Aromatic effects of immobilized enzymatic oxidation of chicken fat on flaxseed (Linum usitatissimum L.) derived Maillard reaction products

Food Chemistry 306 (2020) 125560

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Aromatic effects of immobilized enzymatic oxidation of chicken fat on flaxseed (Linum usitatissimum L.) derived Maillard reaction products Chao-Kun Weia, Zhi-Jing Nia,b, Kiran Thakura, Ai-Mei Liaoc, Ji-Hong Huangc,d, Zhao-Jun Weia,e,

T ⁎

a

School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, People’s Republic of China Biological Science and Engineering College, North Minzu University, Yinchuan 750021, People’s Republic of China c College of Biological Engineering, Henan University of Technology, Zhengzhou 450001, People’s Republic of China d Henan Cooperation Science and Technology Institute, Zhengzhou 450001, People’s Republic of China e Anhui Province Key Laboratory of Functional Compound Seasoning, Anhui Qiangwang Seasoning Food Co., Ltd., Jieshou 236500, People’s Republic of China b

ARTICLE INFO

ABSTRACT

Keywords: Maillard reaction Chicken fat Lipoxygenase Immobilized enzyme Oxidation method Flavor

To control the oxidation in chicken fat by immobilized lipoxygenase (LOX), Maillard reaction products (MRPs) with chicken flavor were prepared and analyzed for flavor mechanism. > 50% activity of immobilized LOX was retained after repeated use for five times or five weeks. The oxidized chicken fats were prepared by thermal, free LOX, and immobilized LOX treatments. After addition of chicken fats, Maillard reaction produced more aliphatic aldehydes and alcohols (126.0–839.5 ng/g and 493.5–2332.4 ng/g, respectively) which resulted in noticeable enhanced reaction, but the content of sulfur compounds such as thiols and thiophenes decreased significantly (870.8–1233.9 ng/g and 1125.0–2880.3 ng/g, respectively), and the structure of sulfur compounds could easily form alkyl side chains. However, there was no significant difference in sensory and flavors between oxidized chicken after treatments, which may be related to oxidized degree. The mechanism was proposed or aromatic effects of oxidized chicken fat on flaxseed derived MRPs.

1. Introduction

methanethiol, thiophene, and dimethyl disulfide in boiled beef; while, in another study, similar components such as 2-methylthiophene and 2furanmethanethiol in the Maillard reaction system of cysteine, ribose, and polyunsaturated fatty acids (Elmore, Mottram, & Hierro, 2001; Elmore, Campo, Enser, & Mottram, 2002). Some animal fats produce a characteristic flavor of the meat when heated in the air; whereas, heating in nitrogen does not lead to the corresponding flavor. This shows that a certain degree of fat oxidation results in the generation of meat flavor. The previous study showed that heating the beef after removing triglycerides and phospholipids led to great alterations in volatile components in terms of flavor characteristics. The changes of volatile compounds were mainly manifested in the decrease of fatty alcohols and aldehydes, and the increase of benzaldehyde and pyrazines (Aaslyng & Meinert, 2017). After removing triglycerides and phospholipids from beef, heat treatment resulted into loss of volatile components and barbecue aroma (Mottram & Edwards, 2010) which was similar to the results obtained in pork and lamb (Guerrero et al., 2014; Song et al., 2017).

The Maillard reaction is responsible for the preparation of meat flavors, and research focus has been primarily directed towards implications of this reaction on food browning and flavor formation (Brehm, Jünger, Frank, & Hofmann, 2019; Hou et al., 2017). Furthermore, various researchers have showed the antioxidant and anti-inflammatory activity of Maillard reaction products (MRPs) from amino acids, protein hydrolysates, and heterocyclic volatile compounds formed during Maillard reaction in vitro and in food products (Qin et al., 2018; Wakamatsu, Stark, & Hofmann, 2019). Flaxseed meal is a protein-rich by-product obtained during flaxseed oil extraction. Despite insignificant applications in foods, flaxseeds proteins are recognized as an important food ingredient (Bekhit et al., 2018). Several food products are fortified using flaxseed meal, especially dairy, cereal-based foods as well as meat products (Teh, Bekhit, Carne, & Birch, 2014). The formation of meat flavor depends on some characteristic flavor substances. For example, sulfur-containing volatile components such as

Abbreviations: AV, acid value; DW, distilled water; DOC, deoxycholate; MDA, malondialdehyde; LOX, lipoxygenase; MRPs, Maillard reaction products; PV, peroxide value ⁎ Corresponding author at: School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, People’s Republic of China. E-mail addresses: [email protected] (K. Thakur), [email protected] (A.-M. Liao), [email protected] (J.-H. Huang), [email protected] (Z.-J. Wei). https://doi.org/10.1016/j.foodchem.2019.125560 Received 20 May 2019; Received in revised form 13 September 2019; Accepted 16 September 2019 Available online 03 October 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.

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Fatty acid oxidation and degradation occur during fat heating and give rise to various degradation products. With the exception of aldehydes, other substances have a smaller contribution to meat odor due to the higher odor threshold value. In fact, lipid degradation produces aldehydes and ketones which interact with the Maillard reaction materials such as amino acids and reducing sugars during heat treatment (Yang et al., 2015). However, the thermal oxidation of oils and fats experiences some drawbacks such as high energy consumption, large equipment volume, low production efficiency, and difficulty in product quality control in the food industry. Lipoxygenase (LOX) are responsible for the formation of hydroperoxides from polyunsaturated fatty acids such as linoleic and arachidonic acids (Fofana et al., 2016). Since vegetable and animal oils contain a certain proportion of unsaturated fatty acids which can be catalyzed by LOX to prepare flavor precursors to participate in Maillard reaction. Since the content of unsaturated fatty acids in vegetable oil is much higher than found in animal oil. There are many reports on the preparation of seasonings by catalytic oxidation of LOX with vegetable oil or unsaturated fatty acid (Aziz, Akacha, Husson, & Kermasha, 2014). On the other hand, there is limited knowledge on the use of animal oils as raw materials for the preparation of seasonings by LOX catalytic oxidation. LOX has limitations in practical applications, such as LOX needs to be inactivated after the oxidation treatment is completed, and the conventional heat treatment inactivation leads to the secondary oxidation of fat. On the other hand, LOX is expensive and also leads to increased production costs. Immobilized enzyme can effectively solve the aforementioned problems. In our previous studies, immobilized Alcalase and Flavourzyme could facilitate the enzymatic hydrolysis of flaxseed protein, and prepare excellent substrates for Maillard reaction (Wei, Thakur, Liu, Zhang, & Wei, 2018). Flaxseed derived MRPs were found acute nontoxic and no adverse effect during 90-days period (Wei et al., 2019a). Therefore, we simplified the oxidation process by using immobilized LOX to generate the oxidized chicken fat with a suitable degree of oxidation. The aim of this study was to utilize the resulting fat in Maillard reaction to obtain the required chicken flavor. At the same time, the effect of oxidized chicken fat on Maillard reaction process, and sensory evaluation were determined. Furthermore, the volatile aromatic components based on molecular sensory science were also studied. In order to reveal the changes in the species, molecular structure, and content of volatile flavors of MRPs after the addition of oxidized chicken fat, the mechanism of chicken flavor formation was elucidated.

height into 0.2 mol/L CaCl2 (400 mL) to form beaded particles at 20 °C. The beads were covered with hexane and tetramethoxyortho-silicate (1:1.5 v/v of the beads) was added. The obtained mixture was allowed to stand for 12 h at 20 °C to complete the polymerization process. Finally, the immobilized LOX was filtered from the solution, air dried at room temperature, and stored at 4 °C. 2.3. Determination of LOX enzyme activity The assay for LOX activity was conducted by measuring the hydroperoxide formation from linoleic acid. LOX was accurately weighed and added to distilled water adjusted to experimental pH for free LOX (1:500, w/v) or immobilized LOX (1:100, w/v). Then after, the peroxide value (PV) was determined after addition of 1.0 g linoleic acid and 0.6 g Tween 20 in order to react for 1.0 h at required temperature. Enzyme activity (U/g) was calculated as substrate concentration. Our study measured the enzyme activity in the temperature range of 15–35 °C at pH 9.0; pH range of 8–11 at 25 °C; a usage count and storage time at 25 °C and pH 9.0. In order to measure the peroxide value, an inactivated enzyme of similar quality was taken as a blank group after the above operation. Catalytic production of 1.0 mmol hydroperoxide per hour by 1.0 g enzyme can be defined as 1.0 U/g.

X=

(PV PV0) m × t m0

(1)

where X represented enzyme activity value; PV and PV0 represented peroxide values of samples and blank group, respectively; m and m0 corresponded to the quality of samples and blank group; n represented dilution multiples of enzymes. 2.4. Oxidative processing of chicken fat For thermal treatment, 100 mL of chicken fat was heated in a thermostatic bath at 110 °C for 2.5 h at 700 r/min stirring speed in a round bottom flask and samples were collected at 30 min intervals for 150 min. For free enzyme treatment, LOX solution (300 U/g substrates) was mixed with 100 mL of chicken fat. The chicken fat was stirred at 20 °C for 2.5 h; while, samples were collected at 30 min intervals for a total time of 150 min at 700 r/min stirring speed. Each sample was inactivated at 95 °C for 5 min. For immobilized LOX treatment, 100 mL of chicken fat was mixed with 100 mL of distilled water (DW) followed by addition of immobilized LOX (300 U/g substrates). The chicken fat was stirred at 20 °C for 2.5 h; while, samples were collected at 30 min intervals for a total time of 150 min at 700 r/min stirring speed. The immobilized enzyme was recovered after the reaction was completed.

2. Materials and methods 2.1. Materials and chemicals Soy lipoxygenase (LOX) was purchased from Shanghai Zheng Biotechnology Co. Ltd. (China). Chicken fat and flaxseeds were purchased from a local market of Hefei city, China. Chicken fat obtained from subcutaneous fat was separated from chicken carcass (AA Broiler). According to our previous study, fresh chicken fat was boiled and filtered for its use in the experiment (Wei, Fu, Liu, Zhang, & Liu, 2017). The chicken fat is a uniform viscous liquid. After centrifugation, liquid oil (84.5%) and solid fat (15.5%) were obtained. The chicken fat used in this study was whole fat. Defatted flaxseed meal was obtained according to our previous study (Wei et al., 2018, 2019b). The standard compounds (≥95%) which are commonly used to identify the generated volatile flavor compounds were purchased from J&K Chemical Ltd. (Beijing, China).

2.5. Determination of acid value (AV), PV, malondialdehyde (MDA), and fatty acid composition AV, PV, and fatty acid composition were analyzed according to Moghtadaei, Soltanizadeh, and Goli (2018). MDA was analyzed as per the method of Moudache, Nerín, Colon, and Zaidi (2017) 2.6. Preparation of MRPs As reported in our recent study, flaxseed protein hydrolysates were used as starting material, while chemical composition of flaxseed meal and flaxseed protein hydrolysates have been analyzed. Flaxseed meal revealed 38.57% protein content, 11.54% crude fiber content, 1.92% fat content, and 3.12% moisture content, while flaxseed protein hydrolysates consisted of 34.81% protein content, 6.40% crude fiber content, 0.32% fat content, 2.66% moisture content, 40.12% total sugar content, and 6.10% ash content (Wei et al., 2018, 2019b). For the Maillard reaction, four main ingredients such as 10.0 g of flaxseed

2.2. Immobilization of LOX Immobilization of LOX enzyme was performed according to the treatment conditions of Hsu, Foglia, and Piazza (1997) Briefly, the mixture of sodium alginate (4%, w/v) in the form of droplets was added into sodium borate buffer (0.2 mol/L, pH 9.0) and LOX (5 mg/mL in sodium borate buffer). The obtained solution was dropped from 10 cm 2

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protein hydrolysates, 3.0 g of D-xylose, 0.75 g of L-cysteine, and 0.3 g, 0.4 g, or 0.5 g of chicken fat were mixed with DW to obtain the concentration of 10% (w/v) followed by the initial pH of 7.5. The above suspensions were allowed to stand at 120 °C with stirring for 120 min followed by immediate cooling.

volatile compound and internal standard. 2.10. Aromatic components analysis by gas chromatography-olfactometry The volatile compounds were sampled with SPME-fibre (75 μm, carboxen/poly-dimethylsiloxane). Three trained evaluators performed the gas chromatography–olfactometry (GC-O) (DB-5MS capillary column, 30 m × 0.25 mm × 0.25 μm, Agilent) analyses using a DATU 2000 high-resolution olfactometer system (DATU Inc., Durham, NC). Chromatographic conditions were used as per the method given by Eric et al. (2014). The column temperature program was maintained at 40 °C for 2 min, 40–100 °C at 2 °C/min, 100–150 °C at 10 °C/min, and 150–280 °C at 20 °C/min for DB-5MS column. Nitrogen was used as carrier gas at the rate of 1.0 mL/min. The used capillary column, programmed oven temperatures and desorption of the fiber were similar to those in the aforementioned GC–MS analysis.

2.7. Sensory evaluation For sensory evaluation, 14 trained panelists (six males and eight females, aged within 23–48 years) were selected from Laboratory of Sensory Science, Hefei University of Technology (Hefei, China) by confirming the criteria for descriptive analysis. Prior to the formal sensory evaluation of the samples, all panelists discussed flavors (meaty, umami, peculiar smell, and total acceptance) in detail until everyone agreed with the aroma and taste attributes of the study. Sensory evaluation criteria were based on previous research methods such as meaty; umami; and total acceptance (Ogasawara, Katsumata, & Egi, 2006), as well as peculiar smell (Unacceptable taste in the sample). The taste criteria were as follow: The meaty is lean chicken which is cooked at 121 °C for 60 min, which is tasted at normal atmospheric temperature (20 °C). The umami is sodium glutamate solution (1%, w/v). The MRPs sample solutions (0.5%, w/w) were suspended into umami solution (1.0% (w/v) sodium glutamate, and 0.5% (w/v) sodium chloride). The members evaluated 10.0 mL of each sample in a triangle test with two umami soups as two blanks. The sensory evaluation was repeated three times per sample. The panelists were instructed to score on a scale from 0 (not detectable) to 10 (strongly detectable) with 10-point category scales properties. Each panelist was asked to take a rest (10 min) for sensory recovery between each set of four different samples. The evaluation was performed at room temperature (25 °C) under a dim light to mask the color difference between samples. All the samples were evaluated in one-time evaluation within one day.

2.11. Statistical analysis Results were analyzed by using one-way ANOVA through SPSS Statistics 20.0 (SPSS, Inc., Chicago, IL, USA). Data were expressed as mean ± SD (n = 3). Significance was considered at ± 5% (P < 0.05). 3. Results and discussion 3.1. Characteristics of immobilized LOX LOX can specifically oxidize unsaturated fatty acids. Natural oils from animal and vegetable contain large amounts of unsaturated fatty acids. These oils can serve as a good substrate for LOX to produce oxidized fats (Sande, Colen, Santos, Ferraz, & Takahashi, 2017). On the other hand, LOX has few drawbacks such as high cost, and inactivation after the reaction. It is possible to overcome the disadvantages of the above free enzymes based on immobilized enzyme technology. LOX activities were observed as 46.37 × 104 U/g at pH 9.0 and 25 °C for free enzyme. After immobilization, the oxidized effect of immobilized enzyme was evaluated. As shown in Fig. 1, LOX demonstrated similar stability for pH 9.0 and temperature 25 °C before and after immobilization. Altogether, the immobilized LOX could retain half of the potential after subsequent usage (5 times). Consequently, the immobilized LOX showed 60% activities after storage at 4 °C.

2.8. Measurement of reaction progress For sample preparation, same procedure was followed as mentioned in Section 2.6. The freshly prepared samples were diluted 50-fold by using DW and the absorbance was measured at 420 nm and 294 nm representing the browning index (Hou et al., 2017). 2.9. Volatile compounds analysis by gas chromatography-mass spectrometry

3.2. Effects of oxidation methods on AV, PV, MDA, and fatty acid composition

For this, 2 μL of 1,2-dichlorobenzene (50 μg, in 1 mL of methanol) was added to each MRPs sample (3 mL) as an internal control. The volatile components of each MRPs solution were extracted with 75 μm carboxen/ poly-dimethylsiloxane SPME-fibre (50 °C, 5 min) and further identified through comparison of data obtained from gas chromatography-mass spectrometry (GC–MS) (Agilent GC–MS 7890 and DB-WAX 30 m × 0.25 mm × 0.25 μm, or Agilent, DB-5MS 30 m × 0.25 mm × 0.25 μm, Agilent) and NIST 08 (Gaithersburg, MD, USA). Kovats indices (KIs) were obtained by applying C7-C30 standard under the same condition and all the compounds were analyzed with the available standard compounds for identification. Chromatographic conditions were used as per the methods given by Hou et al. (2017) and Eric et al. (2014). The column flow rate was set as 1.0 mL/min, using helium as a carrier gas. The column temperature program was maintained at 40 °C for 2 min, 40–80 °C at 3 °C/min, 80–120 °C at 4 °C/min, and 120–230 °C at 10 °C/min for DB-WAX column; whereas, at 40 °C for 2 min, 40–100 °C at 2 °C/min, 100–150 °C at 10 °C/ min, and 150–280 °C at 20 °C/min for DB-5MS column. The GC was equipped with a mass spectrometric detector which was set at a scanning range of 35 to 450 m/z.

Cv =

Sv × Ci Si

Lipid oxidation utilizes a free radical mechanism to generate volatile compounds such as aldehydes (Tenyang et al., 2013). Based on the pre-experiment, 300 U/g substrate LOX was selected for free enzyme and immobilized enzyme. The effects of thermal, free LOX, and immobilized LOX treatments on the oxidation of chicken fat were characterized by AV, PV, MDA, and fatty acids. The purpose was to select a treatment process with stable oxidation and relatively low oxidation degree on the premise of ensuring that the oxidized chicken fat is suitable for the subsequent Maillard reaction. As shown in Fig. 2 A, the release efficiency for free fatty acids increase linearly within 30 min of thermal treatment. Free and immobilized LOX treatment of chicken fat varied from heat treated chicken fat. Free fatty acids release was stable after 30 min of enzyme treatment. It can be noticed that the thermal treatment of chicken fat was more intense in initial reaction stage, releasing a large amount of free fatty acids; and the enzyme treatment of chicken fat was more stable in releasing free fatty acids. As shown in Fig. 2B, for the initial 30 min of oxidation treatment, the PV of heattreated chicken fat was higher than that of enzyme-treated chicken fat. For 30–60 min of oxidative treatment, the PV of enzyme-treated chicken fat was increased rapidly than heat-treated chicken fat. However, the trend of change in PV had shown a stable rapid increase.

(2)

Where Cv and Ci represented the concentration of volatile compound and internal standard, respectively; Sv and Si corresponded to the peak area of 3

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Fig. 1. Relative enzyme activities of immobilized LOX at different pH (A), temperatures (B), usage count (C), and storage time (D). The values followed by different letters were significantly different (P < 0.05).

products (294 nm) and the final reaction products (420 nm) were accelerated significantly (P < 0.05) by giving different treatments to the oxidized chicken fat compared with the control groups. This may be due to accelerated pathway of the intermediate products of Maillard reaction, such as aldehydes, after adding chicken fat or oxidizing chicken fat in Maillard reaction, which speeds up the reaction system and produces more dicarbonyl compounds (294 nm) and protein melanin (420 nm). The stated inference was further confirmed in Section 3.5. Whereas, the similarity observed between the intermediate reaction products (294 nm) and the final reaction products (420 nm) absorbance (shown in Fig. 3A and B) indicated that the colorless products were the precursors of the brown final reaction products in the reactions. On the other hand, Maillard reaction rate was reduced significantly (P < 0.05) due to the excess addition of the oxidized chicken fat (such as 0.5 g). This may be due to the excessive oxidation of chicken fat which may block the reaction between reducing sugars and peptides. The Maillard reaction rates of chicken fat obtained under different oxidation modes were as follow: thermal treatment > free enzyme treatment > immobilized enzyme treatment. The Maillard reaction involving the oxidized chicken fat after the immobilized LOX treatment had better controllability.

Hydroperoxide was also formed in the early stage. As shown in Fig. 2C, the formation trend of MDA was positively correlated with oxidation time, and the MDA content of free enzyme-treated chicken fat was significantly higher than that of heat treatment and immobilized enzyme treatment (P < 0.05). PV indicates the degree to which chicken fat oxidizes to form peroxides and aldehydes, while MDA indicates the degree to which chicken fat oxidizes to form final products. Therefore, in order to obtain oxidized chicken fat with suitable PV and MDA, the choice of oxidation time is based on the treatment time of PV rising to a stable level and MDA as small as possible. At the same time, taking untreated chicken fat as a control, we determined the content of unsaturated and saturated fatty acids in chicken fat treated with heat treatment for 30 min, chicken fat treated with free enzyme for 60 min, and chicken fat treated with immobilized enzyme for 60 min. The results showed that the unsaturated fatty acids of the oxidized chicken fat decreased significantly compared with the untreated chicken fat (P < 0.05); while, the saturated fatty acids increased significantly (P < 0.05), but there was no significant difference between the three oxidized chicken fats (P > 0.05) (Fig. 2D). Therefore, the heat-treated chicken fat at 30 min and the enzyme-treated chicken fat at 60 min were selected for the Maillard reaction-related experiment.

3.4. Effects of oxidation methods on sensory characteristics

3.3. Effects of oxidation methods on reaction progress

Sensory evaluation is a direct indicator of whether the MRPs can be used as a chicken flavor in the presence of chicken fat. The sensory properties of MRPs with NCF, UCF, chicken fat of thermal treatment, chicken fat of free LOX treatment, and chicken fat of immobilized LOX treatment were assessed by the well-trained team. To prepare chicken flavor using sensory evaluation after heat-treatment, free enzyme and immobilized enzyme treated oxidized chicken fat samples were used as a raw material for Maillard reaction.

The dicarbonyl compounds formed in the intermediate stage were measured at 294 nm, while the protein melanin developed in the advanced stage was measured at 420 nm (Cao et al., 2017). The effects of thermal treatment, free LOX treatment, and immobilized LOX treatment on the Maillard reaction process were studied with the MRPs without chicken fat (NCF) and added untreated chicken fat (UCF) as control. As shown in Fig. 3 A and B, the content of the intermediate reaction 4

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Fig. 2. Effects of oxidation methods of chicken fat on AV (A), PV (B), MDA (C), and fatty acid composition (D). RCF indicated that raw chicken fat was not oxidized. TT indicated that the chicken fat was oxidized by thermal treatment. FET indicated that the chicken fat was oxidized by free enzyme treatment. IET indicated that the chicken fat was oxidized by immobilized enzyme treatment. Saturated fatty acids (ΣSFA) include myristic acid (C14:0), palmitic acid (C16:0) and stearic acid (C18:0), while unsaturated fatty acids (ΣUSFA) include palmitoleic acid (C16:1), oleic acid (C18:1) and linoleic acid (C18:2). The values on the same fatty acid followed by different letters were significantly different (P < 0.05).

degradation, Maillard reaction, and Maillard reaction with fat oxidation degradation interaction (Yang et al., 2015). The oxidation of fat in the air follows the free radical reaction process. The fat is oxygenated to form a primary oxidation product (hydroperoxide), which is then degraded to produce volatile oxides such as aldehydes, ketones, acids, esters, and hydrocarbons (Neff, Warner, & Byrdwell, 2000). Generally, it is believed that aliphatic aldehydes, ketones, alcohols, acids, and lactones are flavor substances produced by fat oxidation. These volatile flavor ingredients have an outstanding contribution to the characteristic flavor of different meats. However, many hydrocarbons are produced during fat oxidation and degradation. These volatile components have high odor activity values and little contribution to flavor which is not listed in Table 1. The present study could detect many aliphatic compounds, including 26 volatile flavor compounds released during lipid oxidation and degradation such as 13 aldehydes, 2 ketones, 7 alcohols, 1 acid, and 3 lactones from the MRPs (Table 1). In terms of content and type, aldehydes and alcohols have the highest content and the most diverse types, followed by ketones, acids, and lactones. According to the previous studies on the volatile components of oxidized chicken fat and oxidized chicken fat added to the “cysteine-glucose” model reaction system, it can be concluded that these compounds should be derived from the oxidative degradation of chicken fat or the further reaction of oxidized chicken fat in the presence of Maillard reaction (Wang, Yang, & Song, 2012). As shown in Table 1, after the addition of UCF in the Maillard reaction system, the content of the aldehydes, alcohols, ketones, acids, and lactones was significantly (P < 0.05) higher as compared to the one without chicken fat (126.0–839.5 ng/g, and 493.5–2332.4 ng/g of

As shown in Fig. 3C–F, by comparing the MRPs of UCF and NCF, it was found that peculiar smell increased significantly (P < 0.05), total acceptance decreased significantly (P < 0.05), while meaty and umami did not change significantly (P > 0.05). By comparing the MRPs with oxidized chicken fat and UCF, the peculiar smell of the chicken fat was significantly reduced (P < 0.05); while, the meat, umami, and total acceptance were improved (P < 0.05). By comparing the effects of different oxidized chicken fats on the meaty flavor of MRPs, there were no significant difference (P > 0.05) in umami, peculiar smell, and total acceptance between the MRPs supplemented with free enzyme-treated chicken fat and with immobilized enzymetreated chicken fat. To illuminate the effect of the amount of added oxidized chicken fat, as the amount in the reaction system increased from 0.3 g to 0.5 g, the meaty, umami, and peculiar smell increased significantly (P < 0.05); however, there was no significant change in overall acceptability (P > 0.05). The results showed that oxidized chicken fat was prepared by thermal and enzymatic treatment, and Maillard reaction in the presence of oxidized chicken fat, did not cause the difference in sensory evaluation of MRPs in general. 3.5. Aromatic components change derived from lipid oxidation and degradation Based on the flaxseed protein hydrolysates, the Maillard reactions with NCF and UCF were used as controls to study the influence of various oxidative methods on the generation of flavor components in Maillard reaction. The volatile components produced by the reaction system are mainly derived from three aspects: fat oxidative 5

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Fig. 3. Effects of oxidation methods of chicken fat at 294 nm (A) and 420 nm (B) and on meaty (C), umami (D), peculiar smell (E), and total acceptance (F). NCF indicated no chicken fat was added to the MRPs. UCF indicated addition of non-oxidized chicken fat 0.4 g to the MRPs. CF 0.3 g indicated adding 0.3 g oxidized chicken fat to the MRPs. Others indicated the corresponding samples. The values followed by different letters were significantly different (P < 0.05).

aldehydes and alcohols); whereas, for TT 0.4 g, FET 0.4 g, and IET 0.4 g, high significant (P < 0.05) difference was observed in these aromatic components compared with UCF. This data was further confirmed in the following Section 3.3 which demonstrates that the addition of chicken fat could accelerate the reaction rate by producing more intermediate products such as aldehydes and alcohols in Maillard reaction. On the other hand, the difference in content between TT 0.4 g, FET 0.4 g, and IET 0.4 g was more complicated. Linoleic acid (C18:2) is one of the main fatty acids in chicken fat. As shown in Fig. 4 A, linoleic acid oxidizes to form 9-OOOH and 13-OOOH hydrogen peroxides, which further degrades to form aldehydes such as (E,E)-2,4-decadienal, 2-octenal, and hexanal (Jayasena, Ahn, Nam, & Jo, 2013). We identified 2-octenal (No. 8, Table 1) and hexanal (No. 3, Table 1) in the reaction system. On the other hand, many unsaturated aldehydes were found in the reaction system, which have high reactivity and are prone to retro-aldol reaction to produce many kinds of saturated aldehydes (Adams, Kitryte, Venskutonis, & De Kimpe, 2011).

Retro-aldol reaction of (E)-2-heptenal (No. 6, Table 1) was shown in Fig. 4 B. Aldehydes are very important flavor substances, which represent one of the key factors causing different aroma to food, and serve as the intermediates for continuation of the Maillard reaction. The smell of short-chain aliphatic aldehydes is usually grass-like, while that of longchain aliphatic aldehydes is usually greasy. Among aldehydes, the contents of saturated aldehydes such as hexanal, octanal, and nonanal are higher, while the contents of unsaturated aldehydes such as (E)-2heptenal, (E)-2-octenal, and (E,E)-2,4-hexadienal are lower. Because the unsaturated aldehyde has high reactivity and ease to participate in the Maillard reaction. In the presence of Maillard reaction, unsaturated aldehydes such as (E)-2-heptenal, (E)-2-octenal, and (E,E)-2,4-hexadienal can react with hydrogen sulfide or ammonia to form thiophenes, thiols or pyridines with alkyl side chains (Elmore et al., 2002). In the reaction system, the content of alcohols derived from lipid oxidation was also higher. In particular, Fan et al reported that 1-octene-36

– – 1045 1156 1255 1325 1367 1406 1471 1502 1598 1612 1811 1222 1284 – 1336 1427 1459 1566 1148 1353 1816 1653 1760 1986 1024 1114 1275 1407 1548 1076 – 1133 1143 1334 1437 1644

Aldehydes 3-methyl-butanal Pentanal Hexanal Heptanal Octanal (E)-2-heptenal Nonanal (E)-2-octenal Decanal (E)-2-nonenal (E)-2-decenal 2-dodecenal (E,E)-2,4-hexadienal

Alcohols 1-pentanol 3-hexanol 4-methyl-2-pentanol 1-hexanol 1-octen-3-ol 1-heptanol 1-octanol

Ketones 2-heptanone 2-nonanone

Acids hexanoic acid

Lactones γ-hexalactone γ-heptalactone γ-nonalactone

Thiols 1-pentanethiol 1-hexanethiol 1-heptanethiol 2-furfuraylthiol 3-thiophenethiol

Thiophenes 2-methylthiophene 3-methylthiophene 2,5-dimethylthiophene 2-ethylthiophene 2-butylthiophene 2-pentylthiophene 2-heptylthiophene

1 2 3 4 5 6 7 8 9 10 11 12 13

14 15 16 17 18 19 20

21 22

23

24 25 26

27 28 29 30 31

32 33 34 35 36 37 38

WAX

KIs

Flavor compounds

No.

1

7 766 780 886 865 1067 1168 1369

845 911 856 910 947

1150 1235 1367

974

877 1145

751 789 804 850 955 957 1034

– 725 778 864 978 946 1089 1030 1182 1127 1130 1215 1352

5MS

5680.1 ± 476.2a 345.5 ± 26.0a 42.7 ± 4.6 36.0 ± 5.7 68.4 ± 4.3 – – 58.2 ± 5.5

1665.4 ± 141.2a 241.3 ± 15.5a 207.4 ± 16.3a 167.3 ± 9.7a 212.6 ± 23.6a 836.8 ± 76.4a

0 – – –

96.4 ± 7.2a 96.4 ± 7.2a

0 – –

787.4 ± 80.7a 135.2 ± 15.2a 45.3 ± 3.7a 37.8 ± 2.5a 68.9 ± 7.4a 150.5 ± 16.7a 166.7 ± 14.2a 183.0 ± 21.3a

605.2 ± 44.2a – – – – 264.9 ± 21.2a – 287.7 ± 18.6a – – – – 52.6 ± 4.7a –

NCF

2

Amouts (ng/g) UCF

4555.1 ± 393.1b 277.1 ± 30.6b – – 38.8 ± 2.9 26.7 ± 3.2a – 41.3 ± 2.7

794.6 ± 66.8b 163.6 ± 11.4b 144.2 ± 9.2b 89.2 ± 6.7b 125.7 ± 13.8b 271.9 ± 25.7b

97.3 ± 9.1a 37.9 ± 2.3a 33.3 ± 3.8a 26.1 ± 3.0a

176.7 ± 20.1b 176.7 ± 20.1b

96.9 ± 6.9a 45.2 ± 2.7a 51.7 ± 4.2a

1280.9 ± 126.6b 172.0 ± 16.1b 57.4 ± 6.3b 45.3 ± 5.1b 246.1 ± 27.0b 224.4 ± 17.4b 289.5 ± 32.4b 246.2 ± 22.3b

731.2 ± 69.5b – – 58.5 ± 4.3a – 294.7 ± 26.0a – 265.8 ± 28.7a – – – 37.6 ± 2.8a 42.5 ± 3.9b 32.1 ± 3.5a

2

2954.7 ± 279.5c 161.8 ± 11.6c – – – 21.1 ± 3.5b – –

431.5 ± 37.8c 45.8 ± 3.5c 97.2 ± 10.8c 31.3 ± 2.4c 74.5 ± 5.4c 182.7 ± 15.7c

205.4 ± 18.2b 112.3 ± 9.4b 54.5 ± 6.2b 38.6 ± 2.7c

251.0 ± 17.4c 251.0 ± 17.4c

259.3 ± 18.2b 137.4 ± 7.9b 121.9 ± 10.3b

2794.2 ± 266.1c 336.7 ± 28.7c – 73.1 ± 6.9c 351.0 ± 33.2c 417.3 ± 39.4c 772.6 ± 81.8c 843.5 ± 76.3c

1319.1 ± 115.1c 52.7 ± 4.5 33.5 ± 2.6a 144.7 ± 15.1b 36.8 ± 2.8 439.6 ± 36.6b – 414.0 ± 35.4b – 36.4 ± 3.4 51.4 ± 4.6 62.7 ± 5.3b – 47.3 ± 5.1b

TT 0.4 g

2

2799.8 ± 246.7c 207.4 ± 22.5d – – – 38.4 ± 4.3c 23.4 ± 1.7 –

493.0 ± 36.7d 54.1 ± 3.3c 139.4 ± 9.5b 45.1 ± 3.1d 97.6 ± 6.5d 156.8 ± 14.3 cd

249.0 ± 25.6c 135.4 ± 14.5c 68.3 ± 7.2c 45.3 ± 4.1d

222.3 ± 21.6c 222.3 ± 21.6c

319.6 ± 25.9c 168.3 ± 12.7c 151.3 ± 13.2c

2924.4 ± 282.2 cd 420.6 ± 38.3d 56.8 ± 6.8b 33.8 ± 4.2a 327.5 ± 36.6c 486.6 ± 41.3d 862.9 ± 74.8 cd 736.2 ± 80.2c

1282.1 ± 105.8c – 35.8 ± 3.2a 195.3 ± 20.6c – 404.4 ± 32.3b 33.6 ± 2.6 382.9 ± 25.5b 32.2 ± 3.0 – 35.8 ± 2.7 86.8 ± 9.7c 75.3 ± 6.5c –

FET 0.4 g

2

IET 0.4 g

2976.9 ± 244.5c 187.9 ± 15.3 cd – – – 20.7 ± 4.0b – –

502.3 ± 46.1d 52.7 ± 6.2c 111.3 ± 8.7d 37.5 ± 2.3c 105.5 ± 8.6d 195.3 ± 20.4c

203.0 ± 15.3b 96.2 ± 7.7b 74.4 ± 5.2c 32.4 ± 2.4ab

275.1 ± 26.3 cd 275.1 ± 26.3 cd

290.6 ± 19.6d 157.2 ± 11.2c 133.4 ± 8.4b

3119.8 ± 293.7d 376.4 ± 43.0e 71.3 ± 5.2c 57.2 ± 6.1b 432.7 ± 42.8d 457.1 ± 38.7 cd 934.6 ± 102.1d 790.5 ± 55.8c

1444.7 ± 134.5c 36.3 ± 2.7 58.7 ± 6.3b 178.1 ± 19.3c 63.0 ± 5.2 476.3 ± 38.7bc – 427.8 ± 44.0b – 54.7 ± 4.9 – 81.5 ± 7.6c – 68.3 ± 5.8c

2

sulfury sulfury garlic, sulfury garlic meaty meaty meaty

garlic garlic garlic meaty meaty

sweet sweet sweet, coconut

sweat, pungent

green, sweet fruity, fatty

green, fruity green, fruity green, fruity green, fruity mushroom green, fruity fatty, citrus

fruity, green grass, pungent grass, green fatty, green citrus, green fatty green, fatty green, fatty green, fatty green, fatty fatty, tallowy fatty, pungent green, fatty

Odors

3

Identification methods

(continued on next page)

MS/KI/O/S KI/O/S MS/KI/O/S MS/KI/O/S MS/KI/O MS/KI/O/S MS/KI/O

MS/KI/O MS/KI/O MS/KI/O MS/KI/O/S MS/KI/O/S

MS/KI/O/S MS/KI/O MS/KI/O/S

MS/KI/O

MS/KI/O/S MS/KI/O/S

MS/KI/O/S MS/KI/O KI/O/S MS/KI/O/S MS/KI/O/S MS/KI/O MS/KI/O

MS/O/S KI/O/S MS/KI/O/S MS/KI/O/S MS/KI/O/S MS/KI/O/S MS/KI/O/S MS/KI/O/S MS/KI/O/S MS/KI/O/S MS/KI/O/S MS/KI/O/S MS/KI/O/S

4

Table 1 Flavor compounds identified from “chicken fat, xylose, cysteine, and flaxseed protein hydrolysates” thermal reaction products, which were generated from lipid oxidation and degradation or the Maillard reaction or the interaction between the Maillard reaction and lipid oxidation and degradation.

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Food Chemistry 306 (2020) 125560

1621 1252 1267 1301 1564 1945 1208 1440 1477 1545 1648 1689

Thiazoles 2-acetylthiazole

Nitrogen-containing heterocyclics 2-methylpyrazine 2-pentylpyridine 2,5-dimethylpyrazine 2-hexylpyridine 2-acetylpyrrole

Oxygen-containing heterocyclics 2-pentylfuran Furfural 2-acetylfuran 5-methyl-2-furancarboxaldehyde 2-furanmethanol 5-methyl-2-furanmethanol

42

43 44 45 46 47

48 49 50 51 52 53

967 842 876 959 950 966

808 904 908 962 1041

1007

1084 1131 1136

5MS

4981.0 ± 457.3a 396.4 ± 31.2a 1587.5 ± 166.1a 1656.8 ± 134.7a 552.3 ± 45.8a 274.9 ± 17.3a 513.1 ± 62.2a

749.8 ± 69.9a 427.8 ± 37.1a 67.5 ± 5.2a 56.2 ± 6.8a 92.7 ± 11.2a 105.6 ± 9.6a

212.3 ± 16.9a 212.3 ± 16.9a

544.3 ± 78.2a 1895.2 ± 127.4a 2689.8 ± 224.4a

NCF

2

Amouts (ng/g) UCF

5425.3 ± 478.8ab 487.1 ± 38.6b 1897.8 ± 204.5b 1388.3 ± 95.7b 634.2 ± 56.1b 380.5 ± 28.0b 637.4 ± 55.9b

1258.1 ± 106.2b 233.1 ± 25.6b 312.4 ± 17.9b 45.7 ± 3.2b 177.4 ± 16.4b 489.5 ± 43.1b

127.5 ± 9.7b 127.5 ± 9.7b

416.5 ± 29.5b 1527.4 ± 136.8b 2227.3 ± 187.4b

2

9488.3 ± 810.7c 675.7 ± 72.3c 3276.4 ± 269.5c 2673.5 ± 220.8c 1476.6 ± 113.3c 523.8 ± 41.6c 862.3 ± 93.3c

1910.3 ± 171.3c 252.8 ± 13.2b 426.9 ± 57.1c 49.8 ± 6.7ab 556.5 ± 42.9c 624.3 ± 51.4c

68.4 ± 7.3c 68.4 ± 7.3c

382.2 ± 42.3bc 873.1 ± 73.8c 1516.5 ± 148.4c

TT 0.4 g

2

9113.2 ± 865.5c 522.5 ± 60.9b 2933.1 ± 320.4c 2791.4 ± 239.7c 1236.5 ± 95.4d 607.6 ± 53.1d 1022.4 ± 96.0d

1824.3 ± 160.5c 210.6 ± 23.5c 467.0 ± 40.3c 42.4 ± 2.5b 527.8 ± 48.7c 576.5 ± 45.5c

45.2 ± 2.8d 45.2 ± 2.8d

378.7 ± 32.8bc 1009.2 ± 84.9c 1142.7 ± 100.5d

FET 0.4 g

2

IET 0.4 g

9265.1 ± 708.4c 638.4 ± 53.2c 3102.9 ± 186.8c 2534.7 ± 214.4c 1515.2 ± 130.3c 577.3 ± 47.2d 896.7 ± 76.5c

1859.8 ± 186.5c 263.5 ± 19.7b 441.1 ± 52.2c 46.3 ± 3.6b 497.4 ± 38.9c 611.5 ± 72.1c

70.8 ± 6.6c 70.8 ± 6.6c

428.3 ± 36.7b 950.1 ± 73.2c 1389.9 ± 115.3c

2

earthy, green caramel sweet caramel sweet sweet

roasted, nutty burnt, fatty roasted, popcorn nutty licorice, nutty

rice

burnt burnt burnt

Odors

3

Identification methods

MS/KI/O/S MS/KI/O/S MS/KI/O/S MS/KI/O MS/KI/O MS/KI/O

MS/KI/O/S MS/KI/O/S MS/KI/O/S MS/KI/O MS/KI/O/S

MS/KI/O/S

MS/KI/O MS/KI/O/S MS/KI/O/S

4

2

KIs, Kovats indices determined using the n-alkanes C7-C30 on DB-Wax and DB-5MS column (30 m × 0.25 mm × 0.25 μm) in the GC–MS and GC-O analysis. NCF indicated MRPs without chicken fat. UCF indicated adding untreated chicken fat 0.4 g to the MRPs. TT 0.4 g indicated adding oxidized chicken fat 0.4 g by thermal treatment to the MRPs. FET 0.4 g indicated adding oxidized chicken fat 0.4 g by free enzyme treatment to the MRPs. IET 0.4 g indicated adding oxidized chicken fat 0.4 g by immobilized enzyme treatment to the MRPs. Means within different letters are significantly (P < 0.05) different in the same line. “–”, not detected. 3 Odor detected by the panelists in GC-O analysis using the DB-5MS column. 4 KI, identified by Kovats indices (KI); MS, identified by search of mass spectra in the NIST 08 database and manual interpretation; O, identified by odor characteristics; and S, identified by comparison of the abovementioned analytical parameters with the authentic chemicals injected.

1

1738 1751 1779

2-thiophenecarboxaldehyde 5-methyl-2-thiophenecarboxaldehyde 3-methyl-2-thiophenecarboxaldehyde

WAX

39 40 41

KIs

1

Flavor compounds

No.

Table 1 (continued)

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Fig. 4. Possible formation pathways. (A) linoleic acid (C18:2) oxidation and degradation. (B) Formation of hexanal, (E)-2-octenal, and (E,E)-2,4-decadienal from (E)-2Heptenal via hydration and retro-aldol reaction. (C) Strecker degradation of amino acids and the production of H2S, NH3, and CH3CHO from cysteine. (D) Alkyl thiols, (E) thiophenethiols, (F) alkyl thiophenes, and (G) thiophenecarboxaldehydes. -R represents an alkyl group (–CnH2n+1, n = 1–7). ARP represents Amadori rearrangement reaction. RA represents retro-aldol reaction.

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alcohol was one of the sources of characteristic flavor of chicken soup (Fan et al., 2019). Aldehydes in oxidized fats can be reduced to alcohols due to the influence of reduced Maillard reaction products. Hydroperoxide of glycerides produced in oxidized fats can also change the degradation pathway to produce reducing alcohols. The main source of aldehydes is Strecker degradation and its reverse aldehyde reaction produces unsaturated aldehydes for NCF as shown in Fig. 4C (Mottram, 1998). Therefore, it can be seen from Table 1 that aldehydes are produced in both types and quantities: oxidized chicken fat > UCF > NCF. And no matter how the chicken fat is prepared, it has strong reaction activity in Maillard reaction system in order to produce more aldehydes and other flavor substances.

assumed that the main source of thiophenes in the system was the Maillard reaction. Thiols are lower than thiophenes in content and species, but many reports indicate that thiophenes have lower odor thresholds. For instance, previous studies on chicken soup found that some thiols such as methanethiol, 2-methyl-3-furylthiol, 2-furylthiol are the key aroma components (Fan et al., 2019). It is worth noting that the identified thiol and thiophene compounds with alkyl chains such as 1-heptanethiol and 2-heptylthiophene are rarely found in the literature report on the ‘peptide/amino acid-reducing sugar’ model reaction. So, these compounds may be formed after lipid oxidation degradation products take part in Maillard reaction. As shown in Fig. 4C, Strecker degradation in the presence of cysteine produces H2S and NH3 (Jayasena et al., 2013; Mottram, 1998). The oxidation and degradation of fatty acids and the retro-aldol reaction of their products are prone to produce large amounts of aliphatic aldehydes. These aliphatic aldehydes occur in the presence of H2S as shown in Fig. 4 D to form a variety of alkyl thiols (No. 27–29, Table 1) (Zhao, Wang, Xie, Xiao, Chenet al., 2019, Zhao, Wang, Xie, Xiao, Du et al., 2019). The formation pathway of 3-thiophenethiol (No. 31, Table 1) was shown in Fig. 4E (Mottram, 1998; Zhao, Wang, Xie, Xiao, Chenet al., 2019). On the other hand, unsaturated aldehydes react with H2S, then cyclize and rearrange to form alkyl thiophenes (No. 32–33, 35–38, Table 1) (Fig. 4F) (Lee et al., 2010; Yu et al., 2012). In addition, 2-thiophenecarboxaldehyde (No. 39, Table 1) with high content were identified in the products, and the formation pathway was shown in Fig. 4G (Lee et al., 2010; Zhao, Wang, Xie, Xiao, Du et al., 2019). Five nitrogen-containing heterocycles (2 pyridines, 1 pyrrole and 2 pyrazines) were detected, of which 2-pentylpyridine and 2-hexylpyridine belong to alkyl pyridines. Pyrazines is usually produced at high temperatures and contributes to roast and nutty aroma (Zou, Liu, & Song, 2018). It is also an important aroma component in roast meat. Like the formation of alkylthiophenes, 2-pentylpyridine and 2-hexylpyridine were mainly derived from the products of lipid oxidation and degradation, which participated in Maillard reaction. After analyzing flavor compounds, a proposed flow diagram involving aromatic effects of oxidation of chicken fat on flaxseed derived Maillard reaction products mechanisms is presented in Fig. 5. The meaty components shown in Fig. 5 were all sulfur-containing flavors, while these sulfur elements were derived from H2S produced by Strecker degradation of cysteine. On the other hand, dicarbonyl compounds that catalyze Strecker degradation were produced during Maillard reaction. Oxidation and degradation of chicken fat, degradation of cysteine, and formation of typical meaty components were discussed in previous detail.

3.6. Aromatic components derived from the Maillard reaction or the interaction between the Maillard reaction, lipid oxidation, and degradation Aromatic components resulting from the interaction of Maillard reaction or Maillard reaction with fat oxidative degradation comprised in Table 1. The main compounds with high content in these flavor mixes were 5-methyl-2-thiophenecarboxaldehyde, 3-methyl-2-thiophenecarboxaldehyde, furfural, 2-acetylfuran, 5-methyl-2-furancarboxaldehyde, and 5-methyl-2-furanmethanol. There were more sulphurcontaining compounds (5 thiols, 10 thiophenes, and 1 thiazoles) than nitrogen-containing and oxygen-containing heterocycles. However, it can be seen from Table 1 in terms of content, the following trend was observed: oxygen-containing heterocycles > sulphur-containing compounds > nitrogen-containing heterocycles. As shown in Table 1, after the addition of UCF in the Maillard reaction system, the contents of the nitrogen-containing and oxygencontaining heterocycles were significantly (P < 0.05) high as compared to the ones without chicken fat; whereas, for TT 0.4 g, FET 0.4 g, and IET 0.4 g, high significant (P < 0.05) difference was observed in these aromatic components compared with UCF. On the other hand, the content of the thiols, thiophenes, and thiazoles were significantly (P < 0.05) lower as compared to the ones without chicken fat (870.8–1233.9 ng/g and 1125.0–2880.3 ng/g of thiols and thiophenes); while, for TT 0.4 g, FET 0.4 g, and IET 0.4 g, slight significant (P < 0.05) difference was observed in these aromatic components compared with UCF. Similarly, the difference in the content between TT 0.4 g, FET 0.4 g, and IET 0.4 g was more complicated. Therefore, fat oxidative degradation products inhibited the formation of thiols, thiophenes, and thiazoles. On the other hand, fat oxidative degradation and Maillard reaction interact to generate some new heterocyclic compounds with alkyl chains or elevated levels of the original heterocyclic compounds in the reaction system with oxidized fat. Oxygen-containing heterocyclics are important intermediate products of Maillard reaction, mainly from the degradation of sugar, which can be detected in many Maillard reaction systems (Yu, Tan, & Wang, 2012). However, many studies have reported that although the content of oxygen-containing heterocycles is high, they generally have high odor thresholds (Cao et al., 2017; Lee, Jo, & Kim, 2010). This results in the fact that oxygen-containing heterocycles have little effect on the flavor of Maillard reaction products. Sulphur-containing compounds play an important role in meat flavor production during Maillard reaction, and these compounds generally have low odor thresholds. The main sulphur-containing compounds identified in the reaction system were thiophenes, the highest content of which was 2-methylthiophene, 2-thiophenecarboxaldehyde, 5-methyl-2-thiophenecarboxaldehyde and 3-methyl-2-thiophenecarboxaldehyde. Previously, thiophenes such as 2-methylthiophene, 5-methyl-2-thiophenecarboxaldehyde, and 3-methyl-2thiophenecarboxaldehyde have been detected in the “glutathione-glucose” system (Lee et al., 2010; Wang et al., 2012). Therefore, it was

4. Conclusion To summarize, the oxidized chicken fats were prepared by thermal, free LOX, and immobilized LOX treatments. > 50% activity of immobilized LOX was maintained after subsequent use and the immobilized enzymes displayed higher functionalities on immobilized LOX properties. Chicken fat or oxidized chicken fat could react with both the amino group-containing precursor in the flaxseed protein hydrolysates as well as with cysteine and reducing sugar. After the addition of chicken fats to the reaction system, the Maillard reaction was noticeably enhanced producing more aliphatic aldehydes and alcohols, especially with the addition of oxidized chicken fat. From the sensory and flavors evaluation, the addition of immobilized LOX and free LOX oxidized chicken fat had shown no obvious differences in the MRPs, which may be related to the degree of oxidation of three kinds of chicken fat. Chicken fat or oxidized chicken fat could react with both the amino group-containing precursor in the flaxseed protein hydrolysates and with cysteine and reducing sugar. This reaction has resulted in the increased amount of flavor ingredients in the system, but the content of sulfur compounds such as

10

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Fig. 5. Proposed mechanism for aromatic effects of oxidation of chicken fat on flaxseed derived Maillard reaction products. FPH represents flaxseed protein hydrolysates. RA represents retro-aldol reaction. SD represents Strecker degradation. AR represents Amadori rearrangement.

thiols and thiophenes decreased significantly, and the structure of sulfur compounds was likely to form alkyl side chains. This study proposed the mechanism for flavor effects of oxidation of chicken fat on flaxseed derived MRPs. Our results concluded that the MRPs imparted a meaty and the characteristic flavor to the chicken.

Technology in Anhui Province (17030701058, 18030701158, 17030701024, and 17030701028), and Zhongyuan Scholars in Henan Province (192101510004).

Funding

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.

Declaration of Competing Interest

This study was supported by the Major Projects of Science and 11

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