Prediction of Japanese green tea ranking by gas chromatography/mass spectrometry-based hydrophilic metabolite fingerprinting

An innovative technique for green tea's quality determination was developed by means of metabolomics. Gas-chromatography coupled with time-of-flight mass spectrometry and multivariate data analysis was employed to evaluate the quality of green tea. Alteration of green tea varieties and manufacturing processes effects a variation in green tea metabolites, which leads to a classification of the green tea's grade. Therefore, metabolic fingerprinting of green tea samples of different qualities was studied. A set of ranked green tea samples from a Japanese commercial tea contest was analyzed with the aim of creating a reliable quality-prediction model. Several multivariate algorithms were performed. Among those, the partial least-squares projections to latent structures (PLS) analysis with the spectral filtering technique, orthogonal signal correction (OCS), was found to be the most practical approach. In addition, metabolites that play an important role in green tea's grade classification were identified.     

1H NMR based metabolic profiling in the evaluation of Japanese green tea quality

Classification of tea quality is now mainly performed according to the sensory results by professional tea tasters. However, this evaluation method is inconsistent in differentiating their qualities. A combination of a (1)H NMR technique and a multivariate analysis was introduced to the quality evaluation of green tea by means of a metabolomic technique. A broad range of metabolites were detected by (1)H NMR spectrometry. The principal component analysis (PCA) was used to reduce the complexity of the (1)H NMR spectra data set and provided the quality discrimination result. It offered an extensive clue for classification and quality assessment without any prepurification method. A set of green teas from a Japanese tea contest were analyzed by (1)H NMR to classify the quality with respect to that judged by tea tasters and to conceive a quality prediction model. Metabolic profiling and fingerprinting of (1)H NMR spectra of green teas with different quality were studied. PCA showed a separation between the high- and the low-quality green teas. The taste marker compounds contributing to the discrimination of tea quality were identified. Reliable prediction models were obtained by the partial least-squares projection to latent structure (PLS) analysis together with a preprocessing filter of both orthogonal signal correction (OSC) and a combination between OSC and wavelet transform algorithms.     

基于电子舌技术的绿茶滋味品质评价

该文研究利用电子舌技术快速评价绿茶的滋味品质。试验以“碧螺春”绿茶为研究对象,以绿茶滋味化学鉴定法作为绿茶滋味品质的评价方法,获得的滋味总得分值作为电子舌评价模型的参考测量值。在数据分析过程中,首先对不同生产日期的碧螺春茶汤滋味总得分值和各传感器响应值进行单因素方差分析;然后对比采用偏最小二乘法和最小二乘支持向量机建立电子舌传感器响应值与滋味总得分值之间的相关模型。结果显示不同生产日期对绿茶滋味品质及各传感器响应信号都具有极显著影响;当采用4个主成分时,建立的最小二乘支持向量机模型最优。用独立样本检验模型精度,模型预测值与参考值的相关系数为0.906,预测集均方根误差为4.077。研究结果可为茶叶品质智能化评价提供参考。     

High-throughput technique for comprehensive analysis of Japanese green tea quality assessment using ultra-performance liquid chromatography with time-of-flight mass spectrometry (UPLC/TOF MS)

Applications of metabolomics techniques along with chemometrics provide an understanding in the relationship between metabolome of green tea and its quality. A coupled of ultra-performance liquid chromatography with time-of-flight mass spectrometry (UPLC/TOF MS) allowed a high-throughput and comprehensive analysis with minimal sample preparation. Using this technique, a wide range of metabolites were investigated. Data analysis was rapid, considering that the fingerprinting technique was performed. A set of green tea samples from 2006 tea contest of the Kansai area was analyzed to prove usefulness of the developed technique. Green tea with different qualities were discriminated through principal component analysis (PCA). Consequently, projection to latent structure by means of partial least-squares (PLS) was performed to create a constructive quality-predictive model by means of metabolic fingerprinting. Beside epigallocatechin, other predominant catechins, including epigallocatechin gallate and epicatechin gallate, detected in green tea were found to be significant biomarkers to the high quality of Japanese green tea (Sencha).     

Identification of processed Japanese green tea based on polymorphisms generated by STS-RFLP analysis

In Japan, tea is generally sold blended, though 90% of the total production is clonal. Due to the increasingly strict consumer need and taste, however, more and more Japanese green teas are being sold under their particular cultivar name. Moreover, tea made from Yabukita, a much appreciated cultivar originally developed in Japan, has recently been produced and imported from a neighboring country. This paper describes a simple and inexpensive methodology capable of identifying fresh and processed Japanese green teas to discourage its fraudulent commercialization. The study was based on 46 main tea cultivars, and polymorphism detected through STS-RFLP analysis of the coding and noncoding DNA regions of three genes, namely phenylalanine ammonia-lyase, chalcone synthase, and dihydroflavonol 4-reductase, for which nucleotide information was available. All 46 tea cultivars analyzed could be easily distinguished using a combination of codominant DNA markers. Yabukita displayed a unique profile when PAL intron was digested with DdeI, thus allowing its rapid authentication at low cost.     

Identification of potent odorants in Japanese green tea (Sen-cha)

Application of aroma extract dilution analysis using the volatile fraction of a Japanese green tea (Sen-cha) sample resulted in the detection of 36 odor-active peaks with flavor dilution (FD) factors between 10 and 5000. Thirty-six potent odorants were identified from 36 odor-active peaks by gas chromatography/mass spectrometry (GC/MS) and/or the multidimensional GC/MS (MDGC/MS) system. Among these components, 4-methoxy-2-methyl-2-butanethiol (meaty), (Z)-1, 5-octadien-3-one (metallic), 4-mercapto-4-methyl-2-pentanone (meaty), (E,E)-2,4-decadienal (fatty), beta-damascone (honey-like), beta-damascenone (honey-like), (Z)-methyl jasmonate (floral), and indole (animal-like) showed the highest FD factors. Therefore, these odorants were the most important components of the Japanese green tea odor. In addition, 4-methoxy-2-methyl-2-butanethiol, 4-mercapto-4-methyl-2-pentanone, methional, 2-ethyl-3, 5-dimethylpyrazine, (Z)-4-decenal, beta-damascone, maltol, 5-octanolide, 2-methoxy-4-vinylphenol, and 2-aminoacetophenone were newly identified compounds in the green tea.     

A new LC/MS/MS rapid and sensitive method for the determination of green tea catechins and their metabolites in biological samples

A new rapid and sensitive method has been developed, using liquid chromatography in tandem mass spectrometry (LC-ESI-MS/MS) to identify green tea catechin metabolites in plasma and urine after oral intake of a green tea extract. (-)-Epigallocatechin-3-gallate (EGCG), (-)-epicatechin-3-gallate (ECG), (-)-epigallocatechin (EGC)-glucuronide, (-)-epicatechin (EC)-glucuronide, and EC-sulfate were identified in plasma, whereas in urine only the conjugated catechins were detected (EGC-glucuronide, EGC-sulfate, EC-glucuronide, and EC-sulfate). Standard calibration curves prepared in plasma were found to be linear in the range of 10.9-1379.3 nmol/L for EGCG, EGC, ECG, and EC. The accuracy and precision of this assay showed a coefficient of variation of <15%. The method allowed the detection and quantification limits (for 20 microL injection) from 1.1 to 2.6 nmol/L and 3.8-8.7 nmol/L, respectively, in plasma and 0.8-1.8 nmol/L and 2.6-6.0 nmol/L, respectively, in urine. This method can be applied for future clinical and epidemiological studies, allowing the identification of the active metabolites that will reach the target tissues.     

Prediction of Japanese green tea ranking by fourier transform near-infrared reflectance spectroscopy

A rapid and easy determination method of green tea's quality was developed by using Fourier transform near-infrared (FT-NIR) reflectance spectroscopy and metabolomics techniques. The method is applied to an online measurement and an online prediction of green tea's quality. FT-NIR was employed to measure green tea metabolites' alteration affected by green tea varieties and manufacturing processes. A set of ranked green tea samples from a Japanese commercial tea contest was analyzed to create a reliable quality-prediction model. As multivariate analyses, principal component analysis (PCA) and partial least-squares projections to latent structures (PLS) were used. It was indicated that the wavenumber region from 5500 to 5200 cm(-1) had high correlation with the quality of the tea. In this study, a reliable quality-prediction model of green tea has been achieved.     

Molecular and sensory studies on the umami taste of Japanese green tea

Aimed at defining the key drivers for the quality-determining umami taste of a high-grade powdered green tea, called mat-cha, a bioactivity-guided fractionation using solvent extraction, solvent precipitation, preparative chromatographic separations, and human psychophysical experiments was applied on freshly prepared mat-cha. Liquid chromatography-tandem mass spectrometry and one-/two-dimensional nuclear magnetic resonance studies on isolated fractions led to the identification of l-theanine, succinic acid, 3,4,5-trihydroxybenzoic acid (gallic acid), and (1R,2R,3R,5S)-5-carboxy-2,3,5-trihydroxycyclohexyl-3,4,5-trihydroxybenzoate (theogallin) as umami-enhancing compounds in the green tea beverage, and it can be shown by sensory studies that these compounds are able to raise the umami intensity of sodium l-glutamate proportionally.     

Analysis of agricultural residues on tea using d-SPE sample preparation with GC-NCI-MS and UHPLC-MS/MS

This study presents new sample preparation and analytical procedures for the quantification of pesticides on processed tea leaves. The new method includes tea extraction and dispersive solid phase extraction (d-SPE) to prepare gas chromatography (GC) and ultrahigh-performance liquid chromatography (UHPLC)-ready samples, providing a fast and cost-effective solution for time-sensitive industrial analysis to fulfill regulatory requirements. Both GC-negative chemical ionization mass spectrometry (GC-NCI-MS) and UHPLC-tandem mass spectrometry (UHPLC-MS/MS) were employed to produce highly sensitive and reproducible data. Excellent limits of detection (typically below 1 μg/kg for GC and 10 μg/kg for UHPLC), wide linearity ranges, and good recoveries (mostly >70%) were achieved on the selected pesticides. Twenty-seven tea samples purchased from local grocery stores were analyzed using the newly developed methods. Among the pesticides analyzed, endosulfan sulfate and kelthane were the most frequently detected by GC-NCI-MS and imidacloprid and acetamiprid by UHPLC-MS/MS in these teas. The samples were found to be relatively clean, with <1 mg/kg of total pesticide residues. The organic-labeled teas were significantly cleaner than nonorganic ones. The cost per gram of tea did not correlate with pesticide residue levels detected.     

Improved method for quantifying the avicide 3-chloro-p-toluidine hydrochloride in bird tissues using a deuterated surrogate/GC/MS method

A method using a deuterated surrogate of the avicide 3-chloro-p-toluidine hydrochloride (CPTH) was developed to quantify the CPTH residues in the gastrointestinal (GI) tract and breast muscle tissues in birds collected in CPTH-baited sunflower and rice fields. This method increased the range of a previous surrogate/gas chromatography/mass spectroscopy method from 0-2 to 0-20 microg/g in tissue samples and greatly simplified the extraction procedure. The modified method also sought to increase recoveries over a range of matrix effects introduced by analyzing tissues from birds collected in the field, where the GI tract contents would be affected by varying diet. The new method was used to determine the CPTH concentration in GI tract samples fortified with CPTH-treated rice bait to simulate the consumption of varying amounts of treated bait by two nontargeted bird species, pigeon (Columbia livia) and house sparrow (Passer domesticus). The new method was then used to examine the CPTH concentrations in the gizzard contents of the targeted bird species, red-winged black bird (Agelaius phoeniceus) and brown-headed cowbird (Molothrus ater), that were collected after feeding at a treated bait site. The method proved sufficiently sensitive to quantify CPTH in the breast muscle tissues and the gizzard contents of red-winged blackbirds and brown-headed cowbirds during an operational baiting program. The levels of CPTH determined for these birds in both tissue samples were determined to be highly correlated. The appearance of CPTH in the breast muscle tissue immediately after feeding was not anticipated. The potential secondary hazard posed by the targeted birds to potential scavengers and predators was also evaluated.     

Identification of potent odorants in different green tea varieties using flavor dilution technique

Two kinds of pan-fired green teas (Japanese Kamairi-cha and Chinese Longing tea) were compared with the common Japanese green tea (Sen-cha). Application of the aroma extract dilution analysis (AEDA) using the volatile fraction of the Sen-cha, Kamairi-cha and Longing tea infusions revealed 32, 51, and 52 odor-active peaks with flavor dilution factors between 16 and 1024, respectively. (Z)-1,5-Octadien-3-one (metallic, geranium-like), 4-mercapto-4-methyl-2-pentanone (meaty, black currant-like), methional (potato-like), (E,Z)-2,6-nonadienal (cucumber-like), and 3-methylnonane-2,4-dione (green, fruity, hay-like) showed high flavor dilution factors in all varieties. In addition, 2-acetyl-1-pyrroline (popcorn-like), 2-ethyl-3,5-dimethylpyrazine (nutty), 2,3-diethyl-5-methylpyrazine (nutty), and 2-acetyl-2-thiazoline (popcorn-like) belonged to the most potent odorants only in the pan-fired green teas. Among these odorants, 2-acetyl-1-pyrroline and 2-acetyl-2-thiazoline were identified for the first time among the tea volatiles.     

Improved GC/MS method for quantitation of n-alkanes in plant and fecal material

A gas chromatography-mass spectrometry (GC/MS) method for the quantitation of n-alkanes (carbon backbones ranging from 21 to 36 carbon atoms) in forage and fecal samples has been developed. Automated solid-liquid extraction using elevated temperature and pressure minimized extraction time to 30 min per sample as compared to more than 24 h for traditional GC-flame ionization detection methods that use saponification and liquid-liquid extraction. Extraction solvent requirements were also minimized to 10 mL per sample. Under optimal conditions, complete method recoveries, including extraction efficiency, were greater than 91%. The linear dynamic range was 5 to 100 nmol injected onto the column, with a limit of quantitation of 5 nmol. Intra-assay coefficients of variation for the analysis of annual ryegrass (Lolium rigidum), subterranean clover (Trifolium subterranean), and bovine feces ranged from 0.1%-12.9%, where lower concentrations of n-alkane produced a higher degree of imprecision. The reported GC/MS method permits simple, rapid, and precise quantitation of n-alkanes in plant and fecal material and reduces reagent and labor requirements.     

Application of near-infrared reflectance spectroscopy to the simultaneous prediction of alkaloids and phenolic substances in green tea leaves

A near-infrared reflectance spectroscopic (NIRS) method for the prediction of polyphenol and alkaloid compounds in the leaves of green tea [Camellia sinensis (L.) O. Kuntze] was developed. Reference measurements of the individual catechins, gallic acid, caffeine, and theobromine were performed by reversed-phase HPLC. The total polyphenols were determined according to the colorimetric Folin-Ciocalteu assay. Using the partial least-squares algorithm, very good calibration statistics were obtained for the prediction of gallic acid, (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin gallate, (-)-epigallocatechin gallate, caffeine, and theobromine (R(2) > 0.85) with standard deviation/standard error of cross-validation (SD/SECV) ratio ranging from 2.00 to 6.27. Simultaneously, the dry matter content of the tea leaves can be analyzed very precisely (R(2) = 0.94; SD/SECV = 4.12). Furthermore, it is possible to discriminate tea leaves of different age by principal component analysis on the basis of the received NIR spectra. Prediction of the total polyphenol content is performed with a lower accuracy, which might be due to the lack of specificity in the colorimetric reference method. The study demonstrates that NIRS technology can be successfully applied as a rapid method not only for breeding and cultivation purposes but also to estimate the quality and taste of green tea and to control industrial processes, for example, decaffeination.     

基于改进YOLOv5s的名优绿茶品质检测

针对实际检测过程中茶叶数量多、体积小、茶叶之间颜色和纹理相似等特点,该研究提出了一种基于YOLOv5s的名优绿茶品质检测算法。首先,该算法在骨干网络层引入膨胀卷积网络,通过增大感受野的方式增强茶叶微小特征的提取。其次,改进特征融合进程,基于通道注意力和空间注意力抑制无关信息的干扰,构建CBAM注意力机制优化检测器。接着根据swin transformer网络结构在多个维度对小尺度茶叶的特征进行交互和融合。最后,配合SimOTA匹配算法动态分配茶叶正样本,提高不同品质茶叶的识别能力。结果表明,改进后的算法精准度、召回率、平均精度均值、模型体积、检测速度分别为97.4%、89.7%、91.9%、7.11MB和51帧/s,相较于基础的YOLOv5s平均精度均值提高了3.8个百分点,检测速度提高了7帧/s。利用相同数据集在不同目标检测模型上进行对比试验,与Faster-RCNN、SSD、YOLOv3、YOLOv4等模型相比,平均精度均值分别提升10.8、22.9、18.6、8.4个百分点,进一步验证了该研究方法的有效性和可靠性。     

Effects of green tea polyphenol (-)-epigallocatechin-3-gallate on newly developed high-fat/Western-style diet-induced obesity and metabolic syndrome in mice

The aim of this study was to investigate the effects of (-)-epigallocatechin-3-gallate (EGCG) on newly developed high-fat/Western-style diet-induced obesity and symptoms of metabolic syndrome. Male C57BL/6J mice were fed a high fat/Western-style (HFW; 60% energy as fat and lower levels of calcium, vitamin D(3), folic acid, choline bitartrate, and fiber) or HFW with EGCG (HFWE; HFW with 0.32% EGCG) diet for 17 wks. As a comparison, two other groups of mice fed a low-fat diet (LF; 10% energy as fat) and high-fat diet (HF; 60% energy as fat) were also included. The HFW group developed more body weight gain and severe symptoms of metabolic syndrome than the HF group. The EGCG treatment significantly reduced body weight gain associated with increased fecal lipids and decreased blood glucose and alanine aminotransferase (ALT) levels compared to those of the HFW group. Fatty liver incidence, liver damage, and liver triglyceride levels were also decreased by the EGCG treatment. Moreover, the EGCG treatment attenuated insulin resistance and levels of plasma cholesterol, monocyte chemoattractant protein-1 (MCP-1), C-reactive protein (CRP), interlukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). Our results demonstrate that the HFW diet produces more severe symptoms of metabolic syndrome than the HF diet and that the EGCG treatment can alleviate these symptoms and body fat accumulation. The beneficial effects of EGCG are associated with decreased lipid absorption and reduced levels of inflammatory cytokines.     

Characterization of isoflavones and their conjugates in female rat urine using LC/MS/MS

Isoflavone phytoestrogens found in soybeans are the most widely studied phytochemicals in human diets and soy infant formulas. The health benefits of the isoflavones daidzein and genistein have been reported, and concerns about potential adverse effects have also been raised. However, the results of direct analysis of isoflavones and their metabolites in biological fluids after consumption of soy-containing diets are scarce. This study describes an LC/MS/MS method for the analysis of isoflavones and their metabolites in the urine of female rats fed diets made with soy protein isolate. Five isoflavones (daidzein, genistein, glycitein, dihydrodaidzein, and O-desmethylangolensin) were identified by comparison with authentic standards. Seventeen conjugates of isoflavones were characterized in the urine, the most unusual being genistein 5-glucuronide and four glucuronide conjugates of reductive metabolites of daidzein. The application of LC/MS/MS to analyze isoflavone metabolites is simple and sensitive, and appears to be an excellent method for determining the bioavailability and metabolism of food phytochemistry.     

基于漫反射光谱的初制绿茶含水率无损检测方法

茶叶含水率是影响茶叶加工品质的一项重要指标。为了实现茶叶加工中含水率的快速检测,该文提出了一种应用漫反射光谱技术的绿茶初制过程中含水率无损检测方法。采用波长范围在325～1 075 nm 的可见-短波近红外光谱仪,对炒青绿茶在8个加工工序中随机抽取的568个茶叶样本进行漫反射光谱扫描,光谱扫描后立即测量样本的含水率。对于得到的光谱数据,采用小波变换降低其信息维度并提取小波系数,比较小波低频系数对于光谱特征信息的提取能力,结果显示,小波低频系数能够有效提取原始光谱数据中的特征信息。采用3种回归算法：偏最小二乘回归、神经网络和支持向量机分别建立含水率的测量模型。比较发现支持向量机回归模型的结果最优,建模相关系数为0.9985,预测相关系数为0.9875。研究结果表明,漫反射光谱可以用于绿茶含水率的无损、快速检测,小波变换是一种有效的光谱特征提取算法,而且支持向量机回归算法具有高精度和强泛化能力,可广泛用于回归分析。     

电磁内热式绿茶毛火工艺参数优化与分析

为探究新型绿茶电磁内热式毛火工艺对绿茶毛火效果的影响,该文应用正交设计和极差分析,以毛火样的生化成分含量和成品样的感官品质为目标值,对电磁内热式毛火工艺参数(热风温度、热风风机转速、链板传动转速等)进行优化,获得最佳参数,并与传统的燃煤式热风毛火、理条机毛火、电热管式热风毛火等在毛火效果和设备性能方面进行比较,结果表明:综合品质生化成分和感官评审的结果,电磁内热式毛火工艺的最佳参数组合为:热风温度115℃,热风风机转速1 200 r/min,链板传动转速1 050 r/min,3因素中以热风风机风速对绿茶毛火效果的影响最为显著;在此毛火条件下所制提香样的氨基酸、茶多酚、叶绿素、可溶性糖、儿茶素和咖啡碱的含量分别达2.31%、17.61%、1.13 mg/g、5.01%、10.74%、2.01%,品质物质加权值为7.66%,均高于其他3种毛火工艺;此工艺下所制成品茶样的外形、汤色、香气、滋味等均得到显著提升,感官总分达86.40,显著优于其他毛火处理;该毛火工艺能耗成本仅0.76元/kg,较电热管式热风毛火减少了70%以上,升温快,预热时间仅10 min,较传统燃煤式热风毛火的预热时间减少约50%,余热回收装置的设计显著提升了热能利用率,热效率提升50%以上;同时,生产效率达150 kg/h,较传统电热管式热风毛火提升3倍以上,而且温控精准和稳定、热风分布均匀。该研究可为茶叶加工提供技术参考和理论指导。