also reflected on

315

textural properties. In comparison to carrageenan, firmness (g), cohesiveness (g•s) and adhesive force

316

(g) of SMGHs/ι-carrageenan increased by 2.9-, 3.6- and 1.4-folds, respectively (Data not shown).

317

These results suggest that ι-carrageenan can be used as co-gelator to enhance the gel strength of

318

SMGHs.

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3.7. The moisture-distribution of SMGHs combined with commercial hydrocolloids

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Water plays an important role in gel system by binding to the functional groups or holding in the

321

pores of the gel network (Shen & Kuo, 2017). LF-NMR is a kind of effective, reliable and

322

non-destructive measurement for the analysis of changes in water-holding capacity as well as water

323

distribution (Shao et al., 2016). Relaxation time T2 has been prevalently used to monitor the dynamic

324

state of water in protein gels (Li et al., 2017). As shown in Fig. 7, the relaxation time T2 curves of

325

SMGHs (50 mg/mL) with or without ι-carrageenan (25 mg/mL) and gelatin (25mg/mL) were

326

determined. Obviously, two peaks were observed in the T2 relaxation spectra indicating the existence of

327

two distinct water populations for each sample. They were defined as bound water (T21, 1-10 ms) and

328

free water (T23, 100-10000 ms), respectively (Gussoni et al., 2007). However, the immobile water (T22,

329

10-100 ms), which exists in the extracellular spaces, were not detected in this study. The dominant T23

330

population was apparent with peak ration over 92%, indicating the free water dominant the moisture

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ACCEPTED MANUSCRIPT component for all examined gel samples. As shown in Table 1, in terms of T21, the relaxation time of

332

the SMGHs/ι-carrageenan and SMGHs/Gelatin mixture were significantly reduced in comparison with

333

that of SMGHs (P<0.05), suggesting a prominently migration to bind bound water in the mixed gel

334

comparing with SMGHs alone. The similar trends were as well observed in T23 for

335

SMGHs/ι-carrageenan, significantly lower than those of SMGHs and ι-carrageenan, respectively. These

336

results suggest the synergistic effect of ι-carrageenan on the gel formation of SMGHs may be

337

contributed to the migration of water in the gel system.

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4. Discussion

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In our previous studies, we have obtained gelation-like hydrolysates with improved water-holding

340

capacity and emulsifying activity from scallop P. yessoensis male gonad by using neutrase (Jin et al.,

341

2012; Jin et al., 2014). At present studies, we prepared the gel of SMGHs by using trypsin. Comparing

342

with the previous studies, the DH of SMGHs (Fig. 1A) was slightly higher than that treated by neutrase

343

at 60 or 180 min with DH levels of 9.48% and 11.86%, respectively (Jin et al., 2012; Jin et al., 2014).

344

The trypsin-treated SMGHs also exhibited evident gel appearance (Fig. 1C), which was similar with

345

the previous studies (Jin et al., 2012). In addition, both SDS-PAGE and Mw distribution measurements

346

confirmed the generation of peptides in SMGHs (Fig. 2) with Mw from 0.5 kDa to 10 kDa. These

347

results above imply that trypsin is an effective enzyme to obtained SMGHs gel.

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Enzymatic hydrolysis is an effective way to reduce the molecular weight of proteins (Jonathan &

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Danielm, 2010). The increased molecular flexibility promotes the intermolecular interactions among

350

molecules to form aggregates and gels (Perez, Sanchez, Rodriguez, Rubiolo & Santiago, 2012). Recent

351

studies have shown that trypsin as a favorable tool enzyme, has been widely used to obtain

352

hydrolysates with improved functionalities. Tamm et al. (2016) demonstrated that the main peptides

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ACCEPTED MANUSCRIPT from pea protein hydrolyzed by trypsin exhibited stronger and more elastic interfacial layers with Mw

354

below 5 kDa in comparison to the pea protein isolate. Pinterits & Arntfield (2007) proposed that the

355

trypsin-treated canola protein exhibited an improved gelation property, accompaning by a decrease in

356

Mw below 7 kDa. Orcajo et al. (2013) produced hydrolyzed egg yolk granules possessing good

357

rheological character with shear thinning behavior through treating with trypsin at a DH of 12%. Our

358

previous report has proved that the improved gel property of neutrase-treated SMGHs was related to

359

hydrophobic interactions, hydrogen bonds and electrostatic interactions (Jin et al., 2014). These results

360

imply that SMGs might expose some functional groups after being treated by trypsin, thus promoting

361

gel formation.

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Rheology is helpful to understand the response of food structure to force and deformation (Yang,

363

Irudayaraj, Otgonchimeg & Walsh, 2004). Oscillation strain sweep and shear rate sweep are two major

364

models to detect the rheological behavior of food materials. In oscillation strain sweep, G′ represents

365

the recoverable energy which stored in the interface (elastic), while G″ shows the loss of energy via

366

relaxation processes (viscous) (Benjamins, Lyklema & Lucassen-Reynders, 2006; Tamm et al., 2016).

367

The LVR and n-LVR obtained from oscillatory stain tests were always used to explore the

368

characteristic of rheology of food ingredients. It has been shown that food in n-LVR catered more for

369

sensory requirements than that in LVR (Guggisberg, Cuthbertsteven, Piccinali, Bütikofer & Eberhard,

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2009).

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In oscillatory strain sweep, G' was much higher than G" under lower strain (below 2%), indicating

372

a solid-like behavior of SMGHs (Fig. 3A and 4A). This strain-independent behavior of SMGHs

373

could be considered as a result of the elasticity exhibited by the structure. At higher strains (2-1000%),

374

SMGHs showed G' began decreasing while G'' increasing followed by decreasing. This specific

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ACCEPTED MANUSCRIPT phenomenon could be characterized as a weak strain overshoot behavior as described in the previous

376

literatures (Hyun, Kim, Ahn & Lee, 2002; Sim, Ahn & Lee, 2003; Lam & Nickerson, 2014 b). The

377

overshoot could be taken as the balance between the formation and the break of gel network (Sim et al.,

378

2003). The final decrease in both moduli might be caused as a result of structure breakdown by

379

shearing, where over the crossover, G" was predominant and SMGHs behaved more like liquid. In

380

shear rate sweep, all SMGHs indicated a decreasing η within the shear rate range (0.5-50 s-1) (Figs. 3B

381

and 4B), suggesting a structure of clusters or aggregates in SMGHs could be deformed and disrupted

382

during shearing to some extent. Shear thinning property is crucial to thickeners in food industry. These

383

thickeners almost exhibited non-Newtonian behavior and existed as weak gel (Lamsal et al., 2007;

384

Witczak, Juszczak & Gałkowska, 2011).

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Moreover, the present study showed that G', G" and η of SMGHs increased significantly with the

386

increasing trypsin-treated time and hydrolysate concentration in oscillatory strain sweep (Figs. 3 and 4).

387

Other published experimental data as also well supported our findings in terms of the concentration

388

effect on rheological characteristics. Sanchez & Burgos (1997) found that the increase in G' of

389

sunflower proteins hydrolysates could be obtained with the increasing concentration from 1.7-2.5% in

390

the oscillatory mode. Diniz & Martin (1997) observed a great increase viscosity in fish protein

391

hydrolysates with the increasing concentration at fixed shear rates. Aberg et al. (2004) also reported

392

that casein hydrolysate possessed a concentration-dependence manner as well as shear thinning

393

behavior. These results suggest SMGHs exhibit a weak strain overshoot behavior as well as

394

non-Newtonian shear thinning flow behavior. The rheological behavior of SMGHs is dependent on

395

trypsin hydrolysis time and hydrolysate concentration.

396

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However, the drastic increases in the rheological properties of SMGHs after 60 min of trypsin

18

ACCEPTED MANUSCRIPT treatment (Fig. 3) were not completely corresponded to the progress of hydrolysis (Figs. 1A and B) and

398

changes in protein degradation (Figs. 2A and B). Such discrepancy indicated that soluble peptides of

399

SMGHs through hydrolysis might play partial role on gel formation. Nevertheless, protein degradation

400

by trypsin treatment also could be an essential cause of enhancement of gel forming ability in SMGHs.

401

Several literatures have demonstrated that enzymatic hydrolysis is an effective tool to degrade the

402

macromolecular protein into small peptides, thus promoting the intermolecular interactions among

403

molecules with increased flexibility to form aggregates and gels with improved functionalities (Jin et

404

al., 2014; Orcajo et al., 2013; Pinterits & Arntfield 2007; Tamm et al., 2016; Selig et al. 2018). In the

405

present study, SDS-PAGE showed the remaining protein band was mainly less than 14.3 kDa after

406

trypsin treatment for 90-180 min (Fig. 2A). Similarly, Selig et al. (2018) have proposed the remaining

407

fraction of whey protein with molecular weight higher than 10 kDa by protease treatment results in gel

408

strength of fully hydrolyzed whey protein gel. Additionally, the soluble protein and TCA-soluble

409

oligopeptide content in SMGHs reached the maximal production only of 18.41% (Data not shown) and

410

10.71 % (Fig. 1B) at 180 min, respectively. Moreover, we further detected the peptide distribution of

411

soluble peptides in SMGHs as depicted in Fig. 2B. Indeed, the proportion of fractions below 3 kDa in

412

soluble peptides of SMGHs gradually increased from 73.1% to 78.9% (P<0.05) (Fig. 2B),

413

corresponding to the TCA-soluble oligopeptide content of SMGHs increasing from 8.53 % to 10.71 %

414

significantly (P<0.05) (Fig. 1B). TCA is able to induce precipitation of protein because of the three

415

chloro groups in the molecule (Sivaraman, Kumar, Jayaraman & Yu, 1997). It has been reported that

416

most of the peptides left in supernatant solution are small-size peptides (Liu et al., 2015). Therefore, as

417

discussed above, the soluble peptides (especially the fractions below 3 kDa) might partially determine

418

the gel forming ability of SMGHs, while the fractions with higher molecular weight may involve in the

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gel strength. Moreover, our previous report has demonstrated that the SMGHs gel is primarily

420

maintained by non-covalent bonds including hydrophobic, electrostatic interactions and hydrogen

421

bonds (Jin et al., 2014) which may result in an enhancement in rheological property of SMGHs. Texture is a common method to study the gel properties of protein with parameters such as

423

firmness, cohesiveness, and adhesive force (Ellouzi et al., 2014; Lamsal et al., 2007; Garcés-Rimón,

424

Sandoval, Molina, López-Fandiño & Miguel 2016). The SMGHs exhibit gel properties in terms of

425

firmness, cohesiveness as well as adhesive force at appropriate concentration (Fig. 5). As described in

426

terms of the typical kinetic curve for evaluation of gel properties by texture analyzer from our previous

427

report (Jin et al., 2014), the firmness denoted the peak value of positive force, while the adhesive force

428

(absolute value) denoted the peak value of negative. Cohesiveness is a measurement to detect how well

429

the structure of a product withstands compression and defined as the work required to deform the gel in

430

the down movement of the probe, namely the area between positive force and time. These parameters

431

to characterize the protein hydrolysates gel properties were as well reported by other studies. Ellouzi et

432

al. (2016) stated that pasta gluten hydrolysates showed a significant improvement in firmness while a

433

slightly weaken in cohesiveness after being treated by commercial proteases (Alcalase or Pancreatin).

434

Lamsal et al. (2007) compared the firmness of hydrolysates obtained from four different kinds soy

435

protein, and observed that hexane-defatted soy flour hydrolyzed by bromelain at a hydrolysis degree of

436

2% showed the best firmness. Garcés-Rimón et al. (2016) examined the texture characteristics of egg

437

protein hydrolysates depending on the parameters of firmness, cohesiveness, and adhesive force,

438

demonstrating that the egg protein hydrolysates obtained from egg white performed the best texture

439

characteristics, followed by whole egg and yolk. Our study suggests that the gel strength of SMGHs is

440

comparable with ι-carrageenan and gelatin (Fig. 5), though it still belonged to weak gels. Therefore, the

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ACCEPTED MANUSCRIPT 441

effective ways to improve the gel strength of SMGHs should be carried out for better prospect of

442

application. Several reports have demonstrated that ι-carrageenan and gelatin have a synergistic effect on gel

444

formation for protein-based food materials, such as milk protein, Alaska pollock fish protein and whey

445

protein (Wang et al., 2016; Hunt & Park, 2013; Lam & Nickerson 2014 ab; Devi et al., 2014). However,

446

there are still limited studies on interaction between hydrolysates and ι-carrageenan or gelatin. In this

447

study, we proved that G' of SMGHs was significantly enhanced by 19- and 17- fold with the addition of

448

ι-carrageenan and gelatin, respectively, at oscillation strain of 1% (Fig. 6). In general, ι-carrageenan

449

usually exists as unstructured random coils above a certain temperature, while forms double helices of

450

ι-carrageenan by a coil-helix transition (Campo, Kawano, Silva Júnior & Carvalho, 2009). However,

451

the hydrogen bonds are the main intermolecular contacts in gelatin gels formation. During the gelation,

452

partial molecules in gelatin with disordered coil conformation gradually transform into helix

453

conformation (Djabourov, 1988; Pang, Deeth, Sopade, Sharma & Bansal, 2014). In present study,

454

ι-carrageenan had greater influence on the gel strength of SMGHs than gelatin (Fig. 6). This might be

455

contributed to the stronger interaction of ι-carrageenan with SMGHs than gelatin, on account of the

456

different mechanism in ι-carrageenan and gelatin.

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To elucidate the different synergistic effect of ι-carrageenan and gelatin on SMGHs gel formation,

458

the LF-NMR was applied to analyze the moisture distribution in the gel systems. It has been described

459

specific information about the distribution and mobility of protons, reflecting the interactions between

460

two matrix of water and protein in various food materials (Li et al., 2014; Shao et al., 2016). LF-NMR

461

could well analyze the effect of additional component on gel property in various food-based system

462

through examining the water distribution (Gravelle, Marangoni & Barbut, 2016; Yang et al., 2016; Niu,

21

ACCEPTED MANUSCRIPT Li, Han, Liu & Kong 2017). In the present study, the T21 fraction (1.75-6.92 ms) might be due to the

464

tight association between water molecules and other biopolymer (SMGHs, ι-carrageenan and gelatin).

465

The T23 (321.09–2175.45 ms) fraction was as a result of the waters in a larger pores or gaps in the gel

466

network. The T21 in SMGHs/gelatin mixture decreased significantly in comparison to SMGHs alone

467

(P<0.05), while the T23 did not change significantly (P>0.05) (Table 1). In SMGHs/ι-carrageenan

468

mixture, both T21 and T23 decreased significantly (P<0.05), showing a trend of migrating to bound

469

water.

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In general, the state of water as well as biopolymers and morphology were all firmly influenced by

471

the proton relaxation behavior (Hills, Takacs & Belton 1990). The constituents with a shorter relaxation

472

time (0-10ms) might correspond to protons that existed in the structures of macromolecular or bound

473

closely with macromolecular (Wang, Zhang, Bhandari & Gao, 2016). The T2 relaxation time also could

474

reflect the bounding strength of a specie interacting with its surrounding chemicals, where shorter T2

475

indicates a stronger interaction among different components (Gravelle et al., 2016). Therefore, strong

476

interaction might occur between the water and SMGHs by addition with ι-carrageenan providing an

477

improved gel property.

478

5. Conclusion

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Trypsin-induced gelation of scallop P. yessoensis male gonad hydrolysates possessed appreciable

480

gel properties. The gel of SMGHs exhibited a trypsin-treated time- as well as hydrolysate

481

concentration-dependent manner. The gel strength of SMGHs at 50 and 75 mg/mL is close to that of

482

ι-carrageenan at 25 mg/mL and gelatin at 25 mg/mL, respectively. Moreover, both ι-carrageenan and

483

gelatin had a synergistic reaction with SMGHs to improve the gel property. The more prior rheological

484

behavior of SMGHs/ι-carrageenan than SMGHs/Gelatin mixture might be accounted for the stronger

22

ACCEPTED MANUSCRIPT water migration with lower T21 and T23 in SMGHs/ι-carrageenan mixture as reflected by LF-NMR. The

486

combined use of SMGHs with ι-carrageenan may be specifically applied in the special foods such as

487

can, sausage and spread with marine flavor. Further studies are currently underway to study the

488

mechanism involved in the synergistic interaction between SMGHs and ι-carrageenan on gel

489

formation.

490

Acknowledgements

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This work was supported by the Natural Science Foundation of China (NSFC) (No. 31671808),

492

and the Innovative Talent Support Program for Colleges and Universities of Liaoning Province (No.

493

LR2017031).

494

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583

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EP

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590

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TE D

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fish

gelatin

29

and

chicken

meat. LWT-Food

Science

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ACCEPTED MANUSCRIPT

640 641 642

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RI PT

639

643 644

SC

645

M AN U

646 647 648 649

653 654 655 656 657

EP

652

AC C

651

TE D

650

658 659 660

30

ACCEPTED MANUSCRIPT Figure legends

662

Fig. 1 The hydrolysis degree (DH), TCA-soluble oligopeptide content and gelation appearance of

663

SMGHs through proteolysis.

664

A: Kinetic curve of the proteolysis for SMGHs by trypsin during 180 min. B: TCA-soluble

665

oligopeptide content (%) of SMGs as a function of trypsin hydrolysis time. Different small letters mean

666

significant differences (P<0.05). C: Pictures of gelation induced appearance for SMGHs at different

667

hydrolysis time.

SC

RI PT

661

M AN U

668 669

Fig. 2 The molecular weight (Mw) change for SMGHs during proteolysis.

670

A: SDS-PAGE profile of SMGHs at different hydrolysis time. B: Molecular weight distribution of

671

SMGHs at different hydrolysis time.

TE D

672

Fig. 3 Rheological behavior of SMGHs at different hydrolysis time.

674

A: Storage modulus (G'), loss modulus (G'')-oscillation strain profiles of SMGHs at different

675

hydrolysis time; B: Viscosity (η)-shear rate profiles of SMGHs at different hydrolysis time.

AC C

676

EP

673

677

Fig. 4 Rheological behavior of SMGHs with different concentration.

678

A: Storage modulus (G'), loss modulus (G'')-oscillation strain profiles of SMGHs with different

679

concentration; B: Viscosity (η)-shear rate profiles of SMGHs with different concentration.

680 681

Fig. 5 The gel strength of SMGHs with different concentration in comparison with ι-carrageenan and

682

Gelatin in terms of changes in A: Firmness; B: Cohesiveness; C: Adhesive force. Different small letters

31

ACCEPTED MANUSCRIPT 683

mean significant differences (P<0.05).

684 685

Fig. 6: Rheological behavior of SMGHs in addition with or without ι-carrageenan and Gelatin

EP

TE D

M AN U

SC

Fig. 7: Relaxation time T2 curves of SMGHs in addition with or without ι-carrageenan and gelatin.

AC C

687

RI PT

686

32

ACCEPTED MANUSCRIPT Table 1 Relaxation time of SMGHs, in the presence or absence of ι-carrageenan and gelatin Relaxation time (ms) sample T21 (ms)

T23 (ms)

4.99±0.40a

533.67±0.00a

ι-carrageenan

4.21±0.55ac

773.10±184.18b

Gelatin

6.92±1.41b

2175.45±348.29c

SMGHs/ι-carrageenan

1.75±0.61d

321.09±60.05d

SMGHs/Gelatin

3.64±0.26c

573.63±46.14ab

RI PT

SMGHs

Data are expressed as means±SD from triplicate determinations. Different letters in the same column

AC C

EP

TE D

M AN U

SC

indicate significant differences (P<0.05).

A

ACCEPTED MANUSCRIPT 14 12

DH (%)

10 8

4 2 0 0

30

90

120

c

10

b

8 6

2 0

c

M AN U

12

180

c

c

b

TE D

TCA-soluble oligopeptide content (%)

14

4

150

SC

Time (min)

B

a

30

60

90 120 Time (min)

150

180

AC C

EP

0

C

60

RI PT

6

Fig. 1

ACCEPTED MANUSCRIPT

A Hydrolysis time (min) 0

30

60

90 120 150 180 LM

RI PT

HM 200 116 97 66.4

97 66.4

44.3

44.3

M AN U

SC

29

0.2-0.5 kDa

0.5-1 kDa

EP

80%

<0.2 kDa

1-3 kDa

60%

3-5 kDa

AC C

Peptide distribution (%)

100%

14.3

TE D

B

20.1

5-10 kDa

40%

>10 kDa

20%

0% 30

60

90 120 Time (min)

150

180

Fig. 2

ACCEPTED MANUSCRIPT

A min G'G′0 0min min G'G′6060min min G'G′9090min 120min min G'G′120 G'G′150 150min min G'G′180 180min min G'' G′′0 0min min G'' G′′6060min min G'' G′′9090min min G'' G′′120 120min min G'' G′′150 150min min G'' 180 min G′′ 180 min

RI PT

40 30 20

SC

G' (Pa) G'' (Pa)

50

0 (1) 1

10

M AN U

10

100

1000

Oscillation strain (%)

B 20

EP

15 10

AC C

η (Pa·s)

min ηη 00 min η 60 min η 60 min min ηη 9090 min ηη 120 min 120 min ηη 150 min 150 min ηη 180 min 180 min

TE D

25

5

0 (0)

0.5

5 Shear rate

50 (s-1)

Fig. 3

ACCEPTED MANUSCRIPT

A

RI PT

系列3 G′ 100 mg/mL

190

G′′ 50 mg/mL 系列4

140

75mg/ml

50mg/ml

G′′ 75 mg/mL 系列5 G′′ 100 mg/mL 系列6

100mg/ml

SC

G' (Pa) G'' (Pa)

系列1 G′ 50 mg/mL 系列2 G′ 75 mg/mL

100mg/ml

240

90

M AN U

40 0 (10) 1

10

100

1000

Oscillation strain (%)

B 50 40

EP

η (Pa·s)

ηη50 50mg/ml mg/mL 75mg/ml mg/mL ηη75 100mg/ml mg/mL ηη100

TE D

60

30

AC C

20 10

0

0.5

5 Shear rate (s-1)

50

Fig. 4

ACCEPTED MANUSCRIPT

Firmness (g)

A A

6000

25 mg/mL

5000

50 mg/mL 75 mg/mL

g

e

4000 3000

f d

c

c

1000

b

b

a

40000

25 mg/mL

35000

50 mg/mL

30000

75 mg/mL

25000 20000 15000

0

c b

a

1600

κ-Carrageenan ι-carrageenan

Gelatin

25 mg/mL

1400

e

50 mg/mL

AC C

Adhesive force (g)

bc d

EP

SMGHs

C

e

c

10000 5000

f

e

TE D

Cohesiveness (g·sec)

B

Gelatin

SC

ι-carrageenan κ-Carrageenan

SMGHs

M AN U

0

RI PT

2000

1200

e

75 mg/mL

1000

d

800

d

600 400

c b

200

0

c b

a SMGHs

κ-Carrageenan ι-carrageenan

Gelatin

Fig. 5

RI PT

ACCEPTED MANUSCRIPT

G'G′PrSMGHs 50 mg/ml

1000

G'G′κ 25 mg/ml ι-Carrageenan

SC

900

700

M AN U

600 500 400 300 200

-100 1

10

100

SMGHs/ι-Carrageenan G'G′Pr+κ 50+25 mg/ml SMGHs/Gelatin G'G′Pr+G 50+25 mg/ml G′′PrSMGHs G'' 50 mg/ml

G′′κ 25 ι-Carrageenan G'' mg/ml G′′ Gelatin G'' G 25 mg/ml G′′Pr+κ SMGHs/ι-Carrageenan G'' 50+25 mg/ml G′′ SMGHs/Gelatin G'' Pr+G 50+25 mg/ml

1000

Oscillation strain (%)

EP

0

TE D

100

G'G′GGelatin 25 mg/ml

AC C

G' (Pa) G'' (Pa)

800

Fig. 6

RI PT

ACCEPTED MANUSCRIPT

600

SMGHs

500

SC

ι-Carrageenan

SMGHs/ ι-Carrageenan

M AN U

SMGHs/Gelatin

300

200

0 1

10 100 Relaxation (ms)

1000

10000

EP

0.1

TE D

100

AC C

Amplitude

400

Gelatin

Fig. 7

ACCEPTED MANUSCRIPT Highlight -SMGHs treated by trypsin showed a specific gel property caused by peptides. -Gel property of SMGHs had hydrolysate time- and hydrolysate dose-dependent manners. -Gel strength of SMGHs can be comparable with ι-carrageenan and gelatin.

RI PT

-Synergistic effect on gelation was occurred between ι-carrageenan and SMGHs.

AC C

EP

TE D

M AN U

SC

-Synergistic effect on water migration was found between ι-carrageenan and SMGHs.