Pu-erh Tea Research Articles

Simultaneous Determination of Pesticide Residues and Mycotoxins in Storage Pu-erh Tea Using Ultra-High-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry.

Mycotoxins and pesticides are the most concerning chemical contaminants that can affect the quality of Pu-erh tea during its production and storage. This study presents a method that can simultaneously determine 31 pesticide residues and six mycotoxins in Pu-erh tea within 11 min using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) after QuEChERS extraction. The lower limit of quantification (LOQ) for all analytes ranged between 0.06 and 50 ppb. Recoveries for each pesticide and mycotoxin ranged between 62.0 and 130.3%, with intra- and inter-day precisions lower than 15%. Good linear relationships were obtained, with correlation coefficients of r2 > 0.991 for all analytes. The established method was applied to 31 Pu-erh tea samples, including raw and ripened Pu-erh tea with different storage times. As a result, pesticide residues were not detected in any of the collected samples, and the mycotoxins detected in the samples were well below the official maximum residue limits (MRLs). Notably, the levels of aflatoxin B1 (AFB1), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) were lower than 1 ppb in the samples stored for more than 30 years.

Analysis of Volatile Profile and Aromatic Characteristics of Raw Pu-erh Tea during Storage Based on GC-MS and Odor Activity Value.

Volatile constituents are critical to the flavor of tea, but their changes in raw Pu-erh tea (RAPT) during storage have not been clearly understood. This work aimed to investigate the volatile composition and their changes at various storage durations. The volatile profile of RAPT was determined using headspace solid-phase microextraction in combination gas chromatography-mass spectrometry. A total of 130 volatile compounds were identified in RAPT samples, and 64 of them were shared by all samples. The aroma attributes of RAPT over a storage period ranging from 0 to 10 years were assessed through the combination of odor activity value (OAV), aroma characteristic influence(ACI) value, and multivariate statistical analysis. The results revealed that RAPT exhibited a distinct floral and fruity aroma profile after storage for approximately 3-4 years. A notable shift in aroma was observed after 3-4 years of storage, indicating a significant turning point. Furthermore, the likely notable shift after 10 years of storage may signify the second turning point. According to the odor activity value (OAV ≥ 100), eight key volatile compounds were identified: linalool, α-terpineol, geraniol, trans-β-ionone, α-ionone, (E,E)-2,4-heptadienal, 1-octanol, and octanal. Combining OAV (≥100) and ACI (≥1), five compounds, namely linalool, (E,E)-2,4-heptadienal, (Z)-3-hexen-1-ol, 2,6,10,10-tetramethyl-1-oxaspiro [4.5]dec-6-ene, and octanal, were identified as significant contributors to the aroma. The results offer a scientific foundation and valuable insights for understanding the volatile composition of RAPT and their changes during storage.

Compound Chenpi Tea consumption reduces obesity-related metabolic disorders by modulating gut microbiota and serum metabolites in mice.

Compound Chenpi Tea (CCT) is a popular herbal beverage made from Citri Reticulatae Pericarpium, Ganoderma lucidum and Pu-erh tea. This study aimed to investigate the anti-obesity effect of CCT in mice fed a high-fat diet (HFD) and to explore the potential mediators by 16S rRNA sequencing and serum metabolomic analysis. The results showed that CCT supplementation can effectively reduce diet-induced obesity, hepatic steatosis, dyslipidemia, and insulin resistance. Further analyses revealed that CCT could reverse the changes of 28 metabolites induced by HFD, mainly affecting, amino acid metabolism, gut microbiota metabolism and glycerophospholipid metabolism. CCT could also alleviate the intestinal dysbiosis by decreasing the abundance of Romboutsia, Gemella, [Eubacterium]_fissicatena_group and Faecalibaculum, and increasing the abundance of Oscillibacter, Blautia and Acetatifactor. Collectively, these findings demonstrated that CCT supplementation modulated gut microbiota and obesity-related metabolic disorders in mice. This article is protected by copyright. All rights reserved.

Investigation on the Quality of Commercially Available GABA Tea in Taiwan

This study collected 220 commercially available samples of γ-Aminobutyric acid (GABA) tea produced in Taiwan from 2016 to 2021. The 220 tea samples were categorized into five types of GABA tea, including 108 GABA Oolong tea, 71 GABA Black tea, 21 GABA Paochong tea, 12 GABA Green tea, and 8 GABA Puerh tea samples. The most common type of GABA tea in Taiwan is GABA Oolong tea, followed by GABA Black tea. The physico-chemical constituents and consumer acceptance of the GABA tea samples were analyzed. The GABA content varied among the different types of GABA tea: GABA Oolong tea ranged from 128–286 mg/100 g, GABA Black tea ranged from 182–360 mg/100 g, GABA Paochong tea ranged from 98–203 mg/100 g, GABA Green tea ranged from 56–174 mg/100 g, and GABA Puerh tea ranged from 142–191 mg/100 g. In terms of the commercial standard of GABA tea, 22 out of the 220 GABA tea samples failed to meet the commercial standard, with a failure rate of 10%. During the fermentation process of GABA tea, the contents of GABA increased significantly, but the total polyphenol and total catechin contents remained stable. In terms of consumer acceptance, GABA Black tea is the most accepted by consumers, followed by GABA Puerh tea, GABA Paochong, and GABA Oolong tea. The sour flavor in GABA tea is similar to the original sour sensory properties found in black tea. It is assumed that this is the main reason GABA Black tea has the highest acceptance.

Dual Signal-Enhanced Electrochemiluminescence Strategy Based on Functionalized Biochar for Detecting Aflatoxin B1.

Metal-organic frameworks (MOFs) are often used as carriers in the preparation of electrochemiluminescent (ECL) materials, and ECL materials stabilized in the aqueous phase can be prepared by encapsulating chromophores inside MOFs by an in situ growth method. In this study, nanocomposites MIL-88B(Fe)-NH2@Ru(py)32+ with excellent ECL response were prepared by encapsulating Tris(2,2'-bipyridine)ruthenium dichloride (Ru(py)32+) inside MIL-88B(Fe)-NH2 using the one-step hydrothermal method. MIL-88B(Fe)-NH2 possesses abundant amino groups, which can accelerate the catalytic activation process of K2S2O8, and its abundant pores are also conducive to the enhancement of the transmission rate of co-reactant agents, ions, and electrons, which effectively improves the ECL efficiency. In order to obtain more excellent ECL signals, we prepared aminated biochar (NH2-biochar) using Pu-erh tea dregs as precursor and loaded gold nanoparticles (Au NPs) on its surface as substrate material for modified electrodes. Both NH2-biochar and Au NPs can also be used as a co-reactant promoter to catalyze the activation process of co-reactant K2S2O8. Therefore, a sandwich-type ECL immunosensor was prepared based on a dual signal-enhanced strategy for the highly sensitive and selective detection of aflatoxin B1 (AFB1). Under the optimal experimental conditions, the sensitive detection of AFB1 was achieved in the range of 1 pg·mL-1~100 ng·mL-1 with a detection limit of 209 fg·mL-1. The proposed dual signal-enhanced ECL immunosensor can provide a simple, convenient, and efficient method for the sensitive detection of AFB1 in food and agricultural products.

Modulatory Effects of Co-Fermented Pu-erh Tea with Aqueous Corn Silk Extract on Gut Microbes and Fecal Metabolites in Mice Fed High-Fat Diet.

Pu-erh tea is recognized for its weight loss effects, but its potential association with gut microbiota and metabolites remains unclear. This research explored the alterations in gut flora and metabolite composition upon treatment with a co-fermented Pu-erh tea with an aqueous corn silk extract (CPC) in obese mice by employing integrated 16S ribosomal RNA gene sequencing and untargeted metabolomics processes. For 8 weeks, mice were fed control, high-fat, and high-fat diets which included a 46 mg/mL CPC extract. The CPC extract the alleviated high-fat diet (HFD), it stimulated systemic chronic inflammation, and it reduced the body weight, daily energy consumption, and adipose tissue weight of the mice. It also modified the gut microbiota composition and modulated the Lactobacillus, Bifidobacterium, Allobaculum, Turicibacter, and Rikenella genera. Fecal metabolomics analysis revealed that the CPC extract influenced the caffeine, cysteine, methionine, tryptophan, biotin metabolism pathways, primary bile acid, and steroid biosynthesis. This research revealed that the CPC extract could inhibit HFD-stimulated abnormal weight gain and adipose tissue accumulation in mice, and modulate mice gut microbiota composition and multiple metabolic pathways.

Simultaneous determination of polycyclic aromatic hydrocarbons and polychlorinated biphenyls in Pu-erh tea by dispersive solid-phase extraction coupled with GC-MS/MS

A novel method for the simultaneous determination of 13 PAHs and 18 PCBs residues in Pu-erh tea samples was established by combining GC-MS/MS with dispersive-solid phase extraction (d-SPE). Sample pretreatment procedure was achieved by NaCl addition and using acetone/ethyl acetate/n-hexane (50:30:20, v/v/v) extraction followed by d-SPE clean-up with strong cationic exchanger (SCX), primary secondary amine (PSA), polyvinylpolypyrrolidone (PVPP) and graphitized carbon black (GCB). The proposed method had good linearity in the range of 0.5–200 μg/L with correlation coefficients (R2 > 0.998). The detection limit ranges (LODs) were from 0.2 to 1.5 µg/kg. The spiked experiments in raw Pu-erh tea and ripened Pu-erh tea at 2.5–50 µg/kg concentrations showed satisfactory recoveries (70.2–114.7%) for all analytes with RSDs of < 10%. The validated methodology was successfully applied to 86 Pu-erh tea samples. No PCBs residue was more than LODs, and the detection frequency and concentration of PAHs in different types of Pu-erh tea varies greatly were found.

Exploring the mysterious effect of piling fermentation on Pu-erh tea quality formation: Microbial action and moist-heat action

Moist-heat action and microbial action are essential to the formation of Pu-erh tea’s quality. To explore the impact of piling fermentation (PF) environment on Pu-erh tea quality, samples subjected to spontaneous PF (SPPF) and sterile PF (STPF) from the same batch of raw material were processed, and their sensory quality and metabolic profile dynamics were determined. Sensory evaluation indicated that infusions of the SPPF samples were mainly brownish-red, stale-mellow taste, and stale aroma, whereas infusions of the STPF samples were mainly orange-red, sweet-mellow taste, and woody aroma. Metabolomics analysis identified 465 key metabolites, including 33 microbial markers and 39 moist-heat activity markers. Enzyme and pathway analyses revealed that the production of microbial enzymes accelerates the degradation of flavonoids, flavonols, and lipids and also indicated an exclusive influence of microorganisms on the lipid metabolite pathway. This study elucidated the mechanism underlying the formation of Pu-erh tea’s stale flavour.

Distinguishing raw pu-erh tea production regions through a combination of HS-SPME-GC-MS and machine learning algorithms

The authenticity of the geographical origin of agricultural products has received widespread attention. Tea tree varieties, processing processes and origins influence the quality and price of raw pu-erh tea (RPT). This study distinguished RPT from 10 different production areas through headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) combined with orthogonal partial least squares–discriminant analysis (OPLS-DA) model and machine learning algorithms. Among the thirty-five types of common volatiles identified, pentanal, heptanal, naphthalene, cedrol, and 2,6-di-tert-butylbenzoquinone were considered the key differential compounds distinguishing the 10 different production areas of the RPT samples through the screening of variable importance in projection values of the OPLS-DA and coefficient weights of the linear discriminant analysis function. Among them, heptanal and 2,6-di-tert-butylbenzoquinone had the highest content in West of Bingdao and the lowest content in Nannuoshan. The random forest algorithm achieved a discrimination accuracy of 98.4% based on the discrimination of five key compounds in 63 RPT samples. The random forest model was demonstrated to be reliable and valid by using receiver operating characteristic curves (area under the curve = 0.7603). The study results serve as a reference for the differentiation of 10 production areas of RPT.

Effect of geographical origins and pile-fermentation on the multi-element profiles of ripen Pu-erh tea revealed by comprehensive elemental fingerprinting

Elemental fingerprinting could improve geographic origin authentication and quality control of tea. A comprehensive elemental fingerprinting method for ripen Pu-erh tea (RPT) using microwave digestion combined with inductively coupled plasma mass spectrometry (ICP-MS) has been developed, which enabled an accurate quantification of 69 elements. The developed method presented wide dynamic ranges (more than 3-order magnitude, R2 > 0.997), high sensitivity (LODs: 0.0001–8.105 μg/g, LOQs: 0.0003–27.015 μg/g), high accuracy (recoveries: 77.7%–118.2%) and repeatability (relative standard deviation: 0.6–18.8%). The effect of major geographical origins and the pile-fermentation process on the multi-element profiles of RPT was investigated by applying this method. 35 and 31 elements were significantly differential in Pu'er vs. Lincang region, and Pu'er vs. Xishuangbanna region (p < 0.05), respectively, and most of them presented lower levels in Pu'er region. Mo, Nd, Ce, Sr, Ba, V, and Tm were identified as the most relevant elements as potential discriminant indicators of different geographical origins. A prevailing elemental accumulation was revealed during pile-fermentation. 36 elements were significantly enriched in RPT after pile-fermentation. In particular, elements Ti, Cs, Nb, Eu, Zr, Tl, Cr, Co, Dy, Y and Si were most prominently enriched after pile-fermentation with folds changes of 1.55–2.02. This study is expected to provide comprehensive data for origin traceability of RPT, and advanced the understanding of the elemental enrichment mechanism during pile-fermentation of RPT.

Pattern recognition assisted linear sweep voltammetry sensor for analysis of tea quality

This study utilized linear sweep voltammetry (LSV) coupled with pattern recognition techniques to investigate the electrochemical behavior and chemical composition of various tea types, including green tea, black tea, oolong tea, pu-erh tea, white tea, and floral tea. LSV curves were obtained for each tea type, revealing distinct oxidation peaks at different potentials. The analysis of the LSV data enabled the differentiation of tea types based on their electrochemical behavior, as well as the identification of variations within the same tea type based on different grades. Furthermore, tests were conducted on catechins and alkaloids present in tea leaves, and their ultraviolet wavelength scans were obtained. Liquid chromatography was employed to separate and quantify the 11 compounds of interest. The concentrations of these components were determined in 30 tea leaf samples. Cluster analysis and principal component analysis (PCA) were performed on the LSV data, resulting in the successful grouping and classification of the tea samples. The findings highlight the potential of electrochemical analysis coupled with pattern recognition techniques for the rapid and non-destructive assessment of tea quality. Further research should focus on refining and expanding the capabilities of this approach for broader applications in the food and beverage industry.

The Anti-inflammatory Mechanism of Pu-erh Tea via Suppression the Activation of NF-κB/HIF-1α in LPS-stimulated RAW264.7 Cells

The Anti-inflammatory Mechanism of Pu-erh Tea via Suppression the Activation of NF-κB/HIF-1α in LPS-stimulated RAW264.7 Cells

Theogallin-to-Gallic-Acid Ratio as a Potential Biomarker of Pu-Erh Teas

There are two types of Pu-erh tea available on the world market: Raw and Ripe. It is not difficult to tell them apart if the Raw version is relatively young. Researchers have already developed various tools to identify Pu-erh teas. However, they are quite complicated and require advanced statistical analyses. In addition, they are characterized by different levels of accuracy. The aim of the work was to identify relationships or differences that would easily give specific results for identifying types of Pu-erh tea. The content of selected methylxanthines was determined by high-performance liquid chromatography (HPLC) on an Agilent 1200 chromatograph with a UV-VIS diode array detector. The total analysis time was 28 min. A combination of liquid chromatography and a triple quadrupole mass spectrophotometer was used to identify gallic acid and theogallin in the analyzed samples. A multivariate cluster analysis was used to compare the results for single samples, and its results were presented in horizontal hierarchical tree plots. The quantitative determination showed that theophylline is present only in Ripe Pu-erh teas. In addition, it was shown that the ratio of theogallin to gallic acid can be an effective tool to verify the authenticity of Pu-erh varieties.

Effects of Pu-erh and Dian Hong tea polyphenols on the gut-liver axis in mice

Tea polyphenols (TP) are the most biologically active components in tea, with antioxidant, antiobesity, and antitumor properties, as well as the ability to modulate the composition and function of intestinal microbiota. This experimental study evaluated the chemical constituents of polyphenols in Pu-erh (PTP) and Dian Hong tea (DHTP). It also investigated the co-regulatory effects of PTP and DHTP on intestinal flora and liver tissues in mice using 16 S rRNA gene and transcriptome sequencing. The results revealed that DHT had higher concentrations of EGC (epigallocatechin), C (catechin), EC (epicatechin), and EGCG (epigallocatechin gallate). In contrast, PT had higher concentrations of GA (gallic acid), ECG (epicatechin-3-gallate), TF (theaflavin), and TB (theabrownin). PTP and DHTP consumption significantly reduced the rates of weight gain in mice. Microbial community diversity was significantly higher in PTP and DHTP-treated mice than in the control group. Notably, beneficial microbes such as Lactobacillus increased significantly in PTP-treated mice, whereas Lachnospiraceae increased significantly in DHTP-treated mice. Both PTP and DHTP improved the activity of the antioxidant enzymes (SOD) and total antioxidant capacity (T-AOC) in the liver. The transcriptome analysis revealed that the beneficial effects of PTP and DHTP were due to changes in various metabolic pathways, the majority of which were related to antioxidant and lipid metabolism. This study discovered that PTP and DHTP had beneficial effects in mice via the gut-liver axis.

Characterization of volatile metabolites in Pu-erh teas with different storage years by combining GC-E-Nose, GC-MS, and GC-IMS.

Storage time is one of the important factors affecting the aroma quality of Pu-erh tea. In this study, the dynamic changes of volatile profiles of Pu-erh teas stored for different years were investigated by combining gas chromatography electronic nose (GC-E-Nose), gas chromatography-mass spectrometry (GC-MS), and gas chromatography-ion mobility spectrometry (GC-IMS). GC-E-Nose combined with partial least squares-discriminant analysis (PLS-DA) realized the rapid discrimination of Pu-erh tea with different storage time (R2Y=0.992, Q2=0.968). There were 43 and 91 volatile compounds identified by GC-MS and GC-IMS, respectively. A satisfactory discrimination (R2Y=0.991, and Q2=0.966) was achieved by using PLS-DA based on the volatile fingerprints of GC-IMS. Moreover, according to the multivariate analysis of VIP>1.2 and univariate analysis of p<0.05, 9 volatile components such as linalool and (E)-2-hexenal were selected as key variables to distinguish Pu-erh teas with different storage years. The results provide theoretical support for the quality control of Pu-erh tea.

A Nomogram Model for Predicting the Polyphenol Content of Pu-Erh Tea.

To investigate different contents of pu-erh tea polyphenol affected by abiotic stress, this research determined the contents of tea polyphenol in teas produced by Yuecheng, a Xishuangbanna-based tea producer in Yunnan Province. The study drew a preliminary conclusion that eight factors, namely, altitude, nickel, available cadmium, organic matter, N, P, K, and alkaline hydrolysis nitrogen, had a considerable influence on tea polyphenol content with a combined analysis of specific altitudes and soil composition. The nomogram model constructed with three variables, altitude, organic matter, and P, screened by LASSO regression showed that the AUC of the training group and the validation group were respectively 0.839 and 0.750, and calibration curves were consistent. A visualized prediction system for the content of pu-erh tea polyphenol based on the nomogram model was developed and its accuracy rate, supported by measured data, reached 80.95%. This research explored the change of tea polyphenol content under abiotic stress, laying a solid foundation for further predictions for and studies on the quality of pu-erh tea and providing some theoretical scientific basis.

Mineral content and antioxidant potential of Pu-erh tea of different storage periods

Pu-erh tea is a health-promoting beverage that is consumed in many countries; however, the nutritional value of the tea is significantly affected by various factors including processing and storage. The objective of this study was to investigate the mineral contents and antioxidant potentials of 2-, 9-, and 21-year-old Pu-erh teas. The results demonstrated that the total mineral content of fermented tea (FT) was higher than those of raw tea (RT) after 9 years and 21 years. The 9-year-old FT had the highest K content, while the 2-year-old FT had the lowest K content among the FT samples. In contrast, the 2-year-old RT had the highest K content, while that of the 21-year-old RT was the lowest among the RT samples. Interestingly, the 9-year-old RT had the highest Na, Ca, Mg, and Cu contents among the all of the sampels. Overall, these results reveal that Pu-erh tea stored for 9 years would be the most beneficial with regards to the mineral content and antioxidant potential. Furthermore, this study provides valuable insights into the changes that occur in the nutrient content of Pu-erh tea during storage.

Revealing the influence of microbiota on the flavor of kombucha during natural fermentation process by metagenomic and GC-MS analysis

In this work, raw Pu-erh tea (RAPT) was employed for kombucha preparation, and the microbial composition and volatile flavor compounds of the fermented tea had been investigated during natural fermentation process. The head space-solid phase microextraction-gas chromatograph mass spectrometry (HS-SPME-GC-MS) was performed for volatiles analysis of unfermented tea and kombucha fermented for 3 days (KF-3) and 6 days (KF-6). Meanwhile, the microbial community of KF-3 and KF-6 were evaluated by metagenomic analysis. A total of 72 volatile compounds were identified and obvious changes in volatiles were observed during the fermentation process based on the results of GC-MS and principal component analysis (PCA). Metagenomic sequencing analysis demonstrated that bacterium Komagataeibacter saccharivorans and unclassified-g-komagataeibacter and yeast Saccharomyces cerevisiae and Brettanomyces bruxellensis were the most common microbes contained in the sampled kombucha communities. Furthermore, the relevance among microbial community and volatile compounds was evaluated through correlation heatmap analysis. The results suggested that the main flavor volatiles of kombucha (i.e., acids, esters and terpenes) were closely related to species of genus Komagataeibacter, Gluconacetobacter, Saccharomyces, Brettanomyces, Acetobacter, Novacetimonas and Pichia microorganisms. The obtained results would help to better understand microbial communities and volatile compounds of kombucha, which could provide useful information for enhancing the flavor quality of kombucha products.

A novel method for Pu-erh tea face traceability identification based on improved MobileNetV3 and triplet loss

Ensuring the traceability of Pu-erh tea products is crucial in the production and sale of tea, as it is a key means to ensure their quality and safety. The common approach used in traceability systems is the utilization of bound Quick Response (QR) codes or Near Field Communication (NFC) chips to track every link in the supply chain. However, counterfeiting risks still persist, as QR codes or NFC chips can be copied and inexpensive products can be fitted into the original packaging. To address this issue, this paper proposes a tea face verification model called TeaFaceNet for traceability verification. The aim of this model is to improve the traceability of Pu-erh tea products by quickly identifying counterfeit products and enhancing the credibility of Pu-erh tea. The proposed method utilizes an improved MobileNetV3 combined with Triplet Loss to verify the similarity between two input tea face images with different texture features. The recognition accuracy of the raw tea face dataset, ripe tea face dataset and mixed tea face dataset of the TeaFaceNet network were 97.58%, 98.08% and 98.20%, respectively. Accurate verification of tea face was achieved using the optimal threshold. In conclusion, the proposed TeaFaceNet model presents a promising approach to enhance the traceability of Pu-erh tea products and combat counterfeit products. The robustness and generalization ability of the model, as evidenced by the experimental results, highlight its potential for improving the accuracy of Pu-erh tea face recognition and enhancing the credibility of Pu-erh tea in the market. Further research in this area is warranted to advance the traceability of Pu-erh tea products and ensure their quality and safety.

The Influence of the Preparation of Tea Infusion on the Content of Fluoride and the Assessment of Health Risk for the Consumer in Poland

Tea infusion is the most popular drink in Poland, and the tea bush is the most fluoride-rich among plants. Excessive fluoride intake can adversely affect human health by causing dental or skeletal fluorosis. This study aimed to determine the fluoride content in tea infusion depending on the tea types, the degree of fragmentation of the leaves, brewing time, and conducting a health risk assessment for the consumer in Poland. Infusions of 28 popular teas (green leafy tea, pu-erh leafy tea, white leaf tea, black leaf tea, black granulated tea, and black in bags) available in the Polish market were analyzed concerning a fluoride amount using the potentiometric method. Tea leaf size is an important parameter affecting fluoride’s release during tea brewing. An increase in fluoride content in the infusion was also shown with the extension of the brewing time. It was also revealed that significantly higher fluoride content was found in infusions of black tea prepared from tea in bags than from “loose” leaves. The conducted health risk assessment indicates the possibility of adverse health effects caused by fluoride consumption, particularly from infusions of black and green tea purchased in bags.