Accepted Manuscript Characterization and functional evaluation of oat protein isolate-Pleurotus ostreatus β-glucan conjugates formed via Maillard reaction Lei Zhong, Ning Ma, Yiliang Wu, Liyan Zhao, Gaoxing Ma, Fei Pei, Qiuhui Hu PII: S0268-005X(18)31261-X DOI: 10.1016/j.foodhyd.2018.08.034 Reference: FOOHYD 4615 To appear in: Food Hydrocolloids Received Date: 10 July 2018 Revised Date: 16 August 2018 Accepted Date: 19 August 2018 Please cite this article as: Zhong, L., Ma, N., Wu, Y., Zhao, L., Ma, G., Pei, F., Hu, Q., Characterization and functional evaluation of oat protein isolate-Pleurotus ostreatus β-glucan conjugates formed via Maillard reaction, Food Hydrocolloids (2018), doi: 10.1016/j.foodhyd.2018.08.034. 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. AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Characterization and functional evaluation of oat protein isolate-Pleurotus 2 ostreatus β-glucan conjugates formed via Maillard reaction 3 Lei Zhonga, Ning Mab, Yiliang Wub, Liyan Zhaoa, Gaoxing Maa, Fei Peib, Qiuhui 4 Hua* 5 a 6 Nanjing, 210095, China*. 7 email@example.com* 8 firstname.lastname@example.org 9 email@example.com RI PT 1 M AN U SC College of Food Science and Technology, Nanjing Agricultural University, 10 firstname.lastname@example.org 11 b 12 Economics, Nanjing, 210023, China. 13 email@example.com 14 firstname.lastname@example.org 15 email@example.com 16 *Corresponding author 19 20 21 TE D EP 18 AC C 17 College of Food Science and Engineering, Nanjing University of Finance and 22 23 24 25 1 ACCEPTED MANUSCRIPT 26 Abstract 28 Oat protein isolate is nutritious but with poor processing functionality. Pleurotus 29 ostreatus β-glucan with good processing functionality can be conjugated with oat 30 protein isolate via Maillard reaction, leading to an improved utilization of protein in 31 food industry. Therefore, we produced conjugate with oat protein isolate and 32 Pleurotus ostreatus β-glucan via Maillard reaction under controlled dry-heating 33 conditions. The formation of conjugates with high molecular weight was identified by 34 a new band of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 35 analysis of amino acid composition showed that cysteine and lysine were the 36 dominant Maillard reaction sites of oat protein isolate and Pleurotus ostreatus 37 β-glucan. Changes in spatial configuration of conjugates caused reduction in their 38 surface hydrophobicity of proteins and intrinsic fluorescence intensity. Fourier 39 transform infrared spectroscopy analysis of conjugates suggested that Maillard 40 reaction could cause the C=O stretching vibration, as well as the C-H and N-H 41 deformation vibration. Circular dichroism analysis indicated that the secondary 42 structure of conjugates was altered by decreasing the contents of α-helix and β-sheet 43 and increasing the contents of β-turn and random coil. The surface structure of 44 conjugates was loose and porous using scanning electron microscope. Furthermore, 45 Maillard reaction could improve the solubility, emulsifying property and thermal 46 stability of oat protein isolate. Our findings confirm the potential of protein- 47 carbohydrate conjugate formed by Maillard reaction, to improve the application of AC C EP TE D M AN U SC RI PT 27 2 ACCEPTED MANUSCRIPT 48 instable but valuable proteins in food industry. 49 Keywords: Oat protein isolate, Processing functionality, Pleurotus ostreatus β-glucan, 50 Conjugate, Maillard reaction. AC C EP TE D M AN U SC RI PT 51 3 ACCEPTED MANUSCRIPT 1. Introduction 53 Oats with better nutrition are superior sources of low-cost dietary proteins (ranging 54 from12 to 20%, w/w) among cereals (Mohamed et al., 2009; Grigg, 1995). Oat 55 protein isolate has abundant essential amino acids such as lysine (Klose & Arendt, 56 2012). It can be used as a promising food ingredient for human consumption (Zhang 57 et al., 2015). However, the poor functional properties of oat protein isolate, such as 58 solubility, emulsibility, foamability and gel property, can restrict its application in 59 food products (Ma, 1985). Therefore, it is necessary to improve the functionality of 60 oat protein isolate for an effective utilization by some modification techniques. 61 Physical, chemical and enzymatic modifications of oat protein isolates are generally 62 used to improve their functional properties for wider application (Zhao et al., 2017). 63 Most food proteins modified by chemical treatment have potential health risks (Guo 64 & Xiong, 2013). Physical treatment is mostly destructive by extrusion or ultrasound 65 (Tian, Chen, & Small, 2011). Enzymatic treatment is mostly time-consuming and 66 expensive, as well as off-flavor caused by protein hydrolysates (Galazka, Dickinson, 67 & Ledward, 2000). In contrast, under controlled conditions, Maillard reaction is easy 68 to perform without the addition of chemical reagents and the generation of harmful 69 secondary compounds (Oliver, Melton, & Stanley, 2006; Zhuo et al., 2013). It can 70 combine the functionalities of protein and polysaccharide to prepare a novel protein– 71 polysaccharide conjugate for more industrial applications (Akhtar & Ding, 2017). In 72 food processing, such conjugates with stable structure can serve as important food 73 ingredients, leading to improved techno-functional properties of food proteins and AC C EP TE D M AN U SC RI PT 52 4 ACCEPTED MANUSCRIPT changes in the texture, taste, flavor and colour of food products (de Oliveira et al., 75 2016). The quality of final food products that can be accepted by consumers depended 76 on functionalities of their proteins. Moreover, conjugates are also used as supporters 77 of active ingredients to enhance the bioavailability of ingredients in gastrointestinal 78 tract. Thus, food industry has growing interest in converting proteins into excellent 79 functional conjugates via Maillard reaction. 80 Pleurotus ostreatus is an important edible mushroom for its active compounds, such 81 as polysaccharides, phenols and lactones (Julita & Marek, 2007). P. ostreatus is rich 82 in β-glucan containing a linear glucose polymer formed by β (1-3) and β (1-6) 83 linkages and P. ostreatus β-glucan can be linked to proteins by some modification 84 techniques (De Silva et al., 2013). P. ostreatus β-glucan has been approved for a novel 85 food ingredient by the European Food Safety Authority due to its excellent 86 functionalities, such as water-holding capacity, emulsibility and swelling power 87 (Singh, 2015; Thammakiti et al., 2004). During the Maillard reaction, P. ostreatus 88 β-glucan can prevent the polymerization of protein and the formation of advanced 89 glycation end products due to its steric hindrance, which could be conducive to 90 enhance the covalent binding of P. ostreatus β-glucan to protein (de Oliveira et al., 91 2016). Taken together, P. ostreatus β-glucan can be a good candidate for improving 92 the functionalities of oat protein isolate via Maillard reaction. 93 Under controlled conditions of Maillard reaction, the structural properties of food 94 protein can be considerably altered and these changes are helpful to enhance the 95 functional properties of protein, such as solubility, emulsibility and thermal stability AC C EP TE D M AN U SC RI PT 74 5 ACCEPTED MANUSCRIPT (Liu, Ru, & Ding, 2012; Usui et al., 2017; Sheng et al., 2017; Jimenez-Castano, 97 Villamiel, & Lopez-Fandino, 2007; Kim & Shin, 2015). For example, Qu et al. (2018) 98 found that the improved solubility and emulsifying property of rapeseed protein 99 isolate-dextran conjugates are related to their loose surface structure and more RI PT 96 hydrophilic groups. Pirestani et al. (2018) showed that canola protein isolate 101 (CPI)-gum arabic conjugates with extended secondary structure have higher thermal 102 stability than CPI alone via Maillard reaction. Mu et al. (2011) suggested that soy 103 protein isolate (SPI)-acacia gum conjugates have higher emulsion stability than SPI 104 alone and SPI-acacia gum mixture because acacia gum in the conjugates can provide 105 a steric hindrance layer around the oil droplets. Therefore, to understand these 106 structural properties of conjugates better can allow their application in the food 107 processing as components to enhance functionalities of final products. 108 In the study, we prepared conjugates between oat protein isolate and P. ostreatus 109 β-glucan under optimized dry-heating conditions. The structural and functional 110 properties of conjugates were further studied to compare with oat protein isolate, 111 heated oat protein isolate and the mixture of oat protein isolate-P. ostreatus β-glucan. AC C EP TE D M AN U SC 100 6 ACCEPTED MANUSCRIPT 2. Materials and methods 113 2.1. Materials 114 Oats were purchased from the local supermarket in Nanjing, Jiangsu, China. 115 Pleurotus ostreatus was purchased from Zhiqingtang Bio-Technique Co., Ltd. 116 (Yancheng, Jiangsu Province, China). Mixed amino acids standard solution and 117 potassium bromide (spectral purity) were purchased from Sigma-Aldrich (Shanghai, 118 China). All other analytical reagents were purchased from Sinopharm Chemical 119 Regent Co., Ltd (Shanghai, China). M AN U 120 SC RI PT 112 2.2. Extraction of oat protein isolate 122 Oat protein isolate was extracted as reported earlier with some modifications (Zhang 123 et al., 2015). Fat-free oat powders were dissolved in distilled water (10%, w/v) and 124 pH was adjusted to 9.5 with 1 mol/L NaOH before centrifugation at 3500 × g for 15 125 min. The obtained supernatant was kept at pH 4.5 and then centrifuged under the 126 same condition. Precipitates were washed three times with distilled water and then 127 freeze-dried. The purity of protein in oat protein isolate was about 90.15% determined 128 by the Kjeldahl method (Jung, et al., 2003). EP AC C 129 TE D 121 130 2.3. Extraction of P. ostreatus β-glucan 131 To remove soluble impurities, the powder of P. ostreatus was washed with 80% 132 ethanol (v/v) for 8 h and extracted with boiling water for further preparation as 133 reported earlier with some modifications (Szwengiel & Stachowiak, 2016). Before 7 ACCEPTED MANUSCRIPT centrifugation at 3500 × g for 20 min, the hot aqueous extractions were concentrated 135 to a reduced volume by rotary evaporator, followed by precipitation using excess 95% 136 ethanol. The obtained precipitates were dialyzed for 48 h (12-14 kDa) and then 137 freeze-dried. After re-dissolving into ultrapure water, the soluble fractions of β-glucan 138 were purified for further study (Khan et al., 2015). The purity of P. ostreatus β-glucan 139 was 90.36% using yeast and mushroom β-glucan enzymatic assay kit (Megazyme 140 International Ireland Ltd., Wicklow, Ireland). 142 2.4. Preparation of oat protein isolate-P. ostreatus β-glucan conjugates 143 Oat protein isolate and P. ostreatus β-glucan in various proportion of 1:1, 1:2, 1:3, 1:4 144 and 1:5 (w/w) were dissolved in 0.05 mol/L sodium phosphate buffer (pH 7.0) 145 containing 0.02% sodium azide. After completely hydration at 4 °C overnight, the 146 mixture was adjusted to desired pH of 4, 6, 8, 10 and 12 using 0.1 mol/L NaOH or 147 HCI and then freeze-dried. The obtained lyophilized powders were incubated at 60 °C, 148 75% relative humidity for 1, 3, 5, 7 and 9 days. Oat protein isolate, heated oat protein 149 isolate and the mixture of oat protein isolate-P. ostreatus β-glucan were treated as 150 comparison samples in the same way. TE D EP AC C 151 M AN U 141 SC RI PT 134 152 2.5. Evaluation of the extent of Maillard reaction 153 The degree of graft (DG) and browning index were considered as major indicators to 154 evaluate the extent of Maillard reaction. DG was measured by spectrophotometry as 155 reported earlier with some modifications (Snyder & Sobocinski, 1975). Diluted 8 ACCEPTED MANUSCRIPT sample was mixed with 1.5 mL trinitrobenzene sulfonic acid (0.1 mg/L) and 3 mL 157 sodium phosphate buffer (0.2 mol/L, pH 8.0) containing 1 mg/mL sodium dodecyl 158 sulfate, followed by dark-incubation at 55 °C. After an hour, the reaction was 159 terminated by adding 1.2 mL Na2SO3 (0.1 mol/L). The absorbance of the sample was 160 measured at 420 nm after cooling at room temperature for 15 min. A control with 161 distilled water instead of the sample was treated under the same condition. The 162 content of free amino groups was calculated from the standard curve of L-leucine at 163 420 nm and the formula is given below: C1 -Ct C1 M AN U DG = SC RI PT 156 Where C1 is the content of free amino groups of oat protein isolate and Ct is the 165 content of free amino groups of conjugates after reacting for t (min). 166 The browning index was measured at 420 nm as reported earlier with some 167 modifications (Lertittikul, Benjakul, & Tanaka, 2007). Before the measurement, the 168 conjugate was diluted to the concentration of 5 mg/mL with distilled water. EP 169 TE D 164 2.6. Confirmation of oat protein isolate-P. ostreatus β-glucan conjugates 171 SDS-PAGE was performed using 5% stacking gel and 12% separating gel (Zhang et 172 al., 2015). After the sample solution and standard protein (11-180 kDa) were loaded 173 into corresponding well, migration was conducted at 90 V and then at constant 174 voltage of 120 V. Gels were dyed by Coomassie blue R250 and then faded with 10% 175 acetic acid and 10% ethanol. AC C 170 176 9 ACCEPTED MANUSCRIPT 2.7. Characterization of oat protein isolate-P. ostreatus β-glucan conjugates 178 2.7.1. Fourier transform infrared spectroscopy (FT-IR) analysis 179 Sample (1 mg) was mixed with 200 mg KBr and measured at the wavelength of 180 4000-400 cm-1 using FT-IR as reported earlier with some modifications (Mohsin et al., 181 2018) (Bruker tensor 27, Bruker Corporation, Karlsruhe, Germany). 182 RI PT 177 2.7.2. Determination of intrinsic fluorescence emission 184 Samples containing 0.1 mg protein/mL were dissolved in 0.05 mol/L phosphate buffer 185 (pH 7.0). The emission wavelength ranged from 300 to 420 nm at the excitation 186 wavelength of 280 nm with a scanning speed of 1200 nm/min (Sheng et al., 2017). M AN U SC 183 187 2.7.3. Determination of the surface hydrophobicity of proteins 189 The fluorescence intensity of samples was determined at the excitation wavelength of 190 370 nm with the emission wavelength ranging from 300 to 650 nm as reported earlier 191 with some modifications (Kato et al., 1990). Protein solutions (4 mL) in 0.04 mg/mL 192 increments from 0 to 0.16 mg/mL were mixed with 20 µL 8.0 mmol/L 193 1-anilinonaphthalene-8-sulfonic acid. The function could be established between the 194 concentration of protein and its fluorescence intensity. The slope of this function 195 indicated the surface hydrophobicity of proteins. AC C EP TE D 188 196 197 2.7.4. Scanning electron microscopy (SEM) analysis 198 The sample was placed on the conductive adhesive and covered with gold of 10 nm. 10 ACCEPTED MANUSCRIPT 199 The surface structure of the sample was observed in different magnifications (×300, × 200 600, ×1200, ×2000) and in secondary electron mode at 15.0 kV using a scanning 201 electron microscope (TM-3000, Hitachi Corporation, Tokyo, Japan) (Qu et al., 2018). RI PT 202 2.7.5 Circular dichroism (CD) analysis 204 As reported earlier with some modifications, the secondary structure of protein 205 samples with different treatment was determined using CD spectrometer (a model 206 J-1500, JASCO Corporation, Tokyo, Japan) (Pirestani et al., 2017). The sample 207 containing 0.2 mg/mL protein was dissolved in 0.05 mol/L phosphate buffer (pH 7.0) 208 and then scanned between 190 and 260 nm with the band-width of 1 nm. The contents 209 of secondary structures including α-helix, β-sheet, β-turn and random coil, were 210 determined using the JASCO software. TE D M AN U SC 203 211 2.7.6. Determination of amino acid composition 213 All the samples were treated with 6 mol/L HCl at 110 °C for 12 h and the amino acid 214 composition was determined as reported earlier with some modifications (Fang et al., 215 2017). The acidolysis products were analyzed by automatic amino-acid analyzer 216 (Hitachi L 8800, Hitachi Ltd., Tokyo, Japan). AC C 217 EP 212 218 2.8. Functional properties of conjugates 219 2.8.1. Solubility 220 The sample was dissolved in the solution (1 mg/mL) with different pH at 3 to 11 and 11 ACCEPTED MANUSCRIPT then stirred at room temperature for 2 h. After centrifugation at 4000 × g for 20 min, 222 the content of protein in the supernatants was determined according to the earlier 223 report with some modifications (Qu et al., 2018). The solubility was showed as the 224 content of protein in supernatants per 100 g of protein. 225 RI PT 221 2.8.2. Emulsifying activity and emulsion stability 227 The emulsifying activity and emulsion stability were measured by the turbidimetric 228 method as reported earlier with some modifications (Wang, Zhao, & Jiang, 2007). To 229 prepare the emulsion, 5 mL soybean oil was mixed with 20 mL 0.1% (w/v) sample 230 solution with different pH (3 to 11) by mechanical homogenizer (Ultra-Turrax T25, 231 IKA, Staufen, Germany) at 12000 rpm for 3 min separately. The emulsion (100 µL) 232 was diluted with 9.9 mL sodium dodecyl sulfate solution (0.1%, w/v). The 233 emulsifying activity and emulsion stability were calculated using the following 234 formula. TE D M AN U SC 226 235 2×2.303×A0 ×D EP Emulsifying activity ( m2 ⁄g )= AC C Emulsion stability (min)= C×V×W×104 A0 ×15 A0 -A15 236 Where A0 and A15 are the absorbance of emulsion at 500 nm after 0 min and 15 min. 237 D is dilution factor (D=100), C is the concentration of protein, V is the ratio of oil in 238 the emulsion (V=0.2). W is the width of optical path (W=0.01 m). 239 240 2.8.3. Differential scanning calorimetry analysis in thermal property 12 ACCEPTED MANUSCRIPT Protein samples were accurately injected into aluminum pans and subjected to 242 differential scanning calorimetry analysis (25 to 120 °C) with the heating rate of 10 243 °C/min (PE-8000, Perkin Elmer Ltd., Waltham, MA) (Kamboj et al., 2015). 244 Denaturation temperature (Td) and enthalpy changes of denaturation (∆H) were 245 calculated by Pyris software (Version 9.0, Perkin Elmer Ltd., Waltham, MA). RI PT 241 246 2.9. Statistical analysis 248 Data are represented as means ± standard deviation for three replications using SPSS 249 software (Vision 22.0, SPSS Inc., Chicago, Illinois). Differences were considered to 250 be significant at p < 0.05 according to Duncan’s Multiple Range Test. M AN U 251 SC 247 3. Results and discussions 253 3.1. Selection of Maillard reaction conditions 254 As shown in Fig. 1, the degree of graft and browning index of conjugates increased 255 first and then decreased as the increase in proportion of oat protein isolate and P. 256 ostreatus β-glucan, reaction time and pH. Under controlled conditions, the reaction 257 could form covalent bonds between protein and polysaccharide, meanwhile inhibiting 258 the formation of melanoidins at later reaction stage and achieving the desired 259 conjugate with excellent functionality finally (Hofmann, 1998; Hong et al., 2010). 260 Native oat protein isolates could react with few polysaccharides, while denatured oat 261 protein isolates could bind to more carbonyl groups of P. ostreatus β-glucan at the 262 proportion of 1:2 due to more exposure of lysine residues (de Oliveira et al., 2016). AC C EP TE D 252 13 ACCEPTED MANUSCRIPT However, the increased proportion of oat protein isolate and P. ostreatus β-glucan 264 from 1:3 to 1:5 could not cause an improved production of such conjugates because 265 the steric hindrance of polysaccharides limited the extent of Maillard reaction (Oliver, 266 Melton, & Stanley, 2006). As reaction time passed (from 1 to 5 days), the ε-amino 267 groups of lysine residues are gradually exposed on the surface of oat protein isolate, 268 leading to the formation of more conjugates. However, as the extension of reaction 269 time, the cross-link formed between oat protein isolate molecules could decrease the 270 content of their available free amino groups, leading to lower conjugation (Martins, 271 Jongen, & Majs, 2000). Therefore, the reaction time of 5 days was chosen as a 272 standard condition for the formation of conjugates. In addition, the formation of final 273 conjugates also depended on acid-base environment. As the increase of pH (from 4 to 274 8), the protonated ε-amino groups of lysine residues converted to active electrophile, 275 which is helpful to form more conjugates (Kato, 2002). However, higher pH (pH > 8) 276 could impel the conversion of Amadori compounds into advanced glycation end 277 products, leading to the poor functionality of oat protein isolate (Lertittikul, Benjakul, 278 & Tanaka, 2007). 279 Taken together, the desired conjugate was formed at pH 8.0, at the proportion of 1:2 280 (oat protein isolate to P. ostreatus β-glucan) and for 5 days (60 °C and 75% relative 281 humidity). Under this optimum condition, the degree of graft and browning index of 282 conjugates reached 26.27% and 0.45, respectively. AC C EP TE D M AN U SC RI PT 263 283 284 3.2. Evidence for the formation of oat protein isolate-P. ostreatus β-glucan conjugates 14 ACCEPTED MANUSCRIPT As shown in Fig. 2, 2 feature bands of lane A (25-35 kDa and 48-63 kDa) were related 286 to globulins. They were major fraction of oat protein isolate (Mirmoghtadaie, Kadivar, 287 & Shahedi, 2009). A new band of lane B appeared at about 135 kDa when oat protein 288 isolate was heated under the optimum condition. The cross-link formed by heat 289 treatment was between oat protein isolate molecules, leading to the formation of 290 dimmers (Chakraborti et al., 2010; Yadav et al., 2010). This phenomenon agreed with 291 the result in 3.1. The obvious band of the mixture (lane C) was similar to that of the 292 oat protein isolate (lane A) and this result was consistent with the previous study (Kim 293 & Shin, 2015). A characteristic band of lane D was visible on the top of the stacking 294 gel, indicating the covalent bond had formed between the amino groups of oat protein 295 isolate and carbonyl groups of P. ostreatus β-glucan. This phenomenon was consistent 296 with previous reports and the increased molecular weight of protein was considered as 297 a key indicator for the formation of conjugates after Maillard reaction 298 (Ledesma-Osuna et al., 2010; Xu et al., 2010; Sheng et al., 2017). Compared with 299 heated oat protein isolate (lane B), the obvious band of conjugate (lane D) at about 300 135 kDa did not disappear completely because part of oat protein isolates could 301 participate in the formation of cross-link. To some extent, it suggested that P. 302 ostreatus β-glucan could suppress the dimerization of oat protein isolate via Maillard 303 reaction (Bi et al., 2017; Loveday, 2016). AC C EP TE D M AN U SC RI PT 285 304 305 3.3. Characterization of oat protein isolate-P. ostreatus β-glucan conjugates 306 Protein-polysaccharide conjugate is a complex polymer and various conjugates can be 15 ACCEPTED MANUSCRIPT produced under different conditions (Maria et al., 2013). Recent reports focused on 308 studying the characterization of conjugates according to changes in the structure of 309 protein, including functional groups, micro-environment of tryptophan, hydrophobic 310 interaction, amino acid composition, secondary structure and microstructure (Alvarez 311 et al., 2012; Bund et al., 2012; Ledesma-Osuna et al., 2010). 312 RI PT 307 3.3.1. FT-IR analysis in functional groups of conjugates 314 As shown in Fig. 3, in the region between 3500 and 3000 cm-1, O-H stretching 315 vibration caused the appearance of absorption peaks of 4 samples. The absorption 316 peaks of oat protein isolate and heated oat protein isolate in the region of 1360-1310 317 cm-1 resulted from C-N stretching vibration. The C-O stretching vibration of 4 318 samples caused the appearance of absorption peaks at 1200-1000 cm-1. The absorption 319 peaks between 1470 and 1430 cm-1 corresponded to the CH3 deformation vibration of 320 the oat protein isolate and mixture. FT-IR could be used for an effective analysis of 321 protein-polysaccharide conjugate (Van Der Ven, et al., 2002). When the modifications 322 of functional groups occurred at a molecular level via Maillard reaction, FT-IR spectra 323 displayed new peaks and changes of the position or intensity (Deygen & Kudryashova, 324 2016). After covalent binding with the carbonyl group of P. ostreatus β-glucan, oat 325 protein isolate displayed a new absorption peak at 2880 cm-1 caused by C-H 326 stretching vibration. This characteristic peak also appeared in Maillard reaction 327 pattern of glucose and alanine (Mohsin et al., 2018). The characteristic absorption 328 peaks of conjugates at 1707 cm-1 and 1595 cm-1 were attributed to the C=O stretching AC C EP TE D M AN U SC 313 16 ACCEPTED MANUSCRIPT vibration and N-H deformation vibration. In the region between 985 and 995 cm-1, the 330 absorption peak of conjugate at 992 cm-1 resulted from the C-H deformation vibration 331 of oat protein isolate side chains (Gu et al., 2010). The absorption peak at 765 cm-1 in 332 the mixture was considered as the characteristic of glucose residues (Zhu, Xue, & 333 Zhang, 2016). Oat protein isolate, heated oat protein isolate and the mixture have 334 broad peaks at 812 cm-1, resulting from the products of protein degradation (Synytsya 335 et al., 2009). Otherwise, the similar phenomena observed above were proven in many 336 other studies (Chang & Tanaka, 2002; Du et al., 2013; Gu et al., 2010; Su et al., 337 2010). M AN U SC RI PT 329 338 3.3.2. Intrinsic fluorescence analysis of the micro-environment of tryptophan in 340 conjugates 341 As shown in Fig. 4A, compared with oat protein isolate, the fluorescence intensity of 342 heated oat protein isolate increased, while conjugate exhibited the lowest intensity 343 among groups. Fluorescence emission of tryptophan was extremely sensitive to 344 changes of the surrounding environment. It could be described as a great indicator for 345 monitoring the conformational transition of protein (Broersen et al., 2004). The 346 conformation of oat protein isolate turned from tight to extensional state after heat 347 treatment, causing that its internal tryptophan residues were gradually exposed to the 348 surface (Laura, Villamiel, & Rosina, 2007; Renard et al., 1998). We speculated that 349 heated oat protein isolate existed as a molten-globule state with a packed hydrophobic 350 core, causing its increased surface hydrophobicity (Kim & Shin, 2015). Compared AC C EP TE D 339 17 ACCEPTED MANUSCRIPT with the oat protein isolate, a slight increase exhibited in the fluorescence intensity of 352 mixture, indicating that hydrophobic environment could be more inclined to surround 353 the tryptophan residues of mixture than that of oat protein isolate (Choi et al., 2005). 354 After covalent binding with P. ostreatus β-glucan, the fluorescence intensity of oat 355 protein isolate decreased but no shift exhibited in such conjugate compared with 356 heated oat protein isolate. The long-chain P. ostreatus β-glucan had an effect of 357 steric-hindrance and could block the fluorescent signal of tryptophan residues from 358 oat protein isolate (Hattori et al., 1997; Jimenez et al., 2005). These results were 359 consistent with the previous studies (Ledesma-Osuna et al., 2010; Sheng et al., 2017). M AN U SC RI PT 351 360 3.3.3. Surface hydrophobicity of proteins in conjugates 362 As shown in Fig. 4B, in comparison with oat protein isolate, the surface 363 hydrophobicity of heated oat protein isolates increased to 2361 and the conjugate 364 decreased to 1207 significantly (p < 0.05). However, no significant difference 365 exhibited in surface hydrophobicity of proteins between the oat protein isolate and 366 mixture (p > 0.05). The surface hydrophobicity indicated the amount of hydrophobic 367 groups exposed on the surface of a protein molecule was used to evaluate the 368 conformational changes of protein. In the native state, most nonpolar amino acids 369 were served as hydrophobic core and polar amino acids often distributed on the 370 surface of protein (Sheng et al., 2017). The heat treatment increased the exposure of 371 native hydrophobic peptides, leading to an increased surface hydrophobicity of oat 372 protein isolate (Pirestani et al., 2017). It was consistent with the result of 3.3.2. The AC C EP TE D 361 18 ACCEPTED MANUSCRIPT conjugate had the lowest surface hydrophobicity of proteins due to the steric 374 hindrance of P. ostreatus β-glucan (Liu, Ru, & Ding, 2012). In addition, the 375 introduction of P. ostreatus β-glucan increased the amount of hydrophilic groups 376 exposed on the surface of conjugates via Maillard reaction, also leading to the 377 decreased surface hydrophobicity of oat protein isolates in conjugate (Mu et al., 2010). 378 This result could also explain that the improved solubility and emulsibility of oat 379 protein isolate after Maillard reaction. SC RI PT 373 M AN U 380 3.3.4. Amino acid composition of conjugates 382 As shown in table 1, the content of hydrophilic amino acids among groups were 383 higher than that of hydrophobic amino acids. The covalently binding to protein with 384 polysaccharide caused the decrease of several amino acids after Maillard reaction 385 (Chen et al., 2013). Compared with other groups, the contents of Cys and Lys in 386 conjugate significantly decreased to 0.138% and 3.037% (p < 0.05), suggesting that 387 Cys and Lys were the dominant binding sites of oat protein isolate and P. ostreatus 388 β-glucan (Thorpe & Baynes, 2003). The content of His in conjugate significantly 389 increased to 3.883% compared with other groups (p < 0.05) but changes in the content 390 of total amino acids were not significant (p > 0.05). The reason was that the increase 391 in the exposure of other unfolded amine groups could counteract the decrease of 392 amines via modification (Guo & Xiong, 2013). In addition, the conformational 393 changes around Lys residues could influence the reaction efficiency between other 394 amino groups and P. ostreatus β-glucan, and affect the content of amino groups AC C EP TE D 381 19 ACCEPTED MANUSCRIPT eventually (Chen et al., 2013). 396 3.3.5. Circular dichroism analysis in the secondary structure of conjugates 397 Fig. 5 shows circular dichroism spectrum of oat protein isolate, heated oat protein 398 isolate, the mixture and conjugation of oat protein isolate-P. ostreatus β-glucan in the 399 far-UV region of 190 to 260 nm. The spectrum reflected the structure of peptide in 400 protein cytoskeleton. The α-helical structure of four samples in the spectrogram had a 401 positive band around 192 nm and a negative band at 208 nm and 222 nm. A negative 402 band around 216 nm and a positive band ranging from 190 to 200 nm indicated the 403 existence of β-sheet. A positive band at about 206 nm suggested β-turn. 404 As shown in table 2, all samples contained four kinds of secondary structures. α- helix 405 and β-sheet are the dominant proportion in oat protein isolate (34.4% α- helix and 406 34.1% β-sheet) and conjugate (26.4% α- helix and 31.3% β-sheet). As for heated oat 407 protein isolate, the primary secondary structure is random coil (31.6%) and β-sheet 408 (30.8%). However, α-helix (34.7%) and random coil (26.9%) accounted for the 409 dominant proportion in mixture. Compared with oat protein isolate, heated oat protein 410 isolate and conjugate had the lower content of α-helix and β-sheet structure, indicating 411 that heat treatment could disturb hydrogen bonds in these two structures and forces 412 between oat protein isolate molecules (Sheng et al., 2017; Pirestani et al., 2017). The 413 mixture had higher contents of β-turn and random coil than oat protein isolate due to 414 the interaction between polysaccharide and protein (Li et al., 2014). Compared with 415 mixture (11.3% β-turn and 25.4% β-sheet), the significant increase showed in the 416 content of β-turn and β-sheet of conjugate (p < 0.05). This was because covalent AC C EP TE D M AN U SC RI PT 395 20 ACCEPTED MANUSCRIPT binding with P. ostreatus β-glucan could enhance the intermolecular interaction 418 between neighboring protein molecules via Maillard reaction (Mangavel et al., 2001). 419 In addition, the conjugate had higher random coil and lower α-helix than oat protein 420 isolate. The reason was that Maillard reaction caused oat protein isolate to unscrew 421 and transform into random coil (Li et al., 2014; Mangavel et al., 2001). These results 422 indicated that Maillard reaction could affect the extended secondary structure of oat 423 protein isolate (Qu et al., 2018). It was consistent with the result of the previous study 424 (Mu et al., 2010). M AN U SC RI PT 417 425 3.3.6. SEM analysis in the microstructure of conjugates 427 As shown in Fig. 6A, due to its sunken surface structure, oat protein isolate tended to 428 aggregate in an aqueous model system, leading to the decrease of solubility. After heat 429 treatment, oat protein isolate with the smooth particle had a crumblier structure and 430 appeared some visible cracks. This phenomenon was similar to structural changes of 431 bull serum albumin induced by heat treatment (Semasaka et al., 2018). The mixture 432 had similar surface structure to oat protein isolate. However, the surface structure of 433 conjugates became looser and more porous than that of other three samples and this 434 result was consistent with the previous finding (Mu et al., 2010). On the one hand, it 435 indicated that oat protein isolate was combined with P. ostreatus β-glucan molecules 436 firmly, leading to the formation of inhomogeneous and accumulated structure 437 (Boostani et al., 2017). On the other hand, this structure could be beneficial to 438 improve the efficiency of Maillard reaction. As confirmed by the results of intrinsic AC C EP TE D 426 21 ACCEPTED MANUSCRIPT 439 fluorescence and circular dichroism analysis in 3.3.2 and 3.3.5, after covalent binding 440 with P. ostreatus β-glucan, oat protein isolate with the extended structure also caused 441 its loose and porous surface structure. RI PT 442 3.4. Functional properties of oat protein isolate-P. ostreatus β-glucan conjugates 444 Functional properties of proteins, including solubility, emulsibility and thermal 445 stability, played a vital role in determining the quality of final products (Liu, Ru, & 446 Ding, 2012). The emulsibility and thermal stability contributed to food texture and 447 flavor. Furthermore, among these properties, solubility was a crucial property in food 448 industry because it directly affected other techno-functional properties (de Oliveira et 449 al., 2016). M AN U SC 443 TE D 450 3.4.1. Solubility 452 As shown in table 3, the solubility of heated oat protein isolate decreased as the 453 increase of pH compared with that of oat protein isolate. Heat treatment could 454 increase the surface hydrophobicity of oat protein isolate as confirmed in the result of 455 3.3.3, leading to the decrease of its solubility. Furthermore, the zeta-potential of oat 456 protein isolate, heated oat protein isolate and the mixture was 0 mV where pH was 457 around 4.7 (Fig. S1), suggesting that the isoelectric point of these three samples was 458 about 4.7. Therefore, their solubility was relatively low when pH (pH 4.0 and 5.0) 459 was near the isoelectric point. After covalent binding with P. ostreatus β-glucan, the 460 solubility of oat protein isolate could be greatly improved. On the one hand, it is due AC C EP 451 22 ACCEPTED MANUSCRIPT to that the introduction of P. ostreatus β-glucan could increase the amount of 462 hydrophilic groups and enhance the steric stabilization of oat protein isolate 463 (Jimenez-Castano, Villamiel, & Lopez-Fandino, 2007; Mu et al., 2010). On the other 464 hand, Maillard reaction could shift the isoelectric point of oat protein isolate to a more 465 acidic region (Fig. S1), causing that the solubility of conjugate could increase in a 466 broad range of pH. In addition, the limited extent of Maillard reaction could increase 467 the solubility of oat protein isolate due to the restricted attack of hydrophilic 468 polysaccharide residues to the protein (de Oliveira et al., 2016). The previous study 469 confirmed that the lowest solubility exhibited in whey protein isolate and whey 470 protein isolate-maltodextrin conjugate at pH 5.0 and pH 4.0, respectively 471 (Martinez-Alvarenga et al., 2014). However, egg white protein-pectin conjugate was 472 reported to have lower solubility than original egg white protein as the increase of 473 reaction time (Al-Hakkak & Al-Hakkak, 2010). This contradiction resulted from the 474 biochemical complexity of reacting proteins and polysaccharide (Oliver, Melton, & 475 Stanley, 2006). In addition, the increased heat time promoted the development of 476 advanced stage of Maillard reaction, then leading to the lower solubility of protein. It 477 suggested that the controlled conditions in our study could prevent the formation of 478 advanced glycation end products. SC M AN U TE D EP AC C 479 RI PT 461 480 3.4.2. Emulsifying activity and emulsion stability 481 As shown in Fig. 6B-1, heated oat protein isolate had lower emulsifying activity than 482 oat protein isolate as the increase of pH. The reason was that heat treatment could 23 ACCEPTED MANUSCRIPT decrease the solubility of oat protein isolate as confirmed by the result of 3.4.1, 484 causing that few oat protein isolates could be adsorbed on the oil-water interface 485 (Zhang et al., 2015). In addition, the emulsifying activity of mixture increased 486 compared with that of oat protein isolate in a broad range of pH. However, after 487 covalent binding with P. ostreatus β-glucan, the emulsifying activity of oat protein 488 isolate could be considerably improved. The macromolecular P. ostreatus β-glucan in 489 conjugate could form long range steric repulsion between the surface of emulsion 490 droplets. In addition, it could also promote the formation of a stable membrane around 491 the oil droplets, which was conducive to increase the emulsifying activity of oat 492 protein isolate (Dickinson, 2009; Guo & Xiong, 2013; Qi et al., 2010). More 493 hydrophilic groups of conjugates could increase their solubility as confirmed by the 494 result of 3.4.1, leading to an improved emulsifying activity of conjugate. 495 As shown in Fig. 6B-2, except from conjugate, the emulsion stability of samples went 496 up first but then it went down as the increase of pH. Compared with oat protein isolate 497 (31.921 min) at pH 7, the highest emulsion stability of heated oat protein isolate 498 decreased to 28.77 min due to its lower solubility. In addition, mixture had the highest 499 emulsion stability (41.84 min) at pH 8.0, indicating that the addition of P. ostreatus 500 β-glucan could improve the emulsibility of original oat protein isolate and this result 501 was consistent with the previous study (Pirestani et al., 2017). Compared with the 502 mixture, the emulsion stability of conjugate arrived at 50.186 min at pH 8.0 owing to 503 its excellent solubility. After covalent binding with P. ostreatus β-glucan, oat protein 504 isolates were adsorbed on the oil-water interface and could be protected against the AC C EP TE D M AN U SC RI PT 483 24 ACCEPTED MANUSCRIPT instability in the acid environment. On the other hand, conjugate could saturate the 506 surface layer because its surface was much more active than that of the protein or 507 polysaccharide, leading to an improved emulsibility of oat protein isolate. 508 Furthermore, as confirmed by the result of SEM in 3.3.6, the incompact structure on 509 the surface of conjugate could also contribute to the adsorption of protein to the 510 oil-water interface. RI PT 505 SC 511 3.4.3. Thermal property 513 As shown in Fig. 6C, the negative peak at 101.387 °C was a main decalescence peak 514 of oat protein isolate as well as one endothermic transition of heated oat protein 515 isolate at 102.75 °C. However, the denaturation temperature of the conjugate (105.645 516 °C) was significantly higher than that of the mixture (103.091 °C) (p < 0.05). Protein 517 denaturation indicated the non-covalent interactions maintaining the tertiary structure 518 were destroyed, including Van der Waals force, hydrogen bond, hydrophobic and 519 disulfide bond (Boye & Alli, 2000). The result revealed that Maillard reaction could 520 enhance the thermal stability of oat protein isolate through the differential interactions 521 and a combination of excluded volume (Timasheff, 1998). In addition, the 522 introduction of P. ostreatus β-glucan could increase the steric exclusion and 523 electrostatic repulsion of conjugates, preventing the aggregation of oat protein isolate 524 at the high temperature (Robitaille & Ayers, 1995). It was consistent with the 525 extended secondary structure as confirmed from the results of FT-IR and circular 526 dichroism in 3.3.1 and 3.3.5. The result also suggested that oat protein isolate had the AC C EP TE D M AN U 512 25 ACCEPTED MANUSCRIPT lowest denaturation temperature among these samples, indicating that it may be easier 528 to expose more available lysine residues during heat treatment (Liu et al., 2012). 529 Enthalpy value (∆H) was related to the ordered structure content, which determined 530 the requirement of energy to overcome these non-covalent interactions during the 531 denaturation of protein (Choi, Mine, & Ma, 2006). As shown in table 4, conjugate had 532 the lowest ∆H of 2.598 J/g and the mixture had the highest enthalpy value, causing the 533 disruption of intramolecular force in oat protein isolate. These results suggested that 534 oat protein isolate modified by P. ostreatus β-glucan maintained thermally stable. It 535 was related to the decreased content of α-helix and β-sheet of conjugates as confirmed 536 in the result of 3.3.5. M AN U SC RI PT 527 537 Conclusions 539 The conjugate was mainly formed between the carbonyl group of P. ostreatus 540 β-glucan and lysine and cysteine of oat protein isolate via Maillard reaction, leading 541 to the stretching and deformation vibration of functional groups. After covalent 542 binding with P. ostreatus β-glucan, the incompact surface structure and decreased 543 surface hydrophobicity of oat protein isolate caused its increased solubility and 544 emulsibility. The introduction of P. ostreatus β-glucan enhanced the thermal stability 545 of oat protein isolate due to its extended secondary structure induced by Maillard 546 reaction. Under the controlled condition, Maillard reaction was an effective way to 547 improve the application potentials of oat protein isolate in food processing. This study 548 established a framework that can be used to achieve excellent functionalities in other AC C EP TE D 538 26 ACCEPTED MANUSCRIPT modification of proteins and polysaccharides. 550 Acknowledgements 551 This work was supported by the State Key Research and Development Plan "Modern 552 food processing, storage and transportation technology and equipment (No. 553 2017YFD0400205), and a project funded by the Priority Academic Program 554 Development of Jiangsu Higher Education Institutions (PAPD). RI PT 549 SC 555 M AN U 556 557 558 559 563 564 565 566 567 EP 562 AC C 561 TE D 560 568 569 570 27 ACCEPTED MANUSCRIPT 571 Reference 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 Al-Hakkak, J., & Al-Hakkak, F. (2010). Functional egg white-pectin conjugates prepared by controlled Maillard reaction. Journal of Food Engineering, 100(1), 152-159. Akhtar, M., & Ding, R. (2017). Covalently cross-linked proteins & polysaccharides: Formation, characterisation and potential applications. Current Opinion in Colloid & Interface Science, 28, 31-36. Alvarez, C., Garcia, V., Rendueles, M., & Diaz, M. (2012). Functional properties of isolated porcine blood proteins modified by Maillard's reaction. Food Hydrocolloids, 28(2), 267-274. Bi, B., Yang, H., Fang, Y., Nishinari, K., & Phillips, G. O. (2017). Characterization and emulsifying properties of beta-lactoglobulin-gum Acacia seyal conjugates prepared via the Maillard reaction. Food Chemistry, 214, 614-621. Boye, J. I., & Alli, I. (2000). Thermal denaturation of mixtures of alpha-lactalbumin and beta-lactoglobulin: A differential scanning calorimetric study. Food Research International, 33(8), 673-682. Boostani, S., Aminlari, M., Moosavi-nasab, M., Niakosari, M., & Mesbahi, G. (2017). Fabrication and characterisation of soy protein isolate-grafted dextran biopolymer: A novel ingredient in spray-dried soy beverage formulation. International Journal of Biological Macromolecules, 102, 297-307. Broersen, K., Voragen, A. G. J., Hamer, R. J., & de Jongh, H. H. J. (2004). Glycoforms of beta-lactoglobulin with improved thermostability and preserved structural packing. Biotechnology and Bioengineering, 86(1), 78-87. Bund, T., Allelein, S., Arunkumar, A., Lucey, J. A., & Etzel, M. R. (2012). Chromatographic purification and characterization of whey protein-dextran glycation products. Journal of Chromatography A, 1244, 98-105. Chakraborti, S., Chatterjee, T., Joshi, P., Poddar, A., Bhattacharyya, B., Singh, S. P., Gupta, V., & Chakrabarti, P. (2010). Structure and activity of lysozyme on binding to ZnO nanoparticles. Langmuir: the ACS Journal of Surfaces and Colloids, 26(5), 3506-3513. Chang, M. C., & Tanaka, J. (2002). FT-IR study for hydroxyapatite/collagen nanocomposite cross-linked by glutaraldehyde. Biomaterials, 23(24), 4811-4818. Chen, H., Wang, P., Wu, F., Xu, J., Tian, Y., Yang, N., Cissouma, A. I., Jin, Z., & Xu, X. (2013). Preparation of phosvitin-dextran conjugates under high temperature in a liquid system. International Journal of Biological Macromolecules, 55, 258-263. Choi, Kim, H. J., Park, K. H., & Moon, T. W. (2005). Molecular characteristics of ovalbumin-dextran conjugates formed through the Maillard reaction. Food Chemistry, 92(1), 93-99. Choi, S., Mine, Y., & Ma, C. (2006). Characterization of heat-induced aggregates of AC C EP TE D M AN U SC RI PT 572 28 ACCEPTED MANUSCRIPT EP TE D M AN U SC RI PT globulin from common buckwheat (Fagopyrum esculentum Moench). International Journal of Biological Macromolecules, 39(4-5), 201-209. de Oliveira, F. C., dos Reis Coimbra, J. S., de Oliveira, E. B., Giraldo Zuniga, A. D., & Garcia Rojas, E. E. (2016). Food protein-polysaccharide conjugates obtained via the Maillard reaction: A review. Critical Reviews in Food Science and Nutrition, 56(7), 1108-1125. De Silva, D. D., Rapior, S., Sudarman, E., Stadler, M., Xu, J., Alias, S. A., & Hyde, K. D. (2013). Bioactive metabolites from macrofungi: ethnopharmacology, biological activities and chemistry. Fungal Diversity, 62(1), 1-40. Deygen, I. M., & Kudryashova, E. V. (2016). New versatile approach for analysis of PEG content in conjugates and complexes with biomacromolecules based on FTIR spectroscopy. Colloids and Surfaces B-Biointerfaces, 141, 36-43. Dickinson, E. (2009). Hydrocolloids as emulsifiers and emulsion stabilizers. Food Hydrocolloids, 23, 1473–1482. Du, Y., Shi, S., Jiang, Y., Xiong, H., Woo, M. W., Zhao, Q., Bai, C., Zhou, Q., & Sun, W. (2013). Physicochemical properties and emulsion stabilization of rice dreg glutelin conjugated with kappa-carrageenan through Maillard reaction. Journal of the Science of Food and Agriculture, 93(1), 125-133. Fang, D., Yang, W., Kimatu, B. M., Zhao, L., An, X., & Hu, Q. (2017). Comparison of flavour qualities of mushrooms (Flammulina velutipes) packed with different packaging materials. Food Chemistry, 232, 1-9. Galazka, V. B., Dickinson, E., & Ledward, D. A. (2000). Influence of high pressure processing on protein solutions and emulsions. Current Opinion in Colloid & Interface Science, 5(3-4), 182-187. Grigg, D. (1995). The pattern of world protein consumption. Geoforum, 26, 1-17. Gu, F.-L., Kim, J. M., Abbas, S., Zhang, X.-M., Xia, S.-Q., & Chen, Z.-X. (2010). Structure and antioxidant activity of high molecular weight Maillard reaction products from casein-glucose. Food Chemistry, 120(2), 505-511. Guo, X., & Xiong, Y. L. (2013). Characteristics and functional properties of buckwheat protein-sugar schiff base complexes. Lwt-Food Science and Technology, 51(2), 397-404. Hattori, M., Ogino, A., Nakai, H., & Takahashi, K. (1997). Functional improvement of beta-lactoglobulin by conjugating with alginate lyase-lysate. Journal of Agricultural and Food Chemistry, 45(3), 703-708. Hofmann, T. (1998). Characterization of the chemical structure of novel colored Maillard reaction products from furan-2-carboxaldehyde and amino acids. Journal of Agricultural and Food Chemistry, 46(3), 932-940. Hong, J. H., Jung, D. W., Kim, Y. S., Lee, S. M., & Kim, K. O. (2010). Impacts of glutathione Maillard reaction products on sensory characteristics and consumer acceptability of beef soup. Journal of Food science, 75(8), 427-434. Jimenez, C. L., Lopez-Fandino, R., Olano, A., & Villamiel, M. (2005). Study on beta-lactoglobulin glycosylation with dextran: Effect on solubility and heat stability. Food Chemistry, 93(4), 689-695. Jimenez-Castano, L., Villameil, M., & Lopez-Fandino, R. (2007). Glycosylation of AC C 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 29 ACCEPTED MANUSCRIPT EP TE D M AN U SC RI PT individual whey proteins by Maillard reaction using dextran of different molecular mass. Food Hydrocolloids, 21, 433-443. Julita, R., & Marek, S. (2007). Dried shiitake (Lentinulla edodes) and oyster (Pleurotus ostreatus) mushrooms as a good source of nutrient. Acta Scientiarum Polonorum Technologia Alimentaria, 6(4), 339-343. Jung, S., Rickert, D. A., Deak, N. A., Aldin, E. D., Recknor, J., Johnson, L. A., & Murphy, P. A. (2003). Comparison of Kjeldahl and Dumas methods for determining protein contents of soybean products. Journal of the American Oil Chemists Society, 80(12), 1169-1173. Kamboj, S., Singh, K., Tiwary, A. K., & Rana, V. (2015). Optimization of microwave assisted Maillard reaction to fabricate and evaluate corn fiber gum-chitosan IPN films. Food Hydrocolloids, 44, 260-276. Kato, A. (2002). Industrial applications of Maillard-type protein-polysaccharide conjugates. Food Science and Technology Research, 8(3), 193-199. Kato, A., Sasaki, Y., Furuta, R., & Kobayashi, K. (1990). Functional protein polysaccharide conjugate prepared by controled dry-heating of ovalbumin dextran mixtures. Agricultural and Biological Chemistry, 54(1), 107-112. Khan, A. A., Gani, A., Shah, A., Masoodi, F. A., Hussain, P. R., Wani, I. A., & Khanday, F. A. (2015). Effect of gamma-irradiation on structural, functional and antioxidant properties of beta-glucan extracted from button mushroom (Agaricus bisporus). Innovative Food Science & Emerging Technologies, 31, 123-130. Kim, D.-Y., & Shin, W.-S. (2015). Characterisation of bovine serum albuminfucoidan conjugates prepared via the Maillard reaction. Food Chemistry, 173, 1-6. Klose, C., & Arendt, E. K. (2012). Proteins in oats; their synthesis and changes during germination: A review. Critical Reviews in Food Science and Nutrition, 52(7), 629-639. Laura, J. C., Villamiel, M., & Rosina, L.-F. (2007). Glycosylation of individual whey proteins by Maillard reaction using dextran of different molecular mass. Food Hydrocolloids, 21(3), 433-443. Ledesma-Osuna, A. I., Ramos-Clamont, G., Guzman-Partida, A. M., & Vazquez-Moreno, L. (2010). Conjugates of bovine serum albumin with chitin oligosaccharides prepared through the Maillard Reaction. Journal of Agricultural and Food Chemistry, 58(22), 12000-12005. Lertittikul, W., Benjakul, S., & Tanaka, M. (2007). Characteristics and antioxidative activity of Maillard reaction products from a porcine plasma protein–glucose model system as influenced by pH. Food Chemistry, 100(2), 669-677. Li, C., Xue, H., Chen, Z., Ding, Q., & Wang, X. (2014). Comparative studies on the physicochemical properties of peanut protein isolate-polysaccharide conjugates prepared by ultrasonic treatment or classical heating. Food Research International, 57, 1-7. Liu, Y., Zhao, G., Zhao, M., Ren, J., & Yang, B. (2012). Improvement of functional properties of peanut protein isolate by conjugation with dextran through AC C 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 30 ACCEPTED MANUSCRIPT EP TE D M AN U SC RI PT Maillard reaction. Food Chemistry, 131, 901-906. Liu, J., Ru, Q., & Ding, Y. (2012). Glycation a promising method for food protein modification: Physicochemical properties and structure, A review. Food Research International, 49(1), 170-183. Loveday, S. M. (2016). Beta-lactoglobulin heat denaturation: a critical assessment of kinetic modelling. International Dairy Journal, 52, 92-100. Ma, C. Y. (1985). Functional properties of oat concentrate treated with linoleate ortrypsin. Canadian Institute of Food Science and Technology Journal, 18, 79–84. Mangavel, C., Barbot, J., Popineau, Y., & Gueguen, J. (2001). Evolution of wheat gliadins conformation during film formation: A fourier transform infrared study. Journal of Agricultural and Food Chemistry, 49(2), 867-872. Martinez-Alvarenga, M. S., Martinez-Rodriguez, E. Y., Garcia-Amezquita, L. E., Olivas, G. I., Zamudio-Flores, P. B., Acosta-Muniz, C. H., & Sepulveda, D. R. (2014). Effect of Maillard reaction conditions on the degree of glycation and functional properties of whey protein isolate-Maltodextrin conjugates. Food Hydrocolloids, 38, 110-118. Maria, J. S., Perduca, M. J., Piagentini, A., Santiago, L. G., Rubiolo, A. C., & Carrara, C. R. (2013). Gel mechanical properties of milk whey protein-dextran conjugates obtained by Maillard reaction. Food Hydrocolloids, 31(1), 26-32. Martins, S., Jongen, W. M. F., & Majs, v. B. (2000). A review of Maillard reaction in food and implications to kinetic modelling. Trends in Food Science & Technology, 11(9-10), 364-373. Mirmoghtadaie, L., Kadivar, M., & Shahedi, M. (2009). Effects of succinylation and deamidation on functional properties of oat protein isolate. Food Chemistry, 114(1), 127-131. Mohamed, A., Biresaw, G., Xu, J., Hojilla-Evangelista, M. P., & Rayas-Duarte, P. (2009). Oats protein isolate: thermal, rheological, surface and functional properties. Food Research International, 42(1), 107-114. Mohsin, G. F., Schmitt, F.-J., Kanzler, C., Epping, J. D., Flemig, S., & Hornemann, A. (2018). Structural characterization of melanoidin formed from D-glucose and L-alanine at different temperatures applying FTIR, NMR, EPR, and MALDI-ToF-MS. Food Chemistry, 245, 761-767. Mu, L., Zhao, H., Zhao, M., Cui, C. and Liu, L. (2011). Physicochemical properties of soy protein isolates-acacia gum conjugates. Czech Journal of Food Sciences, 29(2), 129-136. Mu, L., Zhao, M., Yang, B., Zhao, H., Cui, C., & Zhao, Q. (2010). Effect of ultrasonic treatment on the graft reaction between soy protein isolate and gum acacia and on the physicochemical properties of conjugates. Journal of Agricultural and Food Chemistry, 58(7), 4494-4499. Oliver, C. M., Melton, L. D., & Stanley, R. A. (2006). Creating proteins with novel functionality via the Maillard reaction: A review. Critical Reviews in Food Science and Nutrition, 46(4), 337-350. Pirestani, S., Nasirpour, A., Keramat, J., & Desobry, S. (2017). Preparation of AC C 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 31 ACCEPTED MANUSCRIPT EP TE D M AN U SC RI PT chemically modified canola protein isolate with gum Arabic by means of Maillard reaction under wet-heating conditions. Carbohydrate Polymers, 155, 201-207. Pirestani, S., Nasirpour, A., Keramat, J., Desobry, S., & Jasniewski, J. (2018). Structural properties of canola protein isolate-gum Arabic Maillard conjugate in an aqueous model system. Food Hydrocolloids, 79, 228-234. Qi, J.-r., Liao, J.-s., Yin, S.-w., Zhu, J., & Yang, X.-q. (2010). Formation of acid-precipitated soy protein-dextran conjugates by Maillard reaction in liquid systems. International Journal of Food Science and Technology, 45(12), 2573-2580. Qu, W., Zhang, X., Han, X., Wang, Z., He, R., & Ma, H. (2018). Structure and functional characteristics of rapeseed protein isolate dextran conjugates. Food Hydrocolloids, 82, 329-337. Renard, D., Lefebvre, J., Griffin, M. C. A., & Griffin, W. G. (1998). Effects of pH and salt environment on the association of beta-lactoglobulin revealed by intrinsic fluorescence studies. International Journal of Biological Macromolecules, 22(1), 41-49. Robitaille, G., & Ayers, C. (1995). Effects of kappa-casein glycosylation on heat stability of milk. Food Research International, 28, 17-21. Sheng, L., Su, P., Han, K., Chen, J., Cao, A., Zhang, Z., Lin, Y., & Ma, M. (2017). Synthesis and structural characterization of lysozyme-pullulan conjugates obtained by the Maillard reaction. Food Hydrocolloids, 71, 1-7. Singh, A. S. S. (2015). Physicochemical, thermal, rheological and morphological characteristics of starch from three indian lotus root (Nelumbo Nucifera Gaertn) cultivars. Journal of Food Processing & Technology, 06(2), 1-8. Snyder, S. L., & Sobocinski, P. Z. (1975). An improved 2,4,6-trinitrobenzenesulfonic acid method for the determination of amines. Analytical Biochemistry, 64(1), 284-288. Su, J.-F., Huang, Z., Yuan, X.-Y., Wang, X.-Y., & Li, M. (2010). Structure and properties of carboxymethyl cellulose/soy protein isolate blend edible films crosslinked by Maillard reactions. Carbohydrate Polymers, 79(1), 145-153. Synytsya, A., Mickova, K., Synytsya, A., Jablonsky, I., Spevacek, J., Erban, V., Kovarikova, E., & Copikova, J. (2009). Glucans from fruit bodies of cultivated mushrooms Pleurotus ostreatus and Pleurotus eryngii: Structure and potential prebiotic activity. Carbohydrate Polymers, 76(4), 548-556. Szwengiel, A., & Stachowiak, B. (2016). Deproteinization of water-soluble beta-glucan during acid extraction from fruiting bodies of Pleurotus ostreatus mushrooms. Carbohydrate Polymers, 146, 310-319. Semasaka, C., Mhaske, P., Buckow, R., & Kasapis, S. (2018). Modeling water partition in composite gels of BSA with gelatin following high pressure treatment. Food Chemistry, 265, 32-38. Thammakiti, S., Suphantharika, M., Phaesuwan, T., & Verduyn, C. (2004). Preparation of spent brewer's yeast β-glucans for potential applications in the food industry. International Journal of Food Science & Technologies, 39, 21– AC C 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 32 ACCEPTED MANUSCRIPT EP TE D M AN U SC RI PT 29. Thorpe, S. R., & Baynes, J. W. (2003). Maillard reaction products in tissue proteins: New products and new perspectives. Amino Acids, 25(3-4), 275-281. Tian, S., Chen, J., & Small, D. M. (2011). Enhancement of solubility and emulsifying properties of soy protein isolates by glucose conjugation. Journal of Food Processing and Preservation, 35(1), 80-95. Timasheff, S. N. (1998). Control of protein stability and reactions by weakly interacting cosolvents: the simplicity of the complicated. Advances in Protein Chemistry, 51, 355-432. Usui, M., Tamura, H., Nakamura, K., Ogawa, T., Muroshita, M., Azakami, H., Kanuma, S., & Kato, A. (2004). Enhanced bactericidal action and masking of allergen structure of soy protein by attachment of chitosan through Maillard-type protein-polysaccharide conjugation. Nahrung-Food, 48(1), 69-72. Van Der Ven, C., Muresan, S., Gruppen, H., de Bont, D. B., Merck, K. B., & Voragen, A. G. (2002). FTIR spectra of whey and casein hydrolysates in relation to their functional properties. Journal of Agricultural and Food Chemistry, 50(24), 6943–6950. Wang, J. S., Zhao, M. M., & Jiang, Y. M. (2007). Effects of wheat gluten hydrolysateand its ultrafiltration fractions on dough properties and bread quality. Food Technology and Biotechnology, 45, 410–414. Xu, C.-h., Yu, S.-j., Yang, X.-q., Qi, J.-r., Lin, H., & Zhao, Z.-g. (2010). Emulsifying properties and structural characteristics of beta-conglycinin and dextran conjugates synthesised in a pressurised liquid system. International Journal of Food Science and Technology, 45(5), 995-1001. Yadav, M. P., Parris, N., Johnston, D. B., Onwulata, C. I., & Hicks, K. B. (2010). Corn fiber gum and milk protein conjugates with improved emulsion stability. Carbohydrate Polymers, 81(2), 476-483. Zhang, B., Guo, X., Zhu, K., Peng, W., & Zhou, H. (2015). Improvement of emulsifying properties of oat protein isolate-dextran conjugates by glycation. Carbohydrate Polymers, 127, 168-175. Zhao, C.-B., Zhang, H., Xu, X.-Y., Cao, Y., Zheng, M.-Z., Liu, J.-S., & Wu, F. (2017). Effect of acetylation and succinylation on physicochemical properties and structural characteristics of oat protein isolate. Process Biochemistry, 57, 117-123. Zhu, W., Xue, X., & Zhang, Z. (2016). Ultrasonic-assisted extraction, structure and antitumor activity of polysaccharide from polygonum multiflorum. International Journal of Biological Macromolecules, 91, 132-142. Zhuo, X.-Y., Qi, J.-R., Yin, S.-W., Yang, X.-Q., Zhu, J.-H., & Huang, L.-X. (2013). Formation of soy protein isolate-dextran conjugates by moderate Maillard reaction in macromolecular crowding conditions. Journal of the Science of Food and Agriculture, 93(2), 316-323. AC C 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 33 ACCEPTED MANUSCRIPT 832 833 Figure caption 835 Fig. 1. Effect of different reaction conditions on the degree of graft and browning 836 index from Maillard reaction products. A: effect of the proportion of OPI and GPO 837 (heated at pH 6.0 and for 5 d); B: effect of reaction time (heated at pH 6.0 with OPI to 838 GPO proportion of 1:3); C: Effect of pH (heated for 5 d with OPI to GPO proportion 839 of 1:3); OPI: oat protein isolate; GPO: β-glucan from P. ostreatus. 840 Fig. 2. SDS-PAGE of OPI, heated OPI, the mixture of OPI-GPO and OPI-GPO 841 conjugate (A). Marker (11-180 kDa); lane A: OPI; lane B: Heated OPI; lane C: The 842 mixture of OPI-GPO; lane D: OPI-GPO conjugate. OPI: oat protein isolate; Heated 843 OPI: heated oat protein isolate; The mixture of OPI-GPO: the mixture of oat protein 844 isolate and β-glucan from P. ostreatus; OPI-GPO conjugate: oat protein isolate and 845 β-glucan from P. ostreatus conjugate. 846 Fig. 3. The fourier transform infrared spectroscopy of OPI, heated OPI, the mixture of 847 OPI-GPO and OPI-GPO conjugate. OPI: oat protein isolate; heated OPI: heated oat 848 protein isolate; The mixture of OPI-GPO: the mixture of oat protein isolate and 849 β-glucan from P. ostreatus; OPI-GPO conjugate: oat protein isolate and β-glucan from 850 P. ostreatus conjugate. 851 Fig. 4. The intrinsic fluorescence of OPI, heated-OPI, the mixture of OPI-GPO and 852 OPI-GPO conjugate (A). Surface hydrophobicity of OPI, heated OPI, the mixture of 853 OPI-GPO and OPI-GPO conjugate (B). OPI: oat protein isolate; heated OPI: heated AC C EP TE D M AN U SC RI PT 834 34 ACCEPTED MANUSCRIPT oat protein isolate; The mixture of OPI-GPO: the mixture of oat protein isolate and 855 β-glucan from P. ostreatus; OPI-GPO conjugate: oat protein isolate and β-glucan from 856 P. ostreatus conjugate. 857 Fig. 5. The far-UV CD of OPI, heated OPI, the mixture of OPI-GPO and OPI-GPO 858 conjugate. OPI: oat protein isolate; heated OPI: heated oat protein isolate; The 859 mixture of OPI-GPO: the mixture of oat protein isolate and β-glucan from P. ostreatus; 860 OPI-GPO conjugate: oat protein isolate and β-glucan from P. ostreatus conjugate. 861 Fig. 6. The surface structure of OPI, heated OPI, the mixture of OPI-GPO, OPI-GPO 862 conjugate (A). The emulsifying activity (B-1) and emulsion stability (B-2) of OPI, 863 heated OPI, the mixture of OPI-GPO, OPI-GPO conjugate. The thermal property of 864 OPI, heated OPI, the mixture of OPI-GPO, OPI-GPO conjugate (C). OPI: oat protein 865 isolate; heated OPI: heated oat protein isolate; The mixture of OPI-GPO: the mixture 866 of oat protein isolate and β-glucan from P. ostreatus; OPI-GPO conjugate: oat protein 867 isolate and β-glucan from P. ostreatus conjugate. AC C EP TE D M AN U SC RI PT 854 35 ACCEPTED MANUSCRIPT Table 1. Analysis of amino acid composition of OPI, heated OPI, the mixture of OPI-GPO and OPI-GPO conjugate. Content (%) The mixture of OPI-GPO OPI-GPO conjugate 5.668 ± 0.492 a 5.908 ± 0.421 a 5.780 ± 0.237 a 1.377 ± 0.122 a 4.102 ± 0.393 a 8.901 ± 0.567 a 6.706 ± 0.530 a 4.782 ± 0.461 a 43.224 ± 3.892 a 5.692 ± 0.321 a 5.951 ± 0.443 a 5.660 ± 0.312 a 1.452 ± 0.018 a 3.967 ± 0.226 a 8.667 ± 0.619 a 6.766 ± 0.471 a 4.821 ± 0.365 a 42.975 ± 2.197 a 5.387 ± 0.476 a 5.892 ± 0.413 a 5.721 ± 0.404 a 1.342 ± 0.053 a 4.021 ± 0.271 a 8.762 ± 0.61 a 6.692 ± 0.482 a 4.679 ± 0.371 a 42.496 ± 4.109 a 5.591 ± 0.344 a 5.677 ± 0.314 a 5.881 ± 0.437 a 1.313 ± 0.084 a 3.895 ± 0.198 a 8.950 ± 0.501 a 6.610 ± 0.182 a 4.721 ± 0.329 a 42.638 ± 1.491 a 5.536 ± 0.128 a 4.612 ± 0.363 a 21.706 ± 1.138 a 5.048 ± 0.282 a 2.325 ± 0.183 a 4.516 ± 0.189 a 0.696 ± 0.051 b 5.115 ± 0.409 a 7.223 ± 0.537 a 56.776 ± 3.98 a 5.870 ± 0.342 a 4.494 ± 0.355 a 21.617 ± 1.828 a 4.889 ± 0.364 a 2.303 ± 0.176 a 4.063 ± 0.29 a 0.629 ± 0.011 b 4.879 ± 0.357 a 7.280 ± 0.618 a 57.025 ± 3.196 a 5.652 ± 0.427 a 4.772 ± 0.374 a 22.012 ± 2.129 a 5.128 ± 0.330 a 2.350 ± 0.205 a 4.492 ± 0.357 a 0.895 ± 0.059 b 5.021 ± 0.243 a 7.182 ± 0.705 a 57.504 ± 5.161 a 5.741 ± 0.439 a 4.878 ± 0.119 a 22.471 ± 1.86 a 5.228 ± 0.183 a 0.138 ± 0.09 b 4.609 ± 0.372 a 3.883 ± 0.277 a 3.037 ± 0.301 b 7.376 ± 0.62 a 57.362 ± 3.811 a SC RI PT Heated OPI M AN U Hydrophobicity Thr Ala Val Met Ile Leu Phe Pro Total Hydrophile Asp Ser Glu Gly Cys Tyr His Lys Arg Total OPI TE D Amino acid EP Values were expressed as mean ± standard deviation values (n=3). Different letters within the AC C same amino acid component showed a significant difference (p < 0.05). OPI: oat protein isolate; Heated OPI: Heated oat protein isolate; The mixture of OPI-GPO: The mixture of oat protein isolate and β-glucan from P. ostreatus; OPI-GPO conjugate: Oat protein isolate and β-glucan from P. ostreatus conjugate. ACCEPTED MANUSCRIPT Table 2. Calculation of secondary structures of OPI, heated OPI, the mixture of OPI-GPO and OPI-GPO conjugate. Secondary structure (%) OPI Heated OPI The mixture of OPI-GPO OPI-GPO conjugate α-Helix 34.4 ± 2.6 a 23.9 ± 2.1 c 34.7 ± 3.5 a 26.4 ± 2.5 b β-Sheet 34.1 ± 1.6 a 30.8 ± 3.3 b 25.4 ± 1.5 c 31.3 ± 2.5 b β-Turn 10.1 ± 0.9 c 11.7 ± 1.3 b 11.3 ± 1.4 b 17.6 ± 1.5 a Random coil 19.7 ± 1.4 d 31.6 ± 2.8 a 26.9 ± 1.5 b 24.0 ± 1.2 c RI PT Sample Values were expressed as mean ± standard deviation values (n=3). Different letters indicated SC significant difference (p < 0.05) in the same column. OPI: oat protein isolate; Heated OPI: heated M AN U oat protein isolate; The mixture of OPI-GPO: the mixture of oat protein isolate and β-glucan from P. ostreatus; OPI-GPO conjugate: oat protein isolate and β-glucan from P. ostreatus conjugate. Table 3. The solubility of OPI, heated OPI, the mixture of OPI-GPO and OPI-GPO conjugate. Solubility (%) 73.871 ± 2.012 b 5.511 ± 0.124 c 10.25 ± 0.621 c 51.762 ± 1.98 c 60.821 ± 1.071 c 64.982 ± 2.581 c 69.125 ± 1.231 c 74.092 ± 1.401 c 82.812 ± 1.012 b AC C 3 4 5 6 7 8 9 10 11 Heated OPI TE D OPI 73.481 ± 1.708 b 5.012 ± 0.582 c 10.071 ± 1.209 c 45.201 ± 1.922 d 56.095 ± 1.210 d 60.704 ± 2.058 d 64.652 ± 2.567 d 68.805 ± 1.668 d 79.102 ± 1.291 d EP pH The mixture of OPI-GPO 76.023 ± 1.019 a 10.768 ± 2.062 b 26.672 ± 1.585 b 57.177 ± 1.296 b 68.965 ± 1.56 b 70.529 ± 2.456 b 71.991 ± 2.178 b 80.208 ± 1.339 b 84.931 ± 1.316 c OPI-GPO conjugate 25.943 ± 0.873 c 38.621 ± 1.087 a 50.846 ± 1.119 a 65.823 ± 2.942 a 75.616 ±1.896 a 80.101 ± 2.628 a 83.291 ± 1.397 a 85.209 ± 2.073 a 87.287 ± 1.306 a Values were expressed as mean ± standard deviation values (n=3). Different letters within the same line showed a significant difference (p < 0.05). OPI: oat protein isolate; Heated OPI: Heated oat protein isolate; The mixture of OPI-GPO: The mixture of oat protein isolate and β-glucan from P. ostreatus; OPI-GPO conjugate: Oat protein isolate and β-glucan from P. ostreatus conjugate. ACCEPTED MANUSCRIPT Table 4. Denaturation temperature (Td) and enthalpy value (∆H) of samples obtained by differential scanning calorimeter. Td (°C) ∆H (J/g) OPI 101.387 ± 8.271 c 6.504 ± 0.429 b Heated OPI 102.750 ± 7.329 b 5.285 ± 0.435 c The mixture of OPI 103.091 ± 9.387 b 7.593 ± 0.903 a OPI-GPO conjugate 105.645 ± 3.302 a 2.598 ± 0.729 d RI PT Sample SC Values were expressed as mean ± standard deviation values (n=3). Different letters indicated M AN U significant difference (p < 0.05) in the same column. OPI: oat protein isolate; Heated OPI: heated oat protein isolate; The mixture of OPI-GPO: the mixture of oat protein isolate and β-glucan from AC C EP TE D P. ostreatus; OPI-GPO conjugate: oat protein isolate and β-glucan from P. ostreatus conjugate. ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT Fig. 1 ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT Fig. 2 ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT Fig. 3 ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT Fig. 4 ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT Fig. 5 ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT Fig. 6 AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT • • AC C EP TE D M AN U SC RI PT • Oat protein isolate-Pleurotus ostreatus β-glucan conjugates with high grafting degree were prepared by dry-heating for the first time. After Maillard reaction, changes in structural properties of oat protein isolate improved its solubility, emulsifying property and thermal stability. Maillard reaction could enhance the utilization value of oat protein isolate in food industry under controlled dry-heating conditions.