Tea marinating-induced improvement of quality in roasted chicken: The potential relationship between tea, flavor, and hazardous substances
茶叶腌制可提高烤鸡的品质：茶、风味和有害物质之间的潜在关系

Acetonitrile, methanol, n-hexane, methyl-tert-butyl-ether (MTBE), formic acid, and acetic acid, were all HPLC grade and acquired from Spectrum Chemical Manufacturing Corporation (Gardena, CA, USA) and Aladdin Reagent Co, Ltd. (Shanghai, China). Anhydrous magnesium sulfate and sodium chloride were acquired from Kermel Chemical Reagent Co. Ltd. (Tianjin, China). Folrisil SPE solid phase extraction column was obtained from Agile Technologies Co, Ltd. (Tianjin, China).
乙腈、甲醇、正己烷、甲基叔丁基醚（MTBE）、甲酸和乙酸均为高效液相色谱级，分别购自光谱化学制造公司（美国加利福尼亚州加迪纳）和阿拉丁试剂有限公司（中国上海）。(中国上海）。无水硫酸镁和氯化钠购自 Kermel Chemical Reagent Co.Ltd. （中国天津）。(中国天津）。Folrisil SPE 固相萃取柱购自 Agile Technologies Co, Ltd. （中国天津）。(Folrisil SPE 固相萃取柱购自 Agile Technologies Co.）

D₃-DL-glutamic acid, Cytarabine-13C₃–5′-monophosphate, D₃-DL-alanine, and D₃-DL-aspartic acid were procured from Aladdin Reagent Co, Ltd. (Shanghai, China). 4,8-Di-MeIQx-d3 were procured from TRC Chemical Ltd. (Toronto, Canada). D₉-TMAO and acrylamide-d₃ were procured from Sigma Aldrich (St. Louis, MO, USA). PAHs, including Naphthalene (Na), 3,4-Benzopyrene (BaP), Fluorene (F), Anthracene (Ant), Benzo[ghi]perylene (B[ghi]P), Chrysene (97 %, Chr), Benzo[k]fluoranthene solution (B[k]F), Fluoranthene (Flu), Benz[a]anthracene (BaA), Benzo[b]fluoranthene (98 %, B[b]F) were procured from Macklin Co, Ltd. (Shanghai, China).
D₃-DL -谷氨酸、Cytarabine-13C₃-5′-monophosphate 、D₃-DL -丙氨酸和D₃-DL -天冬氨酸购自阿拉丁试剂有限公司（中国上海）。(中国上海）购买。4,8-Di-MeIQx-d3 购自 TRC 化学有限公司（加拿大多伦多）。(加拿大多伦多）。D₉-TMAO 和丙烯酰胺-d₃ 购自 Sigma Aldrich (St. Louis, MO, USA)。多环芳烃，包括萘（Na）、3,4-苯并芘（BaP）、芴（F）、蒽（Ant）、苯并[ghi]苝（B[ghi]P）、菊烯（97%，Chr）、苯并[k]荧蒽溶液（B[k]F）、氟蒽 (Flu)、苯并[a]anthracene (BaA)、苯并[b]fluoranthene (98 %, B[b]F) 购自麦克林有限公司（中国上海）。(中国上海）购买。

Black tea, oolong tea, yellow tea, green tea, white tea, dark tea were bought at the flagship store of Eight Horses (Shenzhen, China). Chicken samples were purchased from Zheng Da Foods (Beijing, China).
红茶、乌龙茶、黄茶、绿茶、白茶、黑茶在八马旗舰店（中国深圳）购买。鸡肉样品购自正大食品（北京）有限公司。

Five g of each type of tea was weighed separately and 250 mL of water were mixed and boiled. Keeping it boiling for 10 min, then filter out the tea leaves and let the marinades rest until it reaches room temperature. The chicken was marinated with tea marinade at a ratio of 1:1 (g/mL) at 4 °C for 4 h. Control samples were prepared by marinating the meat in water for 4 h at 4 °C. After marinating, the surface of the chicken was drain. Then, the chicken were roasted at 240 °C for 20 min using the oven. The roasted chicken was cooled to room temperature and stored at −20 °C for further study.
分别称取每种茶叶 5 克，加入 250 毫升水混合煮沸。保持沸腾 10 分钟，然后过滤掉茶叶，将腌料静置至室温。在 4 °C 下以 1:1 的比例（克/毫升）用茶叶腌料腌制鸡肉 4 小时。腌制后，沥干鸡肉表面的水分。然后用烤箱将鸡肉在 240 ℃ 下烤 20 分钟。烤好的鸡肉冷却至室温，储存在 -20 °C，以备进一步研究。

Ethical permission for sensory research was not required in our institution (Dalian Polytechnic University). All participants signed an informed consent before the sensory evaluation. The rights and privacy of all participants were protected during the execution of the study. The selection criteria were usability assessment, interest in participating in the study, no aversion to chicken and tea, allergy or intolerance, and normal perception. Descriptive sensory analysis was conducted by 20 members (11 women and 9 men, ages ranging from 20 to 40 years) to assess the effect of tea curing on roasted meat products. The analysis encompasses the assessment of color, taste, aroma, and texture. They received systematic training and had a wealth of experience in the evaluation of foods. Sensory evaluation for each attribute was performed using a non-structured scale ranging from 1 to 5 points.
在我校（大连工业大学）进行感官研究无需获得伦理许可。所有参与者都在感官评估前签署了知情同意书。在研究过程中，所有参与者的权利和隐私都得到了保护。选择标准为可用性评估、有兴趣参与研究、对鸡肉和茶不反感、不过敏或不耐受、感知正常。20 名成员（11 名女性和 9 名男性，年龄在 20 至 40 岁之间）进行了描述性感官分析，以评估茶叶腌制对烤肉产品的影响。分析包括对颜色、味道、香气和质地的评估。他们接受过系统的培训，在食品评估方面拥有丰富的经验。每种属性的感官评估均采用 1 至 5 分的非结构化量表。

The sensory evaluation team underwent a training program divided into four sessions, each lasting one h, to ensure result repeatability. During the initial session, the panelists evaluated various aspects of the chicken sample, including its color, taste, aroma, and texture. They followed clear instructions to accurately describe their observations. During the second, redundant descriptive terms were eliminated and new properties for testing chicken samples were introduced. Third, all the selected attributes were evaluated on an unstructured scale of 1–5. Finally, the team members evaluated different attributes independently according to the representative samples provided, and they knew nothing about the samples before.
为确保结果的可重复性，感官评估小组接受了分为四节课的培训，每节课持续一个小时。在首次培训中，小组成员对鸡肉样品的各个方面进行了评估，包括颜色、味道、香气和质地。他们按照明确的指示准确描述自己的观察结果。在第二个环节中，删除了多余的描述性术语，并引入了用于检测鸡肉样本的新属性。第三步，以 1-5 分的非结构化量表对所有选定属性进行评估。最后，小组成员根据所提供的代表性样本独立评估不同属性，他们之前对样本一无所知。

During the evaluation sessions, all samples were assigned a random numerical label. The team members were then presented with the samples and each member performed three evaluations.
在评估过程中，所有样本都被随机分配了一个数字标签。然后，小组成员拿到样品，每人进行三次评估。

Samples were prepared according to a previously reported procedure (Okumura et al., 2004). Ten g of samples were weighed and homogenized with 20 mL of ultrapure water for 10 min at 7000 rpm (Ultra Turrax homogenizer, IKA Co, Heidelberg, Germany). Following a 15 min immersion in an ultrasonic bath, 10 mL of n-hexane was introduced. Subsequently, the mixture was vortexed for 1 min and centrifuged at 10000 rpm for 10 min (4 °C). The resulting lower liquid layer was then carefully transferred to a new vial. Repeat the previous step. The lower liquid layer was adjusted to a volume of 100 mL with ultrapure water for subsequent analysis.
样品按照之前报道的程序制备（Okumura 等人，2004）。称取 10 克样品，用 20 mL 超纯水以 7000 rpm 的转速匀浆 10 分钟（Ultra Turrax 匀浆器，IKA 公司，德国海德堡）。在超声波浴中浸泡 15 分钟后，加入 10 毫升正己烷。随后，将混合物涡旋 1 分钟，并以 10000 rpm 的转速离心 10 分钟（4 °C）。然后将得到的下层液体小心地转移到一个新的小瓶中。重复上一步。用超纯水将下层液体调整到 100 mL 的体积，以便进行后续分析。

Samples were prepared according to a previously reported procedure (Huang et al., 2019). The PEN 3 E-nose was utilized to measure the odor response value, employing ten distinct types of metal oxide semiconductors that correspond to various volatile compounds. The sensor-compound relationships were established as follows: W1S matched methane and related compounds, W2S matched alcohols, aldehydes, and ketones, W3S matched long-chain alkanes, W5S matched nitrogen compounds, W6S matched hydrocarbons, W1C matched aromatic benzene compounds, W3C matched aromatic compounds with ammonia, W5C matched short-chain alkanes, W1W matched inorganic sulfides, and W2W matched organic sulfur compounds and aromatic compounds. Before testing, the samples underwent preincubation. 5 g of sample were placed in 20 mL airtight vials. The testing conditions consisted of an incubation temperature of 50 ± 1 °C, and the measurement time was set at 100 s.
样品根据先前报告的程序制备（Huang 等，2019）。利用 PEN 3 E-nose 测量气味响应值，采用了十种不同类型的金属氧化物半导体，与各种挥发性化合物相对应。传感器与化合物的关系确定如下：W1S 与甲烷和相关化合物相匹配，W2S 与醇、醛和酮相匹配，W3S 与长链烷烃相匹配，W5S 与氮化合物相匹配，W6S 与碳氢化合物相匹配，W1C 与芳香苯化合物相匹配，W3C 与含氨的芳香化合物相匹配，W5C 与短链烷烃相匹配，W1W 与无机硫化物相匹配，W2W 与有机硫化合物和芳香化合物相匹配。测试前，样品需要进行预培养。将 5 克样品放入 20 毫升密闭小瓶中。测试条件包括孵育温度为 50 ± 1 °C，测量时间设定为 100 秒。

The samples were prepared following the methodology previously reported by our team (Xin et al., 2023). Taste-active compounds were analyzed using HPLC-MS/MS (LC-30 CE, Shimadzu, Japan, 5500 Qtrap System, AB Sciex, America). The separation of target analytes was achieved on an Infinity Lab Poroshell 120 HILIC-Z column (2.1 mm × 150 mm, 2.7 μm particle size, Agilent). The mobile phase, elution gradient, and mass spectrometry conditions were set according to previous reports. Perform qualitative and quantitative analysis of taste-active compounds in the samples using the internal standard method.
样品是按照我们团队之前报告的方法制备的（Xin 等，2023）。使用 HPLC-MS/MS （LC-30 CE，日本岛津公司；5500 Qtrap 系统，美国 AB Sciex 公司）分析味觉活性化合物。目标分析物在 Infinity Lab Poroshell 120 HILIC-Z 色谱柱（2.1 mm × 150 mm，2.7 μm 粒径，安捷伦）上实现分离。流动相、洗脱梯度和质谱条件均根据之前的报告设定。采用内标法对样品中的味觉活性化合物进行定性和定量分析。

The samples were prepared following the methodology previously reported by our team Chen (Chen et al., 2023). The volatile compounds analysis was performed using the 5977 A-7890B GC–MS (Agilent Technologies, Santa Clara, CA, USA) system with an HP-5MS column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies). The temperature gradient and mass spectrometry conditions were set according to previous reports.
样品的制备采用了我们团队 Chen 以前报告的方法（Chen et al.）挥发性化合物分析采用 5977 A-7890B GC-MS 系统（安捷伦科技公司，美国加利福尼亚州圣克拉拉市）和 HP-5MS 色谱柱（30 m × 0.25 mm × 0.25 μm，安捷伦科技公司）。温度梯度和质谱条件根据之前的报告设定。

N-alkanes standards (C7–C30) were determined to calculate linear retention indices of volatile compounds (Vandendool & Kratz, 1963). Identify the volatile compounds and match them with the standards in NIST 14 through mass fragmentation and RI. The relative quantification of volatile compounds was calculated using the internal standard (IS).
测定 N-烷烃标准（C7-C30），计算挥发性化合物的线性保留指数（Vandendool & Kratz，1963）。识别挥发性化合物，并通过质量碎片和 RI 与 NIST 14 中的标准进行比对。使用内标 (IS) 计算挥发性化合物的相对定量。

The samples were prepared following the methodology previously reported by our team and some changes were made (Gao et al., 2021). Three g of samples were weighed and added internal standard (80 μL of 2 μg/mL acrylamide-d₃ and 1 μg/mL 4,8-Di-MeIQx-d₃). The samples were homogenized in 10 mL of ultrapure water and 10 mL of acetonitrile at 8500 r/min for 2 min. After 15 min of ultrasonication, 5 mL of n-hexane, 4 g of anhydrous magnesium sulfate, and 0.5 g of sodium chloride were subsequently added. After homogenization for 2 min, samples underwent centrifugation at 3040 g for 10 min. The acetonitrile layer was collected for 5 mL and subjected to evaporation using a high-speed vacuum concentrator. The resulting dried sample was reconstituted in water/acetonitrile (1:1, v/v) and agitated for 30 s. Following centrifugation at 20000 g (4 °C) for 10 min, the supernatant was extracted for subsequent analysis.
样品的制备采用我们团队之前报告的方法，并做了一些改动（Gao 等，2021）。称取 3 g 样品并加入内标（80 μL 2 μg/mL 丙烯酰胺-d₃ 和 1 μg/mL 4,8-Di-MeIQx-d₃ ）。样品在 10 mL 超纯水和 10 mL 乙腈中以 8500 r/min 的转速匀浆 2 分钟。超声 15 分钟后，加入 5 mL 正己烷、4 g 无水硫酸镁和 0.5 g 氯化钠。匀浆 2 分钟后，样品在 3040 g 下离心 10 分钟。收集 5 mL 乙腈层，使用高速真空浓缩器蒸发。得到的干燥样品在水/乙腈（1:1，v/v）中重构并搅拌 30 秒。在 20000 g（4 °C）离心 10 分钟后，提取上清液用于后续分析。

Quantification of HCAs, 5-HMF, and ACY were performed by 5500 Q TRAP. An Acquity UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm), equipped with an Acquity UPLC HSS T3 Van Guard Pre-column (2.1 mm × 5 mm, 1.8 μm), was utilized to separate ACY, 5-HMF, and HCAs. The analytical column was maintained at 40 °C. mobile phase A was 0.1 % (v/v) formic acid in water; mobile phase B was 0.1 % (v/v) formic acid in acetonitrile. The following elution gradient was applied: 0–1.5 min, 1 % B; 1.5–16.5 min, 1–99 % B; 16.6–20 min, 99–1 % B. The flow rate was 0.3 mL/min. The injected volume was 1 μL. The solvent removal temperature was set to 600 °C. The entry potential and the ion spray voltage of the source were set at 10 and 5500 V. The curtain gas and Ion source were high-purity nitrogen at 35 and 60 psi. The content in the sample was analyzed by the ratio of peak height to the internal standard.
采用 5500 Q TRAP 对 HCA、5-HMF 和 ACY 进行定量。使用 Acquity UPLC HSS T3 色谱柱（2.1 mm × 100 mm，1.8 μm）分离 ACY、5-HMF 和 HCAs，该色谱柱配有 Acquity UPLC HSS T3 Van Guard 预柱（2.1 mm × 5 mm，1.8 μm）。流动相 A 为 0.1 % (v/v) 甲酸水溶液；流动相 B 为 0.1 % (v/v) 甲酸乙腈水溶液。洗脱梯度如下0-1.5 分钟，1 % B；1.5-16.5 分钟，1-99 % B；16.6-20 分钟，99-1 % B。进样量为 1 μL。溶剂去除温度设定为 600 °C。离子源的入口电位和离子喷雾电压分别设定为 10 V 和 5500 V。帘气和离子源为高纯氮气，压力分别为 35 和 60 psi。根据峰高与内标之比分析样品中的含量。

Two g of sample were weighed and vortexed with 10 mL of acetonitrile and 10 mL of hexane saturated with acetonitrile. After ultrasonic for 15 min, the samples were homogenized at 8000 r/min (4 °C) for 10 min, and the acetonitrile was collected. 10 mL of acetonitrile was added and repeat the extraction step. The acetonitrile obtained from two extractions was evaporated to dryness by rotary evaporation. 5 mL of n-hexane was added to dissolve the residue. Five mL of n-hexane was transferred into a Florisil solid-phase extraction column that had been activated with 5 mL of dichloromethane and 10 mL of n-hexane. The chicken heart bottle was washed with 5 mL of n-hexane, and the washing solution was combined with the column. 8 mL mixture of n-hexane and dichloromethane (1:1) was used for elution. All eluates were collected and nearly dried by nitrogen blowing. The dried sample was dissolved in 1 mL of acetonitrile and shaken for 1 min by vertexing. Before analysis, the solution was filtered through a 0.22 mm membrane filter.
称取 2 g 样品，与 10 mL 乙腈和 10 mL 乙腈饱和正己烷混合并涡旋。超声 15 分钟后，以 8000 r/min（4 °C）的速度均质 10 分钟，收集乙腈。再加入 10 mL 乙腈，重复萃取步骤。通过旋转蒸发将两次萃取得到的乙腈蒸干。加入 5 毫升正己烷溶解残留物。将 5 mL 正己烷转移到用 5 mL 二氯甲烷和 10 mL 正己烷活化的 Florisil 固相萃取柱中。用 5 mL 正己烷洗涤鸡心瓶，然后将洗涤液与柱子合并。用 8 mL 正己烷和二氯甲烷（1:1）混合液进行洗脱。收集所有洗脱液，用氮气吹干。将干燥后的样品溶解在 1 mL 乙腈中，顶置振荡 1 分钟。分析前，溶液经 0.22 mm 薄膜过滤器过滤。

The PAHs were analyzed using Agilent 1260 Infinity-FLD (Agilent Technologies, Santa Clara, CA, USA). Waters PAH C18 column (4.6 mm × 250 mm, 5 μm) was performed to chromatographic analysis at 35 °C. The elution solvents consisted of mobile phase A (water) and mobile phase B (acetonitrile) at a flow rate of 1.2 mL/min. The following elution gradient was applied: 0–9 min, 60 % B; 9–12 min, 60–100 % B; 12–28 min, 100 % B; 28–32 min, 100–60 % B; 32–35 min, 60 % B. Sample injection volume was 10 μL. The excitation and emission wavelengths were 279 and 340 nm, 0.0–12.5 min; 248 and 375 nm, 12.5–13.65 min; 280 and 462 nm, 13.65–15.50 min; 270 and 385 nm, 15.50–17.50 min; 270 and 446 nm, 17.50–18.50 min; 292 and 410 nm, 18.50–25.70 min; 305 and 480 nm, 25.70–35.00 min. The sample's qualitative and quantitative assessment of PAHs was conducted through the utilization of an external calibration curve.
使用 Agilent 1260 Infinity-FLD（Agilent Technologies，Santa Clara，CA，USA）分析多环芳烃。Waters PAH C18 色谱柱（4.6 mm × 250 mm，5 μm）在 35 °C 下进行色谱分析。洗脱溶剂包括流动相 A（水）和流动相 B（乙腈），流速为 1.2 mL/min。洗脱梯度如下0-9 分钟，60 % B；9-12 分钟，60-100 % B；12-28 分钟，100 % B；28-32 分钟，100-60 % B；32-35 分钟，60 % B。激发和发射波长分别为：279 和 340 nm，0.0-12.5 分钟；248 和 375 nm，12.5-13.65 分钟；280 和 462 nm，13.65-15.50 分钟；270 和 385 nm，15.50-17.50 分钟；270 和 446 nm，17.50-18.50 分钟；292 和 410 nm，18.50-25.70 分钟；305 和 480 nm，25.70-35.00 分钟。样品中 PAHs 的定性和定量评估采用外部校准曲线进行。

Ten g of tea samples was weighed separately and 500 mL of ultrapure water was weighed and boiled, the boiling state was maintained for 10 min. The solvent was evaporated nearly 100 mL and lyophilized. Seventy mg of samples was weighed, followed by the addition of 225 μL of chilled methanol, vortexed for 15 s. Then, 250 μL of ultrapure water was introduced and vortexed for 15 s. Finally, 750 μL of MTBE was added, and the mixture was vortexed for 6 min. After centrifuging at 8000 g for 10 min (4 °C), 200 μL of the lower liquid layer was collected and 600 μL of ice-cold methanol/isopropanol (1:1, v/v) was added. After mixing for 6 min, the mixture was centrifuged at 20,000 g for 2 min (4 °C), 300 μL of supernatant was collected and dried using a high-speed vacuum concentrator. 100 μL of methanol/water was used to redissolve the dried sample. The solution was shaken for 2 min and centrifuged 20,000 g for 2 min (4 °C) to collect impurities before analysis.
分别称取 10 克茶叶样品，称取 500 毫升超纯水并煮沸，保持沸腾状态 10 分钟。蒸发近 100 mL 溶剂并冻干。称取 70 mg 样品，加入 225 μL 冷甲醇，涡旋 15 秒；然后加入 250 μL 超纯水，涡旋 15 秒；最后加入 750 μL MTBE，涡旋 6 分钟。在 8000 g 下离心 10 分钟（4 °C）后，收集 200 μL 下液层，然后加入 600 μL 冰冷的甲醇/异丙醇（1:1, v/v）。混合 6 分钟后，混合物在 20,000 g 下离心 2 分钟（4 °C），收集 300 μL 上清液并用高速真空浓缩器干燥。用 100 μL 甲醇/水重新溶解干燥的样品。振荡溶液 2 分钟，然后离心 20,000 g 2 分钟（4 °C），收集杂质，再进行分析。

UPLC-Q Extractive X was used to evaluate the compounds of tea (Chen et al., 2023). To assess the detection accuracy and stability, QC samples and blank samples were set up for each of the 10 samples. The Acquity UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm), equipped with an Acquity UPLC HSS T3 Van Guard Pre-column (2.1 mm × 5 mm, 1.8 μm), was used for chromatographic analysis at 25 °C. mobile phase A consisted of 0.1 % Formic acid in water, whereas mobile phase B consisted of 0.1 % Formic acid in acetonitrile at a flow of 0.5 mL/min. A linear gradient was used: 0–1 min, 1 % B; 1–8 min, 1–99 % B; 8–10 min, 99 % B; 10 min, 99–1 %B; 10–12 min, 1 % B. The injection volume was 1 μL. ESI was used to carry out electrospray ionization. The detection was performed in positive mode, and mass spectrometric data were acquired over the range of 120–1200 m/z. The sheath gas flow rate was 60 %, the aux gas flow rate was 25 %, the purge gas flow rate was 2 %. The spray voltage was at 3.6 kV, the capillary temperature was 380 °C, aux gas heater temperature was 370 °C. The metabolites were annotated by MS-DIAL software, HMDB (http://www.hmdb.ca/) based on accurate mass, and fragment matching. Peak intensities were used to show metabolite trends.
采用 UPLC-Q Extractive X 对茶叶中的化合物进行评估（Chen 等，2023）。为了评估检测的准确性和稳定性，对 10 个样品分别设置了质控样品和空白样品。色谱分析使用 Acquity UPLC HSS T3 色谱柱（2.1 mm × 100 mm，1.8 μm），配备 Acquity UPLC HSS T3 Van Guard 预色谱柱（2.1 mm × 5 mm，1.8 μm），分析温度为 25 °C。流动相A 为 0.1 % 甲酸水溶液，流动相 B 为 0.1 % 甲酸乙腈水溶液，流速为 0.5 mL/min。采用线性梯度：0-1 分钟，1 % B；1-8 分钟，1-99 % B；8-10 分钟，99 % B；10 分钟，99-1 % B；10-12 分钟，1 % B。使用 ESI 进行电喷雾离子化。检测采用正离子模式，质谱数据采集范围为 120-1200 m/z 。鞘气流量为 60%，辅助气体流量为 25%，吹扫气体流量为 2%。喷雾电压为 3.6 kV，毛细管温度为 380 ℃，辅助气体加热器温度为 370 ℃。代谢物由 MS-DIAL 软件、HMDB (http://www.hmdb.ca/) 根据精确的质量和片段匹配进行注释。峰强度用于显示代谢物的变化趋势。

All experiments were replicated three times. The data were organized using Microsoft Office 2016, and statistical analyses were performed using the SPSS 20.0 software. ANOVA was used to statistically assess the outcomes (p < 0.05). Metabo Analyst 5.0 was used to carry out VIP score (variable importance in projection), heatmap, PLS-DA (partial least squares discriminant analysis), and PCA (principal component analysis) (Chong et al., 2018). Prismchs was used for boxplots. TBtools is used for correlation heatmap. The Xlstat was used for PLS-R (Partial least squares regression) analysis.
所有实验均重复三次。数据使用 Microsoft Office 2016 整理，并使用 SPSS 20.0 软件进行统计分析。方差分析用于统计评估结果（p<0.05）。Metabo Analyst 5.0 用于进行 VIP 评分（投影中变量重要性）、热图、PLS-DA（偏最小二乘判别分析）和 PCA（主成分分析）（Chong 等人，2018）。Prismchs 用于方框图。TBtools 用于绘制相关热图。Xlstat用于PLS-R（部分最小二乘回归）分析。

The E-tongue response of chicken marinated with different teas after high-temperature roasting is shown in Fig. 1. The negative response values of the sensors indicate their minimal contribution to the overall taste. Sourness and astringency had a limited impact on the taste changes of chicken marinated with different teas after high-temperature roasting. Saltiness played the most significant role in overall taste perception. Marinated with black, oolong, yellow, green, and black tea effectively reduced the saltiness in high-temperature roasted chicken. The chicken marinated with yellow tea and white tea showed a higher response for umami compared to control samples. Marinated with white tea and black tea significantly enhances the richness of high-temperature roasted chicken. The flavor variances may be ascribed to the existence of unique flavor compounds in tea, consequently impacting the aroma of high-temperature roasted chicken marinated with tea. Tea polyphenols, as a predominant constituent in tea leaves, exhibit antioxidative properties. During the processes of marination and roasting, tea polyphenols engage in reactions with proteins, fats, and other constituents in the ingredients, giving rise to the formation of organic acids, alcohols, and other substances, consequently affecting the taste and aroma (Bortolini et al., 2021).
E 用不同茶叶腌制的鸡肉在高温烘烤后的舌头反应如 图 1 所示。传感器的负响应值表明它们对整体口味的影响微乎其微。高温烘焙后，酸味和涩味对用不同茶叶腌制的鸡肉的口味变化影响有限。咸味对整体味觉的影响最大。用红茶、乌龙茶、黄茶、绿茶和黑茶腌制的鸡肉能有效降低高温烘焙鸡肉的咸味。与对照样品相比，用黄茶和白茶腌制的鸡肉对鲜味的反应更高。用白茶和红茶腌制的鸡肉明显提高了高温烤鸡的鲜味。风味差异可能是由于茶叶中存在独特的风味化合物，从而影响了用茶叶腌制的高温烤鸡的香气。茶多酚是茶叶中的主要成分，具有抗氧化特性。在腌制和烘焙过程中，茶多酚会与食材中的蛋白质、脂肪和其他成分发生反应，形成有机酸、醇和其他物质，从而影响口感和香气（Bortolini et al., 2021）。

Fig. 2G represents the heatmap of taste-active compounds change in high-temperature roasted chicken marinated with different teas. High-temperature processed foods contain a significant amount of FAA, which serves as the principal flavor compound in these food products. FAA contributes to various taste sensations, including bitterness, sweetness, umami, and tastelessness. The tea marinade possesses antioxidant properties, effectively scavenging free radicals, and mitigating lipid and protein oxidation in high-temperature roasted chicken. It can inhibit the oxidative degradation of amino acids (Yao et al., 2021). In addition, tea contains large amounts of amino acids. The accumulation of amino acids makes an important contribution to flavor. It can be observed that the bitter-tasting amino acids such as methionine were significantly reduced in high-temperature roasted chicken marinated with teas. Aspartic acid, an umami-tasting amino acid, increases in high-temperature roasted chicken marinated with green tea, white tea, and black tea. Aspartic acid is involved in human tricarboxylic acid metabolism and is frequently utilized in medical interventions for conditions like hypertension. In high-temperature roasted chicken marinated with green tea, white tea, and black tea, the content of sweet-tasting amino acids such as serine and threonine increases.
Fig.高温加工食品中含有大量的 FAA，它是这些食品的主要风味化合物。FAA 有助于产生各种味觉，包括苦味、甜味、鲜味和无味。茶腌料具有抗氧化特性，能有效清除自由基，减轻高温烤鸡的脂质和蛋白质氧化。它能抑制氨基酸的氧化降解（Yao 等，2021）。此外，茶叶中还含有大量的氨基酸。氨基酸的积累对风味有重要贡献。可以观察到，在用茶叶腌制的高温烤鸡中，蛋氨酸等苦味氨基酸明显减少。在用绿茶、白茶和红茶腌制的高温烤鸡中，鲜味氨基酸天门冬氨酸增加。天门冬氨酸参与人体三羧酸代谢，常用于高血压等疾病的医疗干预。在用绿茶、白茶和红茶腌制的高温烤鸡中，丝氨酸和苏氨酸等甜味氨基酸的含量会增加。

Apart from FAA, nucleotides and related chemicals are significant non-volatile taste-active compounds. IMP, GMP, AMP, UMP, and CM are crucial umami flavor components in meat products (Yue et al., 2016). The AMP content in high-temperature roasted chicken experienced an increase after being marinated with tea, thereby enhancing the umami taste or interacting with other compounds. This phenomenon could be attributed to the impact of tea marination on the generation and degradation of nucleotides in high-temperature roasted chicken. AMP, a metabolite of ATP in normal biological metabolism, degrades to IMP, which is further metabolized to Ino and Hx through phosphatase metabolism (Feng et al., 2016). IMP is considered a major precursor in raw meat as it is one of the compounds derived from ATP degradation (Rotola-Pukkila et al., 2015). After being marinated with green tea or white tea marinade, the content of IMP increases in high-temperature roasted chicken. Compounds such as Inosine, Hypoxanthine, Adenine, Adenosine, Guanosine, and Xanthine are metabolites of nucleotides and typically possess a bitter taste. Tea marinating reduces the levels of Ino and Hx in high-temperature roasted chicken.
除 FAA 外，核苷酸和相关化学物质也是重要的非挥发性味觉活性化合物。IMP、GMP、AMP、UMP 和 CM 是肉制品中重要的鲜味成分（Yue 等人，2016）。高温烤鸡经茶叶腌制后，AMP 含量增加，从而增强了鲜味或与其他化合物相互作用。这一现象可能是由于茶叶腌制对高温烤鸡中核苷酸的生成和降解产生了影响。AMP是正常生物新陈代谢中ATP的代谢产物，可降解为IMP，IMP通过磷酸酶代谢进一步代谢为Ino和Hx（Feng 等人，2016）。IMP 被认为是生肉中的主要前体物，因为它是 ATP 降解产生的化合物之一（Rotola-Pukkila 等人，2015）。用绿茶或白茶腌料腌制后，高温烤鸡中的 IMP 含量会增加。肌苷、次黄嘌呤、腺嘌呤、腺苷、鸟苷和黄嘌呤等化合物是核苷酸的代谢产物，通常带有苦味。茶叶腌制可降低高温烤鸡中肌苷和次黄嘌呤的含量。

Ten different compounds (VIP > 1.0) that differential the taste of high-temperature roasted chicken marinated with various teas were identified. These compounds include cis-Aconitic, Malic, GMP, Pvroglutamic, Salicylic, Pyruvic, Taurine, Succinic, Asparaginate, and Uridine. These compounds primarily affect the umami and astringent tastes of high-temperature roasted chicken. GMP (Fig. 2B), as one of the major nucleotides, significantly affects cellular metabolism, contributes to meat flavor, and works in conjunction with IMP to stimulate umami production (Dai et al., 2011). Pvroglutamic (Fig. 2D), an important organic acid, imparts a distinctive umami and sour taste (D.-W. Chen & Zhang, 2007). GMP, Asparaginate (Fig. 2C), and Succinic (Fig. 2E) were umami taste-active compounds that caused differences among the components, with their total amounts in high-temperature roasted chicken following the order: white > dark > green > water > yellow > black > oolong. Uridine (Fig. 2F) is a key compound for the astringent taste, and its level decreases in high-temperature roasted chicken after tea marinated.
确定了十种不同的化合物（VIP > 1.0），这些化合物能使高温烤鸡与各种茶叶腌制后的味道产生差异。这些化合物包括顺式乌头酸、苹果酸、GMP、焦谷氨酸、水杨酸、丙酮酸、牛磺酸、琥珀酸、天冬酰胺和尿苷酸。这些化合物主要影响高温烤鸡的鲜味和涩味。GMP（Fig.2B）作为主要核苷酸之一，对细胞新陈代谢有显著影响，有助于肉类风味的形成，并与 IMP 一起刺激鲜味的产生（Dai et al、2011）。焦谷氨酸（图 2D）是一种重要的有机酸，具有独特的鲜味和酸味（D. -W.Chen & Zhang，2007）。GMP、天冬酰胺（ 图 2C）和丁二酸（Fig.2E）为鲜味活性化合物，它们在高温烧鸡中的总含量依次为：白>深>绿>水>黄>黑>乌龙。乌啶（Fig.

High-temperature roasting generates large amounts of aldehydes, ketones, alcohols, aromatic compounds, phenols, and hydrocarbons. The results of the heatmap for the volatile compounds in high-temperature roasted chicken marinated with different types of tea are shown in Fig. 3B, as observed, the overall volatile compounds in the high-temperature roasted chicken marinated with black tea, oolong tea, and yellow tea increased. Amidst these, the high-temperature roasted chicken marinated with yellow tea exhibited the highest content of volatile compounds. Protein, carbohydrates, amino acids, and other nutrients undergo intense Maillard reactions under high-temperature aerobic conditions. Alpha-dicarbonyl compounds engage with amino acids, undergoing degradation, resulting in aldehyde formation. The roasted chicken marinated with tea showed a lower content of nonanal, which can be attributed to the antioxidative properties of the tea marinade. Yuan et al. (2023) found that antioxidant-rich spices can attenuate the Maillard reaction and diminish the generation of volatile compounds. Alcohols primarily originate from lipid oxidation and Strecker degradation reactions (Yang et al., 2017). Compared to the control samples, the roasted chicken marinated with tea exhibited an increase of the 1-Hexanol 2-ethyl, a compound with the subtly floral. Ketones typically have a higher odor threshold compared to aldehydes. It has been reported that alkenes and alkanes represent representative flavors of fruit, spices, citrus, and sweetness (Yuan et al., 2023). After marinated with tea, their concentrations in the food were significantly enhanced. Spices are rich in flavor compounds that can transfer to chicken. During roasting, these flavor compounds in the spices break down due to heat, resulting in the unique flavors associated with roasted chicken. Additionally, water-soluble compounds from the spices seep into the chicken, offering more substrates for the Maillard reaction while roasting. Furthermore, the compounds in spices can alter the structure of myofibrillar proteins, facilitating the release of flavors from the meat (Sun, Yu, Saleh, et al., 2024).
高温烘焙会产生大量的醛、酮、醇、芳香族化合物、酚和碳氢化合物。图 3B显示了用不同茶叶腌制的高温烤鸡中挥发性化合物的热图结果。其中，用黄茶腌制的高温烤鸡的挥发性化合物含量最高。在高温有氧条件下，蛋白质、碳水化合物、氨基酸和其他营养物质会发生强烈的马氏反应。α-二羰基化合物与氨基酸接触，发生降解，形成醛。用茶叶腌制的烤鸡显示出较低的壬醛含量，这可归因于茶叶腌泡汁的抗氧化特性。Yuan 等人（2023）发现，富含抗氧化剂的香料可减轻 Maillard 反应，减少挥发性化合物的生成。酒精主要来源于脂质氧化和斯特克降解反应（Yang 等人，2017）。与对照样本相比，用茶叶腌制的烤鸡表现出 1-己醇 2-乙基的增加，这是一种具有微妙花香的化合物。酮类化合物的气味阈值通常高于醛类化合物。 据报道，烯类和烷类代表了水果、香料、柑橘和甜味的代表性风味（Yuan 等人，2023）。用茶叶腌制后，这些物质在食物中的浓度明显提高。香料中含有丰富的风味化合物，可以转移到鸡肉中。在烤制过程中，香料中的这些风味化合物会受热分解，从而产生与烤鸡相关的独特风味。此外，香料中的水溶性化合物会渗入鸡肉，为烤制过程中的 Maillard 反应提供更多底物。此外，香料中的化合物还能改变肌原纤维蛋白质的结构，促进肉味的释放（Sun, Yu, Saleh, et al., 2024）。

Based on the classification of PLS-DA (Fig. 3C), compounds exhibiting VIP > 1.0 were chosen as differential volatile compounds for distinguishing high-temperature roasted chicken marinated with different types of tea. Nonanal was present in higher amounts in control samples. Nonanal exhibits aromas of roses, citrus, and possesses a strong greasy odor (Pino & Mesa, 2010). It is an important volatile compound produced through the β-oxidation of fats. In high-temperature roasted chicken marinated with black tea, the contents of 2,3,5-trimethyl-pyrazine and Acetaldehyde were higher. 2,3,5-trimethyl-Pyrazine is a Maillard reaction product that provides a unique barbecue flavor to heat-treated foods. In high-temperature roasted chicken marinated with yellow tea, 4-Dodecene has a higher content compared to other marinades, making it the main volatile compound.
根据 PLS-DA 的分类（Fig.对照样本中的壬醛含量较高。壬醛具有玫瑰和柑橘的香气，并带有强烈的油腻气味（Pino & Mesa，2010）。它是一种重要的挥发性化合物，通过脂肪的β-氧化作用产生。在用红茶腌制的高温烤鸡中，2,3,5-三甲基吡嗪和乙醛的含量较高。2,3,5-三甲基吡嗪是一种马氏反应产物，可为加热处理的食物提供独特的烧烤风味。在用黄茶腌制的高温烤鸡中，4-十二烯的含量比其他腌料高，是主要的挥发性化合物。

As observed from Table 2, compared with the control samples, the content of most hazardous compounds in high-temperature roasted chicken marinated with tea exhibited a decreasing trend. Among them, green tea demonstrated the best effect in inhibiting hazardous compounds. The majority of hazardous compounds are compounds with cyclic structures, and the antioxidant components in tea may interfere with the formation of these substances by reducing or quenching active free radicals (Lu et al., 2018). Applying marinades containing antioxidants from tea to processed meat products at high-temperature is an effective method to reduce the generation of hazardous compounds. Quelhas et al. (2010) found that marinating meatballs with green tea extract during the marination process can reduce the levels of PHIP and AaC. Wang et al. (2018) discovered a pronounced inhibitory effect of green tea on PAHs in marinaded chicken meat. The antioxidant compounds present in tea marinades served as radical scavengers or free radical quenchers. Additionally, the tea marinade acted as a barrier, mitigating the direct contact with the heat source, leading to the effective mitigation of hazardous compounds in high-temperature roasted chicken.
从 表 2中可以看出，与对照样品相比，用茶叶腌制的高温烤鸡中大部分有害化合物的含量呈下降趋势。其中，绿茶对有害化合物的抑制效果最好。大部分有害化合物都是具有环状结构的化合物，而茶叶中的抗氧化成分可通过减少或淬灭活性自由基来干扰这些物质的形成（Lu 等人，2018）。在高温下将含有茶叶抗氧化剂的腌料应用于加工肉制品是减少有害化合物生成的有效方法。Quelhas 等人（2010 年）发现，在腌制过程中用绿茶提取物腌制肉丸可降低 PHIP 和 AaC 的水平。Wang 等人（2018）发现绿茶对腌制鸡肉中的多环芳烃有明显的抑制作用。茶腌料中的抗氧化化合物可作为自由基清除剂或自由基淬灭剂。此外，茶腌料还能起到屏障作用，减少与热源的直接接触，从而有效减少高温烤鸡肉中的有害化合物。

Although the marinade of tea inhibits the generation of hazardous compounds, not all types of hazardous compounds are affected, and some marinades might facilitate the formation of specific hazardous compounds. For example, 8-MEIQx in chicken roasted with black tea marinades significantly increases. Red tea, oolong tea, and yellow tea also had negative impacts on Chr. During the process of eliminating free radicals, antioxidant compounds, by providing hydrogen, can be oxidized into free radicals themselves, which may contribute to the generation of hazardous compounds (Sharma & Hajaligol, 2003). Jongberg et al. (2013) found the concentration of free radicals in steamed sausages supplemented with green tea extract is markedly higher, and the content of free radical compounds has increased accordingly. In the glucose-acrylamide model under high-temperature conditions, the addition of chlorogenic acid leads to an increase of acrylamide in the model (Cai et al., 2014). It has not been specifically explained how antioxidant compounds promote the generation of free radicals, leading to an increase in hazardous compounds. When food undergoes thermal processing, elevated temperatures speed up oxidation reactions, resulting in the creation of hazardous compounds. The antioxidant properties found in teas can help reduce the formation of hazardous compounds by slowing down these oxidation reactions. Furthermore, the hydroxyl and aromatic rings present in phenolic compounds in tea can interact with the precursors of hazardous compounds, thereby inhibiting its production (Bortolini et al., 2021; Quelhas et al., 2010).
虽然茶叶腌制会抑制有害化合物的生成，但并非所有类型的有害化合物都会受到影响，有些腌制可能会促进特定有害化合物的形成。例如，用红茶腌制的烤鸡中的 8-MEIQx 会明显增加。在消除自由基的过程中，抗氧化剂化合物通过提供氢，本身也会被氧化成自由基，这可能会导致有害化合物的产生（Sharma & Hajaligol，2003）。Jongberg 等人（2013 年）发现，添加了绿茶提取物的蒸香肠中自由基的浓度明显升高，自由基化合物的含量也相应增加。在高温条件下的葡萄糖-丙烯酰胺模型中，添加绿原酸会导致模型中丙烯酰胺的增加（Cai 等，2014）。抗氧化化合物如何促进自由基的生成，从而导致有害化合物的增加，目前还没有具体的解释。当食品进行热加工时，温度升高会加速氧化反应，从而产生有害化合物。茶叶中的抗氧化特性可以减缓这些氧化反应，从而有助于减少有害化合物的形成。 此外，茶叶中酚类化合物的羟基环和芳香环可以与有害化合物的前体相互作用，从而抑制有害化合物的产生（Bortolini et al、2021；Quelhas 等人，2010）。

Based on the screening of compounds with a VIP in the project (Fig. 4C), VIP > 1.5, a total of 35 compounds causing differences were identified. These compounds include 11 heterocyclic compounds, 7 lipids, 5 organic acids and their derivatives, 3 amino acids and their derivatives, 5 benzene compounds, 2 nucleosides and nucleotides, 1 alkaloid, and 1 organic oxide. Lipid degradation is one crucial pathway in the tea production process for the generation of aroma. Carbonyl compounds resulting from lipid oxidation can engage in Maillard reactions with amino acid degradation products, giving rise to the production of flavor compounds. In comparison to other tea marinades, green tea marinades contain lower levels of important lipid compounds. This may be attributed to the processing techniques of tea leaves. During green tea processing, chlorophyll decomposes, phospholipids decrease, glycolipids degrade significantly, and the lipid content partially decreases (Li et al., 2021). Amino acids play a crucial role in imparting freshness and sweetness to the tea marinade, while organic acids significantly contribute to its sour taste. Compared with other tea marinades, black tea contains more amino acids, organic acids and their derivatives. This is attributed to the extensive fermentation process of black tea, where polyphenolic compounds undergo predominant oxidation and polymerization reactions. This may explain the variation in taste experienced when high-temperature roasted chicken is marinated with tea marinades.

PLS-R regression results for differential compounds in different tea marinades and the volatile compounds, flavor compounds, and hazardous compounds in chicken roasted at high temperatures after marinating with different teas (Fig. 4D). 77 differential compounds with VIP > 1.0 were identified among six tea marinades (x variables), 51 taste-active compounds, 56 volatile compounds, and 25 hazardous compounds (y variables) were investigated in high-temperature roasted chicken marinated with different tea marinades. The different marinades were closely grouped, indicating strong similarity among differential compounds of tea marinades and taste-active compounds, volatile compounds, and hazardous compounds in high-temperature roasted chicken. Variables nearby within the same quadrant also show higher correlations, whereas variables in the diagonal quadrant tend to exhibit negative correlations (Sherman et al., 2020). The majority of differential compounds that exhibit significant negative correlations with the y variable of volatile compounds are lipid compounds. Lipids in tea are important precursors for tea aroma constituents. The changes in lipid content and composition resulting from lipid degradation often influence the types of aroma present (Pino & Mesa, 2010). Aldehydes and alcohols derived from lipid degradation are important contributors to the fresh and floral aroma of tea. The y-variables of associated hazardous compounds exhibit significant negative correlations with flavonoid and antioxidative benzene compounds located in the fourth quadrant.

Flavonoids are a class of natural compounds found in plants with potent abilities to scavenge free radicals and inhibit oxidation reactions. In yellow tea and green tea marinades, there was a higher content of flavonoid compounds that negatively correlated with hazardous compounds. Green tea and black tea marinades had lower levels of lipid compounds that negatively correlate with volatile compounds. Yellow tea, green tea, and black tea are considered excellent choices as tea marinades.