Effects and mechanism of ultrasound pretreatment of protein on the Maillard reaction of protein-hydrolysate from grass carp (Ctenopharyngodon idella)

Journal Pre-proofs Effects and mechanism of ultrasound pretreatment of protein on the Maillard reaction of protein-hydrolysate from grass carp (Ctenopharyngodon idella) Xue Yang, Yunliang Li, Songtao Li, Xiaofeng Ren, Ayobami Olayemi Oladejo, Feng Lu, Haile Ma PII: DOI: Reference:

S1350-4177(19)31738-9 https://doi.org/10.1016/j.ultsonch.2020.104964 ULTSON 104964

To appear in:

Ultrasonics Sonochemistry

Received Date: Revised Date: Accepted Date:

3 November 2019 28 December 2019 8 January 2020

Please cite this article as: X. Yang, Y. Li, S. Li, X. Ren, A. Olayemi Oladejo, F. Lu, H. Ma, Effects and mechanism of ultrasound pretreatment of protein on the Maillard reaction of protein-hydrolysate from grass carp (Ctenopharyngodon idella), Ultrasonics Sonochemistry (2020), doi: https://doi.org/10.1016/j.ultsonch. 2020.104964

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Effects and mechanism of ultrasound pretreatment of protein on the Maillard

reaction

of

protein-hydrolysate

from

grass

carp

(Ctenopharyngodon idella) Xue Yanga, Yunliang Lib*, Songtao Lia, Xiaofeng Renb, Ayobami Olayemi Oladejoc, Feng Lub, Haile Mab* a

Department of Basic Medicine, Chengde Medical University, Anyuan Road, Chengde, Hebei

067000, China b

Key Laboratory of Food Processing in Jiangsu Province of China, School of Food and Biological

Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu 212013, China c

Department of Agricultural and Food Engineering, University of Uyo, Uyo, Akwa Ibom State,

Nigeria. * [email protected]

(Yunliang Li), [email protected] (Haile Ma)

1

Abstract The effects of two types (energy-divergent/gathered) of ultrasound pretreatment of protein on the Maillard reaction of protein-hydrolysate from grass carp (Ctenopharyngodon idella) were studied. The test and analysis of Fourier transform infrared spectroscopy, surface hydrophobicity and atomic force microscopy of protein, peptide concentration, molecular weight distribution and free amino acid content of protein-hydrolysate were performed to reveal the mechanism. Also, the sensory characteristics of Maillard reaction products were evaluated. Results showed that Maillard reaction products presented higher absorbance value at 294 and 420 nm after pretreated by two types of ultrasound compared to that of control. The grafting degree value of products pretreated by energy-divergent ultrasound increased by 13.87%. Both of these two types of ultrasound pretreatment showed higher (p<0.05) value of grafting degree compared to that of positive control (thermal denaturation). The random coil content and surface hydrophobicity of protein improved significantly (p<0.05), and the depth distribution of protein molecules narrowed down after pretreated by ultrasound, especially energy-divergent type ultrasound. The change of protein structure increased small molecular peptide/amino acid content in protein-hydrolysate, so that it promoted the Maillard reaction process of protein-hydrolysate and glucose. The mouthfulness and overall acceptance of Maillard reaction products increased after pretreated by two types of ultrasound. Results indicated that ultrasound, especially energy-divergent type ultrasound pretreatment of protein was an effective method to promote Maillard reaction evolution of protein-hydrolysate from grass carp protein and improved the flavor of Maillard reaction products. Keywords Grass carp; Protein; Ultrasound; Pretreatment; Hydrolysate; Maillard reaction

2

1. Introduction The Maillard reaction, also known as non-enzymatic browning, refers to a series of complex chemical reactions that occur between amino acids, peptides or proteins which contain free amino groups and carbonyl compounds (sugars), producing a complex array of products that generate aroma, flavor and color[1,2]. Many researches showed that the functional characteristics of protein or protein-hydrolysate could be improved after Maillard reaction [3-6]. Maillard reaction can produce various flavour enhancers and volatile compounds in food, which can improve the unpleasant taste, especially for the protein after hydrolyzed [7-9]. Many factors are involved in the Marillard reaction of protein-hydrolysate, including the ratio of free amino groups and carbonyl groups, temperature, time, pH, as well as the interaction rate between the free amino groups and the carbonyl groups [8,10,11]. Besides, the preparation of protein-hydrolysate and Maillard reaction process are two important stages which influence the Maillard reaction of protein-hydrolysate. Compared to conventional thermal treatment, ultrasound is a new processing technology that has been recently reported to accelerate the reaction speed between molecules. In terms of promoting Maillard reaction by ultrasound, most of the research focused on the application of ultrasound during Maillard reaction process. And researches showed that ultrasound could promote the Marillard reaction to increase antioxidant properties [12,13] and generate fewer sulfur-containing volatile flavor compounds [14,15]. However, the effect of ultrasound pretreatment of protein for promoting Maillard reaction of protein-hydrolyate has not been studied yet. Normally, ultrasound can be divided into two types: energy-gathered ultrasound (EGU) and energy-divergent ultrasound (EDU) according to different energy forms. The typical ultrasound equipments were ultrasonic cell crusher and ultrasonic cleaning machine, respectively. 3

Research showed that ultrasound pretreatment of defatted wheat germ protein with these two types of ultrasound equipments before enzymolysis presented higher peptide concentration in protein-hydrolysate compared to that without ultrasound [16]. This proved that both of these two types of ultrasound pretreatments can promote enzymolysis of protein significantly. Therefore, it is meaningful to investigate the effects of EGU and EDU pretreatments of protein on the Maillard reaction process of protein-hydrolysate. As a traditional fresh water fish, grass carp (Ctenopharyngodon idellus) is rich in protein, which is a useful resource for the Maillard reaction to prepare flavor substance. Therefore, the objectives of this research were (1) to investigate the effect of ultrasound pretreatment with energy-divergent and energy-gathered types of protein on the Maillard reaction of protein-hydrolysate from grass carp; (2) to reveal the mechanism of these two types of ultrasound pretreatments on the Maillard reaction of protein-hydrolysate from grass carp; (3) to evaulate the flavor characteristics of Maillard reaction products pretreated by different ultrasound methods. It is also hoped that the results of this research will be of great value for the application of ultrasound on Maillard reaction for the preparation of flavor substance.

2. Materials and methods 2.1. Materials Grass carp (Ctenopharyngodon idella) (weight of 1500-2000 g) was purchased from a fish market of Zhenjiang (Jiangsu, China) and the muscle was separated according to the method of Tang et al. [17]. Then it was kept in ice box and transferred to the laboratory for further analysis. Flavourzyme (activity of 2.44×104 U/mg) was provided from Novozymes Co. Ltd. (China). 4

1-anilino-8-naphthalene-sulfonate (ANS) was purchased from Sigma-Aldrich Corp (USA). Aprotinin

(6511

Da),

bacitracin

(1422

Da),

Gly-Gly-Tyr-Arg

(451.2

Da)

and

Glycyl-Glycyl-Glycine (189.1 Da) were bought from Beijing Enjiayi Tech CO,. LTD. (China). All reagents used in the experiment were of analytical grade.

2.2. Pretreatment of protein sample The muscle of grass carp (with the protein content of 73.56%, dry weight) was mixed with distilled water to a final protein concentration of 35 g/L and the mixture was homogenized for 2 min using a homogenizer (IKA ULTRA-TURRAX, Jintan Fuhua Instrument Co., Ltd.). Then the sample was pretreated by EGU or EDU. The ultrasound generator of EGU was provided by Wuxi Shangjia Biotechnology Co., Ltd (Wuxi, China; Model GA98-IIIDB). And that of EDU was purchased from Kunshan ultrasound equipment Co., (Kunshan, China; Model KQ-300DE). The ultrasonic conditions were set as follows: ultrasonic power density of 100 W/L, ultrasonic time of 20 min and ultrasonic temperature of 30±2°C. The sample pretreated by thermal denaturation (TD) with an oil bath at 95°C for 20 min instead of ultrasound was set as a positive control.

2.3. Preparation of Maillard reaction products The pretreated sample was hydrolyzed with flavorzyme (5% [E/S]) at 50°C for 6 h with the pH of 7.0. After hydrolysis, the sample was put in boiling water for 15 min to inactivate enzyme. Then the sample was centrifuged at 5300 g for 15 min after cooled to the room temperature. The supernatant of protein-hydrolysate was mixed with glucose according to the volume-quality ratio of 50:1 (mL/g). Then the sample was put in sealed tube and heated at 120°C for 1 h in oil bath. The mixture was immediately cooled in ice bath for 30 min to terminate Maillard reaction. Then it 5

was kept at 4°C for further analysis.

2.4. Measurement of the absorbance The mixtures were diluted 20 and 10-fold with 0.1% (w/v) sodium dodecyl sulfate and absorbance was measured using a spectrophotometer (Unic 7200, Unocal Corporation, Shanghai, China) at 294 nm and 420 nm, as markers at the intermediate and final stages of the reactions, respectively [18].

2.5. The degree of graft of Maillard reaction products The degree of graft (DG) was determined by o-phthaldialdehyde (OPA) method with some modifications [19]. The preparation method of OPA reagent was as follows: a 40 mg of OPA was dissolved in mixture of 1.0 mL of methanol, 25 mL sodium tetraborate (0.1 mol/L), 2.5 mL sodium dodecyl sulfate (20%, w/w) and 100 μL of β-mercaptoethanol. Then it was diluted to a final volume of 50 mL with distilled water. Two hundred microliter sample solution was added in 4 mL OPA reagent. Then it was mixed rapidly and incubated at 37°C for 2 min. The absorbance of sample was read at 340 nm using a spectrophotometer (Unic 7200, Unocal Corporation, Shanghai, China). The DG was calculated according to the following formula:

DG =

A0 - A1 A0

Where, A0 was the absorbance of the supernatant of protein-hydrolysate at 340 nm, A1 was the absorbance of mixture after Maillard reaction at 340 nm.

6

2.6. The mechanism of ultrasound pretreatment on the Maillard reaction of protein-hydrolysate 2.6.1. Fourier transform infrared spectroscopy of grass carp protein

The Fourier transform infrared (FTIR) spectroscopy of grass carp protein pretreated by different methods was measured by Nicolet IS50 spectrum instrument (Thermo Electron Corporation, USA). The protein sample powder was diluted with potassium bromide powder of spectroscopic grade by one-tenth. The scanning wavelength was between 2000 cm-1 and 1000 cm-1 with 36 scans. Background noise was corrected with pure potassium bromide powder. The analysis of FTIR spectral data was conducted by OMNIC and Peakfit software.

2.6.2. Surface hydrophobicity of grass carp protein

Surface hydrophobicity of grass carp protein pretreated by different methods was determined according to the method of Kato and Nakai with some modifications [20]. The samples were dissolved in 0.01 mol/L phosphate buffer (pH 8.0) at 37°C for 1 h. Then the mixture was centrifuged at 12000 g for 10 min and the supernatant was diluted to a final protein concentration of 1.0, 0.8, 0.6, 0.4 and 0.2 mg/mL. A 4 mL of protein solution was mixed with 20 μL 8.0 mmol/L ANS (dissolved in 0.01 mol/L phosphate buffer, pH 8.0) and reacted for 5 min in the dark. Then the relative fluorescence intensity was measured using a Cary Eclipse spectrophotometer (Varian Inc., Palo Alto, USA) at excitation wavelength of 330 nm at scanning speed of 120 nm/min. The grass carp protein sample without ANS was set as blank. The initial slope of fluorescence intensity vs. protein concentration calculated by linear regression analysis was used as surface hydrophobicity (H0). 7

2.6.3. Atomic force microscopy of grass carp protein

Atomic force microscopy image was generated according to the method described by Zhao et al. [21] using multimode microscope (Bruker, Santa Barbara, CA) with some modifications. A 5 mg sample was dissolved with 1 mL deionized water at 35°C for 12 h. Then it was centrifuged at 12,000 g for 10 min. A 5 µL supernatant was deposited on a fresh mica sheet. The mica sheet was placed in sterilized clean bench to evaporate liquid. Then protein particles were detected by Bruker ScanAsyst needle.

2.6.4. Peptide concentration of protein-hydrolysate

The supernatant of protein-hydrolysate was mixed with 15% (W/V) trichloroacetic acid (volume ratio was 2:1) and reacted at 25°C for 1 h afterwards. Then it was centrifuged at 12,000 g for 10 min. Peptide concentration of protein-hydrolysate in supernatant was determined using the Folin-phenol method [22]. The absorbance was read at 680 nm on a spectrophotometer (Unic 7200, Unocal Corporation, Shanghai, China).

2.6.5. Molecular weight distribution of protein-hydrolysate

Molecular weight distribution of peptide in protein-hydrolysate was determined by high performance liquid chromatography with a TSK gel G2000 SWXL 300mm×7.8mm (flow velocity of 0.5 mL/min, sample injection of 20 μL, column temperature of 30ºC). Calibration curve was drawn according to the average elution volume and corresponding molecular weight using the standard reagents of aprotinin, bacitracin, Gly-Gly-Tyr-Arg and Glycyl-Glycyl-Glycine.

8

2.6.6. Amino acid content of protein-hydrolysate

The amino acid content of protein-hydrolysate was analyzed by automatic amino acid analyzer (S433D, Sykam, German). The supernatant of protein-hydrolysate was divided into two parts. One part was used for the determination of free amino acid analyze directly. The other part was sealed in a tube and hydrolyzed with 6 mol/L HCl (110°C, 24 h). Then the samples were centrifuged at 12000 g for 10 min and the supernatants were used to determine total amino acid composition. All the samples were filtered by 0.22 μm water film before injection for amino acid analysis.

2.7. Sensory analysis of Maillard reaction products The sensory analysis of Maillard reaction products of protein-hydrolysate pretreated by different methods was carried out according to the method of Song et al. [23] by eight trained panelists (ages between 25 and 43 years) from the School of Food Science and Technology at Jiangsu University (Zhenjiang, China).

2.8. Statistical analysis The results were analyzed by one-way ANOVA at the significance level of p<0.05 using SPSS 19.0 software (IBM Corporation, NY, USA). The graphs were drawn by OriginPro8 (OriginLab Corporation, MA, USA). The data are expressed as means and standard errors (n=3).

3. Results 3.1. Effects of different pretreatment methods on Maillard reaction In Maillard reaction, glucose can react with free amino groups on the side chains of protein,

9

peptide or amino acid molecules [24]. This process can be divided into primary, intermediate and advanced stages, with browning generally associated with the reaction due to the generation of a class of nitrogen-containing brown polymers-melanoidin [25]. The color of the reaction system is positive correlation with the process of Maillard reaction. Therefore, the degree of glycosylation of the protein-hydrolysate can be reflected by the color change of the reaction system, which can be determined by 420 nm absorbance [18]. The effects of different pretreatment methods on the Maillard reaction products were shown in Fig. 1. As can be seen from the result, after pretreated by two types of ultrasound, the absorbance value of Maillard reaction products of protein-hydrolysate increased significantly (p<0.05) compared to that of control. The result of EDU pretreatment presented the highest absorbance value of 0.564 (increased by 61.14%) and that of EGU increased by 21.43%. Compared to positive control (TD), the absorbance value of Maillard reaction products pretreated by EDU increased by 13.25% and that of EGU decreased by 14.66%. During the Maillard reaction, the sugars cleavage produced colorless, small molecule mixtures, such as ketones, aldehydes. These substances can absorb ultraviolet-visible light at 294 nm, which is used as one indicator to evaluate Maillard reaction intermediate products amount [26]. The effects of different pretreatment methods of protein on the A294nm of Maillard reaction products of protein-hydrolysate were shown in Fig. 1. Results showed that A294nm of Maillard reaction products of protein-hydrolysate pretreated by EDU and EGU increased significantly (p<0.05) with the value of 0.685 (increased by 33.27%) and 0.592 (increased by 15.18%) compared to that of control. Also, the absorption value of Maillard reaction products was much higher (p<0.05) than that of TD after pretreated by EDU. Same with the result of absorbance value at 420 nm, the 10

absorbance value at 294 nm showed the consistent trend, which was that two types of ultrasound pretreatments could promote the generation of Maillard reaction products and EDU presented higher influence. The EDU pretreatment method was more beneficial for the enzymolysis of protein and the subsequent Maillard reaction. The reason might be that ultrasound pretreatment changed the advanced structure of protein so that it promoted the protein enzymolysis, which produced more peptides/amino acids in protein-hydrolysate that was easier to be involved in Maillard reaction. The free amino groups on the side chain of protein molecules are dehydrated and condensed with the carbonyl group of a reducing sugar. Therefore the amino group in a free-state is transformed into a bound-state. The bound-state amino group content (that is, DG) can show the progress of the Maillard reaction and the change of the protein, which can be calculated by measuring the content of free amino groups in the system before and after the Maillard reaction. The effects of different pretreatment methods on the DG of Maillard reaction products of protein-hydrolysate were shown in Fig. 2. The highest DG was obtained by the pretreatment method of EDU with the value of 55.41% and increased by 13.87%. After pretreated by EGU, the DG of sugar and protein-hydrolysate did not decrease significantly (p>0.05), while that pretreated by TD decreased significantly (p<0.05) compared to that of control. It indicated that both of EDU and EGU pretreatments had beneficial effect on the Maillard reaction of sugar and protein-hydrolysate compared to that of TD. In all the pretreatment methods, EDU presented the highest value of DG. This might be because that ultrasound pretreatment could provide more energy and generate more peptide and/or amino acids that were easily to be involved in Maillard reaction for the process of graft reaction [27]. In contrast, TD pretreatment method might increase 11

macromolecular protein or peptide, which was not favorable for Maillard reaction. According to the above experiments, it could be concluded that the pretreatment method of EDU showed the highest A294nm, A420nm and DG. While that of EGU and TD presented different effects in A294nm, A420nm and DG. This indicated that proper protein pretreatment method could increase the DG of reaction drastically and generate more browning products and intermediate products with ultraviolet-visible absorption characteristics. And the results of absorption characteristics (A294nm and A420nm) presented inconsistent trends in contrast to that of DG. The differing results would be explained by the following experimental results.

3.2. Secondary structure of grass carp protein To reveal the mechanism of different pretreatment methods on the Maillard reaction of protein-hydrolysate, the secondary structure of protein sample after pretreatment was measured using FTIR spectroscopy. As shown in Fig. 3, the amide I (1700-1600 cm-1 wavelength) of FTIR spectroscopy was commonly used to analyze the secondary structure of protein. After analysis by software, the secondary structure contents of grass carp protein pretreated by different methods were summarized and listed in Table 1. Results showed that the secondary structure of grass carp protein pretreated by different methods changed significantly (p<0.05) compared to that of control. The pretreatment methods of EDU and EGU decreased the α-helix and β-sheet content

with the increase of random coil content. The protein pretreated by EDU showed the highest random coil content of 36.36%, which increased by 115.02% and 89.47% compared to that of control and TD, respectively. That of EGU increased by 14.55% and 0.94%, respectively, which showed that ultrasound pretreatment could cause unfolding of the protein structure. These findings

12

were similar to the result of our previous research, which showed that ultrasound caused unfolding of the α-helical region and increased the random coil content of rice protein [28]. Also, it was consistent with the literatures [29,30]. Besides, the secondary structure of grass carp protein pretreated by different methods were consistent with the Maillard reaction results (the grass carp protein pretreated by EDU generated more Maillard reaction products, followed by TD and then EGU). These results suggested that EDU pretreatment of grass carp protein changed the protein secondary structure, especially increased the random coil content of grass carp protein dramatically, which promoted the protein enzymolysis to produce suitable peptides that were beneficial for the Maillard reaction process.

3.3. Surface hydrophobicity of grass carp protein The surface hydrophobicity of grass carp protein pretreated by different methods was shown in Fig. 4. That of EGU and EDU increased by 27.46% and 36.85%, respectively, compared to that of control. The pretreatment method of TD increased the surface hydrophobicity of grass carp protein the most with the value of 675 and increased by 58.45%, which showed that ultrasound was less effective than TD treatment in changing surface hydrophobicity of grass carp protein. Researches showed that the increase of protein surface hydrophobicity improved enzymolysis effect of protein [31,32], so that it promoted the Maillard reaction between protein-hydrolysate and glucose. However, these results were not in conformity with A294nm and A420nm (representing browning products and intermediate products content) in Results 3.1. The reason might be that EDU treatment made partial hydrophobic protein molecules buried inside initially exposed, which increased the contact opportunity between protein and enzyme and improved protein enzymolysis

13

[33]. Therefore, it increased the amount of Maillard reaction products. The paper of McClements et al. reported that protein which was too hydrophobic could lower the solubility in water and surface activity because of the aggregation between proteins molecule [34]. The balance of hydrophobic and hydrophilic groups of protein molecules allowed them to be soluble in water but also keep the surface active. TD pretreatment, an extreme treatment method, improved the surface hydrophobicity of grass carp protein dramatically. As the surface hydrophobicity reached a certain high threshold, the protein became aggregate, which decreased the surface area of protein molecules and reduced the contact chance of protein and enzyme. This indicated that grass carp protein pretreated by proper method (in this paper, it referred to EDU method) could lead to a temperate value of surface hydrophobicity, which promoted the enzymolysis of protein and increased the Maillard reaction of protein-hydrolysate.

3.4. Atomic force microscopy of protein Fig. 5 and Fig. 6 showed the microstructure and depth of grass carp protein pretreated by EGU and EDU methods. Results showed that both of EGU and EDU pretreatment methods increased the depth of protein molecules. This might be due to that the hydrophobic groups and regions existed in the interior of the protein molecules were exposed to the surface under the ultrasound pretreatment condition, which was consistent with the results of surface hydrophobicity. Besides, the depth distribution of protein molecules pretreated by EGU and EDU narrowed down. And the range of width of protein pretreated by EDU and EGU was restricted in a special interval, especially EDU, which showed that it could make the depth distribution of protein molecules more uniform than that of control and TD.

14

The results also showed that the application of EDU pretreatment on protein for enzymolysis improved the Maillard reaction of protein-hydrolysate effectively. Unlike EGU generating high and inhomogenous ultrasound intensity, EDU generated low and uniform ultrasound intensity, which made the depth distribution of protein molecules more uniform. This was beneficial for the enzymolysis of grass carp protein and the Maillard reaction of protein-hydrolysate. And these explained the reason why EDU and EGU of grass carp protein showed significant difference (p<0.05) in Maillard reaction of protein-hydrolysate from grass carp. These results illustrated that the uniformity in the structure of protein molecules played a significant role in enzymolysis of grass carp protein and affected the Maillard reaction speed of protein-hydrolysate.

3.5. Peptide concentration of protein-hydrolysate The peptide concentration of protein-hydrolysate pretreated by EGU and EDU were shown in Fig. 7. The peptide concentration of protein-hydrolysate pretreated by EGU increased by 32.14% and decreased by 10.46% compared to that of control and TD, respectively. That of EDU increased by 55.18% and 5.16%, respectively. These results showed that both of EGU and EDU pretreatment increased the peptide concentration of protein-hydrolysate significantly (p<0.05), which was in line with the results of Zou et al. [35] The highest peptide concentration was obtained with the protein pretreated by EDU, and it had significant difference (p<0.05) compared to that of other pretreatment methods. Also, these results were consistent with the indices of Maillard reaction products. It also indicated that the pretreatment of grass carp protein with EDU increased the peptide concentration in protein-hydrolysate the most. Therefore, it could be concluded that EDU pretreatment changed the advanced structure of protein, including the

15

increase of random coil content, surface hydrophobicity to a temperate value and uniform depth ratio of grass carp protein molecules. Then they could be more easily attacked by flavourzyme during the enzymatic process. These resulted in the increase in peptide/amino acid content which related to the Maillard reaction.

3.6. Molecular weight distribution of protein-hydrolysate The result of molecular weight distribution of protein-hydrolysate was listed in Table 2. As can be seen from Table 2, the total peak area of protein-hydrolysate from grass carp pretreated by EGU and EDU increased significantly (p<0.05) compared to that of control, which increased by 9.72% and 16.70%, respectively. In contrast to TD, that of EGU and EDU pretreatment decreased by 1.96% and increased by 4.28%, respectively. The peak area could present the protein/peptide/amino acids content to some extent. This proved that both of EGU and EDU pretreatment methods could increase the content of proteins (or peptides, or amino acids). Molecular weight distribution of protein-hydrolysate was a very important factor that affected Maillard reaction process. Research showed that amino acids and small molecule peptides presented higher reaction speed compared to macromolecular peptides or proteins [12]. The pretreatment methods of EGU and EDU showed the sum percentage of amino acids and small molecule peptides with the value of 66.70% and 70.34%, which increased by 0.89% and 6.40%, respectively, compared to that of TD. And compared to that of control, EGU and EDU showed different trend with the decrease by 3.42% and increase by 1.85%, respectively. These results disagreed with that of Maillard reaction process (TD pretreatment method obtained more Maillard reaction products compared to that of EGU). The reason might be attributed to the fact that TD

16

pretreatment could increase the peptide or amino acids amount, but it reduced the small molecule peptides or amino acids percentage that involved in Maillard reaction easily. This led to higher Maillard reaction products, but lower DG compared to that of EGU pretreatment, which explained the phenomenon appeared in the section of Results 3.1. EDU pretreatment method showed the highest proteins (or peptides, or amino acids) amount, which could be seen from the peak area of protein-hydrolysate. The reason might be that EDU pretreatment of grass carp protein changed the secondary structure of protein, loosen the protein structure and increased the contact chance between enzyme and protein, which increased the proteins (or peptides, or amino acids) amount in protein-hydrolysate. These resulted in more browning products and intermediate products of protein-hydrolysate during Maillard reaction.

3.7. Amino acids content of protein-hydrolysate The amino acid content of protein-hydrolysate pretreated by different pretreatment methods was shown in Table 3. The results showed that the total amino acid and free amino acid content of protein-hydrolysate increased after pretreated by EGU and EDU methods. The free amino acid content of protein-hydrolysate from grass carp protein pretreated by EGU and EDU was 212.95 and 242.83 mg/mL, and increased by 14.84% and 30.96%, respectively, with the control of 185.43 mg/mL. Also, compared to that of TD, the free amino acid content of protein-hydrolysate from grass carp protein pretreated by EGU and EDU increased by 7.06% and 22.70%, respectively. Among all the free amino acids, Lys, Arg and Leu presented higher content in protein-hydrolysate. And Lys and Arg can be involved in Maillard reaction easily [36,37].

17

Total amino acid content can clearly illustrate hydrolysis process by determining all the amino acid in supernatant. After enzymatic hydrolysis, protein-hydrolysate, generally composed of free amino acids, short chain bioactive peptides and some long chain proteins, were obtained. The total amino acid content of protein-hydrolysate pretreated by EGU and EDU were 441.06 and 485.01 mg/mL and increased by 17.72% and 29.45%, respectively, with the control of 374.68 mg/mL. That of EGU and EDU showed different effects with the decrease of 2.24% and increase of 7.51%, respectively, compared to that of TD. These results were in line with the results of peptide concentration and molecular weight distribution of grass carp protein-hydrolysate. The protein structure before enzymolysis was highly correlation with amino acid content in protein-hydrolysate. From the above results, it can be seen that ultrasound pretreatment loosed the protein structure, increased random coil content (Table 1), exposed the hydrophobic groups moderately (Fig. 4) and increased the uniformity of depth distribution (Fig. 5 and 6) of protein, which was beneficial for the attack of flavourzyme. This resulted in the increase of peptide concentration (Fig. 7), molecular weight from 200 Da to 500 Da (Table 2) and amino acid content in protein-hydrolysate. The increase of total amino acid content improved the contact chance between free amino group on the side chain of protein/peptide/amino acid molecule and carbonyl group of reducing sugar. This increased the Maillard reaction speed and products amount between protein-hydrolysate and glucose. Combined with all the experimental results, it could be concluded that EDU pretreatment of grass carp protein changed the protein structure, which was more favorable for proteolysis, and released more peptides and amino acids into the protein-hydrolysate, therefore it promoted the Maillard reaction of protein-hydrolysate and increased the amount of Maillard reaction products. 18

3.8. Flavor characteristics of Maillard reaction products Results of flavor characteristics of Maillard reaction products were shown in Fig. 8. After pretreated by EDU and EGU, the mouthfulness and overall acceptance of products increased significantly (p<0.05). The flavor of caramel and bitterness of sample pretreated by EDU decreased (p<0.05) in contrast to that of control. Other flavor characteristics of Maillard reaction products pretreated by EDU and EGU had no significant influence (p>0.05) compared to that of control and TD. The protein pretreated by EDU caused the decrease of bitterness amino acids (Val, Leu, Ile, Met, Phe, Ser, Arg and His) by 2.04% and increase of sweet amino acids (Gly, Thr, Ala and Pro) by 17.88% in protein-hydrolysate, which might be the reason for the changes of flavor in Maillard reaction products. These results were useful for the application of Maillard reaction products from grass carp protein as flavor substance in food industry, especially after pretreated by EDU.

4. Discussion Many researches have documented the effects of ultrasound treatment on the Maillard reaction of protein-hydrolysate. However, these studies have either been used ultrasound during Maillard reaction process or have not focused on the effects of two types (energy-divergent/gathered) of ultrasound pretreatments of protein to obtain protein-hydrolyate for promoting Maillard reaction. In this study, we researched on the effects of different ultrasound energy fields (divergent/gathered types, which refers to EDU and EGU in this paper) pretreatments of grass carp protein on the Maillard reaction of protein-hydrolysate. Besides, thermal treatment, which was one commonly used method, was set as a positive control. According to our research results, we found 19

that pretreatment of grass carp protein with both EGU and EDU increased the amount of Maillard reaction products in protein-hydrolysate, including browning products and intermediate products. These findings extended those of Corzo-Martínez et al. [38] who applied high-intensity (energy-gathered) ultrasound during Maillard reaction process, Zhao et al. [39] who confirmed that ultrasonic pretreatment of soy protein/sugar mixture before heating had higher DG of Maillard reaction products than that without ultrasound, and Wang et al. [40] who reported that ultrasound-assisted wet heating Maillard reaction between mung bean protein isolates and glucose could be a promising way to improve functional properties of mung bean protein isolates. This study indicates that the grass carp protein pretreated by EDU increases the Maillard reaction effect (products amount and DG). Most notably, this is one novel study to investigate the effects of two types (energy-divergent/gathered) of ultrasound pretreatment of grass carp protein on the Maillard reaction of protein-hydrolysate. Compared to control and positive control (TD), both of these two types of ultrasound pretreatment methods promote the Maillard reaction more effectively. And EDU pretreatment shows the highest value in both ultraviolet-visible absorbance value and DG. Ultrasound with energy-divergent type can generate low-intensity and uniform ultrasound waves, which makes the depth distribution of protein molecules more uniform than that of EGU. Except for uniformity, EDU also increases the random coil content and surface hydrophobicity of grass carp protein. This results in the increase of peptide content, amino acid content and the proportion of small molecular peptides and amino acids in protein-hydrolysate, which promotes the Maillard reaction process (including browning and intermediate products amount and DG). Besides, the flavor of Maillard reaction products improved after pretreated by ultrasound. Our results provide compelling evidence for applying ultrasound, especially EDU, as 20

protein pretreatment method on the Maillard reaction of protein-hydrolysate and suggest that this approach appears to be effective in promoting Maillard reaction process. However, some limitations are worth noting. The ultrasound working modes and parameters were not being considered in this study, and these factors should be discussed in the future researches.

5. Conclusion The absorbance values of Maillard reaction products of protein-hydrolysate pretreated by two types of ultrasound increased significantly (p<0.05) compared to that of control. The EDU pretreatment method presented higher absorbance value in contrast to that of TD. The DG of sugar and protein-hydrolysate pretreated by EGU did not decrease significantly (p>0.05) compared to that of control, while it was higher (p<0.05) than that of TD, and the highest DG was obtained with the pretreatment method of EDU. Both EDU and EGU decreased the α-helix and β-sheet content with the increase of random coil content, which showed that EGU and EDU pretreatments caused unfolding of the protein structure. The protein pretreated by EDU showed the highest random coil content of 36.36%, which increased by 115.02% compared to that of control. The surface hydrophobicity of grass carp protein pretreated by EGU and EDU methods increased by 27.46% and 36.85%, respectively. The pretreatment method of TD increased the surface hydrophobicity of grass carp protein the most. The depth distribution of protein molecules pretreated by EGU and EDU narrowed down and the range of width of protein pretreated by EDU and EGU was restricted in a special interval, especially EDU, than that of control and TD. The peptide concentration and total peak area of protein-hydrolysate pretreated by EGU and EDU increased compared to that of control. And that of EDU increased in contrast with TD. The

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pretreatment methods of EGU and EDU showed the sum percentage of amino acids and small molecule peptides with the value of 66.70% and 70.34%, which increased by 0.89% and 6.40%, respectively, compared to that of TD. The total amino acid content of EGU and EDU showed different effects with the decrease of 2.24% and increase of 7.51% compared to that of TD. After pretreated by EDU, the mouthfulness and overall acceptance of Maillard reaction products increased with the decrease of bitterness and caramel in contrast to that of control. These presented that EDU and EGU pretreatments of protein were effective methods to promote the Maillard reaction evolution of protein-hydrolysate from grass carp and that of EDU showed more beneficial effect.

Acknowledgments The authors would like to thank the Youth Science Fund Project of Natural Science Foundation of Hebei Province (No. C2019406071), Young Talent Scholar Plan of Higher School in Hebei Province (No. BJ2019046) and Foundation for High-level Talents of Chengde Medical University (No. 201902).

Abbreviations EGU

energy-gathered ultrasound

EDU

energy-divergent ultrasound

TD

thermal denaturation

ANS

1-anilino-8-naphthalene-sulfonate

DG

the degree of graft

OPA

o-phthaldialdehyde 22

FTIR

Fourier transform infrared

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Figure captions Fig. 1 The effects of different pretreatment methods on the absorbance of Maillard reaction products of protein-hydrolysate from grass carp. (The same index marked with different letter means significantly different at p<0.05, n = 3). EGU, EDU and TD presented the pretreatment methods of energy-gathered ultrasound, energy-divergent ultrasound and thermal denaturation, respectively. Fig. 2 The effects of different pretreatment methods on DG of Maillard reaction products of protein-hydrolysate from grass carp. (The different letter means significantly different at p<0.05, n = 3). EGU, EDU and TD presented the pretreatment methods of energy-gathered ultrasound, energy-divergent ultrasound and thermal denaturation, respectively. Fig. 3 The effects of different pretreatment methods on the FTIR of grass carp protein. EGU, EDU and TD presented the pretreatment methods of energy-gathered ultrasound, energy-divergent ultrasound and thermal denaturation, respectively. Fig. 4 The effects of different pretreatment methods on the surface hydrophobicity (H0) of grass carp protein. (The different letter means significantly different at p<0.05, n = 3). EGU, EDU and TD presented the pretreatment methods of energy-gathered ultrasound, energy-divergent ultrasound and thermal denaturation, respectively. Fig. 5 The effects of different pretreatment methods (a) control, (b) EGU, (c) EDU and (d) TD on atomic force microscopy of grass carp protein. EGU, EDU and TD presented the pretreatment methods of energy-gathered ultrasound, energy-divergent ultrasound and thermal denaturation, respectively. Fig. 6 The effects of different pretreatment methods on the depth count (%) of grass carp protein. EGU, EDU and TD presented the pretreatment methods of energy-gathered ultrasound, energy-divergent ultrasound and thermal denaturation, respectively. Fig. 7 The effects of different pretreatment methods on the peptide concentration of protein-hydrolysate from grass carp. (The different letter means significantly different at the significance level of p<0.05, n = 3). EGU, EDU and TD presented the pretreatment methods of energy-gathered ultrasound, energy-divergent ultrasound and thermal denaturation, respectively. Fig. 8 The effects of different pretreatment methods on the flavor characteristics of protein-hydrolysate from grass carp. EGU, EDU and TD presented the pretreatment methods of energy-gathered ultrasound, energy-divergent ultrasound and thermal denaturation, respectively.

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Fig. 1 The effects of different pretreatment methods on the absorbance of Maillard reaction products of protein-hydrolysate from grass carp. (The same index marked with different letter means significantly different at p<0.05, n = 3). EGU, EDU and TD presented the pretreatment methods of energy-gathered ultrasound, energy-divergent ultrasound and thermal denaturation, respectively.

Fig. 2 The effects of different pretreatment methods on DG of Maillard reaction products of protein-hydrolysate from grass carp. (The different letter means significantly different at p<0.05, n = 3). EGU, EDU and TD presented the pretreatment methods of energy-gathered ultrasound, energy-divergent ultrasound and thermal denaturation, respectively.

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Fig. 3 The effects of different pretreatment methods on the FTIR of grass carp protein. EGU, EDU and TD presented the pretreatment methods of energy-gathered ultrasound, energy-divergent ultrasound and thermal denaturation, respectively.

Fig. 4 The effects of different pretreatment methods on the surface hydrophobicity (H0) of grass carp protein. (The different letter means significantly different at p<0.05, n = 3). EGU, EDU and TD presented the pretreatment methods of energy-gathered ultrasound, energy-divergent ultrasound and thermal denaturation, respectively.

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a

b

d

c

Fig. 5 The effects of different pretreatment methods (a) control, (b) EGU, (c) EDU and (d) TD on atomic force microscopy of grass carp protein. EGU, EDU and TD presented the pretreatment methods of energy-gathered ultrasound, energy-divergent ultrasound and thermal denaturation, respectively.

Fig. 6 The effects of different pretreatment methods on the depth count (%) of grass carp protein. EGU, EDU and TD presented the pretreatment methods of energy-gathered ultrasound, energy-divergent ultrasound and thermal denaturation, respectively. 30

Fig. 7 The effects of different pretreatment methods on the peptide concentration of protein-hydrolysate from grass carp. (The different letter means significantly different at the significance level of p<0.05, n = 3). EGU, EDU and TD presented the pretreatment methods of energy-gathered ultrasound, energy-divergent ultrasound and thermal denaturation, respectively.

Fig. 8 The effects of different pretreatment methods on the flavor characteristics of protein-hydrolysate from grass carp. EGU, EDU and TD presented the pretreatment methods of energy-gathered ultrasound, energy-divergent ultrasound and thermal denaturation, respectively.

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Table 1 Effects of different pretreatment methods on the secondary structure content of grass carp protein Secondary structure α-helix β-turn β-sheet Random coil

Control 24.99 21.55 36.55 16.91

EGU 11.67 (-53.30%) 37.42 (73.64%) 31.54 (-13.71%) 19.37 (14.55%)

EDU 15.87 (-36.49%) 17.40 (-19.26%) 30.37 (-16.91%) 36.36 (115.02%)

TD 11.70 (-53.18%) 37.61 (74.52%) 31.50 (-3.72%) 19.19 (13.48%)

Values in parentheses indicate the increase or decrease (with negative sign) in secondary structure (β-sheet, random coil, α-helix, β-turn) content of grass carp protein pretreated by EGU, EDU and TD compared to control. EGU, EDU and TD presented the pretreatment methods of energy-gathered ultrasound, energy-divergent ultrasound and thermal denaturation, respectively.

Table 2 Effects of different pretreatment methods on the molecular weight distribution of protein-hydrolysate from grass carp Molecula r >500 weight 200-500 (Da) <200 Total

Control Conten Peak area 30.94 t 1076.21 (mV*s 29.36 5.89 (%) ) 39.70 7.96 100 20.06

EGU Conten Peak area 33.30 t 1077.33 (mV*s 29.36 6.46 (%) ) 37.34 8.22 100 22.01

EDU Conten Peak area 29.66 t 1076.94 (mV*s 30.99 7.26 (%) ) 39.35 9.21 100 23.41

TD Conten Peak area 33.89 t 1077.61 (mV*s 31.01 6.96 (%) ) 35.10 7.88 100 22.45

EGU: energy-gathered ultrasound; EDU: energy-divergent ultrasound; TD: thermal denaturation.

Table 3 Effects of different pretreatment methods on the amino acids content (mg/mL) of protein-hydrolysate from grass carp

Asp Thr Ser Glu Gly Ala Cys Val Met Ile Leu Tyr Phe His Lys Arg Pro Sum

Control FAA TAA 12.17 33.64 9.35 15.93 7.64 8.07 11.85 56.94 2.93 16.33 9.17 20.64 3.02 8.36 11.85 16.21 8.74 13.16 10.68 16.28 25.63 28.74 10.15 12.79 12.28 14.55 6.53 11.92 21.94 38.45 15.62 20.17 5.88 42.50 185.43 374.68

EGU FAA TAA 13.30 40.26 10.65 18.16 9.26 10.85 14.02 62.70 3.65 20.04 10.53 23.85 3.53 11.78 13.74 19.93 10.07 14.10 12.16 21.43 29.62 32.78 11.50 16.09 14.81 17.63 7.63 17.20 22.93 42.87 18.17 25.81 7.39 45.58 212.95* 441.06*

EDU FAA TAA 15.62 43.39 19.48 21.17 10.24 10.08 14.42 72.64 2.96 20.33 12.08 26.83 3.84 10.50 15.34 21.82 11.57 17.72 13.65 21.26 32.98 37.41 12.64 16.28 16.11 19.64 7.95 15.15 27.15 48.76 19.12 26.41 7.67 55.62 242.83* 485.01*

TD FAA TAA 13.28 39.01 10.29 19.14 8.03 10.05 12.34 68.65 3.07 19.77 9.67 25.88 3.14 9.81 12.39 20.60 9.42 15.74 11.34 20.28 26.38 34.63 10.94 14.92 13.15 18.14 7.20 13.77 23.51 45.09 17.75 23.54 6.01 52.14 197.91* 451.15*

EGU: energy-gathered ultrasound; EDU: energy-divergent ultrasound; TD: thermal denaturation. 32

FAA: Free amino acid; TAA: Total amino acid. *indicates

significant difference at p<0.05 compared to that of control.

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Declaration of interests √ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Highlights 1. Ultrasound pretreatment of protein on Maillard reaction of protein-hydrolysate was studied; 2. Two types (energy-divergent/gathered) of ultrasound were used; 3. Mechanism of ultrasound pretreatment on Maillard reaction was investigated; 4. Flavor characteristics of Maillard reaction products were evaluated.

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