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Food and Beverage Chemistry/Biochemistry

Comparative Study on the Cryoprotective Effects of Three Recombinant Antifreeze Proteins from Pichia pastoris GS115 on Hydrated Gluten Proteins During Freezing Mei Liu, Ying Liang, Hui Zhang, Gangcheng Wu, Li Wang, Haifeng Qian, and Xiguang Qi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00910 • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 4, 2018

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Journal of Agricultural and Food Chemistry

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Comparative Study on the Cryoprotective Effects of Three Recombinant

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Antifreeze Proteins from Pichia pastoris GS115 on Hydrated Gluten Proteins

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During Freezing

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Mei Liu1,2,3, Ying Liang4, Hui Zhang1,2,3*, Gangcheng Wu1,2,3, Li Wang1,2,3, Haifeng Qian1,2,3,

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Xiguang Qi1,2,3

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1

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China.

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2

School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.

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3

National Engineering Research Center for Functional Food, Jiangnan University, Wuxi 214122,

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122,

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China.

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4

College of Biological Engineering, Henan University of Technology, Zhengzhou 450001, China.

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* Corresponding author: Hui Zhang, Ph.D.

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Tel: 0510-85919101; Fax: 0510-85919101;

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E-mail address: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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During freezing process, ice crystals formation leads to the deterioration in physicochemical

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properties and networks of gluten proteins. The cryoprotective effects of recombinant carrot

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(Daucus carota) antifreeze protein (rCaAFP), type II antifreeze protein from Epinephelus

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coioides (rFiAFP), and Tenebrio molitor antifreeze protein (rTmAFP) produced from Pichia

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pastoris GS115 on hydrated gluten, glutenin, and gliadin during freezing were investigated. The

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thermal hysteresis (TH) activity and ice crystals morphology modification ability of recombinant

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antifreeze proteins (rAFPs) were analyzed by differential scanning calorimetry (DSC) and

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cryomicroscope, respectively. The freezing and melting properties, water state, rheological

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properties, and microstructure of hydrated gluten proteins were studied by DSC, low field nuclear

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magnetic resonance, rheometer, and scanning electron microscopy, respectively. The rTmAFP

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exhibited strongest TH activity and ice crystals morphology modification ability, followed by

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rFiAFP and rCaAFP. The addition of the three rAFPs caused freezing hysteresis and weakened

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the damage of freezing to the networks of hydrated gluten, glutenin, and gliadin. During freezing,

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the cryoprotective effects of the three rAFPs on the freezable water content, water mobility and

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distribution, and rheological properties of hydrated gluten were achieved by protecting these

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corresponding properties of hydrated glutenin. Among the three rAFPs, rTmAFP was most

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effective in the cryoprotective activities on hydrated gluten proteins during freezing. The results

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demonstrate the potential of these rAFPs, especially rTmAFP, to preserve the above properties of

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hydrated gluten proteins during freezing process.

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Keywords: recombinant antifreeze protein; gluten; glutenin; gliadin; freezing

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INTRODUCTION

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Freezing technology is increasingly being employed for the preservation of dough. The effect of

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freezing and frozen storage on the dough properties is a field of active research aiming to improve

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the quality of final thawed product.1 Notably particularly, deterioration of gluten network caused

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by ice formation or recrystallization in frozen dough is one of the major factors causing the

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quality loss of bakery products.2,3 Wheat gluten proteins, which mainly composed of glutenins

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and gliadins, play a key role in the unique baking quality of dough. Glutenins, which confer

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elasticity and strength of dough, are interchain disulfide-linked aggregated proteins with

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molecular weight ranging from 105 to 107 Da. Gliadins, which impart viscous properties of

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dough, are monomeric proteins with molecular weight ranging from 3 to 8 ×104 Da.4,5 In gluten,

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glutenin forms a network and gliadins are scattered throughout the network filling the space

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around glutenin polymers.6 Freezing involves the transition of water to ice through the

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crystallization process. Both ice content and size are the key parameters in determining the end-

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use quality of frozen product.7 During freezing process, ice crystals formation leads to the

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deterioration in physicochemical properties and networks of gluten proteins.8-12 Therefore, ice

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crystals formation should be properly controlled to protect gluten proteins and obtain good bread

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properties from frozen dough.13

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Antifreeze proteins (AFPs), also called ice structuring proteins, are a family of proteins with

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thermal hysteresis (TH) activity that can bind to ice and decrease the freezing point of solutions in

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a non-colligative manner. They also exhibit ice recrystallization inhibition (IRI) activity that can

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prevent the growth of minute ice crystals to large crystals during frozen storage, and have the

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ability to modify ice crystals morphology.14-16 AFPs are classified as moderately active AFPs or 3



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hyperactive AFPs. Generally, moderately active AFPs bind to prism and/or pyramidal planes of

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an ice crystal and generate a hexagonal bipyramidal ice crystal shape, whereas hyperactive AFPs

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bind to these surfaces and the basal plane of an ice crystal, resulting in a circular disk-like ice

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crystal morphology (Figure 1). The key difference between hyperactive and moderately active

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AFPs is the ability of the former to bind to the basal plane of ice crystals. Hyperactive AFPs give

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better protection from ice growth by providing more complete ice surface coverage than

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moderately active AFPs, thereby exhibiting much higher TH activity than moderately active

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AFPs.17-20 Because of these desirable properties, AFPs could be used as cryoprotectants in frozen

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food.21-23 Although AFPs have been isolated from plants, polar fish, insects and fungi, the low

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yield and high cost of extracting AFPs limit their wide applications in frozen food. The

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biosynthesis of recombinant AFPs (rAFPs) could realize mass production of various AFPs with

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different TH activities.24-26

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The application of AFPs in frozen dough has attracted a great deal of attention. Previous studies

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mainly focused on the effects of AFPs on the physicochemical, rheological, conformational,

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thermal and microscopic properties of frozen dough during frozen storage or freeze-thawed

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cycles, and textural and baking characteristics of the products.27-32 Both Kontogiorgos et al.33 and

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Jia et al.34 have demonstrated the cryoprotective effects of plant AFPs on frozen hydrated gluten

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during frozen storage. Nevertheless, to our knowledge, there has been little information published

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describing the effects of AFPs on the thermal properties, water state, rheological properties, and

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microstructure of hydrated gluten proteins during freezing. Thermal properties, including freezing

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and melting properties, can provide reliable basic parameters for dough freezing process design.

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Freezing treatment exerts detrimental effects on gluten proteins in diverse ways from both gluten 4


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proteins-water interactions and structure-functionality perspectives.3 Specifically, water state,

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including freezable water content, water mobility, and water distribution, can influence the

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physicochemical properties of dough, and vice versa.35

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Therefore, the objective of this study was to investigate and compare the cryoprotective effects of

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three rAFPs on the freezing and melting properties, water state, rheological properties, and

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microstructure of hydrated gluten, glutenin, and gliadin during freezing. Recombinant carrot

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(Daucus carota) AFP (rCaAFP), fish AFP (type II from Epinephelus coioides) (rFiAFP), and

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Tenebrio molitor AFP (rTmAFP) produced from Pichia pastoris GS115 were used in this study.

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This study may gain insights into the effects of different rAFPs on hydrated gluten proteins

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during freezing process from the perspectives of gluten and its components glutenin and gliadin,

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and provide a theoretical basis for the cryopreservation of frozen dough by rAFPs.

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MATERIALS AND METHODS

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Materials. Gluten (7.5% moisture; 91.5% protein on a dry basis) was purchased from Yufeng

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Co., Xinxiang, Henan, China. Methylotrophic yeast Pichia pastoris GS115, E. coli DH5α, and

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pPIC9K vector were purchased from Invitrogen (USA). Restriction enzymes, Taq DNA

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polymerase, T4 DNA ligase, and pMD18-T vector were from Takara (Dalian, China). Deionized

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water was used throughout the experiment. All the chemicals were of analytical grade unless

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otherwise specified.

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Preparation of rAFPs. All three rAFPs (rCaAFP, rFiAFP, and rTmAFP) used in this study were

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produced by recombinant Pichia pastoris GS115. Briefly, based on the amino acid sequences of

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CaAFP (GenBank Accession No. AAC62932.1), FiAFP (GenBank Accession No.

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AGM15882.1), and TmAFP (GenBank Accession No. ABB03885.1), three codon-optimized 5



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AFPs genes were synthesized and cloned into the plasmid pPIC9K to form the recombinant

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plasmids, namely pPIC9K-CaAFP, pPIC9K-FiAFP, and pPIC9K-TmAFP. These three

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recombinant plasmids were transformed into Pichia pastoris GS115, respectively. The positive

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yeast transformants which were cloned with putative multicopy number of pPIC9K-CaAFP,

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pPIC9K-FiAFP, and pPIC9K-TmAFP were selected and named GS115/pPIC9K-CaAFP,

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GS115/pPIC9K-FiAFP, and GS115/pPIC9K-TmAFP strains, respectively. Then, the three

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recombinant strains were used to produce rCaAFP, rFiAFP, and rTmAFP in the methanol-

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induced fermentation, respectively.26,36 Following this, cells were removed by centrifugation, the

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collected supernatants were successively purified by ammonium sulfate precipitation, ice-binding

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protocol, and HPLC methods. Finally, the rAFPs were purified to electrophoretic purity, which

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were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The synthesis and

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purification of the three rAFPs will not be discussed in detail in this paper, but will form part of

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another publication. The protein concentrations were determined by the Bradford method37 using

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bovine serum albumin (BSA) as the standard.

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Determination of TH Activity of rAFPs. The rAFPs were freeze-dried and dissolved in PBS

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solution (50 mM, pH 7.4) at a concentration of 10 mg/mL. Then the TH activity was determined

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using a Q2000 differential scanning calorimetry (DSC) (TA Instruments, New Castle, Delaware,

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USA) according to the method described in Ding et al.21 Briefly, the sample (~10 mg) was cooled

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to -40 °C at a rate of 1 °C/min, equilibrated at -40 °C for 5 min, and then heated to the holding

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temperature for 10 min when the sample was at solid-liquid phase equilibrium with a 10%-12%

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amount of ice remaining. The sample was then cooled from the holding temperature to -40 °C at a

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rate of 1 °C/min. The onset temperature when the exothermic process began was recorded. The 6


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TH activity is defined as the difference between the holding temperature and the onset

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temperature.

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Assay of Ice Crystals Morphology Modification Ability of rAFPs. The rAFPs were dissolved

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in water at a concentration of 1 mg/mL. Direct observation of ice crystals morphology was

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performed using a cold stage (Model THMS600, Linkham Scientific Instruments, Ltd., Surrey,

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UK) mounted on a U-TV0.63C microscope (Olympus Co., Isigawakenn, JP) according to the

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method described in Cao et al.38 A small drop (5 µL) of 1 mg/mL rAFP solution was placed on a

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glass microscope slide and covered with a glass coverslip. 1 mg/mL BSA solution was used as the

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control. The slide was placed inside the chamber of the cooling stage and quickly frozen by

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decreasing the temperature to -50 °C at 20 °C/min. After being held at -50 °C for 5 min, the

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microscopic images of samples were captured at 40× magnification. The ability of rAFPs to

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modify ice growth behavior was assayed by comparing the ice crystals size and shape of rAFPs

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solution with that of BSA solution.

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Extraction of Glutenin and Gliadin. Both glutenin and gliadin were extracted from gluten

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according to the method described in Wang et al.5 Gliadin was extracted in three steps from 200 g

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of gluten with two extractions with 60% ethanol (3 L each) and one extraction with deionized

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water (3 L). Before the second and third extraction step, the cohesive glutenin was mechanically

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disrupted by a spatula. Each extraction was conducted at 20 °C for 3 h and then centrifuged at

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3000 × g, at 4 °C for 10 min. The supernatants were pooled and the containing ethanol was

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removed using a rotary evaporator at 30 °C. The gliadin and glutenin (sediment after ethanol

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extraction) were freeze-dried. The nitrogen contents of glutenin and gliadin, which were

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determined using Kjeldahl nitrogen method, were 95.3% and 94.5%, respectively. 7



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Preparation of Hydrated Gluten Proteins and Freezing Treatment. To prepare the hydrated

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proteins, gluten (40% w/w), glutenin (40% w/w), and gliadin (50% w/w) were mixed with

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deionized water, kneaded with a spatula and allowed for complete hydration at 4 °C for 1 h,

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respectively. BSA (non-AFPs) was used as a negative-control. The addition of BSA, rCaAFP,

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rFiAFP, and rTmAFP were all 0.5% (w/w, gluten proteins basis). All fresh samples were wrapped

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in a plastic membrane.

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Freezing treatment: all fresh samples were immediately placed in a freezer at -35 °C until the core

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temperature reached -20 °C; Thawing treatment: samples after freezing were thawed at 4 °C for 6

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h.

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Before freezing, fresh glutens/glutenins/gliadins without BSA or rAFPs addition, with BSA,

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rCaAFP, rFiAFP, and rTmAFP addition were abbreviated as gluten/glutenin/gliadin-control-B,

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gluten/glutenin/gliadin-BSA-B, gluten/glutenin/gliadin-rCaAFP-B, gluten/glutenin/gliadin-

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rFiAFP-B, and gluten/glutenin/gliadin-rTmAFP-B, respectively; after freezing,

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glutens/glutenins/gliadins without BSA or rAFPs addition, with BSA, rCaAFP, rFiAFP, and

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rTmAFP addition were abbreviated as gluten/glutenin/gliadin-control-A, gluten/glutenin/gliadin-

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BSA-A, gluten/glutenin/gliadin-rCaAFP-A, gluten/glutenin/gliadin-rFiAFP-A, and

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gluten/glutenin/gliadin-rTmAFP-A, respectively.

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Determination of Thermal Properties. The freezing and melting properties of fresh hydrated

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gluten proteins were determined using the aforementioned DSC according to the method

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described in Ding et al.32 with minor modification. The sample (~10 mg) was cooled to -40 °C at

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the rate of 1 °C/min, equilibrated at -40 °C for 5 min, and then heated to 10 °C at 1 °C/min. Tf,o

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and Tf,p were taken as the onset temperature and peak temperature at exothermic curve, 8


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respectively. Tm,o, Tm,p, and Tm,e refered to onset temperature, peak temperature, and complete

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melting temperature at endothermic curve, respectively. The range of melting temperature (Tm,δ)

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was calculated as the difference between Tm,e and Tm,o.

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Determination of Freezable Water Content. The freezable water content of hydrated gluten

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proteins was assayed using the aforementioned DSC.31 The sample (~10 mg) was initially cooled

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to -40 °C at the rate of 1 °C/min, equilibrated at -40 °C for 5 min, and then heated to 10 °C at

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1 °C/min. The melting curves were recorded, and the freezable water content (Fw) was calculated

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using Eq. (1):

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Fw (%) =

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where △Hm was the enthalpy of endothermic peak of melting curve, J/g; △fusHm was the known

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latent heat of fusion of ice, 333.3 J/g; WA was the moisture content of sample, g/g.

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Determination of Water Mobility. The water mobility of hydrated gluten proteins was measured

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using low field nuclear magnetic resonance (LF-NMR) (MesoMR, Niumag Corporation,

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Shanghai, China).32 The sample (~6 g) was placed in a glass weighing bottle and inserted in the

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NMR probe. The transverse relaxation times (T2) were measured using Carr-Purcell-Meiboom-

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Gill (CPMG) sequence. The experimental parameters were as follows: SW=100 kHz, SF=21

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MHz, RFD=0.080 ms, RG1=20.0 db, DRG1=3, TD=75002, PRG=2, TW=2000 ms, NS=16,

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TE=0.5 ms, and NECH=1500. Each measurement was performed in triplicate.

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Determination of Water Distribution. The water distribution of hydrated gluten proteins was

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also measured using the aforementioned LF-NMR.32 The sample (~6 g) was placed in a glass

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weighing bottle and inserted in the NMR probe. Proton density images were acquired using spin-

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echo (SE) sequence. The experimental parameters were as follows: Sinc Pulse Duration=1200 µs,

△Hm ×100 △fus Hm×WA

Eq. (1)

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Sweep Width (SW)=20 kHz, Phase Encoding Duration=3.0 ms, Echo Position=10%, TE=10 ms,

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TR=2000 ms, and Average=6. In the proton density images, colors from blue through bright

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yellow to red represent increasing moisture content in the sample.

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Determination of Rheological Properties. The rheological properties of hydrated gluten

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proteins were measured using a Discovery Hybrid Rheometer (HR-3, TA Instruments Ltd.,

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Leatherhead, UK).39 A small piece (~2 g) was cut from samples and loaded in the plate and

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parallel plate geometry (20 mm diameter and 2 mm gap). After the sample was equilibrated for 5

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min, a frequency sweep test (0.1-10 Hz) was carried out at 25 °C and 0.2% strain. The elastic

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modulus (G′) and viscous modulus (G″) were determined as function frequency in the linear

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viscoelastic region of samples. The measurements were performed in triplicate.

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Scanning Electron Microscopy (SEM) Observation of Hydrated Gluten Proteins Networks.

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Samples were freeze-dried, cut, gold sputter coated for 2 min, and finally observed using SEM

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(Model S3400N VP, Hitachi, Japan) at an accelerating voltage of 5 kV.

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Statistical Analysis. All data are expressed as mean ±standard deviation of the mean from three

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independent experiments. In each experiment, measurements were performed in triplicate. The

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data were analyzed using an SPSS package (version 13.0 for Windows, SPSS Inc., Chicago, IL),

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and statistical significance was analyzed using an analysis of variance (ANOVA) with a Duncan’s

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comparison method test at P < 0.05.

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RESULTS AND DISCUSSION

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TH Activity and Ice Crystals Morphology Modification Ability of rAFPs. As shown in Figure

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2, the TH activities of rCaAFP, rFiAFP, and rTmAFP were 1.62, 4.23, and 7.57 °C (10 mg/mL),

212

respectively. The TH activity is generally attributed to an irreversible adsorption of AFPs to the 10


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surface of ice crystals, resulting in a localized freezing point depression.40,41 In addition, the

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difference in the TH activities between different rAFPs may result from the difference in the ice

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coverage (Figure 1).17,42

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In Figure 3, the ice crystals size of rCaAFP, rFiAFP, and rTmAFP solutions were all smaller than

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that of control, indicating that the three rAFPs had the special function of modifying ice

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morphology. Especially in Figure 3d, many very small overlapping crystals were formed leaving

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a feathery appearance, suggesting that rTmAFP exhibited the strongest modification ability of ice

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crystals morphology.

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Effects of rAFPs on Thermal Properties of Hydrated Gluten Proteins. The DSC method was

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used to mimic freezing/melting process of hydrated gluten proteins and investigate the effects of

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three rAFPs on the thermal characteristics of hydrated gluten proteins during freezing. The typical

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freeze-thawed curves of hydrated gluten proteins are shown in Figure S1 (Supporting

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Information), and the freezing and melting temperature of hydrated gluten proteins with and

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without added rAFPs are summarized in Table 1.

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As shown in Figure S1, when the temperature was lowered than -6.22 °C, the exothermic ice

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forming peak of gluten-control-B appeared, and was distorted as the formation of ice released a

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significant amount of latent heat which effectively caused a “temperature recoil” inside the

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sample pan. The similar trend was observed in glutenin-control-B. Particularly, no “temperature

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recoil” appeared in the exothermic peak of gliadin-control-B, and gliadin-control-B began to

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freeze at -18.47 °C, which was significantly lower than the freezing temperature of gluten-

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control-B (-6.22 °C) and glutenin-control-B (-6.79 °C). Then, the ice melted when the frozen

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gluten proteins were thawed in the heating process, which notably took place in a very wide 11



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temperature range. The heat flow signal gradually decreased over a large temperature range, from

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about -10 °C up to the onset of ice melting, and it did not contain any definite region that could be

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assigned to a glass transition temperature of the frozen hydrated gluten proteins. The similar

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broadness of the endotherm of frozen hydrated gluten was observed in Kontogiorgos and Goff1,

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which has been attributed to the complexity of interactions between protein molecules. Specially,

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a minor peak appeared just before the major peak in the melting curve of gliadin-control-B, the

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minor peak and major peak were assigned to the melting of capillary-confined ice and bulk ice,

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respectively.33

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As shown in Table 1, Tf,o of hydrated gluten/glutenin/gliadin with added rAFPs were all lower

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than that of gluten/glutenin/gliadin-control-B and gluten/glutenin/gliadin-BSA-B, indicating that

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the addition of rAFPs inhibited ice formation to some extent. AFPs are reported 500 times more

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effective at lowering the freezing temperature than any other known solute molecules.43

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Compared with gluten-control-B, the freezing temperature of gluten-rCaAFP-B, gluten-rFiAFP-

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B, and gluten-rTmAFP-B decreased by 2.50, 3.76, and 4.67 °C, respectively, suggesting rTmAFP

249

exhibited maximum ice formation inhibition ability, followed by rFiAFP and rCaAFP. The

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similar trends were found in hydrated glutenin and gliadin. The ice formation inhibition ability of

251

the three rAFPs was found to be probably positively correlated with their TH activities.

252

Both Tm,o and Tm,δ of gluten-rCaAFP-B, gluten-rFiAFP-B, and gluten-rTmAFP-B were all

253

significantly higher than gluten-control-B and gluten-BSA-B. The similar trends were observed in

254

hydrated glutenin and gliadin. These findings suggest that the ice crystals grown in hydrated

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gluten proteins with added rAFPs were protected from melting as well as from freezing, which

256

may arise because rAFPs irreversibly bound to the ice surface, thereby slowing down the melting 12


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of ice crystals.44,45 However, no statistically significant difference (P > 0.05) was observed in

258

both Tm,o and Tm,δ of samples with added rCaAFP, rFiAFP, and rTmAFP. The reasons for this

259

finding are unclear.

260

The addition of the three rAFPs all lowered freezing temperature and increased melting

261

temperature of hydrated gluten proteins, resulting in freezing hysteresis and a small melting

262

hysteresis. Additionally, rTmAFP exhibited maximum ice formation inhibition ability, followed

263

by rFiAFP and rCaAFP.

264

Effects of rAFPs on Freezable Water Content of Hydrated Gluten Proteins. Water is

265

classified into freezable water and unfreezable water according to its response to freezing.32

266

During freezing, only freezable water is turned into ice; thus, freezable water content can reflect

267

the amount of ice crystals formed during freezing,27 and is a key factor affecting the quality of

268

hydrated gluten proteins during freezing.

269

As shown in Figure 4a and b, the freezable water content of both hydrated gluten and glutenin

270

without added rAFPs increased after freezing, this may arise because freezing weakened the

271

interactions between water and the nonpolar and polar amino acids of gluten and glutenin, then

272

some of the bound water was released and converted to freezable water.8,46 After freezing, the

273

addition of the three rAFPs lessened the increment of freezable water content of hydrated gluten

274

and glutenin, thereby reducing the damage of freezing to hydrated gluten and glutenin. For

275

hydrated gluten after freezing, although there was no significant difference (P > 0.05) observed in

276

the freezable water content of hydrated glutens with rFiAFP and rTmAFP, the freezable water

277

content of hydrated gluten with added rFiAFP or rTmAFP was significantly lower (P < 0.05) than

278

that with added rCaAFP; For hydrated glutenin after freezing, the freezable water content of 13



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hydrated glutenin with added rTmAFP was significantly lower (P < 0.05) than that with added

280

rCaAFP.

281

However, no significant difference was observed in the freezable water content of gliadin-control-

282

A and gliadin-control-B, indicating the freezable water content of hydrated gliadin was not

283

susceptible to freezing. This was probably because gliadin reportedly possessed the strongest

284

water-binding capacity followed by gluten and glutenin.6 Furthermore, the freezable water

285

content of hydrated gliadins with and without added rAFPs were also not significantly different

286

(Figure 4c).

287

Effects of rAFPs on Water Mobility of Hydrated Gluten Proteins. LF-NMR is a powerful tool

288

to study water status and mobility of hydrated gluten proteins according to T2 information.47 In

289

order to investigate the effects of rAFPs on the water mobility of hydrated gluten proteins during

290

freezing, T2 relaxations of hydrated gluten, glutenin, and gliadin before or after freezing and with

291

or without added rAFPs were analyzed by LF-NMR.

292

Figure S2 (Supporting Information) shows typical T2 relaxation time distribution curves of

293

hydrated gluten, glutenin, and gliadin without added rAFPs. T2 relaxation time distribution of

294

hydrated gluten showed three proton populations with different T2 values: T21 (0.02-1.32 ms), T22

295

(10.72-86.97 ms), and T23 (114.98-305.39 ms), which were assigned to water populations of

296

different mobilities. The three proton populations of hydrated glutenin appeared at T21 (0.03-2.31

297

ms), T22 (2.66-86.97 ms), and T23 (100-200.92 ms), respectively, and the three proton populations

298

of hydrated gliadin appeared at T21 (0.02-1.32 ms), T22 (7.05-57.22 ms), and T23 (151.99-265.61

299

ms), respectively. Different T2 values, including T21, T22, and T23, reflect different water status in

300

hydrated gluten proteins, and the lower T2 values represent the water molecules with lower 14


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mobility, whereas the higher T2 values indicate the water molecules are more mobile.35 Water

302

mobility is critical for the interaction of gluten and water to form a viscoelastic network.3

303

The integrated area under each peak represents the total number of protons,48 and the peak area

304

proportions of T21, T22, and T23 are summarized in Figure 5. For hydrated gluten and glutenin

305

without added rAFPs, no significant difference was found in T21 peak proportion before and after

306

freezing, whereas after freezing the distinct decrease in T22 peak proportion and the increase in

307

T23 peak proportion were observed, suggesting the water in hydrated gluten and glutenin became

308

more mobile because of freezing. The increase in water mobility of hydrated gluten and glutenin

309

may arise because more hydrophobic moieties were exposed during freezing, resulting in a

310

weakened association between protein and water, i.e. a lower water-binding capacity.6

311

Before and after freezing, no significant differences in T22 peak proportion were found in

312

hydrated gluten with added rTmAFP or rFiAFP, indicating both rTmAFP and rFiAFP could

313

weaken the influence of freezing on the water mobility of hydrated gluten. For hydrated glutenin,

314

only the T22 peak proportion of glutenin-rTmAFP-A and glutenin-rTmAFP-B were not

315

significantly different, indicating the addition of rTmAFP to glutenin reduced the increase in T22

316

peak proportion and consequently restricted water mobility. The rCaAFP had little influence on

317

water mobility of hydrated gluten and glutenin during freezing.

318

For hydrated gliadin before or after freezing, and with or without added rAFPs, no remarkable

319

changes in T21 and T22 peak proportions were observed, which was in good agreement with the

320

results of freezable water content in hydrated gliadin (Figure 4c), further demonstrating gliadin

321

exhibited the highest water-binding capacity.

15



Page 16 of 41

322

Effects of rAFPs on Water Distribution of Hydrated Gluten Proteins. Proton weighted

323

imaging could monitor the spatial distribution of water. The 2D proton density images from

324

transverse sections of hydrated gluten proteins before or after freezing, and with or without added

325

rAFPs, are presented in Figure 6.

326

Before and after freezing, the water distribution of both hydrated gluten and glutenin without

327

added rAFPs were obviously different. The red signal densities of gluten-control-B and glutenin-

328

control-B were well distributed, suggesting that the water distribution of both hydrated gluten and

329

glutenin before freezing were homogeneous on the transverse sections; however, the red signal

330

densities of gluten-control-A and glutenin-control-A were not evenly distributed, which means

331

water was clustered in certain areas, further demonstrating that after freezing water migrated from

332

parts of the transverse section to other areas. This may arise because the vapor pressure difference

333

between samples and the freezer, and changes in the water-binding capacity of gluten and

334

glutenin during freezing drove the water migration and redistribution.

335

Notably, after freezing, the water distributions of hydrated gluten in the presence of rAFPs were

336

more even than gluten-control-A and gluten-BSA-A. Especially the water distribution of gluten-

337

rTmAFP-A was more even than gluten-rFiAFP-A and gluten-rCaAFP-A. The similar trend was

338

observed in hydrated glutenin. These findings imply that rTmAFP restricted the water migration

339

and redistribution of hydrated gluten and glutenin best, followed by rFiAFP and rCaAFP.

340

The proton densities of gliadin-control-B and gliadin-control-A were not obviously different, and

341

the addition of rAFPs did not affect the proton densities, indicating the water distribution of

342

hydrated gliadin before or after freezing, and with or without added rAFPs did not obviously

16


Page 17 of 41


343

change, which was in agreement with the results of freezable water content and water mobility of

344

hydrated gliadin.

345

Mechanistically, the nucleation and growth of ice crystals are correlated with the mobility and

346

distribution of freezable water in hydrated gluten.10 Therefore, based on the abilities of rAFPs to

347

restrict water mobility and redistribution, the addition of rAFPs may change the normal growth

348

pattern of ice crystals, and minimize the damage of freezing to hydrated gluten and glutenin. The

349

results of Figure 5 and 6 demonstrate that rTmAFP was most effective in restricting water

350

mobility and redistribution of hydrated gluten and glutenin.

351

Effects of rAFPs on Rheological Properties of Hydrated Gluten Proteins. Gluten is of great

352

importance in maintaining viscoelastic properties of dough. Generally, glutenins impart strength

353

and elasticity whilst gliadins confer viscous properties.49 Figure 7 presents rheological modulus

354

changes vs frequency for hydrated gluten proteins. G′ and G″ describe the character elastic and

355

viscous state of samples, respectively.39

356

As shown in Figure 7a and b, the values of G′ and G″ of hydrated gluten without added rAFPs

357

significantly decreased after freezing, indicating the rheological properties of hydrated gluten

358

decreased because of freezing. After freezing, compared with gluten-control-A, the addition of

359

the three rAFPs all significantly enhanced the values of G′ and G″. The values of G′ and G″ of

360

gluten-rTmAFP-A were the highest, followed by gluten-rFiAFP-A and gluten-rCaAFP-A, but still

361

lower than that of the samples before freezing. The similar trends were observed in hydrated

362

glutenin (Figure 7c and d). Rheological changes of hydrated gluten and glutenin occurred during

363

freezing was mainly due to the mechanical damage of ice crystals formation to hydrated gluten

364

and glutenin,34 as found by Kontogiorgos et al.33 and Meziani et al.11 The addition of rAFPs 17



Page 18 of 41

365

resulted in a noticeable deceleration of the adverse effect of ice crystals on the functionality of

366

hydrated gluten and glutenin. In addition, water content plays a major role in dough rheology and

367

changes in water dynamics could also affect the mechanical properties;50 thus, the increase in

368

freezable water content, water mobility, and water redistribution of hydrated gluten and glutenin

369

after freezing also led to the deterioration of their rheological properties. The addition of rAFPs

370

lessened the increment of freezable water content, restricted water mobility and redistribution,

371

thereby protecting the rheological properties of hydrated gluten and glutenin from freezing. In

372

addition, rTmAFP was most effective in protecting the rheological properties of hydrated gluten

373

and glutenin during freezing.

374

As shown in Figure 7e and f, no significant changes were observed in the values of G′ and G″ of

375

hydrated gliadin before or after freezing and with or without added rAFPs, suggesting that the

376

viscoelastic properties of hydrated gliadin after freezing were not significantly altered, and that

377

the addition of rAFPs did not affect the viscoelastic properties of hydrated gliadin. The reasons

378

for this finding may be related to the strong water-binding capacity of gliadin, and agreed well

379

with the results of freezable water content, water mobility and distribution of hydrated gliadin

380

during freezing. However, these findings on the other hand demonstrate that the cryoprotective

381

effects of rAFPs on the freezable water content, water mobility and distribution, and rheological

382

properties of hydrated gluten were achieved by protecting these corresponding properties of

383

hydrated glutenin.

384

Microstructure of Hydrated Gluten Proteins Networks. The conformational integrity of gluten

385

network plays a key role in the baking quality of dough. The disruption of gluten network

386

integrity results in the loss of gas retention, poor loaf volume and strong alteration in the textural 18


Page 19 of 41


387

properties of the baked products.6,10 Therefore, protecting gluten network is essential for the

388

improvement of freezing process. The effects of three rAFPs on the microstructure of hydrated

389

gluten proteins networks during freezing were evaluated (Figure 8). The voids formed by

390

sublimation can reflect the shape and size of ice crystals in hydrated gluten proteins.51

391

The networks of both gluten-control-A and gluten-BSA-A were less continuous and disrupted,

392

and the angular voids were less uniform with some very large voids presented. The mechanical

393

damage from ice crystallization during freezing process could cause less continuous and disrupted

394

gluten network.27 Compared with gluten-control-A, the networks of gluten-rCaAFP-A, gluten-

395

rFiAFP-A, and gluten-rTmAFP-A were more continuous and less disrupted with more uniform

396

voids; moreover, the voids of gluten-rTmAFP-A network were most uniform and smallest,

397

followed by gluten-rFiAFP-A and gluten-rCaAFP-A. The same trends were noticed in glutenin

398

and gliadin. These observations confirmed the ability of these rAFPs to modify ice crystal size, as

399

reported by Kong et al.52 For hydrated gluten, glutenin, and gliadin, rTmAFP was most effective

400

in controlling ice crystals size, followed by rFiAFP and rCaAFP. Final thawed products quality

401

was closely related to the ice crystals size formed during freezing.53 These three rAFPs changed

402

the normal growth pattern of ice crystals, considerably controlled ice crystals size and

403

morphology, contributing to the protection of gluten proteins networks during freezing.

404

In conclusion, this study clearly demonstrates the utility of rAFPs as potent agents in preservation

405

of the functional quality of hydrated gluten proteins during freezing process. The addition of the

406

three rAFPs caused freezing hysteresis, controlled ice crystals size, and weakened the damage of

407

freezing to the networks of hydrated gluten, glutenin, and gliadin. During freezing, the

408

cryoprotective effects of rAFPs on the freezable water content, water mobility and distribution, 19



Page 20 of 41

409

and rheological properties of hydrated gluten were achieved by protecting these corresponding

410

properties of hydrated glutenin. No significant changes were observed in the freezable water

411

content, water mobility and distribution, and rheological properties of hydrated gliadins before or

412

after freezing and with or without rAFPs. Among the three rAFPs, rTmAFP was most effective in

413

the cryoprotective activities on hydrated gluten proteins during freezing. Therefore, the

414

information presented may provide an alternative approach to protect hydrated gluten proteins

415

from freezing injuries with rTmAFP addition, and open the possibility of exploring the potential

416

of rTmAFP in the protection of cells, tissues, and organs from freezing injuries. Meanwhile, this

417

study gains new insights into the cryoprotective effects of rAFPs on frozen dough properties

418

during freezing from the perspectives of gluten and its components glutenin and gliadin, and

419

provides a theoretical basis for the cryopreservation of frozen dough by rAFPs. Additionally, the

420

biosynthesis of rAFPs will contribute to addressing the cost issue to realize their potential in

421

cryopreservation and accelerating further application studies.

422

Abbreviations Used

423

AFPs, antifreeze proteins; rAFPs, recombinant antifreeze proteins; rCaAFP, recombinant carrot

424

antifreeze protein; rFiAFP, recombinant fish antifreeze protein; rTmAFP, recombinant Tenebrio

425

molitor antifreeze protein; TH, thermal hysteresis; IRI, ice recrystallization inhibition; DSC,

426

differential scanning calorimetry; LF-NMR, low field nuclear magnetic resonance; T2, transverse

427

relaxation time; G′, elastic modulus; G″, viscous modulus; SEM, scanning electron microscopy;

428

Tf,o, onset temperature of freezing; Tf,p, peak temperature of freezing; Tm,o, onset temperature of

429

melting; Tm,p, peak temperature of melting; Tm,e, complete melting temperature; Tm,δ, temperature

430

range of melting process; BSA, bovine serum albumin; gluten/glutenin/gliadin-control-B, 20


Page 21 of 41


431

gluten/glutenin/gliadin without added BSA or rAFPs before freezing; gluten/glutenin/gliadin-

432

BSA-B, gluten/glutenin/gliadin with added BSA before freezing; gluten/glutenin/gliadin-rCaAFP-

433

B, gluten/glutenin/gliadin with added rCaAFP before freezing; gluten/glutenin/gliadin-rFiAFP-B,

434

gluten/glutenin/gliadin with added rFiAFP before freezing; gluten/glutenin/gliadin-rTmAFP-B,

435

gluten/glutenin/gliadin with added rTmAFP before freezing; gluten/glutenin/gliadin-control-A,

436

gluten/glutenin/gliadin without added BSA or rAFPs after freezing; gluten/glutenin/gliadin-BSA-

437

A, gluten/glutenin/gliadin with added BSA after freezing; gluten/glutenin/gliadin-rCaAFP-A,

438

gluten/glutenin/gliadin with added rCaAFP after freezing; gluten/glutenin/gliadin-rFiAFP-A,

439

gluten/glutenin/gliadin with added rFiAFP after freezing; gluten/glutenin/gliadin-rTmAFP-A,

440

gluten/glutenin/gliadin with added rTmAFP after freezing.

441

Funding

442

This work was supported by National Natural Science Foundation of China [grant numbers

443

31671891, 31471617, and 31471679]; National Key Research and Development Plan Program

444

[grant number 2016YFD0401204]; and National High Technology Research and Development

445

Program 863 [grant number 2013AA102203-7].

446

Notes

447

The authors declare no competing financial interest.

448

Supporting Information

449

Figure S1. Typical freezing and melting curves of gluten-control-B, glutenin-control-B and

450

gliadin-control-B samples. Figure S2. Typical T2 relaxation time distribution curves of hydrated

451

gluten, glutenin, and gliadin without added BSA or rAFPs.

21



Page 22 of 41

452

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Figure captions Figure 1. (a) Schematic representation of a hexagonal ice crystal without added AFPs: basal plane, prism planes, and the c- and three a-axes (a1, a2, a3); (b) Schematic representation of a circular disk-like hexagonal ice crystal with added hyperactive AFPs; (c) Schematic representation of a classical hexagonal bipyramidal ice crystal with added moderately active AFPs. Redrawn on the basis of several similar figures published in the literature.17-20 Figure 2. TH activities of (a) rCaAFP, (b) rFiAFP, and (c) rTmAFP. Figure 3. Ice crystals morphology of (a) control, (b) rCaAFP, (c) rFiAFP, and (d) rTmAFP solutions. Figure 4. Changes in the freezable water content of hydrated (a) gluten, (b) glutenin, and (c) gliadin before or after freezing and with or without added rAFPs. Figure 5. Changes in T2 peak area proportions of hydrated gluten, glutenin, and gliadin before or after freezing and with or without added rAFPs. (a) T21 peak area proportion of hydrated gluten; (b) T22 peak area proportion of hydrated gluten; (c) T23 peak area proportion of hydrated gluten; (d) T21 peak area proportion of hydrated glutenin; (e) T22 peak area proportion of hydrated glutenin; (f) T23 peak area proportion of hydrated glutenin; (g) T21 peak area proportion of hydrated gliadin; (h) T22 peak area proportion of hydrated gliadin; (i) T23 peak area proportion of hydrated gliadin. Figure 6. Changes in water distribution of hydrated gluten, glutenin, and gliadin before or after freezing and with or without added rAFPs. Colors from blue through bright yellow to red represent increasing moisture content in the sample. 29



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Figure 7. Frequency sweeps of G′ and G″ for hydrated gluten, glutenin, and gliadin before or after freezing and with or without added rAFPs. (a) G′ of hydrated gluten; (b) G″ of hydrated gluten; (c) G′ of hydrated glutenin; (d) G″ of hydrated glutenin; (e) G′ of hydrated gliadin; (f) G″ of hydrated gliadin. Figure 8. SEM observations of hydrated gluten, glutenin, and gliadin after freezing. (a) glutencontrol-A; (b) gluten-BSA-A; (c) gluten-rCaAFP-A; (d) gluten-rFiAFP-A; (e) gluten-rTmAFP-A; (f) glutenin-control-A; (g) glutenin-BSA-A; (h) glutenin-rCaAFP-A; (i) glutenin-rFiAFP-A; (j) glutenin-rTmAFP-A; (k) gliadin-control-A; (l) gliadin-BSA-A; (m) gliadin-rCaAFP-A; (n) gliadin-rFiAFP-A; (o) gliadin-rTmAFP-A.

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Table 1. Freezing Temperature and Melting Temperature of Fresh Hydrated Gluten, Glutenin, and Gliadin with or without Added rAFPsa sample

Tf,o (°C)

Tf,p (°C)

Tm,o (°C)

Tm,p (°C)

Tm,δ (°C)

gluten-control-B

-6.22 ±0.07a

-4.93 ±0.19a

-0.63 ±0.02b

1.70 ±0.15a 9.54 ±0.26b

gluten-BSA-B

-6.26 ±0.05a

-4.97 ±0.08a

-0.62 ±0.01b

1.66 ±0.18a 9.66 ±0.27b

gluten-rCaAFP-B

-8.72 ±0.09b

-8.45 ±0.14b

-0.42 ±0.05a

1.63 ±0.08a 11.92 ±0.13a

gluten-rFiAFP-B

-9.98 ±0.15c

-7.41 ±0.11c

-0.47 ±0.04a

1.69 ±0.13a 12.09 ±0.28a

gluten-rTmAFP-B

-10.89 ±0.12d

-10.89 ±0.03d

-0.49 ±0.03a

1.64 ±0.21a 11.84 ±0.18a

glutenin-control-B

-6.79 ±0.27a

-5.20 ±0.09a

-0.68 ±0.04b

2.43 ±0.16a 8.92 ±0.15b

glutenin-BSA-B

-6.81 ±0.15a

-5.23 ±0.05a

-0.64 ±0.05b

2.39 ±0.09a 8.99 ±0.12b

glutenin-rCaAFP-B -7.92 ±0.22b

-5.67 ±0.18b

-0.44 ±0.07a

2.28 ±0.07a 10.97 ±0.29a

glutenin-rFiAFP-B

-9.84 ±0.15c

-9.81 ±0.11c

-0.49 ±0.03a

2.37 ±0.13a 10.87 ±0.17a

glutenin-rTmAFP-

-10.97 ±0.19d

-11.01 ±0.26d

-0.51 ±0.03a

2.36 ±0.22a 10.95 ±0.23a

gliadin-control-B

-18.47 ±0.28a

-18.78 ±0.22a

-3.12 ±0.08b

-0.02±0.14a

7.66 ±0.19b

gliadin-BSA-B

-18.49 ±0.13a

-18.83 ±0.15a

-3.13 ±0.05b

-0.09±0.12a

7.83 ±0.11b

gliadin-rCaAFP-B

-19.62 ±0.25b

-19.01 ±0.17a

-2.84 ±0.12a

-0.17±0.22a

10.38 ±0.12a

gliadin-rFiAFP-B

-21.91 ±0.31c

-22.73 ±0.25c

-2.88 ±0.07a

0.19 ±0.13a 10.26 ±0.23a

gliadin-rTmAFP-B

-23.56 ±0.19d

-22.15 ±0.13b

-2.85 ±0.09a

0.03 ±0.25a 10.49 ±0.27a

B

a

Data are expressed as mean ±standard deviation (n = 3). Values with different lowercase letters

in the same column for hydrated gluten/glutenin/gliadin are significantly different (P < 0.05). Tf,o and Tf,p were taken as the onset temperature and peak temperature of freezing, respectively. Tm,o 31



and Tm,p refered to the onset temperature and peak temperature of melting, respectively. Tm,δ refered to the temperature range of melting process.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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For Table of Contents Only

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