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Hydration, water distribution and microstructure of gluten during freeze thaw process: Role of a high molecular weight dextran produced by Weissella confusa QS813
Accepted Manuscript Hydration, water distribution and microstructure of gluten during freeze thaw process: Role of a high molecular weight dextran produced by Weissella confusa QS813 Xiaojuan Tang, Binle Zhang, Weining Huang, Zilin Ma, Fengwen Zhang, Feng Wang, Qibo Zou, Jianxian Zheng PII:
S0268-005X(18)30895-6
DOI:
10.1016/j.foodhyd.2018.10.025
Reference:
FOOHYD 4706
To appear in:
Food Hydrocolloids
Received Date: 16 May 2018 Revised Date:
4 September 2018
Accepted Date: 16 October 2018
Please cite this article as: Tang, X., Zhang, B., Huang, W., Ma, Z., Zhang, F., Wang, F., Zou, Q., Zheng, J., Hydration, water distribution and microstructure of gluten during freeze thaw process: Role of a high molecular weight dextran produced by Weissella confusa QS813, Food Hydrocolloids (2018), doi: https://doi.org/10.1016/j.foodhyd.2018.10.025. 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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Hydration, water distribution and microstructure of gluten during freeze thaw process: role of a high molecular weight dextran produced by Weissella confusa QS813
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Xiaojuan Tang1, Binle Zhang1, Weining Huang1*, Zilin Ma1, Fengwen Zhang1, Feng Wang2, Qibo Zou2, Jianxian Zheng3
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1 State Key Laboratory of Food Science and Technology, Laboratory of Baking Science, Sourdough and Ingredient Functionality Research, Jiangnan University, Wuxi 214122, China;
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2 MagiBake International Inc., Wuxi, Jiangsu 214131, China;
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3 Institute of Food and Bioengineering, South China University of Technology, Guangzhou, Guangdong 510640, China
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*Corresponding author. Tel.: +86(510)8591 9139; fax: +86(510)8591 9139.
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E-mail address: [email protected] (W. Huang).
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Abstract
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The effect of exopolysaccharide (EPS) produced by Weissella confusa QS813 on the hydration, water distribution,
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rheology and microstructure of wheat gluten during freeze–thaw cycles (FTC) was investigated. Addition of EPS
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increased the water content of fresh gluten and delayed the dehydration of gluten during FTC. Low field-nuclear
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magnetic resonance showed that the presence of EPS reduced the mobility of both confined and bulk water in the
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fresh gluten matrix. Proton distribution changes in gluten during FTC indicated that deterioration of the gluten
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network was attributable to ice recrystallization of capillary confined water and bulk water. The presence of EPS
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effectively reduced the release of bulk water and retarded the redistribution of confined water induced by FTC.
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Dynamic oscillatory studies indicated that the presence of EPS induced a softening effect on the fresh gluten. FTC
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significantly decreased the rheological parameters, (storage modulus (G′) and loss modulus (G″), of all gluten
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samples. Gluten with higher concentrations of EPS (above 0.5%) exhibited a delayed decrease in G′ and G″ and
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maintained a constant tanδ value after FTC. Laser scanning confocal microscope and scanning electron
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microscope revealed that EPS maintained the structural integrity of gluten during FTC, suggesting an inhibitory
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effect on the recrystallization of ice crystals. These results in the present study indicated that the bacterial dextran
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is a promising cryoprotectant for the frozen dough industry.
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Keywords: Exopolysaccharide, Wheat gluten, Freeze–thaw cycles, Hydration, Water distribution, Microstructure
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1. Introduction
Frozen dough technology has been widely used in bakery goods to facilitate centralized manufacturing and
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distribution processes as well as the standardization of product quality (Selomulyo & Zhou, 2007). Nevertheless,
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the final quality of frozen dough is often negatively affected by disruption of the gluten network and loss of yeast
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viability owing to ice formation and temperature fluctuation-driven ice recrystallization (Hsu, Hoseney, & Seib,
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1979; Phimolsiripol, Siripatrawan, Tulyathan, & Cleland, 2008; Wang, Jin, & Xu, 2015). To retard frozen dough
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quality deterioration, addition of functional agents to the matrix aimed at improving the physico-chemical and
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thermo-mechanical properties of frozen dough has been reported. Additives such as ice structuring proteins,
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enzymes, emulsifiers and hydrocolloids have been used in frozen dough to regulate the hydration properties of
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gluten, thereby further inhibiting ice recrystallization (Rosell & Gomez, 2007; Rosell, Rojas, & De Barber, 2001;
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Zhang, Zhang, & Wang, 2007).
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The cryoprotective effects of hydrocolloids in the frozen dough bakery industry have been demonstrated by
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several researchers, and recently reviewed by Maity, Saxena and Raju (2016). Hydrocolloids have a high
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water-retention capacity, which confers stability to products undergoing successive freeze–thaw cycles (FTC)
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(Bárcenas, Benedito, & Rosell, 2004). Currently, most hydrocolloids used in frozen dough are derived from
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seaweeds (e.g. guar gum and carrageenan), plants (e.g. cellulose, pectin, and locust bean gum), and bacteria (e.g.,
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xanthan). Microbial exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) during sourdough
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fermentation, especially those with high molecular weight, have the potential to replace commercial hydrocolloids
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in baking applications (Galle & Arendt, 2014). However, although LAB EPS is a novel food ingredient in bakery
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applications, its functionality as a bread ingredient has been less frequently explored (Zannini, Waters, & Arendt,
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2014).
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In our earlier study, a high-molecular-mass, low-branched dextran with 97% α-(1, 6) linkages was produced
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by LAB Weissella confusa QS813, isolated from Chinese traditionally fermented sourdough. The dextran
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produced by W. confusa QS813 significantly improved the freeze–thaw stability of wheat starch gel (Tang et al.,
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2018). Although dextran has been used as a cryoprotectant in frozen dairy products (Mccurdy, Goff, & Stanley,
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1994), the use of dextran in the field of frozen dough has been minimally explored. Tieking and Ganzle (2005)
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suggested the beneficial influence of EPS on dough stability during frozen storage. Therefore, studies on the effect
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of EPS addition on the properties of wheat gluten in frozen dough and the related mode of action are necessary.
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Therefore, the aim of this study was to characterize the effect of LAB EPS on the hydration, polymerization, and
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microstructure of wheat gluten during the freeze–thaw process.
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2. Materials and methods
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2.1 Isolation and purification of EPS W. confusa QS813 was obtained from the culture collection of the Laboratory of Baking Science, Sourdough
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and Ingredient Functionality Research, Jiangnan University. The strain was cultivated at 37 °C for 24 h in de Man,
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Rogosa, and Sharpe (MRS) medium and sub-cultured at least twice until a cell concentration of 109 CFU/mL was
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reached prior to experimental use. The production of EPS by W. confusa QS813 was performed in modified MRS
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(mMRS) medium (50 g/L sucrose instead of 20 g/L glucose). One hundred milliliters of mMRS medium was
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inoculated with 2% (v/v) culture (2 × 107 CFU/mL) and incubated without shaking at 20 ℃ for 24 h. Cells were
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removed by centrifugation at 12,000×g for 50 min at 4 °C. A final concentration of 4% (w/v) trichloroacetic acid
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(TCA) was used for protein precipitation and incubated with the cells at 4 ℃ overnight. Proteins were removed by
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centrifugation at 12,000×g for 30 min at 4 °C. Three volumes of 95% (v/v) cold ethyl alcohol was then added to
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the supernatant, followed by centrifugation at 12,000×g for 30 min at 4 ℃. The precipitate was re-suspended in
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ultrapure water and dialyzed for 48 h at 4 °C (8,000–12,000 Da) with water changes every 8 h. Finally, the
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dialyzed EPS preparations were freeze dried and stored at -18 °C.
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2.2 Preparation of fresh and frozen–thawed gluten–EPS mixtures
Purified EPS (0.1, 0.5, 1.0, and 1.5 g) was sprinkled into 100 mL deionized water and continuously stirred
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for complete dissolution. Five grams of wheat gluten (81.20% protein, 7.51% water, and 0.71% fat; Sigma–
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Aldrich Chemical Co., USA) was then dispersed in the EPS solution and stirred for 1 h at room temperature. The
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resulting mixture was centrifuged for 20 min at 4,000×g. The supernatant was discarded and the pellet obtained
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was fresh gluten. Afterwards, the gluten pellet was sealed in a polyethylene bag (50 × 70 mm). For freeze–thaw
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processing, the gluten was frozen at -20 °C for 22 h and then thawed at 30 °C for 2 h in a water bath. Five FTC
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were conducted.
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2.3 Water content of fresh and frozen–thawed gluten
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To determine the water content of the fresh and frozen–thawed prepared gluten, samples were removed from
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the hermetically sealed polyethylene bag. The water content of the gluten was determined according to AACC
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(2000).
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2.4 Low-field nuclear magnetic resonance (LF-NMR) measurements of gluten–EPS mixtures
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Determination of the proton distribution in fresh and frozen–thawed gluten was performed with an NMR
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spectrometer (MesoMR23-060 V-I; Niumag Electric Corporation, China) at a resonance frequency of 23 MHz.
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The fresh and frozen–thawed gluten samples were prepared as described in 2.2. Before LF-NMR measurements,
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gluten samples kept a the sealed polyethylene bag were thawed for 2 h in a water bath at 30°C, which was also the
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temperature of the magnet. The polyethylene bag containing the gluten pellet was transferred into the NMR probe
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(25 mm external diameter). Spin−spin (T2) relaxation time was acquired using a Carr–Purcell–Meiboom–Gill
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(CPMG) pulse sequence. The pulse parameters were as follows: TR=2,000 ms, TE=0.250ms, NECH=10,000,
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SW=100 KHz, PRG=2, NS=16. The SIRT algorithm (NMR analysis software provided by Niumag Electric
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Corporation) was used to invert the collected signals to the T2 relaxation time plots. The integrated area under the
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curves of the populations with certain T2 represents the number of protons.
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2.5 Rheology of fresh and frozen–thawed gluten
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Dynamic rheology of the gluten was determined according to the method described by Bárcenas, De la
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O-Keller, and Rosell (2009) with modifications. Fresh and frozen–thawed gluten samples were subjected to an
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oscillatory frequency sweep test using a controlled-stress rheometer (DHR-3, TA Instruments, USA) with
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parallel-plate geometry (20 mm diameter, 1 mm gap). The gluten was equilibrated for 15 min before measurement.
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Frequency sweep (0.1–10 Hz) experiments were performed with a strain of 1% at 30 °C. The storage modulus
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(G′), loss modulus (G″), and tanδ were recorded. Measurements were performed in triplicate.
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2.6 Confocal laser scanning microscopy (CLSM)
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Fresh and frozen–thawed gluten–EPS mixture samples were prepared as described in 2.2, cut to roughly 5 ×
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5 × 5 mm with a sharp blade, embedded in tissue freezing medium (Leica Biosystems, USA) and maintained at
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-20 ℃ for 30 min. Fifteen-micrometer sections were cut with a cryostat microtome (CM1950, Leica Biosystems,
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Germany) at -20℃, and fragment slice were discarded to avoid misinterpretation of the structural changes. Protein
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was labeled with rhodamine B (1.3 × 105 g/mL) for 2 min, followed by washing three times with deionized water.
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Macro photos of the frozen sections were acquired with a microscope (ZEISS Axio vert. A1, Germany) equipped
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with a computer. Images were recorded at 200× magnification.
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2.7 Scanning electron microscopy (SEM)
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The microstructures of fresh and frozen–thawed gluten was observed with a QUANTA-200 SEM (FEI,
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Eindhoven, The Netherlands). Before SEM observation, samples were pretreated according to Li et al. (2010). The
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gluten was fixed with 2.5% glutaraldehyde and post-fixed with 1% osmiumtetroxide. Samples were dehydrated
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with ethanol of concentrations ranging from 30% to 100%, followed by displacement with isoamyl acetate. The
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final dehydrated samples were obtained by the critical point drying method. For SEM observation, samples were
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coated with gold. Micrographs were acquired at 1,200× magnification.
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2.8 Data analysis
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The results are presented as average values with a confidence interval. Statistical analyses of all data were
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performed with SPSS, version 7.1 using one-way analysis of variance (ANOVA). The means were compared by
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Duncan’s multiple range test. A level of 0.05 probability was set to determine statistical significance. All tests
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were performed in triplicate.
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3. Results and discussion
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3.1 Effect of EPS on water content of gluten during FTC
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In the present study, gluten powder was dispersed homogeneously in excessive water and then subjected to
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centrifugation. As a result of hydration and mechanical energy input, the gluten network was formed by the
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interaction between gliadins and glutenins through numerous hydrogen and disulfide bonds (Frederic,
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Mariehelene, Jacques, Muriel, & Andreas. 2008). The effect of EPS on the water content of gluten pellet during
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FTC is shown in Table 1. The presence of small amounts of EPS (0.1%) did not impact the water–binding
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capacity of gluten, suggesting the weak influence of low concentrations of EPS on the hydration properties of
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gluten. However, a steady increase in water content of the fresh wheat gluten was achieved upon addition of
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increasing amounts of EPS, indicating the higher binding capacity of gluten with added EPS. This is in agreement
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with the observations in previous studies after the addition of hydroxypropyl methylcellulose (HPMC) to gluten
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(Bárcenas et al., 2009; Rosell & Foegeding, 2007). In the presence of sufficient water and adequate hydration, the
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water content was not a limiting factor for determining the effect of the hydrocolloids (Rosell & Foegeding, 2007).
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The molecular structure of the EPS produced by W. confusa QS813 was determined in our previous study, and
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described as a high-molecular-mass, low-branched dextran with 97% α-(1,6) linkages (Tang et al., 2018). As a
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highly linear polysaccharide with very flexible random coil conformations in solution, the EPS could be highly
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capable of forming hydrogen bonds and steric interactions with proteins; this might be responsible for the
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increased water binding ability of gluten with added EPS (Ross, McMaster, Tomlinson, & Cheetham, 1992;
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Ribotta, Ausar, Beltramo, & León, 2005).
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The water content of gluten decreased in all samples as the number of FTC increased (Table 1). After the
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fifth FTC treatment, the water content of control gluten decreased from 62.13% to 59.48, representing a decrease
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of 4.27%. However, the presence of EPS at a concentration above 0.5% significantly reduced the water loss of
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gluten after repeated FTC treatment. This result might be ascribed to the ability of the high molecular weight
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linear dextran to trap water. According to Phimolsiripol et al. (2008), moisture loss in frozen dough is due to
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transfer of water/ice in the frozen matrix to frost inside the polyethylene bag. Larger temperature fluctuations will
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induce a higher water loss in frozen dough. Repeated freezing and thawing diminishes the total number of crystals
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and increases the mean crystal size, thus further damaging the gluten network and increasing the release of bound
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water from the dough (Ding et al., 2015; Xu, Huang, Jia, Kim, & Liu, 2009). In this study, during the FTC,
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addition of EPS to gluten tended to improve moisture retention, inhibit ice crystal formation and subsequently
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reduce the freeze damage to gluten. Therefore, the water content of the gluten with added EPS decreased at a
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slower pace.
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3.2 Effect of EPS on water mobility of gluten during FTC
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The interaction of biopolymers with water reduces water mobility and results in different molecular motilities
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in the gluten system (Bosmans et al., 2012). In the present study, the molecular mobility of water in fresh and
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frozen–thawed gluten was studied with LF-NMR and the relaxation time distributions are shown in Figure 1. For
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the fresh gluten, two CPMG populations were deduced from the relaxation signals (Fig. 1a). The first one, likely
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assigned to protons that are strongly associated with the gluten matrix (amino acids and low- mobility water
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molecules) (Kontogiorgos, Goff & Kasapis, 2007), represents population A with relaxation time (T2) less than
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0.66 ms. Similar results were reported by Kontogiorgos, Goff and Kasapis (2007), Peters et al. (2016) and Liu et
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al. (2018), who found only one CPMG population with a T2 less than 10 ms in hydrated gluten with a moisture
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content above 60%. However, two CPMG populations with a T2 less than 10ms were distinguished in hydrated
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gluten with the similar moisture content (Wang et al., 2014; Xuan et al., 2017). The difference in the distribution
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pattern of T2 in gluten may be explained by the use of different sampling methods, NMR instruments, test
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conditions, and inversion algorithms. The second population B was the predominant proton population in the
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CPMG spectrum, largely consisting of mobile exchanging protons, and had a higher mobility with a T2 range of
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28.48–114.98 ms. The porous structure of the gluten network has been conceptualized as a sheet–like structure
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(Kontogiorgos & Goff, 2006; Kontogiorgos, 2011). According to the assignments of CPMG proton populations of
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the gluten–water model system based on the sheet model by Bosmans et al. (2012), population A contained
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protons of gluten in the sheets in contact with confined and bulk water, whereas population B was assigned to bulk
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water surrounding the sheet, which can exchange with gluten protons on the outside of the sheet.
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As shown in Fig. 1a and Table 2, addition of different concentrations of EPS changed the NMR profile of
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fresh gluten. T2 of population A and B decreased with increasing amounts of EPS. However, no significant change
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in the area of populations was observed. The differences showed that EPS addition to the fresh gluten resulted in
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the presence of more rigid protons. Their reduced mobility was caused by more intense contact between water and
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gluten. As a type of unfolded macromolecules that can occupy a large volume, EPS produced by W. confusa
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QS813 might have limited the mobility in the solutions, thereby inducing decreased mobility of bulk water in the
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parallel sheet space of gluten (population B). The decreased mobility of population A might have resulted from
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arrangement changes in gluten sheets induced by EPS addition, an outcome that may reflect enhanced associations
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between the proteinaceous macromolecules. Non-ionic polysaccharide is reported to interact with gluten proteins
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through hydrogen bonding and steric interactions (Ribotta et al., 2005; Rosell & Foegeding, 2007; Ross et al.,
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1992). Based on the change in population A, we reasonably suggest that the final conformation of gluten was
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modified by the present EPS. This was also consistent with the results of Linlaud, Ferrer, Cecilia Puppo, and
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Ferrero (2011), who found that the final conformation of the combined gluten–hydrocolloid–water matrix
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determines the degree of water binding.
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FTC changed the NMR profile of the gluten (Fig. 1b, 1c and Table 2), indicating a redistribution of water in
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the gluten mix. After one FTC, a new population C appeared at high T2 in the CPMG spectrum of all gluten
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samples. With the increasing number of FTC, the area of population C increased, whereas the area of population B
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decreased, indicating the release of bulk water from the gluten network. However, addition of EPS delayed the
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transformation from population B to C. The frozen gluten network is considered a porous matrix in which the
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pores are filled with confined water and surrounded by bulk ice after hydration (Kontogiorgos & Goff, 2006).
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During freezing, bulk water in the space of the parallel sheet transforms to ice through crystallization.
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Temperature fluctuation drives ice recrystallization and bulk water release due to the partial disruption of the
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gluten network (Wang et al., 2015). In the present study, addition of EPS effectively inhibited bulk water release
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from the gluten matrix and controlled recrystallization of hydrated gluten during FTC. This was in line with
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previous findings regarding water content changes during FTC. Population A with a low T2 value was also
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sensitive to FTC. After one FTC, a new distinct proton population A1 at a lower T2 value was distinguished in
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control gluten without EPS. As shown in Table 2, areas of both population A and A1 in all experimental groups
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significantly increased after five FTC. At the same time, FTC increased the mobility of population A and
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decreased the mobility of population A1, indicating a more heterogeneous arrangement of gluten structure. During
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the FTC, more hydrophobic binding sites were exposed, resulting in a weakened association between protein and
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water (Wang et al., 2014). Zhao et al. (2017) identified the formation of gluten protein aggregates during
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long-term frozen storage. We therefore deduced that changes in population A and population A1 could be due to
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the more hydrophobic and rigid structure induced by FTC as a result of gluten depolymerization. The results of
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the present study also confirm the mechanism proposed by Kontogiorgos, Goff, and Kasapis (2007), who reported
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that the ice confined in pores can act as secondary sources of deterioration of the gluten network along with the
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growth of the bulk water. In the presence of EPS, area changes in population A were delayed. EPS at high
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concentration (1.5%) significantly controlled mobility changes in population A and showed no signal in
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population A1 even after being subjected to five FTC, an outcome that could retard the ice recrystallization and
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deterioration of the gluten structure induced by FTC.
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3.3 Effect of EPS on rheology of gluten during FTC As a water-insoluble proteinaceous component of wheat flour, hydrated gluten forms a viscoelastic mass that
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is mainly responsible for the mechanical properties of dough (Delcour et al., 2012). The rheological properties of
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hydrated gluten are strongly dependent on the water content in the matrix solid phase, and therefore the water
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distribution in the gluten, which has already been recognized by various research (Almutawah, Barker, & Belton,
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2007; Dekkers et al., 2016; Kontogiorgos, 2017). The effect of EPS addition on the viscoelastic properties of fresh
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gluten is shown in Fig. 2. The presence of increasing amounts of EPS in the fresh gluten resulted in a reduction in
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both G′ and G″, indicating a softening effect on the gluten. Bárcenas et al. (2009), Rosell and Foegeding (2007),
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and Xuan et al. (2017) also observed similar elastic behavior of gluten when mixed with HPMC. However, an
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opposite trend was observed when gluten was mixed with hydrocolloids such as arabic gum, pectin, or
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water-soluble pentosan (Bárcenas et al., 2009; Ma, Xu, Xu, & Guo, 2012). These results were attributed to the
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wide variations in biopolymer structure and the amounts incorporated into gluten. In the case of non-ionic
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polymers, only weak inter-biopolymer complexes can be formed with the proteins (Ribotta et al., 2005). Hence,
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we suggested that the change in viscoelastic properties of fresh gluten with added EPS was related to the
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hydration changes induced by the added EPS (Song & Zheng, 2007). On a molecular scale, a “loop and train”
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model has been proposed by Belton (1999). Logically, upon higher gluten protein hydration, more loop regions
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are formed, hence increasing the water content and resulting in a more easily deformable system (Shewry,
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Popineau, Lafiandra, & Belton, 2000).
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The viscoelastic parameters of gluten with different concentration of EPS during FTC at 1 Hz were extracted
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from the frequency sweeps (Table 3). Both the G′ and G″ both decreased significantly after FTC in the presence
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and absence of EPS, indicating a reduction in gluten elasticity and viscosity. Extensive research has revealed that
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decreased viscoelasticity of gluten is induced by loss of polymer cross-linking and gluten network structural
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deterioration caused by water crystallization and ice recrystallization (Kontogiorgos et al., 2007; Ribotta, León, &
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Añón, 2001; Ribotta, Pérez, León, &Añón, 2004; Wang et al., 2014, 2015). During FTC, the rate of decrease of
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gluten viscoelasticity was retarded by added EPS. The loss tangent (tanδ = G′/ G″) of gluten was kept constant
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with or without EPS after one FTC. However, after five FTC, a significant (p<0.05) increase in tanδ value was
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observed in glutens containing EPS concentrations lower than 0.5%, indicating a declining contribution of the
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elastic component in gluten with repeated FTC. This result is in agreement with that of Wang et al. (2014), who
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demonstrated that depolymerization of GMP was the main indicator of deterioration of gluten during frozen
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storage. Glutens with higher concentrations of EPS (above 0.5%) exhibited a delayed decrease in G′ and G″ and
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maintained a constant tanδ value after five FTC. Therefore, the addition of high concentrations of EPS improved
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the rheological properties of gluten during FTC.
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3.4 Effect of EPS on microstructure of gluten during FTC
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A frozen section technique and CLSM were combined to observe the morphological changes in gluten during
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FTC. As shown in Fig. 3, the red color represented proteins labeled by rhodamine B, and the dark regions
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represented the voids in the structure. Fresh gluten displayed a continuous and uniform structure. A more
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connected and dense structure was observed in fresh gluten with 1.5% EPS (Fig. 3b). After five FTC, more dark
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regions appeared, and the continuous gluten structure was found to be destroyed in gluten with or without EPS.
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The structural deterioration was attributed to the mechanical damage induced by ice recrystallization (Ribotta et
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al., 2004). As shown in Fig. 3c, control gluten appeared more porous and irregular after being subjected to five
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FTC. However, addition of 1.5% EPS maintained the structural integrity during FTC, suggesting an inhibitory
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effect on the recrystallization of ice crystals.
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In the present study, morphological changes in gluten were successfully observed by CLSM of frozen
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sections. Compared with SEM, CLSM observation of frozen sections technique is a rapid and easy method to
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observe the morphology of gluten because it does not require complicated sample pretreatment before observation.
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However, the accuracy of the observation might be influenced by the embedding and slicing technique. Therefore,
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the results of CLSM were compared with those of SEM, and the gluten structures were further observed in more
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details by SEM (Fig. 4). A continuous membranous structure was observed in fresh glutens, and clear differences
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were observed between the control gluten and EPS–gluten after five FTC. An irregular “cell-wall” structure was
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also observed after five FTC (indicated by the white arrow in Fig. 4c, 4d), especially in gluten samples lacking
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EPS. Compared with gluten in the presence of EPS, the microstructure of control gluten was coarser and more
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fractured after five FTC. These observations suggest that the incorporation of EPS reduced the mobility of water
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in the gluten system, thereby inhibiting ice recrystallization and thus stabilizing the microstructure of gluten. This
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is consistent with the results obtained from optical microscopy of frozen sections.
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Conclusions
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FTC induced an increase in water loss and a decrease in the viscoelasticity of gluten and altered water
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mobility and distribution. The continuous gluten structure was destroyed because of the mechanical damage
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induced by ice recrystallization. Addition of EPS increased the water content and decreased the water mobility
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and elasticity parameters of fresh gluten. The presence of EPS effectively delayed the dehydration of gluten and
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retarded water redistribution induced by FTC. Gluten with a higher concentration of EPS (above 0.5%) exhibited
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a delayed decrease in G′ and G″ and maintained a constant tanδ value after five FTC. EPS maintained the
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structural integrity of gluten during FTC, suggesting an inhibitory effect on the recrystallization of ice crystals.
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The high-molecular- weight, linear EPS dextran produced by W. confusa QS813 was shown to be an effective
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agent for improving gluten during FTC. These results suggested that this novel LAB dextran is appropriate for use
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as a cryoprotectant in wheat gluten-based frozen food, thus contributing new knowledge and application potential
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to the frozen dough and food industry, trending with the consumer demand for clean label products.
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Acknowledgments
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We are grateful for the financial support of this research from Grants (31071595, 31571877) from the
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National Natural Science Foundation of China, the National High Technology Research and Development
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Program of China (863 Program, 2012AA022200), Fujian “Hundreds of Talents Expert” Program of China
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(20172022), and the Science and Technology “LiaoYuan Plan” Program of Quanzhou, Fujian Province, China
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(2015G46), of BaihoBake Biotechnology International, Inc. (Nanjing, China), and MagiBake International, Inc.
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(Wuxi, China).
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Xu, H. N., Huang, W., Jia, C., Kim, Y., & Liu, H. (2009). Evaluation of water holding capacity and breadmaking properties for frozen dough containing ice structuring proteins from winter wheat. Journal of Cereal Science, 49(2), 250-253. Xuan, Y. F., Zhang, Y., Zhao, Y. Y., Zheng, Z., Jiang, S.T., & Zhong, X. Y. (2017). Effect of hydroxypropylmethylcellulose on transition of water status and physicochemical properties of wheat gluten upon frozen storage. Food Hydrocolloids, 63, 35-42.
Zannini, E., Waters, D. M., & Arendt, E. K. (2014). The application of dextran compared to other hydrocolloids as a novel food ingredient to compensate for low protein in biscuit and wholemeal wheat flour. European Food Research and Technology, 238(5), 763-771. Zhang, C., Zhang, H., & Wang, L. (2007). Effect of carrot (Daucus carota) antifreeze proteins on the fermentation capacity of frozen dough. Food Research International, 40(6), 763-769. Zhao, L., Liu, X., Hu, Z., Li, L., & Li, B. (2016). Molecular Structure Evaluation of Wheat Gluten during Frozen Storage. Food Biophysics, 1-9.
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409 410 Figure Captions
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Fig. 1. Carr−Purcell−Meiboom−Gill (CPMG) (a) proton distributions of fresh wet gluten with different content of
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EPS (b) proton distributions of wet gluten with different content of EPS after one cycle of freeze-thaw treatment
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and (c) proton distributions of wet gluten with different content of EPS after five cycles of frozen-thaw treatment.
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Amplitudes are given in arbitrary units (au). The different proton populations are indicated with capital letters in
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order of their increasing mobility. Inset shows a magnification of the population A.
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Fig. 2. Viscoelastic behavior of fresh wet gluten with different content of EPS. (a) The storage modulus of gluten
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(G′); (b) The loss modulus of gluten (G″); (c) The loss tangents values of gluten (tanδ= G″/ G′).
Fig. 3. Laser scanning confocal microscopy (CLSM) images of (a) fresh gluten without EPS (the control); (b) fresh gluten with 1.5% EPS; (c) gluten without EPS (the control) after five cycles of freeze-thaw treatment; (d) gluten with 1.5% EPS after 5 cycles of freeze-thaw treatment.
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Fig. 4. Scanning electron microscope (SEM) images of (a) fresh gluten without EPS (the control); (b) fresh gluten with 1.5% EPS; (c) gluten without EPS (the control) after five cycles of freeze-thaw treatment; (d) gluten with 1.5% EPS after five cycles of freeze-thaw treatment.
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Fig. 2.
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Sample
Fresh
1FTC
2FTC
3FTC
4FTC
5FTC
Control
62.13±0.06a
61.68±0.11a
61.50±0.06a
60.80±0.09a
60.20±0.13a
59.48±0.33a
0.1%
62.12±0.08a
61.80±0.11a
61.52±0.09a
60.87±0.08ab
60.23±0.06a
59.74±0.35a
0.5%
62.71±0.15
b
b
a
b
60.66±0.08b
1.0%
62.83±0.05b
62.63±0.13b
61.87±0.43ab
61.55±0.10c
61.32±0.09c
60.90±0.18b
1.5%
63.52±0.10c
63.32±0.11c
62.79±0.61b
62.33±0.38d
62.25±0.11d
61.72±0.11c
61.84±0.09
61.17±0.15
b
60.82±0.15
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Means±SD (n=3) with different letters within a column are significantly different (P<0.05).
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Moisture content of gluten during freeze-thaw cycles
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Table2
T2 relaxation times (ms) and relative areas of the Carr-Purcell-Meiboom-Gill (CPMG) populations A, A1, B and C of gluten during freeze-thaw cycles Sample
Population A
Population A1
Population B
Population C
T2 (ms)
Area (%)
T2 (ms)
Area (%)
T2 (ms)
Area (%)
T2 (ms)
Area (%)
0.54bc
4.14ab
nd
nd
57.22c
95.86d
nd
nd
0.35
ab
4.29
ab
nd
57.22
c
95.71
d
nd
nd
0.35
ab
4.56
ab
54.74
b
95.44
d
nd
nd
0.22
a
3.88
a
49.77
a
96.12
d
nd
nd
0.21
a
3.81
a
a
96.19
d
nd
nd
Control 0.1% EPS 0.5% EPS 1.0% EPS 1.5% EPS
nd nd
nd
nd
nd
nd
nd
49.77
0.36c
0.71a
49.77a
93.21c
a
d
1FTC Control
2.31d c
5.22bc a
nd
nd
49.77
567
0.58b
0.5% EPS
0.80c
3.60a
nd
nd
49.77a
96.01d
705cd
0.39a
1.0% EPS
0.33ab
3.25a
nd
nd
49.77a
96.43d
560ab
0.31a
1.5% EPS
0.32ab
3.70a
nd
nd
49.77a
95.99d
510ab
0.31a
Control
2.67e
7.98d
0.25a
0.1% EPS
2.92e
6.17c
0.5% EPS
2.79e
1.0% EPS
2.79e
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0.87c
cd
1.21
5FTCs
95.70
955e
0.1% EPS
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3.71
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Fresh
2.44b
49.77a
85.18a
785d
4.41f
0.33bc
1.76b
49.77a
88.27b
643bc
3.80e
5.86c
0.29ab
2.37b
49.77a
88.48b
510ab
3.28d
6.19c
0.31c
1.63ab
49.77a
89.05b
487a
3.13d
4.05ab nd nd 49.77a 93.12c 464a 1.5% EPS 0.72c nd = not detectable. Means±SD (n=3) with different letters within a column are significantly different (P<0.05).
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2.82d
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Table3
Viscoelastic parameters (1.0Hz) of gluten during freeze-thaw cycles
Samples
Fresh G’(Pa)
1FTC
G” (Pa)
tanδ
G’ (Pa)
G” (Pa)
5FTC tanδ
G’ (Pa)
G” (Pa)
tanδ
0.45±0.02a
2017.0±55.37b 940.8±51.94b
0.47±0.02a
1394.0±38.05a 710.7±45.28a
0.51±0.02b
0.1% EPS 2259.9±26.33b 1040.0±51.22b
0.44±0.01a
1999.5±25.92b 911.6±29.48b
0.46±0.02a
1418.3±10.50ab 697.7±29.79a
0.49±0.01ab
0.5% EPS 2170.3±45.5a 944.9±14.88a
0.44±0.01a
1856.8±30.67a 836.1±11.53a
0.45±0.01a
1461.7±32.96b 709.2±11.6a
0.49±0.01ab
1.0% EPS 2137.8±42.2a
913.3±8.50a
0.43±0.01a
1861.7±37.88a 823.4±6.59a
0.44±0.01a
1556.2±21.91c 723.2±6.15a
0.46±0.01a
1.5% EPS 2143.9±42.3a 935.0±10.15a
0.44±0.01a
1916.3±33.00a 851.0±13.26a
0.44±0.01a
1670.6±29.17d 781.6±3.26b
0.46±0.01a
571
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Control 2400.8±67.99b 1081.0±62.50b
FTC=freeze-thaw cycle. Means±SD (n=3) with different letters within a column are significantly different (P<0.05).
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EPS maintained the structure integrity of gluten during freeze-thaw cycles.
S0268-005X(18)30895-6
DOI:
10.1016/j.foodhyd.2018.10.025
Reference:
FOOHYD 4706
To appear in:
Food Hydrocolloids
Received Date: 16 May 2018 Revised Date:
4 September 2018
Accepted Date: 16 October 2018
Please cite this article as: Tang, X., Zhang, B., Huang, W., Ma, Z., Zhang, F., Wang, F., Zou, Q., Zheng, J., Hydration, water distribution and microstructure of gluten during freeze thaw process: Role of a high molecular weight dextran produced by Weissella confusa QS813, Food Hydrocolloids (2018), doi: https://doi.org/10.1016/j.foodhyd.2018.10.025. 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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Hydration, water distribution and microstructure of gluten during freeze thaw process: role of a high molecular weight dextran produced by Weissella confusa QS813
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Xiaojuan Tang1, Binle Zhang1, Weining Huang1*, Zilin Ma1, Fengwen Zhang1, Feng Wang2, Qibo Zou2, Jianxian Zheng3
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1 State Key Laboratory of Food Science and Technology, Laboratory of Baking Science, Sourdough and Ingredient Functionality Research, Jiangnan University, Wuxi 214122, China;
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2 MagiBake International Inc., Wuxi, Jiangsu 214131, China;
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3 Institute of Food and Bioengineering, South China University of Technology, Guangzhou, Guangdong 510640, China
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*Corresponding author. Tel.: +86(510)8591 9139; fax: +86(510)8591 9139.
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E-mail address: [email protected] (W. Huang).
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Abstract
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The effect of exopolysaccharide (EPS) produced by Weissella confusa QS813 on the hydration, water distribution,
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rheology and microstructure of wheat gluten during freeze–thaw cycles (FTC) was investigated. Addition of EPS
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increased the water content of fresh gluten and delayed the dehydration of gluten during FTC. Low field-nuclear
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magnetic resonance showed that the presence of EPS reduced the mobility of both confined and bulk water in the
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fresh gluten matrix. Proton distribution changes in gluten during FTC indicated that deterioration of the gluten
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network was attributable to ice recrystallization of capillary confined water and bulk water. The presence of EPS
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effectively reduced the release of bulk water and retarded the redistribution of confined water induced by FTC.
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Dynamic oscillatory studies indicated that the presence of EPS induced a softening effect on the fresh gluten. FTC
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significantly decreased the rheological parameters, (storage modulus (G′) and loss modulus (G″), of all gluten
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samples. Gluten with higher concentrations of EPS (above 0.5%) exhibited a delayed decrease in G′ and G″ and
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maintained a constant tanδ value after FTC. Laser scanning confocal microscope and scanning electron
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microscope revealed that EPS maintained the structural integrity of gluten during FTC, suggesting an inhibitory
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effect on the recrystallization of ice crystals. These results in the present study indicated that the bacterial dextran
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is a promising cryoprotectant for the frozen dough industry.
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Keywords: Exopolysaccharide, Wheat gluten, Freeze–thaw cycles, Hydration, Water distribution, Microstructure
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1. Introduction
Frozen dough technology has been widely used in bakery goods to facilitate centralized manufacturing and
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distribution processes as well as the standardization of product quality (Selomulyo & Zhou, 2007). Nevertheless,
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the final quality of frozen dough is often negatively affected by disruption of the gluten network and loss of yeast
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viability owing to ice formation and temperature fluctuation-driven ice recrystallization (Hsu, Hoseney, & Seib,
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1979; Phimolsiripol, Siripatrawan, Tulyathan, & Cleland, 2008; Wang, Jin, & Xu, 2015). To retard frozen dough
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quality deterioration, addition of functional agents to the matrix aimed at improving the physico-chemical and
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thermo-mechanical properties of frozen dough has been reported. Additives such as ice structuring proteins,
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enzymes, emulsifiers and hydrocolloids have been used in frozen dough to regulate the hydration properties of
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gluten, thereby further inhibiting ice recrystallization (Rosell & Gomez, 2007; Rosell, Rojas, & De Barber, 2001;
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Zhang, Zhang, & Wang, 2007).
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The cryoprotective effects of hydrocolloids in the frozen dough bakery industry have been demonstrated by
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several researchers, and recently reviewed by Maity, Saxena and Raju (2016). Hydrocolloids have a high
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water-retention capacity, which confers stability to products undergoing successive freeze–thaw cycles (FTC)
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(Bárcenas, Benedito, & Rosell, 2004). Currently, most hydrocolloids used in frozen dough are derived from
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seaweeds (e.g. guar gum and carrageenan), plants (e.g. cellulose, pectin, and locust bean gum), and bacteria (e.g.,
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xanthan). Microbial exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) during sourdough
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fermentation, especially those with high molecular weight, have the potential to replace commercial hydrocolloids
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in baking applications (Galle & Arendt, 2014). However, although LAB EPS is a novel food ingredient in bakery
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applications, its functionality as a bread ingredient has been less frequently explored (Zannini, Waters, & Arendt,
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2014).
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In our earlier study, a high-molecular-mass, low-branched dextran with 97% α-(1, 6) linkages was produced
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by LAB Weissella confusa QS813, isolated from Chinese traditionally fermented sourdough. The dextran
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produced by W. confusa QS813 significantly improved the freeze–thaw stability of wheat starch gel (Tang et al.,
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2018). Although dextran has been used as a cryoprotectant in frozen dairy products (Mccurdy, Goff, & Stanley,
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1994), the use of dextran in the field of frozen dough has been minimally explored. Tieking and Ganzle (2005)
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suggested the beneficial influence of EPS on dough stability during frozen storage. Therefore, studies on the effect
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of EPS addition on the properties of wheat gluten in frozen dough and the related mode of action are necessary.
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Therefore, the aim of this study was to characterize the effect of LAB EPS on the hydration, polymerization, and
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microstructure of wheat gluten during the freeze–thaw process.
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2. Materials and methods
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2.1 Isolation and purification of EPS W. confusa QS813 was obtained from the culture collection of the Laboratory of Baking Science, Sourdough
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and Ingredient Functionality Research, Jiangnan University. The strain was cultivated at 37 °C for 24 h in de Man,
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Rogosa, and Sharpe (MRS) medium and sub-cultured at least twice until a cell concentration of 109 CFU/mL was
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reached prior to experimental use. The production of EPS by W. confusa QS813 was performed in modified MRS
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(mMRS) medium (50 g/L sucrose instead of 20 g/L glucose). One hundred milliliters of mMRS medium was
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inoculated with 2% (v/v) culture (2 × 107 CFU/mL) and incubated without shaking at 20 ℃ for 24 h. Cells were
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removed by centrifugation at 12,000×g for 50 min at 4 °C. A final concentration of 4% (w/v) trichloroacetic acid
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(TCA) was used for protein precipitation and incubated with the cells at 4 ℃ overnight. Proteins were removed by
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centrifugation at 12,000×g for 30 min at 4 °C. Three volumes of 95% (v/v) cold ethyl alcohol was then added to
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the supernatant, followed by centrifugation at 12,000×g for 30 min at 4 ℃. The precipitate was re-suspended in
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ultrapure water and dialyzed for 48 h at 4 °C (8,000–12,000 Da) with water changes every 8 h. Finally, the
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dialyzed EPS preparations were freeze dried and stored at -18 °C.
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2.2 Preparation of fresh and frozen–thawed gluten–EPS mixtures
Purified EPS (0.1, 0.5, 1.0, and 1.5 g) was sprinkled into 100 mL deionized water and continuously stirred
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for complete dissolution. Five grams of wheat gluten (81.20% protein, 7.51% water, and 0.71% fat; Sigma–
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Aldrich Chemical Co., USA) was then dispersed in the EPS solution and stirred for 1 h at room temperature. The
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resulting mixture was centrifuged for 20 min at 4,000×g. The supernatant was discarded and the pellet obtained
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was fresh gluten. Afterwards, the gluten pellet was sealed in a polyethylene bag (50 × 70 mm). For freeze–thaw
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processing, the gluten was frozen at -20 °C for 22 h and then thawed at 30 °C for 2 h in a water bath. Five FTC
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were conducted.
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2.3 Water content of fresh and frozen–thawed gluten
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To determine the water content of the fresh and frozen–thawed prepared gluten, samples were removed from
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the hermetically sealed polyethylene bag. The water content of the gluten was determined according to AACC
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(2000).
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2.4 Low-field nuclear magnetic resonance (LF-NMR) measurements of gluten–EPS mixtures
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Determination of the proton distribution in fresh and frozen–thawed gluten was performed with an NMR
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spectrometer (MesoMR23-060 V-I; Niumag Electric Corporation, China) at a resonance frequency of 23 MHz.
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The fresh and frozen–thawed gluten samples were prepared as described in 2.2. Before LF-NMR measurements,
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gluten samples kept a the sealed polyethylene bag were thawed for 2 h in a water bath at 30°C, which was also the
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temperature of the magnet. The polyethylene bag containing the gluten pellet was transferred into the NMR probe
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(25 mm external diameter). Spin−spin (T2) relaxation time was acquired using a Carr–Purcell–Meiboom–Gill
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(CPMG) pulse sequence. The pulse parameters were as follows: TR=2,000 ms, TE=0.250ms, NECH=10,000,
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SW=100 KHz, PRG=2, NS=16. The SIRT algorithm (NMR analysis software provided by Niumag Electric
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Corporation) was used to invert the collected signals to the T2 relaxation time plots. The integrated area under the
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curves of the populations with certain T2 represents the number of protons.
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2.5 Rheology of fresh and frozen–thawed gluten
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Dynamic rheology of the gluten was determined according to the method described by Bárcenas, De la
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O-Keller, and Rosell (2009) with modifications. Fresh and frozen–thawed gluten samples were subjected to an
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oscillatory frequency sweep test using a controlled-stress rheometer (DHR-3, TA Instruments, USA) with
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parallel-plate geometry (20 mm diameter, 1 mm gap). The gluten was equilibrated for 15 min before measurement.
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Frequency sweep (0.1–10 Hz) experiments were performed with a strain of 1% at 30 °C. The storage modulus
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(G′), loss modulus (G″), and tanδ were recorded. Measurements were performed in triplicate.
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2.6 Confocal laser scanning microscopy (CLSM)
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Fresh and frozen–thawed gluten–EPS mixture samples were prepared as described in 2.2, cut to roughly 5 ×
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5 × 5 mm with a sharp blade, embedded in tissue freezing medium (Leica Biosystems, USA) and maintained at
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-20 ℃ for 30 min. Fifteen-micrometer sections were cut with a cryostat microtome (CM1950, Leica Biosystems,
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Germany) at -20℃, and fragment slice were discarded to avoid misinterpretation of the structural changes. Protein
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was labeled with rhodamine B (1.3 × 105 g/mL) for 2 min, followed by washing three times with deionized water.
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Macro photos of the frozen sections were acquired with a microscope (ZEISS Axio vert. A1, Germany) equipped
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with a computer. Images were recorded at 200× magnification.
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2.7 Scanning electron microscopy (SEM)
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The microstructures of fresh and frozen–thawed gluten was observed with a QUANTA-200 SEM (FEI,
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Eindhoven, The Netherlands). Before SEM observation, samples were pretreated according to Li et al. (2010). The
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gluten was fixed with 2.5% glutaraldehyde and post-fixed with 1% osmiumtetroxide. Samples were dehydrated
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with ethanol of concentrations ranging from 30% to 100%, followed by displacement with isoamyl acetate. The
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final dehydrated samples were obtained by the critical point drying method. For SEM observation, samples were
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coated with gold. Micrographs were acquired at 1,200× magnification.
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2.8 Data analysis
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The results are presented as average values with a confidence interval. Statistical analyses of all data were
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performed with SPSS, version 7.1 using one-way analysis of variance (ANOVA). The means were compared by
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Duncan’s multiple range test. A level of 0.05 probability was set to determine statistical significance. All tests
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were performed in triplicate.
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3. Results and discussion
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3.1 Effect of EPS on water content of gluten during FTC
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In the present study, gluten powder was dispersed homogeneously in excessive water and then subjected to
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centrifugation. As a result of hydration and mechanical energy input, the gluten network was formed by the
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interaction between gliadins and glutenins through numerous hydrogen and disulfide bonds (Frederic,
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Mariehelene, Jacques, Muriel, & Andreas. 2008). The effect of EPS on the water content of gluten pellet during
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FTC is shown in Table 1. The presence of small amounts of EPS (0.1%) did not impact the water–binding
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capacity of gluten, suggesting the weak influence of low concentrations of EPS on the hydration properties of
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gluten. However, a steady increase in water content of the fresh wheat gluten was achieved upon addition of
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increasing amounts of EPS, indicating the higher binding capacity of gluten with added EPS. This is in agreement
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with the observations in previous studies after the addition of hydroxypropyl methylcellulose (HPMC) to gluten
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(Bárcenas et al., 2009; Rosell & Foegeding, 2007). In the presence of sufficient water and adequate hydration, the
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water content was not a limiting factor for determining the effect of the hydrocolloids (Rosell & Foegeding, 2007).
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The molecular structure of the EPS produced by W. confusa QS813 was determined in our previous study, and
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described as a high-molecular-mass, low-branched dextran with 97% α-(1,6) linkages (Tang et al., 2018). As a
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highly linear polysaccharide with very flexible random coil conformations in solution, the EPS could be highly
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capable of forming hydrogen bonds and steric interactions with proteins; this might be responsible for the
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increased water binding ability of gluten with added EPS (Ross, McMaster, Tomlinson, & Cheetham, 1992;
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Ribotta, Ausar, Beltramo, & León, 2005).
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The water content of gluten decreased in all samples as the number of FTC increased (Table 1). After the
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fifth FTC treatment, the water content of control gluten decreased from 62.13% to 59.48, representing a decrease
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of 4.27%. However, the presence of EPS at a concentration above 0.5% significantly reduced the water loss of
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gluten after repeated FTC treatment. This result might be ascribed to the ability of the high molecular weight
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linear dextran to trap water. According to Phimolsiripol et al. (2008), moisture loss in frozen dough is due to
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transfer of water/ice in the frozen matrix to frost inside the polyethylene bag. Larger temperature fluctuations will
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induce a higher water loss in frozen dough. Repeated freezing and thawing diminishes the total number of crystals
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and increases the mean crystal size, thus further damaging the gluten network and increasing the release of bound
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water from the dough (Ding et al., 2015; Xu, Huang, Jia, Kim, & Liu, 2009). In this study, during the FTC,
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addition of EPS to gluten tended to improve moisture retention, inhibit ice crystal formation and subsequently
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reduce the freeze damage to gluten. Therefore, the water content of the gluten with added EPS decreased at a
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slower pace.
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3.2 Effect of EPS on water mobility of gluten during FTC
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The interaction of biopolymers with water reduces water mobility and results in different molecular motilities
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in the gluten system (Bosmans et al., 2012). In the present study, the molecular mobility of water in fresh and
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frozen–thawed gluten was studied with LF-NMR and the relaxation time distributions are shown in Figure 1. For
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the fresh gluten, two CPMG populations were deduced from the relaxation signals (Fig. 1a). The first one, likely
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assigned to protons that are strongly associated with the gluten matrix (amino acids and low- mobility water
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molecules) (Kontogiorgos, Goff & Kasapis, 2007), represents population A with relaxation time (T2) less than
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0.66 ms. Similar results were reported by Kontogiorgos, Goff and Kasapis (2007), Peters et al. (2016) and Liu et
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al. (2018), who found only one CPMG population with a T2 less than 10 ms in hydrated gluten with a moisture
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content above 60%. However, two CPMG populations with a T2 less than 10ms were distinguished in hydrated
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gluten with the similar moisture content (Wang et al., 2014; Xuan et al., 2017). The difference in the distribution
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pattern of T2 in gluten may be explained by the use of different sampling methods, NMR instruments, test
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conditions, and inversion algorithms. The second population B was the predominant proton population in the
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CPMG spectrum, largely consisting of mobile exchanging protons, and had a higher mobility with a T2 range of
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28.48–114.98 ms. The porous structure of the gluten network has been conceptualized as a sheet–like structure
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(Kontogiorgos & Goff, 2006; Kontogiorgos, 2011). According to the assignments of CPMG proton populations of
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the gluten–water model system based on the sheet model by Bosmans et al. (2012), population A contained
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protons of gluten in the sheets in contact with confined and bulk water, whereas population B was assigned to bulk
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water surrounding the sheet, which can exchange with gluten protons on the outside of the sheet.
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As shown in Fig. 1a and Table 2, addition of different concentrations of EPS changed the NMR profile of
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fresh gluten. T2 of population A and B decreased with increasing amounts of EPS. However, no significant change
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in the area of populations was observed. The differences showed that EPS addition to the fresh gluten resulted in
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the presence of more rigid protons. Their reduced mobility was caused by more intense contact between water and
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gluten. As a type of unfolded macromolecules that can occupy a large volume, EPS produced by W. confusa
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QS813 might have limited the mobility in the solutions, thereby inducing decreased mobility of bulk water in the
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parallel sheet space of gluten (population B). The decreased mobility of population A might have resulted from
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arrangement changes in gluten sheets induced by EPS addition, an outcome that may reflect enhanced associations
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between the proteinaceous macromolecules. Non-ionic polysaccharide is reported to interact with gluten proteins
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through hydrogen bonding and steric interactions (Ribotta et al., 2005; Rosell & Foegeding, 2007; Ross et al.,
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1992). Based on the change in population A, we reasonably suggest that the final conformation of gluten was
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modified by the present EPS. This was also consistent with the results of Linlaud, Ferrer, Cecilia Puppo, and
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Ferrero (2011), who found that the final conformation of the combined gluten–hydrocolloid–water matrix
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determines the degree of water binding.
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FTC changed the NMR profile of the gluten (Fig. 1b, 1c and Table 2), indicating a redistribution of water in
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the gluten mix. After one FTC, a new population C appeared at high T2 in the CPMG spectrum of all gluten
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samples. With the increasing number of FTC, the area of population C increased, whereas the area of population B
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decreased, indicating the release of bulk water from the gluten network. However, addition of EPS delayed the
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transformation from population B to C. The frozen gluten network is considered a porous matrix in which the
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pores are filled with confined water and surrounded by bulk ice after hydration (Kontogiorgos & Goff, 2006).
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During freezing, bulk water in the space of the parallel sheet transforms to ice through crystallization.
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Temperature fluctuation drives ice recrystallization and bulk water release due to the partial disruption of the
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gluten network (Wang et al., 2015). In the present study, addition of EPS effectively inhibited bulk water release
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from the gluten matrix and controlled recrystallization of hydrated gluten during FTC. This was in line with
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previous findings regarding water content changes during FTC. Population A with a low T2 value was also
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sensitive to FTC. After one FTC, a new distinct proton population A1 at a lower T2 value was distinguished in
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control gluten without EPS. As shown in Table 2, areas of both population A and A1 in all experimental groups
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significantly increased after five FTC. At the same time, FTC increased the mobility of population A and
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decreased the mobility of population A1, indicating a more heterogeneous arrangement of gluten structure. During
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the FTC, more hydrophobic binding sites were exposed, resulting in a weakened association between protein and
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water (Wang et al., 2014). Zhao et al. (2017) identified the formation of gluten protein aggregates during
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long-term frozen storage. We therefore deduced that changes in population A and population A1 could be due to
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the more hydrophobic and rigid structure induced by FTC as a result of gluten depolymerization. The results of
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the present study also confirm the mechanism proposed by Kontogiorgos, Goff, and Kasapis (2007), who reported
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that the ice confined in pores can act as secondary sources of deterioration of the gluten network along with the
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growth of the bulk water. In the presence of EPS, area changes in population A were delayed. EPS at high
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concentration (1.5%) significantly controlled mobility changes in population A and showed no signal in
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population A1 even after being subjected to five FTC, an outcome that could retard the ice recrystallization and
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deterioration of the gluten structure induced by FTC.
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3.3 Effect of EPS on rheology of gluten during FTC As a water-insoluble proteinaceous component of wheat flour, hydrated gluten forms a viscoelastic mass that
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is mainly responsible for the mechanical properties of dough (Delcour et al., 2012). The rheological properties of
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hydrated gluten are strongly dependent on the water content in the matrix solid phase, and therefore the water
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distribution in the gluten, which has already been recognized by various research (Almutawah, Barker, & Belton,
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2007; Dekkers et al., 2016; Kontogiorgos, 2017). The effect of EPS addition on the viscoelastic properties of fresh
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gluten is shown in Fig. 2. The presence of increasing amounts of EPS in the fresh gluten resulted in a reduction in
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both G′ and G″, indicating a softening effect on the gluten. Bárcenas et al. (2009), Rosell and Foegeding (2007),
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and Xuan et al. (2017) also observed similar elastic behavior of gluten when mixed with HPMC. However, an
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opposite trend was observed when gluten was mixed with hydrocolloids such as arabic gum, pectin, or
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water-soluble pentosan (Bárcenas et al., 2009; Ma, Xu, Xu, & Guo, 2012). These results were attributed to the
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wide variations in biopolymer structure and the amounts incorporated into gluten. In the case of non-ionic
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polymers, only weak inter-biopolymer complexes can be formed with the proteins (Ribotta et al., 2005). Hence,
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we suggested that the change in viscoelastic properties of fresh gluten with added EPS was related to the
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hydration changes induced by the added EPS (Song & Zheng, 2007). On a molecular scale, a “loop and train”
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model has been proposed by Belton (1999). Logically, upon higher gluten protein hydration, more loop regions
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are formed, hence increasing the water content and resulting in a more easily deformable system (Shewry,
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Popineau, Lafiandra, & Belton, 2000).
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The viscoelastic parameters of gluten with different concentration of EPS during FTC at 1 Hz were extracted
259
from the frequency sweeps (Table 3). Both the G′ and G″ both decreased significantly after FTC in the presence
260
and absence of EPS, indicating a reduction in gluten elasticity and viscosity. Extensive research has revealed that
261
decreased viscoelasticity of gluten is induced by loss of polymer cross-linking and gluten network structural
262
deterioration caused by water crystallization and ice recrystallization (Kontogiorgos et al., 2007; Ribotta, León, &
263
Añón, 2001; Ribotta, Pérez, León, &Añón, 2004; Wang et al., 2014, 2015). During FTC, the rate of decrease of
264
gluten viscoelasticity was retarded by added EPS. The loss tangent (tanδ = G′/ G″) of gluten was kept constant
265
with or without EPS after one FTC. However, after five FTC, a significant (p<0.05) increase in tanδ value was
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observed in glutens containing EPS concentrations lower than 0.5%, indicating a declining contribution of the
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elastic component in gluten with repeated FTC. This result is in agreement with that of Wang et al. (2014), who
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demonstrated that depolymerization of GMP was the main indicator of deterioration of gluten during frozen
269
storage. Glutens with higher concentrations of EPS (above 0.5%) exhibited a delayed decrease in G′ and G″ and
270
maintained a constant tanδ value after five FTC. Therefore, the addition of high concentrations of EPS improved
271
the rheological properties of gluten during FTC.
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3.4 Effect of EPS on microstructure of gluten during FTC
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A frozen section technique and CLSM were combined to observe the morphological changes in gluten during
274
FTC. As shown in Fig. 3, the red color represented proteins labeled by rhodamine B, and the dark regions
275
represented the voids in the structure. Fresh gluten displayed a continuous and uniform structure. A more
276
connected and dense structure was observed in fresh gluten with 1.5% EPS (Fig. 3b). After five FTC, more dark
277
regions appeared, and the continuous gluten structure was found to be destroyed in gluten with or without EPS.
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The structural deterioration was attributed to the mechanical damage induced by ice recrystallization (Ribotta et
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al., 2004). As shown in Fig. 3c, control gluten appeared more porous and irregular after being subjected to five
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FTC. However, addition of 1.5% EPS maintained the structural integrity during FTC, suggesting an inhibitory
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effect on the recrystallization of ice crystals.
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In the present study, morphological changes in gluten were successfully observed by CLSM of frozen
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sections. Compared with SEM, CLSM observation of frozen sections technique is a rapid and easy method to
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observe the morphology of gluten because it does not require complicated sample pretreatment before observation.
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However, the accuracy of the observation might be influenced by the embedding and slicing technique. Therefore,
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the results of CLSM were compared with those of SEM, and the gluten structures were further observed in more
287
details by SEM (Fig. 4). A continuous membranous structure was observed in fresh glutens, and clear differences
288
were observed between the control gluten and EPS–gluten after five FTC. An irregular “cell-wall” structure was
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also observed after five FTC (indicated by the white arrow in Fig. 4c, 4d), especially in gluten samples lacking
290
EPS. Compared with gluten in the presence of EPS, the microstructure of control gluten was coarser and more
291
fractured after five FTC. These observations suggest that the incorporation of EPS reduced the mobility of water
292
in the gluten system, thereby inhibiting ice recrystallization and thus stabilizing the microstructure of gluten. This
293
is consistent with the results obtained from optical microscopy of frozen sections.
294
Conclusions
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FTC induced an increase in water loss and a decrease in the viscoelasticity of gluten and altered water
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mobility and distribution. The continuous gluten structure was destroyed because of the mechanical damage
297
induced by ice recrystallization. Addition of EPS increased the water content and decreased the water mobility
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and elasticity parameters of fresh gluten. The presence of EPS effectively delayed the dehydration of gluten and
299
retarded water redistribution induced by FTC. Gluten with a higher concentration of EPS (above 0.5%) exhibited
300
a delayed decrease in G′ and G″ and maintained a constant tanδ value after five FTC. EPS maintained the
301
structural integrity of gluten during FTC, suggesting an inhibitory effect on the recrystallization of ice crystals.
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The high-molecular- weight, linear EPS dextran produced by W. confusa QS813 was shown to be an effective
303
agent for improving gluten during FTC. These results suggested that this novel LAB dextran is appropriate for use
304
as a cryoprotectant in wheat gluten-based frozen food, thus contributing new knowledge and application potential
305
to the frozen dough and food industry, trending with the consumer demand for clean label products.
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Acknowledgments
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We are grateful for the financial support of this research from Grants (31071595, 31571877) from the
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National Natural Science Foundation of China, the National High Technology Research and Development
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Program of China (863 Program, 2012AA022200), Fujian “Hundreds of Talents Expert” Program of China
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(20172022), and the Science and Technology “LiaoYuan Plan” Program of Quanzhou, Fujian Province, China
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(2015G46), of BaihoBake Biotechnology International, Inc. (Nanjing, China), and MagiBake International, Inc.
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(Wuxi, China).
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Tang, X., Liu, N., Huang, W., Cheng, X., Wang, F., Zhang, B., Chen, J., Jiang, H., Omedi, J. O., & Li, Z. (2018). Syneresis rate,
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water distribution, and microstructure of wheat starch gel during freeze-thaw process: Role of a high molecular weight dextran produced by Weissella confusa QS813 from traditional sourdough. Cereal Chemistry, 95(1), 117-129. Tieking, M., & Ganzle, M. G. (2005). Exopolysaccharides from cereal-associated lactobacilli. Trends in Food Science & Technology, 16(1-3), 79-84.
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physico-chemistry of wheat gluten proteins: Studies on gluten-, glutenin-and gliadin-rich fractions. Food Hydrocolloids, 39,
Wang, P., Jin, Z., & Xu, X. (2015). Physicochemical alterations of wheat gluten proteins upon dough formation and frozen storage–A review from gluten, glutenin and gliadin perspectives. Trends in Food Science & Technology, 46(2), 189-198.
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Xu, H. N., Huang, W., Jia, C., Kim, Y., & Liu, H. (2009). Evaluation of water holding capacity and breadmaking properties for frozen dough containing ice structuring proteins from winter wheat. Journal of Cereal Science, 49(2), 250-253. Xuan, Y. F., Zhang, Y., Zhao, Y. Y., Zheng, Z., Jiang, S.T., & Zhong, X. Y. (2017). Effect of hydroxypropylmethylcellulose on transition of water status and physicochemical properties of wheat gluten upon frozen storage. Food Hydrocolloids, 63, 35-42.
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409 410 Figure Captions
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Fig. 1. Carr−Purcell−Meiboom−Gill (CPMG) (a) proton distributions of fresh wet gluten with different content of
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EPS (b) proton distributions of wet gluten with different content of EPS after one cycle of freeze-thaw treatment
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and (c) proton distributions of wet gluten with different content of EPS after five cycles of frozen-thaw treatment.
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Amplitudes are given in arbitrary units (au). The different proton populations are indicated with capital letters in
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order of their increasing mobility. Inset shows a magnification of the population A.
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Fig. 2. Viscoelastic behavior of fresh wet gluten with different content of EPS. (a) The storage modulus of gluten
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(G′); (b) The loss modulus of gluten (G″); (c) The loss tangents values of gluten (tanδ= G″/ G′).
Fig. 3. Laser scanning confocal microscopy (CLSM) images of (a) fresh gluten without EPS (the control); (b) fresh gluten with 1.5% EPS; (c) gluten without EPS (the control) after five cycles of freeze-thaw treatment; (d) gluten with 1.5% EPS after 5 cycles of freeze-thaw treatment.
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Fig. 4. Scanning electron microscope (SEM) images of (a) fresh gluten without EPS (the control); (b) fresh gluten with 1.5% EPS; (c) gluten without EPS (the control) after five cycles of freeze-thaw treatment; (d) gluten with 1.5% EPS after five cycles of freeze-thaw treatment.
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(c)
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Fig. 1.
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(a)
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460 461 (c)
463 464
Fig. 2.
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Fig. 3.
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Fig. 4.
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Sample
Fresh
1FTC
2FTC
3FTC
4FTC
5FTC
Control
62.13±0.06a
61.68±0.11a
61.50±0.06a
60.80±0.09a
60.20±0.13a
59.48±0.33a
0.1%
62.12±0.08a
61.80±0.11a
61.52±0.09a
60.87±0.08ab
60.23±0.06a
59.74±0.35a
0.5%
62.71±0.15
b
b
a
b
60.66±0.08b
1.0%
62.83±0.05b
62.63±0.13b
61.87±0.43ab
61.55±0.10c
61.32±0.09c
60.90±0.18b
1.5%
63.52±0.10c
63.32±0.11c
62.79±0.61b
62.33±0.38d
62.25±0.11d
61.72±0.11c
61.84±0.09
61.17±0.15
b
60.82±0.15
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Means±SD (n=3) with different letters within a column are significantly different (P<0.05).
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62.48±0.07
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530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563
Moisture content of gluten during freeze-thaw cycles
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Table1
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564 565 566
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Table2
T2 relaxation times (ms) and relative areas of the Carr-Purcell-Meiboom-Gill (CPMG) populations A, A1, B and C of gluten during freeze-thaw cycles Sample
Population A
Population A1
Population B
Population C
T2 (ms)
Area (%)
T2 (ms)
Area (%)
T2 (ms)
Area (%)
T2 (ms)
Area (%)
0.54bc
4.14ab
nd
nd
57.22c
95.86d
nd
nd
0.35
ab
4.29
ab
nd
57.22
c
95.71
d
nd
nd
0.35
ab
4.56
ab
54.74
b
95.44
d
nd
nd
0.22
a
3.88
a
49.77
a
96.12
d
nd
nd
0.21
a
3.81
a
a
96.19
d
nd
nd
Control 0.1% EPS 0.5% EPS 1.0% EPS 1.5% EPS
nd nd
nd
nd
nd
nd
nd
49.77
0.36c
0.71a
49.77a
93.21c
a
d
1FTC Control
2.31d c
5.22bc a
nd
nd
49.77
567
0.58b
0.5% EPS
0.80c
3.60a
nd
nd
49.77a
96.01d
705cd
0.39a
1.0% EPS
0.33ab
3.25a
nd
nd
49.77a
96.43d
560ab
0.31a
1.5% EPS
0.32ab
3.70a
nd
nd
49.77a
95.99d
510ab
0.31a
Control
2.67e
7.98d
0.25a
0.1% EPS
2.92e
6.17c
0.5% EPS
2.79e
1.0% EPS
2.79e
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0.87c
cd
1.21
5FTCs
95.70
955e
0.1% EPS
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3.71
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Fresh
2.44b
49.77a
85.18a
785d
4.41f
0.33bc
1.76b
49.77a
88.27b
643bc
3.80e
5.86c
0.29ab
2.37b
49.77a
88.48b
510ab
3.28d
6.19c
0.31c
1.63ab
49.77a
89.05b
487a
3.13d
4.05ab nd nd 49.77a 93.12c 464a 1.5% EPS 0.72c nd = not detectable. Means±SD (n=3) with different letters within a column are significantly different (P<0.05).
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568
2.82d
569 570
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Table3
Viscoelastic parameters (1.0Hz) of gluten during freeze-thaw cycles
Samples
Fresh G’(Pa)
1FTC
G” (Pa)
tanδ
G’ (Pa)
G” (Pa)
5FTC tanδ
G’ (Pa)
G” (Pa)
tanδ
0.45±0.02a
2017.0±55.37b 940.8±51.94b
0.47±0.02a
1394.0±38.05a 710.7±45.28a
0.51±0.02b
0.1% EPS 2259.9±26.33b 1040.0±51.22b
0.44±0.01a
1999.5±25.92b 911.6±29.48b
0.46±0.02a
1418.3±10.50ab 697.7±29.79a
0.49±0.01ab
0.5% EPS 2170.3±45.5a 944.9±14.88a
0.44±0.01a
1856.8±30.67a 836.1±11.53a
0.45±0.01a
1461.7±32.96b 709.2±11.6a
0.49±0.01ab
1.0% EPS 2137.8±42.2a
913.3±8.50a
0.43±0.01a
1861.7±37.88a 823.4±6.59a
0.44±0.01a
1556.2±21.91c 723.2±6.15a
0.46±0.01a
1.5% EPS 2143.9±42.3a 935.0±10.15a
0.44±0.01a
1916.3±33.00a 851.0±13.26a
0.44±0.01a
1670.6±29.17d 781.6±3.26b
0.46±0.01a
571
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Control 2400.8±67.99b 1081.0±62.50b
FTC=freeze-thaw cycle. Means±SD (n=3) with different letters within a column are significantly different (P<0.05).
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572 573 574 575 576 577 578 579 580
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ACCEPTED MANUSCRIPT EPS delayed dehydration of gluten during freeze-thaw cycles. EPS retarded water redistribution induced by freeze-thaw cycles. EPS stabilized the rheological parameters of gluten during freeze-thaw cycles.
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EPS maintained the structure integrity of gluten during freeze-thaw cycles.