Pile Fermentation Research Articles

Effects of Microbial Proteins on Qingzhuan Tea Sensory Quality during Pile Fermentation.

To determine the effects of microbial proteins on Qingzhuan tea sensory quality during tea pile fermentation, tea leaf metabolomic and microorganism proteomic analyses were performed. In total, 1835 differential metabolites and 443 differentially expressed proteins of the microorganisms were identified. Correlation analysis between metabolomics and proteomics data revealed that the levels of microbial proteins EG II and CBH I cellulase may play important roles in cell wall construction and permeability, which were crucial for the interaction between tea leaves and microorganisms. Microbial proteins heat shock proteins (HSP), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and CuAO related to detoxification and stress responses showed a positive correlation with tea theanine, glutamine, γ-aminobutyric acid, glutamic acid, catechin, (-)-gallocatechin gallate, and (-)-catechin gallate, suggesting their effects on tea characteristic compound accumulation, thus affecting Qingzhuan tea sensory quality.

Insights into the airborne microorganisms in a Sichuan south-road dark tea pile fermentation plant during production.

Sichuan south-road dark tea (SSDT) is generally produced through a series of processes, including fixing, rolling, pile fermentation, and drying, with microbial action during pile fermentation playing a crucial role in determining tea quality. The air within the SSDT pile fermentation plant (SSDTPP) is considered an important source of these microbes, but research in this area has been limited. In this study, air samples from SSDTPP were collected on the 1st (SSDT1), 12th (SSDT2), and 24th (SSDT3) days of pile fermentation and comprehensively analyzed by high-throughput sequencing. The results revealed the presence of 2 and 24 phyla, 9 and 49 classes, 18 and 88 orders, 28 and 153 families, 38 and 253 genera, and 47 and 90 species of fungi and bacteria, respectively, across all samples. SSDT1 and SSDT2 individually had the highest fungal and bacterial diversity, while Aspergillus was the dominant genus throughout the pile fermentation with an abundance of 34.6%, 91.17%, and 67.86% in SSDT1, SSDT2, and SSDT3, respectively. Microbial populations in SSDT1 were predominantly involved in xenobiotic biodegradation and metabolism, amino acid metabolism, the biosynthesis of other secondary metabolites, etc. However, SSDT2 exhibited a higher prevalence of human disease-related functions. SSDT3 primarily focused on the metabolism of other amino acids and carbohydrate metabolism. Additionally, 104 genera and 22 species coexisted in both SSDTPP air and piled SSDT, suggesting that frequent microbial exchange may occur between them. These findings pave the way for microbial traceability during SSDT production and provide a foundation for further functional microbial research.

Co-occurrence network and functional profiling of the bacterial community in the industrial pile fermentation of Qingzhuan tea: Understanding core functional bacteria

The distinct quality of Qingzhuan tea is greatly influenced by the bacterial community but was poorly characterized. Therefore, this study investigated the Co-occurrence network and functional profiling of the bacterial community, with special attention paid to core functional bacteria in the industrial pile fermentation. Microbiomics analysis indicated that Klebsiella and Pantoea dominated raw tea leaves, and were rapidly replaced by Pseudomonas in pile fermentation, but substituted mainly by Burkholderia and Saccharopolyspora in final fermented tea. Bacterial taxa were grouped into 7 modules with the dominant in module I, III, and IV, which were involved in flavor formation and biocontrol production. Functional profiling revealed that “penicillin and cephalosporin biosynthesis” increased in pile fermentation. Twelve bacterial genera were identified as core functional bacteria, in which Klebsiella, Pantoea, and Pseudomonas also dominated the pile fermentation. This work would provide theoretical basis for its chemical biofortification and quality improvement by controlling bacterial communities.

Degradation of Cell Wall Polysaccharides during Traditional and Tank Fermentation of Chinese Liupao Tea.

The increase of polysaccharides in the dark tea pile process is thought to be connected to the cell wall polysaccharides' breakdown. However, the relationship between tea polysaccharides (TPSs) and tea cell wall polysaccharides has not been further explored. In this study, the structural changes in the cell wall polysaccharides [e.g., cellulose, hemicellulose (HC), and pectin] in Liupao tea were characterized before and after traditional fermentation and tank fermentation. Additionally, the degradation mechanism of tea cell wall polysaccharides during fermentation was assessed. The results showed that cellulose crystallinity decreased by 11.9-49.6% after fermentation. The molar ratio of monosaccharides, such as arabinose, rhamnose, and glucose in HC, was significantly reduced, and the molecular weight decreased. The esterification degree and linearity of water-soluble pectin (WSP) were reduced. TPS content increases during pile fermentation, which may be due to HC degradation and the increase in WSP caused by cell wall structure damage. Microorganisms were shown to be closely associated with the degradation of cell wall polysaccharides during fermentation according to correlation analyses. Traditional fermentation had a greater effect on the cellulose structure, while tank fermentation had a more noticeable impact on HC and WSP.

Exploring core functional fungi driving the metabolic conversion in the industrial pile fermentation of Qingzhuan tea

The distinct sensory quality of Qingzhuan tea is mainly formed in pile fermentation by a group of functional microorganisms but the core functional ones was poorly characterized. Therefore, this study investigated the dynamic changes in the fungal community and metabolic profile by integrating microbiomics and metabolomics, and explored the core functional fungi driving the metabolic conversion in the industrial pile fermentation of Qingzhuan tea. Indicated by microbiomics analysis, Aspergillus dominated the entire pile-fermentation process, while Thermoascus, Rasamsonia, and Cylindrium successively abounded in the different stages of the pile fermentation. A total of 50 differentially changed metabolites were identified, with the hydrolysis of galloyl/polymeric catechins, biosynthesis of theabrownins, oxidation of catechins, N-ethyl-2-pyrrolidinone substitution of catechins, and deglycosylation of flavonoid glucosides. Nine fungal genera were identified as core functional fungi, in which Aspergillus linked to the hydrolysis of polymeric catechins and insoluble polysaccharides as well as biosynthesis of theabrownins, while Thermoascus participated in the biosynthesis of theabrownins, deglycosylation of flavonoid glucosides, and N-ethyl-2-pyrrolidinone substitution of catechins. These findings would advance our understanding of the quality formation of Qingzhuan tea and provide a benchmark for precise inoculation for its quality improvement.

Deciphering the underlying core microorganisms and the marker compounds of Liupao tea during the pile-fermentation process.

Pile fermentation is one of the key steps in developing the Liupao tea (LBT) quality and unique characteristics. The complex biochemical profile of LBT results from microorganisms present during the pile-fermentation process. However, the critical underlying microorganisms and the marker compounds still need to be determined. Staphylococcus, Brevibacterium, Kocuria, Aspergillus, and Blastobotrys were the common dominant microorganisms at the end of the pile fermentation of LBT. Staphylococcus, Aspergillus, Blastobotrys, and nine other genera carried by raw tea are the core microorganisms in the LBT during pile fermentation. A total of 29 critical compounds contributed to the metabolic changes caused by the processing of LBT. Of these, gallic acid, adenine, hypoxanthine, uridine, betaine, 3,4-dihydroxybenzaldehyde, and α-linolenic acid could be characterized as potential marker compounds. Correlation analysis showed that the core microorganisms, including Sphingomonas, Staphylococcus, Kocuria, Aureobasidium, Blastobotrys, Debaryomyce, and Trichomonascus, were closely related to major chemical components and differential compounds. Moreover, the mutually promoting Staphylococcus, Kocuria, Blastobotrys, and Trichomonascus were correlated with the enrichment of marker compounds. Integrated molecular networking and metabolic pathways revealed relevant compounds and enzymes that possibly affect the enrichment of marker compounds. This study analyzed the LBT fermentation samples by omics analysis to reveal the stable microbial community structure, critical microorganisms, and markers compounds affecting the quality of LBT, which contributes to a better understanding of pile fermentation of LBT and the fermentation theory of dark tea. © 2023 Society of Chemical Industry.

Dynamic Changes in the Microbial Community and Metabolite Profile during the Pile Fermentation Process of Fuzhuan Brick Tea.

The pile fermentation process of Fuzhuan brick tea is unique in that it involves preheating without the use of starter cultures. The detailed metabolite changes and their drivers during this procedure are not known. Characterizing these unknown changes that occur in the metabolites and microbes during pile fermentation of Fuzhuan brick tea is important for industrial modernization of this traditional fermented food. Using microbial DNA amplicon sequencing, mass spectrometry-based untargeted metabolomics, and feature-based molecular networking, we herein reveal that significant changes in the microbial community occur before changes in the metabolite profile. These changes were characterized by a decrease in Klebsiella and Aspergillus, alongside an increase in Bacillus and Eurotium. The decrease in lysophosphatidylcholines, unsaturated fatty acids, and some astringent flavan-3-ols and bitter amino acids, as well as the increase in some less astringent flavan-3-ols and sweet or umami amino acids, contributed importantly to the overall changes observed in the metabolite profile. The majority of these changes was caused by bacterial metabolism and the corresponding heat generated by it.

Microbial Diversity and Characteristic Quality Formation of Qingzhuan Tea as Revealed by Metagenomic and Metabolomic Analysis during Pile Fermentation.

In order to analyze the changes in the microbial community structure during the pile fermentation of Qingzhuan tea and their correlation with the formation of quality compounds in Qingzhuan tea, this study carried out metagenomic and metabolomic analyses of tea samples during the fermentation process of Qingzhuan tea. The changes in the expression and abundance of microorganisms during the pile fermentation were investigated through metagenomic assays. During the processing of Qingzhuan tea, there is a transition from a bacterial dominated ecosystem to an ecosystem enriched with fungi. The correlation analyses of metagenomics and metabolomics showed that amino acids and polyphenol metabolites with relatively simple structures exhibited a significant negative correlation with target microorganisms, while the structurally complicated B-ring dihydroxy puerin, B-ring trihydroxy galloyl puerlin, and other compounds showed a significant positive correlation with target microorganisms. Aspergillus niger, Aspergillus glaucus, Penicillium in the Aspergillaceae family, and Talaromyces and Rasamsonia emersonii in Trichocomaceae were the key microorganisms involved in the formation of the characteristic qualities of Qingzhuan tea.

Isolation, identification, and community diversity of microorganisms during tank fermentation of Liupao tea.

Tank fermentation is a novel approach to fermenting teas; however, the species of microorganisms present remain unclear. The microbial community composition of Liupao tea at various stages of tank fermentation was analyzed using high-throughput sequencing. Sphingomonas, Aquabacterium, Pelomonas, Acinetobacter, Blastobotrys, Aspergillus, Debaryomyces, and Aureobasidium were the predominant genera, which is different from pile fermentation. Fifteen genera (including Lactobacillus, Debaryomyces, Candida, Allobaculum, Flavobacterium, Caulobacter, Blastobotrys, Aspergillus, and Rasamsonia) were identified as biomarkers. PICRUSt analysis predicted that the most abundant functional genes were related to metabolism of carbohydrates, amino acids, cofactors, vitamins, and other secondary metabolites. Using the pure culture method, 283 strains were isolated at various stages of fermentation, representing 20 genera and 29 species of bacteria, and 11 genera and 18 species of fungi. Most of the dominant Sphingomonas, Staphylococcus, Aspergillus, and Blastobotrys identified by sequencing were also isolated. Of these, Sphingomonas olei, Aspergillus luchuensis, Aspergillus niger, Aspergillus aculeatus, Aspergillus amstelodami, Blastobotrys adeninivorans, Candida metapsilosis, and Candida blankii were beneficial strains that might be used to ferment Liupao tea. This study provides a basis for the development of processing technologies and utilization of microbial strains in the production of dark teas. PRACTICAL APPLICATION: Microbial diversity in tank-fermented Liupao tea was reported for the first time. 8 microorganisms were the predominant genera. The species, functions and potential risks of microorganisms was revealed. We clarified the differences between tank and pile fermentation.

Metagenomics reveal the role of microorganism and GH genes contribute to Sichuan South-road dark tea quality formation during pile fermentation

Microbes are critical for the flavor development of Sichuan South-road Dark Tea (SSDT) during pile-fermentation. However, their mechanism of action has not been fully elucidated yet. In this study, the SSDT microbial metagenome obtained from the different locations of piled tea center during the pile-fermentation metaphase were functionally annotated, and the glycoside hydrolase (GH) genes of the core functional strain were comprehensively analyzed. The results revealed that carbohydrate metabolism (8.2%) were predominant in the total SSDT microbial metagenome. GHs (39.8%) and glycosyl transferases (GTs, 37.3%) were abundant in the SSDT microbes. Moreover, Aspergillus niger was prevailing in SSDT, with 220 GH genes being distributed in 43 GH subfamilies and GH3, GH5, GH13, GH16, GH18, and GH28 being highly abundant. The expression patterns of the GH genes of Aspergillus niger M10 varied among different stages of submerged and solid-state fermentation. Notably, ANI_1_1714074 (xylosidase gene belongs to GH31) and ANI_1_2704024 (β-N-acetylglucosaminidase gene belongs to GH3) might be the potential functional genes degrading polysaccharides in Aspergillus niger M10. It was hypothesized that SSDT microbes, especially Aspergillus niger M10, have an abundant repertoire of carbohydrate-degrading enzymes, and may actively participate in tea taste formation through GHs during pile-fermentation.

Effects of pile fermentation on the physicochemical, functional, and biological properties of tea polysaccharides

This study investigated the influence of pile fermentation on the physicochemical, functional, and biological properties of tea polysaccharides (TPS). Results indicated that the extraction yield, uronic acid content, and polyphenol content of TPS greatly increased from 1.8, 13.1 and 6.3 % to 4.1, 27.9, and 7.8 %, respectively, but the molecular weight markedly decreased from 153.7 to 76.0 kDa after pile fermentation. Additionally, the interfacial, emulsion formation, and emulsion stabilization properties of TPS were significantly improved after pile fermentation. For instance, 1.0 wt% TPS isolated from dark tea (D-TPS) can fabricate 8.0 wt% MCT oil-in-water nanoemulsion (d32 ≈ 159 nm) with potent storage stability. Moreover, the antioxidant and α-glucosidase inhibitory activities of D-TPS was higher than that of TPS isolated from sun-dried raw tea (R-TPS). Overall, this study indicated that pile fermentation markedly affected the physicochemical and structural characteristics of TPS, thereby improving their functional and biological properties.

Qualitative and quantitative analysis of the pile fermentation degree of Pu-erh tea

During the pile fermentation (PF) process, changes to the polyphenol composition directly affect the quality of Pu-erh tea. In this study, we have applied laboratory-made computer vision system (CVS) and miniature near-infrared spectroscopy (NIRS) to the at-line rapid detection of the PF degree of Pu-erh tea at an industrial scale. High-performance liquid chromatography was used for determining the content of catechins (EGCG, EGC …) and gallic acid (GA). Based on the least-square support vector machine (LSSVM) qualitative model analysis, the texture features and color information extracted by CVS better predicted the degree of PF with a prediction set of 99.30% and calibration set of 100.00% compared to the spectral information extracted by NIRS. The best quantitative models for total catechins (TC), GA/TC, and red and green values of the tea infusion (R-TI, G-TI) were obtained with residual prediction deviations (RPD) of 4.76, 2.36, 5.18, and 4.71, respectively, based on CVS fusion data. And the optimal PF degree of Pu-erh tea was defined when the predicted values of TC, GA/TC, R-TI, and G-TI were in the ranges of 0.46 ± 0.08 mg/g, 19.04 ± 6.67, 74.81 ± 6.37, and 29.81 ± 2.46, respectively. In-situ quality monitoring of Pu-erh tea PF was realized.

Lipid metabolic characteristics and marker compounds of ripened Pu-erh tea during pile fermentation revealed by LC-MS-based lipidomics

Ripened Pu-erh tea (RPT) is a unique microbial fermented tea. Herein, we investigated the lipid composition of RPT and its metabolic changes during pile fermentation, by nontargeted lipidomics profiling and quantitative analysis using liquid chromatography-mass spectrometry (LC-MS). A total of 485 individual lipid species covering 26 subclasses were detected, and fatty acid ester of hydroxy fatty acid (FAHFA) was detected in tea for the first time. Among them, 362 species were significantly altered during fermentation. Chlorophylls decomposition, phospholipids degradation (especially phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine), formation of free fatty acid (FFA) (especially FFA18:3, FFA18:2), and formation of FAHFA, were annotated as the key pathways. Particularly, FAHFAs were undetected in raw tea and gradually enriched to 227.0 ± 9.6 nmol/g after fermentation (p < 0.001), which could serve as marker compounds of RPT associated with microbial fermentation. This study will advance understanding the lipid metabolic fate in microbial fermentation and its role in RPT quality.Chemical compounds studied in this article: Linolenic acid (PubChem CID: 5280934); Linoleic acid (PubChem CID: 5280450); Oleic acid (PubChem CID: 445639); PS(22:0/18:2) (PubChem CID: 52925820); PS(20:0/18:3) (PubChem CID: 52925629); Pheophytin a (PubChem CID: 135398712); Pheophorbide a (PubChem CID: 253193)

Microflora for improving the Auricularia auricula spent mushroom substrate for Protaetia brevitarsis production

SummaryMushroom cultivation is a sustainable agricultural waste utilization method, but the lack of high-value utilization of the produced spent mushroom substrate (SMS) has hindered the development of mushroom cultivation-based circular agricultural systems. Conversion and utilization of SMS via Protaetia brevitarsis larvae (PBL) have proven to be a high-value AASMS utilization strategy. However, Auricularia auricula SMS (AASMS), which contains woodchips, is less palatable and digestible for PBL. To solve this problem, in this investigation, we screened out microflora (MF) for AASMS fermentation by comparing the fermentation performance as well as the effect on PBL feed intake, weight gain, and AASMS phytotoxic compound removal efficiency. In addition, by bacterial community analysis, the genera Luteimonas, Moheibacter, and Pseudoxanthomonas were predicted to be functional bacteria for AASMS fermentation and contribute to palatability and digestibility improvement.

Mellow and Thick Taste of Pu-Erh Ripe Tea Based on Chemical Properties by Sensory-Directed Flavor Analysis.

The mellow and thick taste is a unique characteristic of pu−erh ripe tea infusion, and it is closely related to the chemical composition of pu−erh ripe tea, which is less studied. This paper clarifies and compares the chemical composition of pu−erh ripe tea to that of the raw materials of sun−dried green tea, and uses membrane separation technology to separate pu−erh ripe tea into the rejection liquid and the filtration liquid. The results show that microorganisms transformed most physicochemical components, except caffeine, during the pile fermentation. It was found that total tea polyphenols, soluble proteins, total soluble sugars, theabrownin, and galloylated catechins became enriched in the rejection liquid, and the rejection liquid showed a more obvious mellow and thick characteristic. Taste interactions between crude protein, crude polysaccharide, and theabrownin were determined. They illustrated that the mellow and thick taste of pu−erh ripe tea with the addition of theabrownin increased from 4.45 to 5.13. It is of great significance to explore the chemical basis of the mellow and thick taste in pu−erh tea for guiding the pu−erh tea production process and for improving the quality of pu−erh tea.

Penicillium simplicissimum possessing high potential to develop decaffeinated Qingzhuan tea

The excessive intake of caffeine might adversely affect health, so decaffeinated food becomes a hot topic for investigation. Penicillium simplicissimum 4–17 was inoculated into sun-dried green tea for the pile fermentation (QZTWP) at a pilot scale to develop decaffeinated Qingzhuan tea (QZT). Through comparisons in chemical compositions, fungal community structure and sensory qualities, the results showed that after 21-d pile fermentation, most quality components in QZTWP were same or similar to those in naturally fermented QZT, except caffeine. Caffeine decreased nearly 50%, and some quality components were better in QZTWP than those in naturally fermented QZT. The addition of P. simplicissimum 4–17 couldn't change the dominant strains in the pile fermentation of QZT, but was conducive to increasing the amount of Saccharomyces, Candida, and Penicillium and to inhibiting the unfavorable strains. The results confirmed that P. simplicissimum 4–17 not only contributed high caffeine degradation ability of pilot fermentation of QZT, but also helped to improve the comprehensive quality of QZT. It means that there is a great potential of P. simplicissimum 4–17 for industrial production of decaffeinated QZT.

Microbial and Nonvolatile Chemical Diversities of Chinese Dark Teas Are Differed by Latitude and Pile Fermentation.

Understanding the microbial and chemical diversities, as well as what affects these diversities, is important for modern manufacturing of traditional fermented foods. In this work, Chinese dark teas (CDTs) that are traditional microbial fermented beverages with relatively high sample diversity were collected. Microbial DNA amplicon sequencing and mass spectrometry-based untargeted metabolomics show that the CDT microbial β diversity, as well as the nonvolatile chemical α and β diversities, is determined by the primary impact factors of geography and manufacturing procedures, in particular, latitude and pile fermentation after blending. A large number of metabolites sharing between CDTs and fungi were discovered by Feature-based Molecular Networking (FBMN) on the Global Natural Products Social Molecular Networking (GNPS) web platform. These molecules, such as prenylated cyclic dipeptides and B-vitamins, are functionally important for nutrition, biofunctions, and flavor. Molecular networking has revealed patterns in metabolite profiles on a chemical family level in addition to individual structures.

Dynamic evolution and correlation between microorganisms and metabolites during manufacturing process and storage of Pu-erh tea

The quality and safety of Pu-erh tea are largely determined by microorganisms, processing and storage conditions. The systemic co-relationship between microbiome and metabolomics during manufacturing of Pu-erh tea is not fully clarified. We comprehensively explored the microbial and metabolic dynamic evolution and revealed the decisive factors for the quality and safety of Pu-erh tea based on creative methods. The results showed that fungal abundance increased in the middle of pile fermentation period and then decreased during storage, but the bacterial abundance increased in the initial stage, subsequently decreased and finally increased. All microorganism species as well as dominant fungi and bacteria were identified during different processing steps. A total of 209 metabolites were identified and 50 of them were characterized as critical metabolites responsible for metabolic changes. Multivariate analysis verified that these critical metabolites were generated by specific dominant species. The environmental condition exhibited a great effect on the growth and reproduction of bacteria, but less than 10% of the dominant fungi exhibited significant correlation with temperature and humidity. In conclusion, our results revealed the microbial composition is the critical factor in changing the metabolic profile and provided a theoretical basis for improving the quality and safety of Pu-erh tea.

Chemical profile of a novel ripened Pu-erh tea and its metabolic conversion during pile fermentation

Ripened Pu-erh tea is a unique tea type produced from microbial fermentation. Recently, a novel ripened Pu-erh tea (NPT) produced using a patented pile fermentation method has become increasingly popular due to its improved flavor and enriched bioactive gallic acid (GA). However, the detailed chemical features of NPT and their formation during pile fermentation remain unclear. Herein, untargeted metabolomics revealed enrichment of GA, amino acids, free sugars and reduction in catechins and flavonol glycosides in NPT. Mainly, GA was 1.99 times higher in NPT than traditional Pu-erh tea (p < 0.001). The metabolic changes were tracked during pile fermentation, and possible pathways were mapped. GA enrichment may be produced from enhanced hydrolysis of galloyl catechins and phenolic acid esters. Degradation of flavonol glycosides and formation of other metabolites were observed. This study will advance our understanding of conversions during pile fermentation and provide new insights into directional manufacturing of high-quality ripened tea.

Dynamic changes in the aroma profile of Qingzhuan tea during its manufacture

Changes in key odorants and aroma profiles of Qingzhuan tea (QZT) during its manufacture were determined using headspace solid-phase microextraction gas chromatography-mass spectrometry/olfactometry. An aroma profile was constructed to illustrate sensory changes during manufacture. The characteristic aroma of QZT was aged fragrance, which was mostly developed during pile fermentation and was enhanced during the aging and drying stages. Using volatile compounds found in the raw materials, sun-dried green tea and QZT finished product were compared by orthogonal partial least square-discriminant analysis. Among 108 detected volatiles, 19 were significantly upregulated and 15 were downregulated. (E)-β-Ionone, (E,Z)-2,6-nonadienal, 1-octen-3-one, (E,E)-2,4-heptadienal, (E,E)-2,4-nonadienal, safranal, (E)-2-nonenal, α-ionone, and 1,2,3-trimethoxybenzene were found to be significant contributors to the aged QZT fragrance, reflecting their high odor-activity values and aroma intensities. Finally, the metabolic transformation of key aroma-active compounds was systematically analyzed. This study provided a theoretical basis for improving the processing and quality of QZT.